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1
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Lee MA, Brown JS, Farquhar CE, Loas A, Pentelute BL. Affinity selection-mass spectrometry with linearizable macrocyclic peptide libraries. SCIENCE ADVANCES 2025; 11:eadr1018. [PMID: 40106557 PMCID: PMC11922053 DOI: 10.1126/sciadv.adr1018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/14/2024] [Accepted: 02/11/2025] [Indexed: 03/22/2025]
Abstract
Despite their potential, the preparation of large synthetic macrocyclic libraries for ligand discovery and development has been limited. Here, we produce 100-million-membered macrocyclic libraries containing natural and nonnatural amino acids. Near-quantitative intramolecular disulfide formation is facilitated by rapid oxidation with iodine in solution. After use in affinity selection, treatment with dithiothreitol enables near-quantitative reduction, rendering linear peptide analogs for standard tandem mass spectrometry. We use these libraries to discover macrocyclic binders to cadherin-2 and anti-hemagglutinin antibody clone 12ca5. Structure-activity relationship studies of an initial cadherin-binding peptide [CBP; apparent dissociation constant (Kd) = 53 nanomolar] reveal residues responsible for driving affinity (hotspots) and mutation-tolerant residues (coldspots). Two original macrocyclic libraries are prepared in which these hotspots and coldspots are derivatized with nonnatural amino acids. Following discovery and validation, high-affinity ligands are discovered from the coldspot library, with NCBP-4 demonstrating improved affinity (Kd = 29 nanomolar). Overall, we expect that this work will improve the use of macrocyclic libraries in therapeutic peptide development.
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Affiliation(s)
- Michael A. Lee
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Joseph S. Brown
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Charlotte E. Farquhar
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Andrei Loas
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Bradley L. Pentelute
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
- Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
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2
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Brown JS, Lee MA, Vuong W, Loas A, Pentelute BL. pyBinder: Quantitation to Advance Affinity Selection-Mass Spectrometry. Anal Chem 2025; 97:3855-3863. [PMID: 39949296 DOI: 10.1021/acs.analchem.4c04445] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/26/2025]
Abstract
Affinity selection-mass spectrometry (AS-MS) is a ligand discovery platform that relies upon mass spectrometry to identify molecules bound to a biomolecular target. When utilized with large peptide libraries (108 members), AS-MS sample complexity can surpass the sequencing capacity of modern mass spectrometers, resulting in incomplete data, identification of few target-specific ligands, and/or incomplete sequencing. To address this challenge, we introduce pyBinder to perform quantitation on AS-MS data to process primary MS1 data and develop two scores to rank the peptides from the integration of their peak area: target selectivity and concentration-dependent enrichment. We benchmark pyBinder utilizing AS-MS data developed against antihemagglutinin antibody 12ca5, revealing that peptides that contain a motif known for target-specific high-affinity binding are well characterized by these two scores. AS-MS data from a second protein target, WD Repeat Domain 5 (WDR5), is analyzed to confirm the two pyBinder scores reliably capture the target-specific motif-containing peptides. From the results delivered by pyBinder, a list of target-selective features is developed and fed back into subsequent MS experiments to facilitate expanded data generation and the targeted discovery of selective ligands. pyBinder analysis resulted in a 4-fold increase in motif-containing sequence identification for WDR5 (from 3 to 14 ligands discovered), showing the utility of the two scores. This work establishes an improved approach for AS-MS to enable discovery outcomes (i.e., more ligands identified), but also a way to compare AS-MS data across samples, protocols, and conditions broadly.
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Affiliation(s)
- Joseph S Brown
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
| | - Michael A Lee
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
| | - Wayne Vuong
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
| | - Andrei Loas
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
| | - Bradley L Pentelute
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
- The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, United States
- Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States
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3
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Orsi M, Reymond J. Navigating a 1E+60 Chemical Space of Peptide/Peptoid Oligomers. Mol Inform 2025; 44:e202400186. [PMID: 39390672 PMCID: PMC11733718 DOI: 10.1002/minf.202400186] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2024] [Revised: 08/21/2024] [Accepted: 08/27/2024] [Indexed: 10/12/2024]
Abstract
Herein we report a virtual library of 1E+60 members, a common estimate for the total size of the drug-like chemical space. The library is obtained from 100 commercially available peptide and peptoid building blocks assembled into linear or cyclic oligomers of up to 30 units, forming molecules within the size range of peptide drugs and potentially accessible by solid-phase synthesis. We demonstrate ligand-based virtual screening (LBVS) using the peptide design genetic algorithm (PDGA), which evolves a population of 50 members to resemble a given target molecule using molecular fingerprint similarity as fitness function. Target molecules are reached in less than 10,000 generations. Like in many journeys, the value of the chemical space journey using PDGA lies not in reaching the target but in the journey itself, here by encountering non-obvious analogs. We also show that PDGA can be used to generate median molecules and analogs of non-peptide target molecules.
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Affiliation(s)
- Markus Orsi
- Department of ChemistryBiochemistry and Pharmaceutical SciencesUniversity of BernFreiestrasse 33012BernSwitzerland
| | - Jean‐Louis Reymond
- Department of ChemistryBiochemistry and Pharmaceutical SciencesUniversity of BernFreiestrasse 33012BernSwitzerland
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4
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Xing Y, Zhang H, Wang Y, Zong Z, Bogyo M, Chen S. DNA encoded peptide library for SARS-CoV-2 3CL protease covalent inhibitor discovery and profiling. RSC Chem Biol 2024; 5:691-702. [PMID: 38966676 PMCID: PMC11221529 DOI: 10.1039/d4cb00097h] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2024] [Accepted: 05/29/2024] [Indexed: 07/06/2024] Open
Abstract
Covalent protease inhibitors serve as valuable tools for modulating protease activity and are essential for investigating the functions of protease targets. These inhibitors typically consist of a recognition motif and a covalently reactive electrophile. Substrate peptides, featuring residues capable of fitting into the substrate pockets of proteases, undergo chemical modification at the carbonyl carbon of the P1 residue with an electrophile and have been widely applied in the development of covalent inhibitors. In this study, we utilized a DNA-encoded peptide library to replicate peptide binder sequences and introduced a vinyl sulfone warhead at the C-termini to construct the DNA-encoded peptide covalent inhibitor library (DEPCIL) for targeting cysteine proteases. Screening results toward 3CL protease demonstrated the efficacy of this library, not only in identifying protease inhibitors, but also in discovering amino acids that can conform to aligned protease pockets. The identified peptide sequences provide valuable insight into the amino acid preferences within substrate binding pockets, and our novel technology is indicative of the potential for similar strategies to discover covalent inhibitors and profile binding preferences of other proteases.
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Affiliation(s)
- Yuyu Xing
- Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences Shanghai 201203 China
- University of Chinese Academy of Sciences No. 19A Yuquan Road Beijing 100049 China
| | - Huiya Zhang
- Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences Shanghai 201203 China
| | - Yanhui Wang
- Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences Shanghai 201203 China
| | - Zhaoyun Zong
- Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences Shanghai 201203 China
| | - Matthew Bogyo
- Department of Pathology, Stanford University School of Medicine Stanford CA USA
| | - Shiyu Chen
- Biotech Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences Shanghai 201203 China
- University of Chinese Academy of Sciences No. 19A Yuquan Road Beijing 100049 China
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5
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Keller M, Petrov D, Gloger A, Dietschi B, Jobin K, Gradinger T, Martinelli A, Plais L, Onda Y, Neri D, Scheuermann J. Highly pure DNA-encoded chemical libraries by dual-linker solid-phase synthesis. Science 2024; 384:1259-1265. [PMID: 38870307 DOI: 10.1126/science.adn3412] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Accepted: 05/07/2024] [Indexed: 06/15/2024]
Abstract
The first drugs discovered using DNA-encoded chemical library (DEL) screens have entered late-stage clinical development. However, DEL technology as a whole still suffers from poor chemical purity resulting in suboptimal performance. In this work, we report a technique to overcome this issue through self-purifying release of the DEL after magnetic bead-based synthesis. Both the first and last building blocks of each assembled library member were linked to the beads by tethers that could be cleaved by mutually orthogonal chemistry. Sequential cleavage of the first and last tether, with washing in between, ensured that the final library comprises only the fully complete compounds. The outstanding purity attained by this approach enables a direct correlation of chemical display and encoding, allows for an increased chemical reaction scope, and facilitates the use of more diversity elements while achieving greatly improved signal-to-noise ratios in selections.
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Affiliation(s)
- Michelle Keller
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
| | - Dimitar Petrov
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
| | - Andreas Gloger
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
| | - Bastien Dietschi
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
| | - Kilian Jobin
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
| | - Timon Gradinger
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
| | | | - Louise Plais
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
| | - Yuichi Onda
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
| | - Dario Neri
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
| | - Jörg Scheuermann
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
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6
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Selicharová I, Fabre B, Soledad Garre Hernández M, Lubos M, Pícha J, Voburka Z, Mitrová K, Jiráček J. Combinatorial Libraries of Bipodal Binders of the Insulin Receptor. ChemMedChem 2024; 19:e202400145. [PMID: 38445366 DOI: 10.1002/cmdc.202400145] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2024] [Accepted: 03/05/2024] [Indexed: 03/07/2024]
Abstract
The binding process of insulin to its transmembrane receptor entails a sophisticated interplay between two proteins, each possessing two binding sites. Given the difficulties associated with the use of insulin in the treatment of diabetes, despite its remarkable efficacy, there is interest in smaller and more stable compounds than the native hormone that would effectively activate the receptor. Our study adopts a strategy focused on synthesizing extensive combinatorial libraries of bipodal compounds consisting of two distinct peptides linked to a molecular scaffold. These constructs, evaluated in a resin bead-bound format, were designed to assess their binding to the insulin receptor. Despite notable nonspecific binding, our approach successfully generated and tested millions of compounds. Rigorous evaluations via flow cytometry and specific antibodies revealed peptide sequences with specific interactions at either receptor binding Site 1 or 2. Notably, these sequences bear similarity to peptides discovered through phage display by other researchers. This convergence of chemical and biological methods underscores nature's beauty, revealing general principles in peptide binding to the insulin receptor. Overall, our study deepens the understanding of molecular interactions in ligand binding to the insulin receptor, highlighting the challenges of targeting large proteins with small synthetic peptides.
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Affiliation(s)
- Irena Selicharová
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 16610, Praha 6, Czech Republic
| | - Benjamin Fabre
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 16610, Praha 6, Czech Republic
| | - María Soledad Garre Hernández
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 16610, Praha 6, Czech Republic
| | - Marta Lubos
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 16610, Praha 6, Czech Republic
| | - Jan Pícha
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 16610, Praha 6, Czech Republic
| | - Zdeněk Voburka
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 16610, Praha 6, Czech Republic
| | - Katarína Mitrová
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 16610, Praha 6, Czech Republic
| | - Jiří Jiráček
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 16610, Praha 6, Czech Republic
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7
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Hansen T, Danková D, Bæk M, Grlaš L, Olsen CA. Sulfur(VI) Fluoride Exchange Chemistry in Solid-Phase Synthesis of Compound Arrays: Discovery of Histone Deacetylase Inhibitors. JACS AU 2024; 4:1854-1862. [PMID: 38818074 PMCID: PMC11134391 DOI: 10.1021/jacsau.4c00042] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/10/2024] [Revised: 03/18/2024] [Accepted: 03/19/2024] [Indexed: 06/01/2024]
Abstract
Multistep synthesis performed on solid support is a powerful means to generate small-molecule libraries for the discovery of chemical probes to dissect biological mechanisms as well as for drug discovery. Therefore, expansion of the collection of robust chemical transformations amenable to solid-phase synthesis is desirable for achieving chemically diverse libraries for biological testing. Here, we show that sulfur(VI) fluoride exchange (SuFEx) chemistry, exemplified by pairing phenols with aryl fluorosulfates, can be used for the solid-phase synthesis of biologically active compounds. As a case study, we designed and synthesized a library of 84 hydroxamic acid-containing small molecules, providing a rich source of inhibitors with diverse selectivity profiles across the human histone deacetylase enzyme family. Among other discoveries, we identified a scaffold that furnished inhibitors of HDAC11 with exquisite selectivity in vitro and a selective inhibitor of HDAC6 that was shown to affect the acetylation of α-tubulin over histone sites H3K18, H3K27, as well as SMC3 in cultured cells. Our results encourage the further use of SuFEx chemistry for the synthesis of diverse small-molecule libraries and provide insight for future design of selective HDAC inhibitors.
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Affiliation(s)
| | | | | | - Linda Grlaš
- Center for Biopharmaceuticals
and Department of Drug Design and Pharmacology, Faculty of Health
and Medical Sciences, University of Copenhagen, Jagtvej 160, DK-2100 Copenhagen, Denmark
| | - Christian A. Olsen
- Center for Biopharmaceuticals
and Department of Drug Design and Pharmacology, Faculty of Health
and Medical Sciences, University of Copenhagen, Jagtvej 160, DK-2100 Copenhagen, Denmark
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8
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Castanha PMS, McEnaney PJ, Park Y, Bouwer A, Chaves EJF, Lins RD, Paciaroni NG, Dickson P, Carlson G, Cordeiro MT, Magalhaes T, Craigo J, Marques ETA, Kodadek T, Burke DS. Identification and characterization of a nonbiological small-molecular mimic of a Zika virus conformational neutralizing epitope. Proc Natl Acad Sci U S A 2024; 121:e2312755121. [PMID: 38743628 PMCID: PMC11127016 DOI: 10.1073/pnas.2312755121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2023] [Accepted: 03/26/2024] [Indexed: 05/16/2024] Open
Abstract
Antigenic similarities between Zika virus (ZIKV) and other flaviviruses pose challenges to the development of virus-specific diagnostic tools and effective vaccines. Starting with a DNA-encoded one-bead-one-compound combinatorial library of 508,032 synthetic, non-natural oligomers, we selected and characterized small molecules that mimic ZIKV epitopes. High-throughput fluorescence-activated cell sorter-based bead screening was used to select molecules that bound IgG from ZIKV-immune but not from dengue-immune sera. Deep sequencing of the DNA from the "Zika-only" beads identified 40 candidate molecular structures. A lead candidate small molecule "CZV1-1" was selected that correctly identifies serum specimens from Zika-experienced patients with good sensitivity and specificity (85.3% and 98.4%, respectively). Binding competition studies of purified anti-CZV1-1 IgG against known ZIKV-specific monoclonal antibodies (mAbs) showed that CZV1-1 mimics a nonlinear, neutralizing conformational epitope in the domain III of the ZIKV envelope. Purified anti-CZV1-1 IgG neutralized infection of ZIKV in cell cultures with potencies comparable to highly specific ZIKV-neutralizing mAbs. This study demonstrates an innovative approach for identification of synthetic non-natural molecular mimics of conformational virus epitopes. Such molecular mimics may have value in the development of accurate diagnostic assays for Zika, as well as for other viruses.
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Affiliation(s)
- Priscila M. S. Castanha
- Department of Infectious Diseases and Microbiology, School of Public Health, University of Pittsburgh, Pittsburgh, PA15261
| | - Patrick J. McEnaney
- Department of Chemistry, The Herbert Wertheim University of Florida Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL33458
| | - Yongseok Park
- Department of Biostatistics, School of Public Health, University of Pittsburgh, Pittsburgh, PA15261
| | - Anthea Bouwer
- Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, PA15219
| | - Elton J. F. Chaves
- Department of Virology, Aggeu Magalhaes Institute, Oswaldo Cruz Foundation, Cidade Universitearia, Recife, Pernambuco50740-465, Brazil
| | - Roberto D. Lins
- Department of Virology, Aggeu Magalhaes Institute, Oswaldo Cruz Foundation, Cidade Universitearia, Recife, Pernambuco50740-465, Brazil
| | | | - Paige Dickson
- Department of Chemistry, The Herbert Wertheim University of Florida Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL33458
| | | | - Marli T. Cordeiro
- Department of Virology, Aggeu Magalhaes Institute, Oswaldo Cruz Foundation, Cidade Universitearia, Recife, Pernambuco50740-465, Brazil
| | - Tereza Magalhaes
- Department of Entomology, Texas A&M University, College Station, TX77843
- Department of Preventive and Social Medicine, School of Medicine, Universidade Federal da Bahia, Bahia40026-010, Brazil
| | - Jodi Craigo
- Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, PA15219
| | - Ernesto T. A. Marques
- Department of Infectious Diseases and Microbiology, School of Public Health, University of Pittsburgh, Pittsburgh, PA15261
- Department of Virology, Aggeu Magalhaes Institute, Oswaldo Cruz Foundation, Cidade Universitearia, Recife, Pernambuco50740-465, Brazil
| | - Thomas Kodadek
- Department of Chemistry, The Herbert Wertheim University of Florida Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL33458
| | - Donald S. Burke
- Department of Epidemiology, School of Public Health, University of Pittsburgh, Pittsburgh, PA15261
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9
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Kekessie I, Wegner K, Martinez I, Kopach ME, White TD, Tom JK, Kenworthy MN, Gallou F, Lopez J, Koenig SG, Payne PR, Eissler S, Arumugam B, Li C, Mukherjee S, Isidro-Llobet A, Ludemann-Hombourger O, Richardson P, Kittelmann J, Sejer Pedersen D, van den Bos LJ. Process Mass Intensity (PMI): A Holistic Analysis of Current Peptide Manufacturing Processes Informs Sustainability in Peptide Synthesis. J Org Chem 2024; 89:4261-4282. [PMID: 38508870 PMCID: PMC11002941 DOI: 10.1021/acs.joc.3c01494] [Citation(s) in RCA: 13] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2023] [Revised: 01/17/2024] [Accepted: 03/01/2024] [Indexed: 03/22/2024]
Abstract
Small molecule therapeutics represent the majority of the FDA-approved drugs. Yet, many attractive targets are poorly tractable by small molecules, generating a need for new therapeutic modalities. Due to their biocompatibility profile and structural versatility, peptide-based therapeutics are a possible solution. Additionally, in the past two decades, advances in peptide design, delivery, formulation, and devices have occurred, making therapeutic peptides an attractive modality. However, peptide manufacturing is often limited to solid-phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS), and to a lesser extent hybrid SPPS/LPPS, with SPPS emerging as a predominant platform technology for peptide synthesis. SPPS involves the use of excess solvents and reagents which negatively impact the environment, thus highlighting the need for newer technologies to reduce the environmental footprint. Herein, fourteen American Chemical Society Green Chemistry Institute Pharmaceutical Roundtable (ACS GCIPR) member companies with peptide-based therapeutics in their portfolio have compiled Process Mass Intensity (PMI) metrics to help inform the sustainability efforts in peptide synthesis. This includes PMI assessment on 40 synthetic peptide processes at various development stages in pharma, classified according to the development phase. This is the most comprehensive assessment of synthetic peptide environmental metrics to date. The synthetic peptide manufacturing process was divided into stages (synthesis, purification, isolation) to determine their respective PMI. On average, solid-phase peptide synthesis (SPPS) (PMI ≈ 13,000) does not compare favorably with other modalities such as small molecules (PMI median 168-308) and biopharmaceuticals (PMI ≈ 8300). Thus, the high PMI for peptide synthesis warrants more environmentally friendly processes in peptide manufacturing.
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Affiliation(s)
- Ivy Kekessie
- Early Discovery
Biochemistry - Peptide Therapeutics, Genentech,
Inc., A Member of the Roche Group, 1 DNA Way, South San Francisco, California 94080, United States
| | - Katarzyna Wegner
- Active Pharmaceutical
Ingredient Development, Ipsen Manufacturing
Ireland Ltd., Blanchardstown
Industrial Park, Dublin 15, Ireland
| | - Isamir Martinez
- Green Chemistry
Institute, American Chemical Society, 1155 16th St North West, Washington, District of Columbia, 20036, United
States
| | - Michael E. Kopach
- Synthetic
Molecule Design and Development, Eli Lilly
and Company, Indianapolis, Indiana 46285, United States
| | - Timothy D. White
- Synthetic
Molecule Design and Development, Eli Lilly
and Company, Indianapolis, Indiana 46285, United States
| | - Janine K. Tom
- Drug Substance
Technologies, Amgen, Inc., 1 Amgen Center Drive, Thousand
Oaks, California 91320, United States
| | - Martin N. Kenworthy
- Chemical
Development, Pharmaceutical Technology & Development, Operations, AstraZeneca, Macclesfield, SK10 2NA, United Kingdom
| | - Fabrice Gallou
- Chemical
& Analytical Development, Novartis Pharma
AG, 4056 Basel, Switzerland
| | - John Lopez
- Chemical
& Analytical Development, Novartis Pharma
AG, 4056 Basel, Switzerland
| | - Stefan G. Koenig
- Small
Molecule
Pharmaceutical Sciences, Genentech, Inc.,
A Member of the Roche Group, 1 DNA Way, South San Francisco, California 94080, United States
| | - Philippa R. Payne
- Outsourced
Manufacturing, Pharmaceutical Development & Manufacturing, Gilead Alberta ULC, 1021 Hayter Rd NW, Edmonton, T6S 1A1, Canada
| | - Stefan Eissler
- Bachem
AG, Hauptstrasse 144, 4416 Bubendorf, Switzerland
| | - Balasubramanian Arumugam
- Chemical
Macromolecule Division, Asymchem Life Science
(Tianjin) Co., Ltd., 71 Seventh Avenue, TEDA Tianjin 300457, China
| | - Changfeng Li
- Chemical
Macromolecule Division, Asymchem Life Science
(Tianjin) Co., Ltd., 71 Seventh Avenue, TEDA Tianjin 300457, China
| | - Subha Mukherjee
- Chemical
Process Development, Bristol Myers Squibb, New Brunswick, New Jersey 08903, United States
| | | | | | - Paul Richardson
- Chemistry, Pfizer, 10578 Science Center Drive (CB6), San Diego, California 09121, United States
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10
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Whitely C, Winburn H, Li Y. Synthesis and Identification of Heterobivalent Anticancer Compounds Containing Urea and 5-Arylidene-2-Thiohydantoin Motifs. ChemistrySelect 2024; 9:e202304688. [PMID: 38585446 PMCID: PMC10993625 DOI: 10.1002/slct.202304688] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2023] [Accepted: 02/27/2024] [Indexed: 04/09/2024]
Abstract
Urea and thiohydantoin are among the important privileged structures for drug discovery. We have developed a synthetic approach to the high-throughput synthesis of the heterobivalent compounds containing both urea and 5-arylidene-2-thiohydantoin functional groups. This synthetic methodology was applied to the synthesis of a mixture-based library containing a total of 5280 compounds in a positional scanning format. The library was screened for its antiproliferative activity against cancer cells using a tetrazolium dye (MTT) based assay. Deconvolution of the library identified six hit compounds exhibiting moderate inhibitory potency against cancer cell proliferation.
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Affiliation(s)
- Chelsi Whitely
- Department of Drug Discovery & Biomedical Sciences, University of South Carolina, 517 Sumter St., CLS 617, Columbia, SC 29208
| | - Haley Winburn
- Department of Drug Discovery & Biomedical Sciences, University of South Carolina, 517 Sumter St., CLS 617, Columbia, SC 29208
| | - Yangmei Li
- Department of Drug Discovery & Biomedical Sciences, University of South Carolina, 517 Sumter St., CLS 617, Columbia, SC 29208
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11
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Trier NH. Characterization of Peptide Antibodies by Epitope Mapping Using Resin-Bound and Soluble Peptides. Methods Mol Biol 2024; 2821:179-193. [PMID: 38997489 DOI: 10.1007/978-1-0716-3914-6_14] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/14/2024]
Abstract
Characterization of peptide antibodies through identification of their target epitopes is of utmost importance, as information about epitopes provide important knowledge, among others, for discovery and development of new therapeutics, vaccines, and diagnostics.This chapter describes a strategy for mapping of continuous peptide antibody epitopes using resin-bound and soluble peptides. The approach combines three different types of peptide sets for full characterization of peptide antibodies; (i) overlapping peptides, used to locate antigenic regions; (ii) truncated peptides, used to identify the minimal peptide length required for antibody binding; and (iii) substituted peptides, used to identify the key residues important for antibody binding and to determine the specific contribution of key residues. For initial screening, resin-bound peptides are used for epitope estimation, while soluble peptides subsequently are used for final epitope characterization and identification of critical hot spot residues. The combination of resin-bound peptides and soluble peptides for epitope mapping provides a time-saving and straightforward approach for characterization of antibodies recognizing continuous epitopes, which applies to peptide antibodies and occasionally antibodies directed to larger proteins as well.
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12
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Sikora H, Gruba N, Wysocka M, Piwkowska A, Lesner A. Optimization of fluorescent substrates for ADAM17 and their utility in the detection of diabetes. Anal Biochem 2023; 681:115337. [PMID: 37783443 DOI: 10.1016/j.ab.2023.115337] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2023] [Revised: 09/28/2023] [Accepted: 09/29/2023] [Indexed: 10/04/2023]
Abstract
ADAM17 (a disintegrin and metalloproteinase 17) is a sheddase that releases various types of membrane-associated proteins, including adhesive molecules, cytokines and their receptors, and inflammatory mediators. Evidence suggests that the enzyme is involved in the proteolytic cleavage of antiaging transmembrane protein Klotho (KL). What is more, reduced serum and urinary KL levels are observed in the early stages of chronic kidney disease. This study aimed to optimise the ADAM17 specific and selective fluorescent substrates. Then, the obtained substrate was used to detect the enzyme in urine samples of patients diagnosed with diabetes. It turned out that in all cases we were able to detect proteolytic activity, which was the opposite of the healthy samples.
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Affiliation(s)
- Honorata Sikora
- Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdańsk, Poland
| | - Natalia Gruba
- Department of Environmental Technology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63 Street, PL, 80-308, Gdańsk, Poland.
| | - Magdalena Wysocka
- Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdańsk, Poland
| | - Agnieszka Piwkowska
- Laboratory of Molecular and Cellular Nephrology, Mossakowski Medical Research Institute Polish Academy of Sciences, Wita Stwosza 63, 80-308, Gdansk, Poland; Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdansk, Poland
| | - Adam Lesner
- Department of Environmental Technology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63 Street, PL, 80-308, Gdańsk, Poland
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13
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Gruba N, Piwkowska A, Lesner A. Initial study of the detection of ADAM 10 in the urine of type-2 diabetic patients. Bioorg Chem 2023; 140:106826. [PMID: 37666108 DOI: 10.1016/j.bioorg.2023.106826] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2023] [Revised: 08/17/2023] [Accepted: 08/29/2023] [Indexed: 09/06/2023]
Abstract
Diabetes mellitus (DM) is a disease of civilization. If left untreated, it can cause serious complications and significantly shortens the life time. DM is one of the leading causes of end-stage renal disease (uremia) worldwide. Early diagnosis is a prerequisite for successful treatment, preferably before the first symptoms appear. In this paper, we describe the optimization and synthesis of the internally quenched fluorescent substrate disintegrin and metalloproteinase 10 (ADAM10). Using combinatorial chemistry methods with iterative deconvolution, the substrate specificity of the enzyme in non-primed and primed positions was determined. We used the ABZ-Lys-Ile-Ile-Asn-Leu-Lys-Arg-Tyr(3-NO2)-NH2 peptide to study ADAM10 activity in urine samples collected from patients diagnosed with type 2 diabetes, compared to urine samples from healthy volunteers. The proteolytically active enzyme was present in diabetes samples, while in the case of healthy people we did not observe any activity. In conclusion, our study provides a possible basis for further research into the potential role of ADAM10 in the diagnosis of type 2 diabetes.
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Affiliation(s)
- Natalia Gruba
- Department of Environmental Technology, Faculty of Chemistry University of Gdansk, Wita Stwosza 63 Street, PL 80-308 Gdańsk, Poland.
| | - Agnieszka Piwkowska
- Laboratory of Molecular and Cellular Nephrology, Mossakowski Medical Research Institute, Polish Academy of Sciences, Wita Stwosza 63, 80-308 Gdansk, Poland; Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland
| | - Adam Lesner
- Department of Environmental Technology, Faculty of Chemistry University of Gdansk, Wita Stwosza 63 Street, PL 80-308 Gdańsk, Poland
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14
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Huang H, Dong X, Sun Y, Shi Q. Biomimetic affinity chromatography for antibody purification: Host cell protein binding and impurity removal. J Chromatogr A 2023; 1707:464305. [PMID: 37607431 DOI: 10.1016/j.chroma.2023.464305] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Revised: 08/04/2023] [Accepted: 08/16/2023] [Indexed: 08/24/2023]
Abstract
Peptide affinity chromatography has received increasing attention as an alternative to protein A chromatography in antibody purification. However, its lower selectivity than protein A chromatography has impeded its success in practical applications. In particular, efficient removal of contaminants, including host cell proteins (HCPs) and DNA, is a great challenge for peptide affinity chromatography in monoclonal antibody (mAb) manufacturing. In this work, a biomimetic peptide ligand (bPL), FYWHCLDE, was coupled onto Sepharose 6 Fast Flow (SepFF) to synthesize a peptide affinity gel, SepFF-bPL, for the investigation of the binding mechanism of HCP as well as the feasibility of antibody capture. The results showed that the SepFF-bPL column exhibited effective removal of mAb aggregates as well as mAb capture from feedstocks of various origins, whereas poor removal of HCP and DNA was found. Mechanistic studies of HCP binding indicated that electrostatic interactions dominated HCP binding on the SepFF-bPL gel and that ionic conductivity had a significant influence on HCP binding at low salt concentrations. Thus, combined chromatin extraction and anion exchange adsorption were introduced prior to SepFF-bPL chromatography for initial contaminant removal to reduce mAb aggregation induced by HCP and the loading burden of contaminants in SepFF-bPL chromatography. A proof-of-concept study of the purification train demonstrated a high recovery of mAb (68.7%) and low levels of HCP (23 ppm) and DNA (below the limit of detection) in the final product, which were acceptable for the mandatory requirements in clinical applications. This research provided a deep understanding of HCP binding on the peptide affinity column and led to the development of an effective purification train.
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Affiliation(s)
- Haotian Huang
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China
| | - Xiaoyan Dong
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China; Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin 300350, China
| | - Yan Sun
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China; Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin 300350, China
| | - Qinghong Shi
- Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China; Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin 300350, China.
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15
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Alboreggia G, Udompholkul P, Baggio C, Pellecchia M. Mixture-Based Screening of Focused Combinatorial Libraries by NMR: Application to the Antiapoptotic Protein hMcl-1. J Med Chem 2023. [PMID: 37464766 PMCID: PMC10388297 DOI: 10.1021/acs.jmedchem.3c01073] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/20/2023]
Abstract
We report on an innovative ligand discovery strategy based on protein NMR-based screening of a combinatorial library of ∼125,000 compounds that was arranged in 96 distinct mixtures. Using sensitive solution protein NMR spectroscopy and chemical perturbation-based screening followed by an iterative synthesis, deconvolutions, and optimization strategy, we demonstrate that the approach could be useful in the identification of initial binding molecules for difficult drug targets, such as those involved in protein-protein interactions. As an application, we will report novel agents targeting the Bcl-2 family protein hMcl-1. The approach is of general applicability and could be deployed as an effective screening strategy for de novo identification of ligands, particularly when tackling targets involved in protein-protein interactions.
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Affiliation(s)
- Giulia Alboreggia
- Division of Biomedical Sciences, School of Medicine, University of California Riverside, 900 University Avenue, Riverside, California 92521, United States
| | - Parima Udompholkul
- Division of Biomedical Sciences, School of Medicine, University of California Riverside, 900 University Avenue, Riverside, California 92521, United States
| | - Carlo Baggio
- Division of Biomedical Sciences, School of Medicine, University of California Riverside, 900 University Avenue, Riverside, California 92521, United States
| | - Maurizio Pellecchia
- Division of Biomedical Sciences, School of Medicine, University of California Riverside, 900 University Avenue, Riverside, California 92521, United States
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16
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Monti A, Vitagliano L, Caporale A, Ruvo M, Doti N. Targeting Protein-Protein Interfaces with Peptides: The Contribution of Chemical Combinatorial Peptide Library Approaches. Int J Mol Sci 2023; 24:7842. [PMID: 37175549 PMCID: PMC10178479 DOI: 10.3390/ijms24097842] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2023] [Revised: 04/22/2023] [Accepted: 04/23/2023] [Indexed: 05/15/2023] Open
Abstract
Protein-protein interfaces play fundamental roles in the molecular mechanisms underlying pathophysiological pathways and are important targets for the design of compounds of therapeutic interest. However, the identification of binding sites on protein surfaces and the development of modulators of protein-protein interactions still represent a major challenge due to their highly dynamic and extensive interfacial areas. Over the years, multiple strategies including structural, computational, and combinatorial approaches have been developed to characterize PPI and to date, several successful examples of small molecules, antibodies, peptides, and aptamers able to modulate these interfaces have been determined. Notably, peptides are a particularly useful tool for inhibiting PPIs due to their exquisite potency, specificity, and selectivity. Here, after an overview of PPIs and of the commonly used approaches to identify and characterize them, we describe and evaluate the impact of chemical peptide libraries in medicinal chemistry with a special focus on the results achieved through recent applications of this methodology. Finally, we also discuss the role that this methodology can have in the framework of the opportunities, and challenges that the application of new predictive approaches based on artificial intelligence is generating in structural biology.
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Affiliation(s)
- Alessandra Monti
- Institute of Biostructures and Bioimaging (IBB), National Research Council (CNR), 80131 Napoli, Italy; (A.M.); (L.V.); (M.R.)
| | - Luigi Vitagliano
- Institute of Biostructures and Bioimaging (IBB), National Research Council (CNR), 80131 Napoli, Italy; (A.M.); (L.V.); (M.R.)
| | - Andrea Caporale
- Institute of Crystallography (IC), National Research Council (CNR), Strada Statale 14 km 163.5, Basovizza, 34149 Triese, Italy;
| | - Menotti Ruvo
- Institute of Biostructures and Bioimaging (IBB), National Research Council (CNR), 80131 Napoli, Italy; (A.M.); (L.V.); (M.R.)
| | - Nunzianna Doti
- Institute of Biostructures and Bioimaging (IBB), National Research Council (CNR), 80131 Napoli, Italy; (A.M.); (L.V.); (M.R.)
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17
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Pianka J, Gruba N, Lesner A. Novel tools to study West Nile virus NS3 protease activity. Bioorg Chem 2023; 133:106426. [PMID: 36801793 DOI: 10.1016/j.bioorg.2023.106426] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2022] [Revised: 01/11/2023] [Accepted: 02/12/2023] [Indexed: 02/17/2023]
Abstract
West Nile Virus (WNV) belongs to a group of pathogenic viruses called flaviviruses. West Nile virus infection can be mild, causing so-called West Nile Fever (WNF) or severe neuroinvasive form of the disease (WNND), and ultimately even death. There are currently no known medications to prevent West Nile virus infection. Only symptomatic treatment is used. To date, there are no unequivocal tests enabling a quick and unambiguous assessment of WN virus infection. The aim of the research was to obtain specific and selective tools for determining the activity of the West Nile virus serine proteinase. Using the methods of combinatorial chemistry with iterative deconvolution, the substrate specificity of the enzyme in non-primed and primed positions was determined. The FRET ABZ-Ala-Lys-Gln-Arg-Gly-Gly-Thr-Tyr(3-NO2)-NH2 substrate was obtained, characterized by kinetic parameters (KM = 4.20 ± 0.32 × 10-5 M) as for the majority of proteolytic enzymes. The obtained sequence was used to develop and synthesize highly sensitive functionalized quantum dot-based protease probes (QD). A QD WNV NS3 protease probe was obtained to detect an increase in fluorescence of 0.05 nmol enzyme in the assay system. This value was at least 20 times lower than that observed with the optimized substrate. The obtained result may be the basis for further research on the potential use of the WNV NS3 protease in the diagnosis of West Nile virus infection.
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Affiliation(s)
- Joanna Pianka
- University of Gdansk, Faculty of Chemistry, Wita Stwosza 63 Street, PL 80-308 Gdańsk, Poland
| | - Natalia Gruba
- University of Gdansk, Faculty of Chemistry, Wita Stwosza 63 Street, PL 80-308 Gdańsk, Poland.
| | - Adam Lesner
- University of Gdansk, Faculty of Chemistry, Wita Stwosza 63 Street, PL 80-308 Gdańsk, Poland
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18
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Gitlin-Domagalska A, Dębowski D, Maciejewska A, Samsonov S, Maszota-Zieleniak M, Ptaszyńska N, Łęgowska A, Rolka K. Cyclic Peptidic Furin Inhibitors Developed by Combinatorial Chemistry. ACS Med Chem Lett 2023; 14:458-465. [PMID: 37077382 PMCID: PMC10107917 DOI: 10.1021/acsmedchemlett.3c00008] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2023] [Accepted: 03/10/2023] [Indexed: 03/17/2023] Open
Abstract
Furin is a human serine protease responsible for activating numerous physiologically relevant cell substrates and is also involved in the development of various pathological conditions, including inflammatory diseases, cancers, and viral and bacterial infections. Therefore, compounds with the ability to inhibit furin's proteolytic action are regarded as potential therapeutics. Here we took the combinatorial chemistry approach (library consisting of 2000 peptides) to obtain new, strong, and stable peptide furin inhibitors. The extensively studied trypsin inhibitor SFTI-1 was used as a leading structure. A selected monocylic inhibitor was further modified to finally yield five mono- or bicyclic furin inhibitors with values of K i in the subnanomolar range. Inhibitor 5 was the most active (K i = 0.21 nM) and significantly more proteolytically resistant than the reference furin inhibitor described in the literature. Moreover, it reduced furin-like activity in PANC-1 cell lysate. Detailed analysis of furin-inhibitor complexes using molecular dynamics simulations is also reported.
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Affiliation(s)
- Agata Gitlin-Domagalska
- Department of Molecular Biochemistry, Faculty of Chemistry, University of Gdańsk, 80-308 Gdańsk, Poland
| | - Dawid Dębowski
- Department of Molecular Biochemistry, Faculty of Chemistry, University of Gdańsk, 80-308 Gdańsk, Poland
| | - Aleksandra Maciejewska
- Department of Molecular Biochemistry, Faculty of Chemistry, University of Gdańsk, 80-308 Gdańsk, Poland
| | - Sergey Samsonov
- Department of Theoretical Chemistry, Faculty of Chemistry, University of Gdańsk, 80-308 Gdańsk, Poland
| | - Martyna Maszota-Zieleniak
- Department of Theoretical Chemistry, Faculty of Chemistry, University of Gdańsk, 80-308 Gdańsk, Poland
| | - Natalia Ptaszyńska
- Department of Molecular Biochemistry, Faculty of Chemistry, University of Gdańsk, 80-308 Gdańsk, Poland
| | - Anna Łęgowska
- Department of Molecular Biochemistry, Faculty of Chemistry, University of Gdańsk, 80-308 Gdańsk, Poland
| | - Krzysztof Rolka
- Department of Molecular Biochemistry, Faculty of Chemistry, University of Gdańsk, 80-308 Gdańsk, Poland
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19
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Mini-αA-Crystallin Stifled Melittin-Induced Haemolysis and Lymphocyte Lysis. Int J Pept Res Ther 2023. [DOI: 10.1007/s10989-023-10502-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/05/2023]
Abstract
AbstractMelittin, the most potent pharmacological ingredient of honey bee venom, induces haemolysis, lymphocyte lysis, long-term pain, localised inflammation, and hyperalgesia. In this study, efforts were made to subdue the melittin’s ill effects using a chaperone peptide called ‘mini-αA-crystallin’ (MAC) derived from eye lens αA-crystallin. Haemolytic test on human red blood cells, percentage viability, and DNA diffusion assay on Human peripheral blood lymphocytes (HPBLs) were performed with melittin in the presence or absence of MAC. Propidium iodide and Annexin V-FITC dual staining were performed to analyse quantitative levels of necrotic and apoptotic induction by melittin in the presence or absence of MAC on HPBLs using a flow cytometer. A computational study to find out the interactions between MAC and melittin was undertaken by modelling the structure of MAC using a PEP-FOLD server. The result showed that MAC inhibited melittin-induced lysis in nucleated (lymphocytes) and enucleated (RBC) cells. Flow cytometric analysis revealed a substantial increase in the necrotic and late apoptotic cells after treating HPBLs with melittin (4 µg/ml) for 24 h. Treatment with MAC at a 2:1 molar ratio prevented HPBLs from developing melittin-induced necrosis and late apoptosis. In the docking study, hydrogen, van der Waals, π-π stacking, and salt bridges were observed between the MAC and melittin complex, confirming a strong interaction between them. The MAC-melittin complex was stable during molecular dynamics simulation. These findings may be beneficial in developing a medication for treating severe cases of honeybee stings.
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20
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Shen Y, Wang J, Li Y, Yang CT, Zhou X. Modified Bacteriophage for Tumor Detection and Targeted Therapy. NANOMATERIALS (BASEL, SWITZERLAND) 2023; 13:nano13040665. [PMID: 36839030 PMCID: PMC9963578 DOI: 10.3390/nano13040665] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/15/2023] [Revised: 02/05/2023] [Accepted: 02/06/2023] [Indexed: 05/07/2023]
Abstract
Malignant tumor is one of the leading causes of death in human beings. In recent years, bacteriophages (phages), a natural bacterial virus, have been genetically engineered for use as a probe for the detection of antigens that are highly expressed in tumor cells and as an anti-tumor reagent. Furthermore, phages can also be chemically modified and assembled with a variety of nanoparticles to form a new organic/inorganic composite, thus extending the application of phages in biological detection and tumor therapeutic. This review summarizes the studies on genetically engineered and chemically modified phages in the diagnosis and targeting therapy of tumors in recent years. We discuss the advantages and limitations of modified phages in practical applications and propose suitable application scenarios based on these modified phages.
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Affiliation(s)
- Yuanzhao Shen
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou 225009, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou 225009, China
| | - Jingyu Wang
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou 225009, China
| | - Yuting Li
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou 225009, China
| | - Chih-Tsung Yang
- Future Industries Institute, Mawson Lakes Campus, University of South Australia, Adelaide, SA 5095, Australia
- Correspondence: (X.Z.); (C.-T.Y.)
| | - Xin Zhou
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou 225009, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou 225009, China
- Correspondence: (X.Z.); (C.-T.Y.)
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21
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Kugler M, Hadzima M, Dzijak R, Rampmaier R, Srb P, Vrzal L, Voburka Z, Majer P, Řezáčová P, Vrabel M. Identification of specific carbonic anhydrase inhibitors via in situ click chemistry, phage-display and synthetic peptide libraries: comparison of the methods and structural study. RSC Med Chem 2023; 14:144-153. [PMID: 36760748 PMCID: PMC9890587 DOI: 10.1039/d2md00330a] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2022] [Accepted: 11/10/2022] [Indexed: 11/19/2022] Open
Abstract
The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.
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Affiliation(s)
- Michael Kugler
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám. 2 16000 Prague Czech Republic
| | - Martin Hadzima
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám. 2 16000 Prague Czech Republic
- Department of Organic Chemistry, Faculty of Science, Charles University Albertov 6 12800 Praha 2 Czech Republic
| | - Rastislav Dzijak
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám. 2 16000 Prague Czech Republic
| | - Robert Rampmaier
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám. 2 16000 Prague Czech Republic
| | - Pavel Srb
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám. 2 16000 Prague Czech Republic
| | - Lukáš Vrzal
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám. 2 16000 Prague Czech Republic
| | - Zdeněk Voburka
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám. 2 16000 Prague Czech Republic
| | - Pavel Majer
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám. 2 16000 Prague Czech Republic
| | - Pavlína Řezáčová
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám. 2 16000 Prague Czech Republic
| | - Milan Vrabel
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám. 2 16000 Prague Czech Republic
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22
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Zwillinger M, Fischer L, Sályi G, Szabó S, Csékei M, Huc I, Kotschy A. Isotope Ratio Encoding of Sequence-Defined Oligomers. J Am Chem Soc 2022; 144:19078-19088. [PMID: 36206533 DOI: 10.1021/jacs.2c08135] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Information storage at the molecular level commonly entails encoding in the form of ordered sequences of different monomers and subsequent fragmentation and tandem mass spectrometry analysis to read this information. Recent approaches also include the use of mixtures of distinct molecules noncovalently bonded to one another. Here, we present an alternate isotope ratio encoding approach utilizing deuterium-labeled monomers to produce hundreds of oligomers endowed with unique isotope distribution patterns. Mass spectrometric recognition of these patterns then allowed us to directly readout encoded information with high fidelity. Specifically, we show that all 256 tetramers composed of four different monomers of identical constitution can be distinguished by their mass fingerprint using mono-, di-, tri-, and tetradeuterated building blocks. The method is robust to experimental errors and does not require the most sophisticated mass spectrometry instrumentation. Such isotope ratio-encoded oligomers may serve as tags that carry information, but the method mainly opens up the capability to write information, for example, about molecular identity, directly into a pure compound via its isotopologue distribution obviating the need for additional tagging and avoiding the use of mixtures of different molecules.
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Affiliation(s)
- Márton Zwillinger
- Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary.,Hevesy György PhD School of Chemistry, Eötvös Loránd University, H-1053 Budapest, Hungary
| | - Lucile Fischer
- CBMN UMR5248, University of Bordeaux-CNRS-IPB, F-33600 Pessac, France
| | - Gergő Sályi
- Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
| | - Soma Szabó
- Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
| | - Márton Csékei
- Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
| | - Ivan Huc
- Department of Pharmacy and Center for Integrated Protein Science, Ludwig-Maximilians-University, D-81377 Munich, Germany
| | - András Kotschy
- Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
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23
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Garrido-Mesa J, Brown MA. T cell Repertoire Profiling and the Mechanism by which HLA-B27 Causes Ankylosing Spondylitis. Curr Rheumatol Rep 2022; 24:398-410. [PMID: 36197645 PMCID: PMC9666335 DOI: 10.1007/s11926-022-01090-6] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/25/2022] [Indexed: 11/25/2022]
Abstract
Purpose of Review Ankylosing spondylitis (AS) is strongly associated with the HLA-B27 gene. The canonical function of HLA-B27 is to present antigenic peptides to CD8 lymphocytes, leading to adaptive immune responses. The ‘arthritogenic peptide’ theory as to the mechanism by which HLA-B27 induces ankylosing spondylitis proposes that HLA-B27 presents peptides derived from exogenous sources such as bacteria to CD8 lymphocytes, which subsequently cross-react with antigens at the site of inflammation of the disease, causing inflammation. This review describes findings of studies in AS involving profiling of T cell expansions and discusses future research opportunities based on these findings. Recent Findings Consistent with this theory, there is an expanding body of data showing that expansion of a restricted pool of CD8 lymphocytes is found in most AS patients yet only in a small proportion of healthy HLA-B27 carriers. Summary These exciting findings strongly support the theory that AS is driven by presentation of antigenic peptides to the adaptive immune system by HLA-B27. They point to new potential approaches to identify the exogenous and endogenous antigens involved and to potential therapies for the disease.
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Affiliation(s)
- Jose Garrido-Mesa
- Department of Medical and Molecular Genetics, Faculty of Life Sciences and Medicine, King's College London, London, England
| | - Matthew A Brown
- Department of Medical and Molecular Genetics, Faculty of Life Sciences and Medicine, King's College London, London, England.
- Genomics England, Charterhouse Square, London, EC1M 6BQ, England.
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24
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Gruba N, Musielak M, Rejmak W, Lesner A. Detection of ADAM15 in urine from patients with bladder cancer. Anal Biochem 2022; 654:114805. [PMID: 35810783 DOI: 10.1016/j.ab.2022.114805] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Revised: 07/04/2022] [Accepted: 07/04/2022] [Indexed: 11/01/2022]
Abstract
Cancer is one of the leading causes of death in the United States and Europe. Of the cancers, bladder cancer is the 10th most frequently diagnosed cancer and the 13th most frequently diagnosed cancer in men. There are many studies showing that proteolytic enzymes, e.g. A Disintegrin and Metalloproteinases (ADAMs), play a key role in the development and progression of neoplasms. In this paper, we present the use of chromogenic substrate of ADAM15 for the qualitative determination of specific activity of enzyme in urine of patients with confirmed bladder cancer. In the first step, we optimized the substrate molecule in non-primed positions using combinatorial chemistry. By means of the obtained ABZ-His-Ala-Arg-Gly-ANB-NH2 peptide, we detected ADAM15 activity in urine samples collected from patients diagnosed with bladder cancer. In contrast, we did not observe such activity in urine obtained from healthy volunteers.
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Affiliation(s)
- Natalia Gruba
- Department of Environmental Technology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63 Street, PL 80-308, Gdańsk, Poland.
| | - Monika Musielak
- Department of Environmental Technology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63 Street, PL 80-308, Gdańsk, Poland
| | - Wiktoria Rejmak
- Department of Environmental Technology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63 Street, PL 80-308, Gdańsk, Poland
| | - Adam Lesner
- Department of Environmental Technology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63 Street, PL 80-308, Gdańsk, Poland
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25
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Kalita D, Sahariah B, Mookerjee SP, Sarma BK. Strategies to Control the cis-trans Isomerization of Peptoid Amide Bonds. Chem Asian J 2022; 17:e202200149. [PMID: 35362652 DOI: 10.1002/asia.202200149] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2022] [Revised: 03/30/2022] [Indexed: 11/11/2022]
Abstract
Peptoids are oligomers of N-substituted glycine units. They structurally resemble peptides but, unlike natural peptides, the side chains of peptoids are present on the amide nitrogen atoms instead of the α-carbons. The N-substitution improves cell-permeability of peptoids and enhance their proteolytic stability over natural peptides. Therefore, peptoids are ideal peptidomimetic candidates for drug discovery, especially for intracellular targets. Unfortunately, most peptoid ligands discovered so far possess moderate affinity towards their biological targets. The moderate affinity of peptoids for biomacromolecules is linked to their conformational flexibility, which causes substantial entropic loss during the peptoid-biomacromolecule binding process. The conformational flexibility of peptoids is caused by the lack of backbone chirality, absence of hydrogen bond donors (NH) in their backbone to form CO···HN hydrogen bonds and the facile cis-trans isomerization of their tertiary amide bonds. In recent years, many investigators have shown that the incorporation of specific side chains with unique steric and stereoelectronic features can favourably shift the cis-trans equilibria of peptoids towards one of the two isomeric forms. Such strategies are helpful to design homogenous peptoid oligomers having well defined secondary structures. Herein, we discuss the strategies developed over the years to control the cis-trans isomerization of peptoid amide bonds.
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Affiliation(s)
- Debajit Kalita
- Jawaharlal Nehru Centre for Advanced Scientific Research, New Chemistry Unit, INDIA
| | - Biswajit Sahariah
- Jawaharlal Nehru Centre for Advanced Scientific Research, New Chemistry Unit, INDIA
| | | | - Bani Kanta Sarma
- Jawaharlal Nehru Centre for Advanced Scientific Research, New Chemistry Unit, Rachenahalli Lake Road, Jakkur, 560064, India, 560064, Bangalore, INDIA
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26
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Affinity Selection from Synthetic Peptide Libraries Enabled by De Novo MS/MS Sequencing. Int J Pept Res Ther 2022. [DOI: 10.1007/s10989-022-10370-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
AbstractRecently, de novo MS/MS peptide sequencing has enabled the application of affinity selections to synthetic peptide mixtures that approach the diversity of phage libraries (> 108 random peptides). In conjunction with ‘split-mix’ solid phase synthesis to access equimolar peptide mixtures, this approach provides a straightforward means to examine synthetic peptide libraries of considerably higher diversity than has been feasible historically. Here, we offer a critical perspective on this work, report emerging data, and highlight opportunities for further methods refinement. With continued development, ‘affinity selection–mass spectrometry’ may become a complimentary approach to phage display, in vitro selection, and DNA-encoded libraries for the discovery of synthetic ligands that modulate protein function.
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27
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Tran N, Shiveshwarkar P, Jaworski J. Peptide Linked Diacetylene Amphiphiles for Detection of Epitope Specific Antibodies. CHEMOSENSORS (BASEL, SWITZERLAND) 2022; 10:62. [PMID: 36540572 PMCID: PMC9762857 DOI: 10.3390/chemosensors10020062] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Antibodies produced in response to adaptive immunity provide a receptor with multiple sites for binding to a distinct epitope of an antigen. Determining antibody levels to specific antigens has important clinical applications in assessing immune status or deficiency, monitoring infectious or autoimmune diseases, and diagnosing allergies. Leveraging that a specific antibody will bind to a distinct small peptide epitope without requiring the entire antigen to be present, we demonstrate in this work a proof-of-concept assay to detect the presence of an antibody by using peptide epitopes linked to an amphiphile to generate a vesicle-based sensing system. By affording multiple copies of the epitope site on the vesicle, we revealed that the vesicles visibly aggregate in response to an antibody specific for that epitope due to multivalent binding provided by the antibody. We also uncovered the role of peptide surface density in providing accessible epitopes on the vesicles for antibody binding. In summary, using a peptide derived from the coat protein of human influenza virus directly linked to a diacetylene-containing amphiphile afforded peptide-laden vesicles that proved capable of detecting the presence of antibodies specific for human influenza hemagglutinin.
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Affiliation(s)
- Natalie Tran
- Department of Bioengineering, University of Texas at Arlington, Arlington, TX 76010, USA
| | - Priyanka Shiveshwarkar
- Department of Bioengineering, University of Texas at Arlington, Arlington, TX 76010, USA
| | - Justyn Jaworski
- Department of Bioengineering, University of Texas at Arlington, Arlington, TX 76010, USA
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28
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Perry K, Sui B, Li Y. Mercapto-functionalized polyhedral oligomeric silsesquioxane as a soluble support for the synthesis of peptide thioesters. Tetrahedron Lett 2021; 85:153483. [PMID: 35153339 PMCID: PMC8833314 DOI: 10.1016/j.tetlet.2021.153483] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The revival of peptide-based drugs has led to the increasing demand for the development of large-scale synthesis of these complex molecules. To meet this demand, the use of mercapto-functionalized polyhedral oligomeric silsesquioxane (POSS-SH) as a soluble support for the synthesis of a model pentapeptide POSS-thioester is reported. The synthetic process provided a total yield of 62% for the pentapeptide POSS-thioester and the 1H NMR spectra validated the high purity of the products. The successful synthesis of the pentapeptide POSS-thioester with high yield and purity provides a promising way to the scale-up chemical synthesis of peptide thioesters, peptides, peptide amides, cyclic peptides, and even proteins.
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Affiliation(s)
- Kimberly Perry
- Department of Drug Discovery & Biomedical Sciences, University of South Carolina, 715 Sumter St, Columbia, SC 29208, USA
| | - Binglin Sui
- Department of Drug Discovery & Biomedical Sciences, University of South Carolina, 715 Sumter St, Columbia, SC 29208, USA
| | - Yangmei Li
- Department of Drug Discovery & Biomedical Sciences, University of South Carolina, 715 Sumter St, Columbia, SC 29208, USA
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29
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Gruba N, Rachubik P, Piwkowska A, Lesner A. Analysis of urinary kallikrein-related peptidase 13 for monitoring bladder cancer. Biomarkers 2021; 26:770-779. [PMID: 34704886 DOI: 10.1080/1354750x.2021.1999502] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
BACKGROUND Bladder cancer (BC) is one of the 10 most common types of cancer worldwide, with approximately 550,000 new cases each year. Early detection and appropriate diagnosis are important factors in successful treatment of the disease. MATERIAL AND METHODS We used specific fluorogenic substrate for the quantitative determination of urine kallikrein 13 (KLK13) activity in healthy (n = 15) and BC (n = 54) patients. The proteolytic activity in individual urine samples was determined by fluorescence measurements. Then, immunoenzymatic analyses (ELISA, Western blot) were performed to confirm the presence of KLK13 in the tested samples. RESULTS Urine samples from patients with G2 and G3 grade BC contained proteolytically active KLK13, as confirmed by kinetic analysis and immunochemical detection. KLK13 was not detected in the urine of patients with G1 grade BC. DISCUSSION Previous clinical study reveals the KLK13 significance for BC prognosis as increased KLK13 expression was highlighted in bladder tumours compared to normal adjacent tissues. Our findings correlate to the report. KLK13 activity was confirmed in BC patients with G2 and G3 stage of disease development. CONCLUSIONS Using specific chromogenic/fluorogenic peptides could be useful for the non-invasive disease monitoring of BC progress.
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Affiliation(s)
- Natalia Gruba
- Department of Environmental Technology, Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland
| | - Patrycja Rachubik
- Laboratory of Molecular and Cellular Nephrology, Mossakowski Medical Research Institute, Polish Academy of Sciences, Gdańsk, Poland
| | - Agnieszka Piwkowska
- Laboratory of Molecular and Cellular Nephrology, Mossakowski Medical Research Institute, Polish Academy of Sciences, Gdańsk, Poland.,Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland
| | - Adam Lesner
- Department of Environmental Technology, Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland
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30
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Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification. Int J Pept Res Ther 2021; 27:2905-2921. [PMID: 34690622 PMCID: PMC8525457 DOI: 10.1007/s10989-021-10299-5] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/07/2021] [Indexed: 12/12/2022]
Abstract
The interest in therapeutic monoclonal antibodies (mAbs) has significantly grown in the pharmaceutical industry, exceeding 100 FDA mAbs approved. Although the upstream processing of their industrial production has been significantly improved in the last years, the downstream processing still depends on immobilized protein A affinity chromatography. The high cost, low capacity and short half-life of immobilized protein A chromatography matrices, encouraged the design of alternative short-peptide ligands for mAb purification. Most of these peptides have been obtained by screening combinatorial peptide libraries. These low-cost ligands can be easily produced by solid-phase peptide synthesis and can be immobilized on chromatographic supports, thus obtaining matrices with high capacity and selectivity. Furthermore, matrices with immobilized peptide ligands have longer half-life than those with protein A due to the higher stability of the peptides. In this review the design and synthesis of peptide ligands, their immobilization on chromatographic supports and the evaluation of the affinity supports for their application in mAb purification is described.
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31
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Guha S, Ferrie RP, Ghimire J, Ventura CR, Wu E, Sun L, Kim SY, Wiedman GR, Hristova K, Wimley WC. Applications and evolution of melittin, the quintessential membrane active peptide. Biochem Pharmacol 2021; 193:114769. [PMID: 34543656 DOI: 10.1016/j.bcp.2021.114769] [Citation(s) in RCA: 82] [Impact Index Per Article: 20.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2021] [Revised: 09/14/2021] [Accepted: 09/15/2021] [Indexed: 12/12/2022]
Abstract
Melittin, the main venom component of the European Honeybee, is a cationic linear peptide-amide of 26 amino acid residues with the sequence: GIGAVLKVLTTGLPALISWIKRKRQQ-NH2. Melittin binds to lipid bilayer membranes, folds into amphipathic α-helical secondary structure and disrupts the permeability barrier. Since melittin was first described, a remarkable array of activities and potential applications in biology and medicine have been described. Melittin is also a favorite model system for biophysicists to study the structure, folding and function of peptides and proteins in membranes. Melittin has also been used as a template for the evolution of new activities in membranes. Here we overview the rich history of scientific research into the many activities of melittin and outline exciting future applications.
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Affiliation(s)
- Shantanu Guha
- University of Texas Health Science Center at Houston, Department of Microbiology and Molecular Genetics, Houston, TX, USA
| | - Ryan P Ferrie
- Tulane University School of Medicine, Department of Biochemistry and Molecular Biology, New Orleans, LA, USA
| | - Jenisha Ghimire
- Tulane University School of Medicine, Department of Biochemistry and Molecular Biology, New Orleans, LA, USA
| | - Cristina R Ventura
- Seton Hall University, Department of Chemistry and Biochemistry, South Orange, NJ, USA
| | - Eric Wu
- Tulane University School of Medicine, Department of Biochemistry and Molecular Biology, New Orleans, LA, USA
| | - Leisheng Sun
- Tulane University School of Medicine, Department of Biochemistry and Molecular Biology, New Orleans, LA, USA
| | - Sarah Y Kim
- Duke University, Department of Biomedical Engineering, Durham, NC, USA
| | - Gregory R Wiedman
- Seton Hall University, Department of Chemistry and Biochemistry, South Orange, NJ, USA
| | - Kalina Hristova
- Johns Hopkins University, Department of Materials Science and Engineering, Baltimore, MD, USA.
| | - Wimley C Wimley
- University of Texas Health Science Center at Houston, Department of Microbiology and Molecular Genetics, Houston, TX, USA.
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32
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Guzmán F, Gauna A, Roman T, Luna O, Álvarez C, Pareja-Barrueto C, Mercado L, Albericio F, Cárdenas C. Tea Bags for Fmoc Solid-Phase Peptide Synthesis: An Example of Circular Economy. Molecules 2021; 26:5035. [PMID: 34443624 PMCID: PMC8399505 DOI: 10.3390/molecules26165035] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Revised: 08/15/2021] [Accepted: 08/16/2021] [Indexed: 12/30/2022] Open
Abstract
Peptide synthesis is an area with a wide field of application, from biomedicine to nanotechnology, that offers the option of simultaneously synthesizing a large number of sequences for the purpose of preliminary screening, which is a powerful tool. Nevertheless, standard protocols generate large volumes of solvent waste. Here, we present a protocol for the multiple Fmoc solid-phase peptide synthesis in tea bags, where reagent recycling steps are included. Fifty-two peptides with wide amino acid composition and seven to twenty amino acid residues in length were synthesized in less than three weeks. A clustering analysis was performed, grouping the peptides by physicochemical features. Although a relationship between the overall yield and the physicochemical features of the sequences was not established, the process showed good performance despite sequence diversity. The recycling system allowed to reduce N, N-dimethylformamide usage by 25-30% and reduce the deprotection reagent usage by 50%. This protocol has been optimized for the simultaneous synthesis of a large number of peptide sequences. Additionally, a reagent recycling system was included in the procedure, which turns the process into a framework of circular economy, without affecting the quality of the products obtained.
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Affiliation(s)
- Fanny Guzmán
- Núcleo Biotecnología Curauma, Pontificia Universidad Católica de Valparaíso, Valparaíso 2373223, Chile;
| | - Adriana Gauna
- Doctorado en Biotecnología, Pontificia Universidad Católica de Valparaíso, Universidad Técnica Federico Santa María, Valparaíso 2373223, Chile; (A.G.); (T.R.)
| | - Tanya Roman
- Doctorado en Biotecnología, Pontificia Universidad Católica de Valparaíso, Universidad Técnica Federico Santa María, Valparaíso 2373223, Chile; (A.G.); (T.R.)
| | - Omar Luna
- Núcleo Biotecnología Curauma, Pontificia Universidad Católica de Valparaíso, Valparaíso 2373223, Chile;
- Networking Centre on Bioengineering, Department of Organic Chemistry and CIBER-BBN, Biomaterials and Nanomedicine, University of Barcelona, 08028 Barcelona, Spain;
- Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain
| | - Claudio Álvarez
- Laboratorio de Fisiología y Genética Marina (FIGEMA), Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Coquimbo 1781421, Chile;
- Facultad de Ciencias del Mar, Universidad Católica del Norte, Coquimbo 1781421, Chile
| | - Claudia Pareja-Barrueto
- Department of Hematology and Oncology, Pontificia Universidad Católica de Chile, Santiago 8320000, Chile;
| | - Luis Mercado
- Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso 2373223, Chile;
| | - Fernando Albericio
- Networking Centre on Bioengineering, Department of Organic Chemistry and CIBER-BBN, Biomaterials and Nanomedicine, University of Barcelona, 08028 Barcelona, Spain;
- Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain
- School of Chemistry, University of KwaZulu-Natal, Durban 4001, South Africa
| | - Constanza Cárdenas
- Núcleo Biotecnología Curauma, Pontificia Universidad Católica de Valparaíso, Valparaíso 2373223, Chile;
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33
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Zhong C, Zhang F, Yao J, Zhu Y, Zhu N, Zhang J, Ouyang X, Zhang T, Li B, Xie J, Ni J. New Antimicrobial Peptides with Repeating Unit against Multidrug-Resistant Bacteria. ACS Infect Dis 2021; 7:1619-1637. [PMID: 33829758 DOI: 10.1021/acsinfecdis.0c00797] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
With the aim of tackling the increasingly serious antimicrobial resistance and improving the clinical potential of AMPs, a facile de novo strategy was adopted in this study, and a series of new peptides comprising repeating unit (WRX)n (X represents I, L, F, W, and K; n = 2, 3, 4, or 5) and amidation at C-terminus were designed. Most of the newly designed peptides exhibited a broad range of excellent antimicrobial activities against various bacteria, especially difficult-to-kill multidrug-resistant bacteria clinical isolates. Among (WRK)4 and (WRK)5, with n = 4 and n = 5 of repeating unit WRK, the highest selectivity for anionic bacterial membranes over a zwitterionic mammalian cell membrane is presented with strong antimicrobial potential and low toxicity. Additionally, both (WRK)4 and (WRK)5 emerged with fast killing speed and low tendency of resistance in sharp contrast to the conventional antibiotics ciprofloxacin, gentamicin, and imipenem, as well as having antimicrobial activity through multiple mechanisms including a membrane-disruptive mechanism and an intramolecular mechanism (nucleic acid leakage, DNA binding and ROS generation) characterized by a series of assays. Furthermore, (WRK)4 exerted impressive therapeutic effects in vivo similarly to polymyxin B but displayed much lower toxicity in vivo than polymyxin B. Taken together, the newly designed peptides (WRK)4 and (WRK)5 presented tremendous potential as novel antimicrobial candidates in response to the growing antimicrobial resistance.
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Affiliation(s)
- Chao Zhong
- Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China
- School of Pharmacy, Lanzhou University, Lanzhou 730000, China
| | - Fangyan Zhang
- School of Pharmacy, Lanzhou University, Lanzhou 730000, China
| | - Jia Yao
- The First Hospital of Lanzhou University, Lanzhou 730000, China
| | - Yuewen Zhu
- School of Pharmacy, Lanzhou University, Lanzhou 730000, China
| | - Ningyi Zhu
- School of Pharmacy, Lanzhou University, Lanzhou 730000, China
| | - Jingying Zhang
- Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China
| | - Xu Ouyang
- School of Pharmacy, Lanzhou University, Lanzhou 730000, China
| | - Tianyue Zhang
- School of Pharmacy, Lanzhou University, Lanzhou 730000, China
| | - Beibei Li
- School of Pharmacy, Lanzhou University, Lanzhou 730000, China
| | - Junqiu Xie
- Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China
| | - Jingman Ni
- Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China
- School of Pharmacy, Lanzhou University, Lanzhou 730000, China
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macao 999078, China
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34
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Nichols PL. Automated and enabling technologies for medicinal chemistry. PROGRESS IN MEDICINAL CHEMISTRY 2021; 60:191-272. [PMID: 34147203 DOI: 10.1016/bs.pmch.2021.01.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Having always been driven by the need to get new treatments to patients as quickly as possible, drug discovery is a constantly evolving process. This chapter will review how medicinal chemistry was established, how it has changed over the years due to the emergence of new enabling technologies, and how early advances in synthesis, purification and analysis, have provided the foundations upon which the current automated and enabling technologies are built. Looking beyond the established technologies, this chapter will also consider technologies that are now emerging, and their impact on the future of drug discovery and the role of medicinal chemists.
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Affiliation(s)
- Paula L Nichols
- Synple Chem AG, Kemptthal, Switzerland; ETH, Zurich, Switzerland.
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35
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Roy A, Koesema E, Kodadek T. High-Throughput Quality Control Assay for the Solid-Phase Synthesis of DNA-Encoded Libraries of Macrocycles*. Angew Chem Int Ed Engl 2021; 60:11983-11990. [PMID: 33682283 PMCID: PMC8193836 DOI: 10.1002/anie.202100230] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2021] [Indexed: 12/18/2022]
Abstract
There is considerable interest in the development of libraries of scaffold-diverse macrocycles as a source of ligands for difficult targets, such as protein-protein interaction surfaces. A classic problem in the synthesis of high-quality macrocyclic libraries is that some linear precursors will cyclize efficiently while some will not, depending on their conformational preferences. We report here a powerful quality control method that can be employed to readily distinguish between scaffolds that do and do not cyclize efficiently during solid-phase synthesis of thioether macrocycles without the need for tedious liquid chromatography/mass spectrometry analysis. We demonstrate that this assay can be employed to identify linear impurities in a DNA-encoded library of macrocycles. We also use the method to establish a useful quality control protocol for re-synthesis of putative macrocyclic screening hits.
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Affiliation(s)
- Animesh Roy
- Deluge Biotechnologies, 6671 W. Indiantown Rd., Suite 50-325, Jupiter, FL 33458 (USA)
| | - Eric Koesema
- Deluge Biotechnologies, 6671 W. Indiantown Rd., Suite 50-325, Jupiter, FL 33458 (USA)
| | - Thomas Kodadek
- Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458 (USA)
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36
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Roy A, Koesema E, Kodadek T. High‐Throughput Quality Control Assay for the Solid‐Phase Synthesis of DNA‐Encoded Libraries of Macrocycles**. Angew Chem Int Ed Engl 2021. [DOI: 10.1002/ange.202100230] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Affiliation(s)
- Animesh Roy
- Deluge Biotechnologies 6671 W. Indiantown Rd., Suite 50–325 Jupiter FL 33458 USA
| | - Eric Koesema
- Deluge Biotechnologies 6671 W. Indiantown Rd., Suite 50–325 Jupiter FL 33458 USA
| | - Thomas Kodadek
- Department of Chemistry The Scripps Research Institute 130 Scripps Way Jupiter FL 33458 USA
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37
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Structural Determinants of Substrate Specificity of SplF Protease from Staphylococcus aureus. Int J Mol Sci 2021; 22:ijms22042220. [PMID: 33672341 PMCID: PMC7926377 DOI: 10.3390/ijms22042220] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Revised: 02/16/2021] [Accepted: 02/19/2021] [Indexed: 11/17/2022] Open
Abstract
Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.
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38
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Siriwardena T, Gan BH, Köhler T, van Delden C, Javor S, Reymond JL. Stereorandomization as a Method to Probe Peptide Bioactivity. ACS CENTRAL SCIENCE 2021; 7:126-134. [PMID: 33532575 PMCID: PMC7845017 DOI: 10.1021/acscentsci.0c01135] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/24/2020] [Indexed: 06/01/2023]
Abstract
Solid-phase peptide synthesis (SPPS) is usually performed with optically pure building blocks to prepare peptides as single enantiomers. Herein we report that SPPS using racemic amino acids provides stereorandomized (sr) peptides, containing up to billions of different stereoisomers, as well-defined single HPLC peaks, single mass products with high yield, which can be used to investigate peptide bioactivity. To exemplify our method, we show that stereorandomization abolishes the membrane-disruptive effect of α-helical amphiphilic antimicrobial peptides but preserves their antibiofilm effect, implying different mechanisms involving folded versus disordered conformations. For antimicrobial peptide dendrimers by contrast, stereorandomization preserves antibacterial, membrane-disruptive, and antibiofilm effects but reduces hemolysis and cytotoxicity, thereby increasing their therapeutic index. Finally, we identify partially stereorandomized analogues of the last resort cyclic peptide antibiotic polymyxin B with preserved antibacterial activity but lacking membrane-disruptive and lipopolysaccharide-neutralizing activity, pointing to the existence of additional targets.
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Affiliation(s)
- Thissa
N. Siriwardena
- Department
of Chemistry and Biochemistry, University
of Bern, Freiestrasse 3, 3012 Bern, Switzerland
| | - Bee-Ha Gan
- Department
of Chemistry and Biochemistry, University
of Bern, Freiestrasse 3, 3012 Bern, Switzerland
| | - Thilo Köhler
- Department
of Microbiology and Molecular Medicine, University of Geneva, Service
of Infectious Diseases, University Hospital
of Geneva, 1211 Geneva, Switzerland
| | - Christian van Delden
- Department
of Microbiology and Molecular Medicine, University of Geneva, Service
of Infectious Diseases, University Hospital
of Geneva, 1211 Geneva, Switzerland
| | - Sacha Javor
- Department
of Chemistry and Biochemistry, University
of Bern, Freiestrasse 3, 3012 Bern, Switzerland
| | - Jean-Louis Reymond
- Department
of Chemistry and Biochemistry, University
of Bern, Freiestrasse 3, 3012 Bern, Switzerland
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Schütznerová E, Přibylka A, Medran NS, Krchňák V. Greening Solid-Phase Organic Synthesis: Environmentally Conscious Synthesis of Pharmaceutically Relevant Privileged Structures 5,6-Dihydropyridin-2(1 H)-ones and Quinolin-2(1 H)-ones. J Org Chem 2020; 85:11867-11881. [PMID: 32841557 DOI: 10.1021/acs.joc.0c01623] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Solid-phase organic synthesis (SPOS) is a very efficient methodology for the synthesis of diverse organic molecules, particularly exploited in drug discovery. Here, we present the transformation of the traditional SPOS to an eco-friendlier methodology on examples of pharmacologically relevant privileged structures 5,6-dihydropyridin-2(1H)-ones and quinolin-2(1H)-ones. The green approach is primarily based on the utilization of environmentally friendly solvent 2-MeTHF in all steps of the synthesis. Target heterocycles were synthesized by extending our previously published synthesis of five-membered tetramic acid analogues to six-membered cycles. The crucial step of the synthesis is cyclization via nonclassical Wittig olefination of resin-bound esters. Traditional and green protocols provided comparable results with respect to purity and yield of products, thus opening the way for greener access to a variety of diverse heterocycles.
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Affiliation(s)
- Eva Schütznerová
- Department of Organic Chemistry, Faculty of Science, Palacký University, 17. Listopadu 12, 771 46 Olomouc, Czech Republic
| | - Adam Přibylka
- Department of Organic Chemistry, Faculty of Science, Palacký University, 17. Listopadu 12, 771 46 Olomouc, Czech Republic
| | - Noelia S Medran
- Instituto de Quı́mica Rosario-IQUIR (CONICET), Facultad de Ciencias Bioquı́micas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario S2002LRK, Argentina
| | - Viktor Krchňák
- Department of Organic Chemistry, Faculty of Science, Palacký University, 17. Listopadu 12, 771 46 Olomouc, Czech Republic.,Department of Chemistry and Biochemistry, 251 Nieuwland Science Center, University of Notre Dame, Notre Dame, Indiana 46556, United States
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40
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Pikus G, Tyszka-Gumkowska A, Jurczak J. The influence of high pressure on static combinatorial libraries of chiral BINOL-based macrocyclic amides. Tetrahedron 2020. [DOI: 10.1016/j.tet.2020.131438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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41
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McEnaney P, Balzarini M, Park H, Kodadek T. Structural characterization of a peptoid-inspired conformationally constrained oligomer (PICCO) bound to streptavidin. Chem Commun (Camb) 2020; 56:10560-10563. [PMID: 32785302 DOI: 10.1039/d0cc02588g] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
A high affinity Streptavidin ligand was mined from a DNA-encoded library of non-peptidic oligimers and characterized structurally.
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Affiliation(s)
- Patrick McEnaney
- Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 3345, USA.
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42
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Ramamourthy G, Park J, Seo C, J. Vogel H, Park Y. Antifungal and Antibiofilm Activities and the Mechanism of Action of Repeating Lysine-Tryptophan Peptides against Candida albicans. Microorganisms 2020; 8:E758. [PMID: 32443520 PMCID: PMC7285485 DOI: 10.3390/microorganisms8050758] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2020] [Revised: 05/06/2020] [Accepted: 05/16/2020] [Indexed: 11/29/2022] Open
Abstract
The rapid increase in the emergence of antifungal-resistant Candida albicans strains is becoming a serious health concern. Because antimicrobial peptides (AMPs) may provide a potential alternative to conventional antifungal agents, we have synthesized a series of peptides with a varying number of lysine and tryptophan repeats (KWn-NH2). The antifungal activity of these peptides increased with peptide length, but only the longest KW5 peptide displayed cytotoxicity towards a human keratinocyte cell line. The KW4 and KW5 peptides exhibited strong antifungal activity against C. albicans, even under conditions of high-salt and acidic pH, or the addition of fungal cell wall components. Moreover, KW4 inhibited biofilm formation by a fluconazole-resistant C. albicans strain. Circular dichroism and fluorescence spectroscopy indicated that fungal liposomes could interact with the longer peptides but that they did not release the fluorescent dye calcein. Subsequently, fluorescence assays with different dyes revealed that KW4 did not disrupt the membrane integrity of intact fungal cells. Scanning electron microscopy showed no changes in fungal morphology, while laser-scanning confocal microscopy indicated that KW4 can localize into the cytosol of C. albicans. Gel retardation assays revealed that KW4 can bind to fungal RNA as a potential intracellular target. Taken together, our data indicate that KW4 can inhibit cellular functions by binding to RNA and DNA after it has been translocated into the cell, resulting in the eradication of C. albicans.
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Affiliation(s)
- Gopal Ramamourthy
- Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada; (G.R.); (H.J.V.)
- Department of Biomedical Science and BK21-Plus Research Team for Bioactive Control Technology, Chosun University, Gwangju 61452, Korea
| | - Jonggwan Park
- Department of Bioinformatics, Kongju National University, Kongju 38065, Korea; (J.P.); (C.S.)
| | - Changho Seo
- Department of Bioinformatics, Kongju National University, Kongju 38065, Korea; (J.P.); (C.S.)
| | - Hans J. Vogel
- Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada; (G.R.); (H.J.V.)
| | - Yoonkyung Park
- Department of Biomedical Science and BK21-Plus Research Team for Bioactive Control Technology, Chosun University, Gwangju 61452, Korea
- Research Center for Proteineous Materials, Chosun University, Gwangju 61452, Korea
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43
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Sandomenico A, Caporale A, Doti N, Cross S, Cruciani G, Chambery A, De Falco S, Ruvo M. Synthetic Peptide Libraries: From Random Mixtures to In Vivo Testing. Curr Med Chem 2020; 27:997-1016. [PMID: 30009695 DOI: 10.2174/0929867325666180716110833] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2018] [Revised: 06/22/2018] [Accepted: 06/29/2018] [Indexed: 01/13/2023]
Abstract
Combinatorially generated molecular repertoires have been largely used to identify novel bioactive compounds. Ever more sophisticated technological solutions have been proposed to simplify and speed up such process, expanding the chemical diversity space and increasing the prospect to select new molecular entities with specific and potent activities against targets of therapeutic relevance. In this context, random mixtures of oligomeric peptides were originally used and since 25 years they represent a continuous source of bioactive molecules with potencies ranging from the sub-nM to microM concentration. Synthetic peptide libraries are still employed as starting "synthetic broths" of structurally and chemically diversified molecular fragments from which lead compounds can be extracted and further modified. Thousands of studies have been reported describing the application of combinatorial mixtures of synthetic peptides with different complexity and engrafted on diverse structural scaffolds for the identification of new compounds which have been further developed and also tested in in vivo models of relevant diseases. We briefly review some of the most used methodologies for library preparation and screening and the most recent case studies appeared in the literature where compounds have reached at least in vivo testing in animal or similar models. Recent technological advancements in biotechnology, engineering and computer science have suggested new options to facilitate the discovery of new bioactive peptides. In this instance, we anticipate here a new approach for the design of simple but focused tripeptide libraries against druggable cavities of therapeutic targets and its complementation with existing approaches.
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Affiliation(s)
- Annamaria Sandomenico
- Istituto di Biostrutture e Bioimmagini del CNR and CIRPeB, Universita Federico II di Napoli, via Mezzocannone, 16, 80134 Napoli, Italy
| | - Andrea Caporale
- Istituto di Biostrutture e Bioimmagini del CNR and CIRPeB, Universita Federico II di Napoli, via Mezzocannone, 16, 80134 Napoli, Italy
| | - Nunzianna Doti
- Istituto di Biostrutture e Bioimmagini del CNR and CIRPeB, Universita Federico II di Napoli, via Mezzocannone, 16, 80134 Napoli, Italy
| | - Simon Cross
- Molecular Discovery Ltd, Unit 501 Centennial Park, Centennial Avenue Elstree, Borehamwood, Hertfordshire WD6 3FG, United Kingdom
| | - Gabriele Cruciani
- Molecular Discovery Ltd, Unit 501 Centennial Park, Centennial Avenue Elstree, Borehamwood, Hertfordshire WD6 3FG, United Kingdom.,Dipartimento di Chimica, Biologia e Biotecnologia, Via Elce di Sotto 8, 06123 Perugia, Italy
| | - Angela Chambery
- Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Università della Campania "Luigi Vanvitelli", via Vivaldi, 43, 81100 Caserta, Italy
| | - Sandro De Falco
- Istituto di Genetica e Biofisica del CNR, via Pietro Castellino, 111, 80131, Napoli, Italy
| | - Menotti Ruvo
- Istituto di Biostrutture e Bioimmagini del CNR and CIRPeB, Universita Federico II di Napoli, via Mezzocannone, 16, 80134 Napoli, Italy
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44
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Kupai A, Vaughan RM, Dickson BM, Rothbart SB. A Degenerate Peptide Library Approach to Reveal Sequence Determinants of Methyllysine-Driven Protein Interactions. Front Cell Dev Biol 2020; 8:241. [PMID: 32328492 PMCID: PMC7160673 DOI: 10.3389/fcell.2020.00241] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2020] [Accepted: 03/23/2020] [Indexed: 11/19/2022] Open
Abstract
Lysine methylation facilitates protein-protein interactions through the activity of methyllysine (Kme) “reader” proteins. Functions of Kme readers have historically been studied in the context of histone interactions, where readers aid in chromatin-templated processes such as transcription, DNA replication and repair. However, there is growing evidence that Kme readers also function through interactions with non-histone proteins. To facilitate expanded study of Kme reader activities, we developed a high-throughput binding assay to reveal the sequence determinants of Kme-driven protein interactions. The assay queries a degenerate methylated lysine-oriented peptide library (Kme-OPL) to identify the key residues that modulate reader binding. The assay recapitulated methyl order and amino acid sequence preferences associated with histone Kme readers. The assay also revealed methylated sequences that bound Kme readers with higher affinity than histones. Proteome-wide scoring was applied to assay results to help prioritize future study of Kme reader interactions. The platform was also used to design sequences that directed specificity among closely related reader domains, an application which may have utility in the development of peptidomimetic inhibitors. Furthermore, we used the platform to identify binding determinants of site-specific histone Kme antibodies and surprisingly revealed that only a few amino acids drove epitope recognition. Collectively, these studies introduce and validate a rapid, unbiased, and high-throughput binding assay for Kme readers, and we envision its use as a resource for expanding the study of Kme-driven protein interactions.
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Affiliation(s)
- Ariana Kupai
- Center for Epigenetics, Van Andel Institute, Grand Rapids, MI, United States
| | - Robert M Vaughan
- Center for Epigenetics, Van Andel Institute, Grand Rapids, MI, United States
| | - Bradley M Dickson
- Center for Epigenetics, Van Andel Institute, Grand Rapids, MI, United States
| | - Scott B Rothbart
- Center for Epigenetics, Van Andel Institute, Grand Rapids, MI, United States
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45
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Wysocka M, Romanowska A, Gruba N, Michalska M, Giełdoń A, Lesner A. A Peptidomimetic Fluorescent Probe to Detect the Trypsin β2 Subunit of the Human 20S Proteasome. Int J Mol Sci 2020; 21:ijms21072396. [PMID: 32244300 PMCID: PMC7177456 DOI: 10.3390/ijms21072396] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Revised: 03/25/2020] [Accepted: 03/29/2020] [Indexed: 01/20/2023] Open
Abstract
This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (β2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building blocks, the sequence ABZ–Dap(O2(Cbz))–Dap(GO1)–Dap(O2(Cbz))–Arg–ANB–NH2, where ABZ is 2-aminobenzoic acid, and ANB- 5 amino 2- nitro benzoic acid was selected. Its cleavage followed sigmoidal kinetics, characteristic for allosteric enzymes, with Km = 3.22 ± 0.02 μM, kcat = 245 s−1, and kcat/Km = 7.61 × 107 M−1 s−1. This process was practically halted when a selective inhibitor of the β2 subunit of the 20S human proteasome was supplemented to the reaction system. Titration of the substrate resulting in decreased amounts of proteasome 20S produced a linear signal up to 10−11 M. Using this substrate, we detected human proteasome 20S in human urine samples taken from the bladders of cancer patients. This observation could be useful for the noninvasive diagnosis of this severe disease.
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Affiliation(s)
| | | | | | | | | | - Adam Lesner
- Correspondence: ; Tel.: +48-585-235-095; Fax: +48-585-235-472
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46
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Farrera-Soler L, Daguer JP, Raunft P, Barluenga S, Imberty A, Winssinger N. PNA-Based Dynamic Combinatorial Libraries (PDCL) and screening of lectins. Bioorg Med Chem 2020; 28:115458. [PMID: 32241620 DOI: 10.1016/j.bmc.2020.115458] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Revised: 03/13/2020] [Accepted: 03/17/2020] [Indexed: 11/16/2022]
Abstract
Selections from dynamic combinatorial libraries (DCL) benefit from the dynamic nature of the library that can change constitution upon addition of a selection pressure, such as ligands binding to a protein. This technology has been predominantly used with small molecules interacting with each other through reversible covalent interaction. However, application of this technology in biomedical research and drug discovery has been limited by the reversibility of covalent exchange and the analytical deconvolution of small molecule fragments. Here we report a supramolecular approach based on the use of a constant short PNA tag to direct the combinatorial pairing of fragment. This PNA tag yields fast exchange kinetics, while still delivering the benefits of cooperativity, and provides favourable properties for analytical deconvolution by MALDI. A selection from >6,000 assemblies of glycans (mono-, di-, tri-saccharides) targeting AFL, a lectin from pathogenic fungus, yielded a 95 nM assembly, nearly three orders of magnitude better in affinity than the corresponding glycan alone (41 µM).
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Affiliation(s)
- Lluc Farrera-Soler
- Department of Organic Chemistry, National Centre of Competence in Research (NCCR) in Chemical Biology, Faculty of Science, University of Geneva, 1211 Geneva, Switzerland
| | - Jean-Pierre Daguer
- Department of Organic Chemistry, National Centre of Competence in Research (NCCR) in Chemical Biology, Faculty of Science, University of Geneva, 1211 Geneva, Switzerland
| | - Patrick Raunft
- Department of Organic Chemistry, National Centre of Competence in Research (NCCR) in Chemical Biology, Faculty of Science, University of Geneva, 1211 Geneva, Switzerland
| | - Sofia Barluenga
- Department of Organic Chemistry, National Centre of Competence in Research (NCCR) in Chemical Biology, Faculty of Science, University of Geneva, 1211 Geneva, Switzerland
| | - Anne Imberty
- Université Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France
| | - Nicolas Winssinger
- Department of Organic Chemistry, National Centre of Competence in Research (NCCR) in Chemical Biology, Faculty of Science, University of Geneva, 1211 Geneva, Switzerland.
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47
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Singh Y, Rodriguez Benavente MC, Al-Huniti MH, Beckwith D, Ayyalasomayajula R, Patino E, Miranda WS, Wade A, Cudic M. Positional Scanning MUC1 Glycopeptide Library Reveals the Importance of PDTR Epitope Glycosylation for Lectin Binding. J Org Chem 2019; 85:1434-1445. [PMID: 31799848 PMCID: PMC7012140 DOI: 10.1021/acs.joc.9b02396] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
One of the main barriers to explaining the functional significance of glycan-based changes in cancer is the natural epitope heterogeneity found on the surface of cancer cells. To help address this knowledge gap, we focused on designing synthetic tools to explore the role of tumor-associated glycans of MUC1 in the formation of metastasis via association with lectins. In this study, we have synthesized for the first time a MUC1-derived positional scanning synthetic glycopeptide combinatorial library (PS-SGCL) that vary in number and location of cancer-associated Tn antigen using the "tea bag" approach. The determination of the isokinetic ratios necessary for the equimolar incorporation of (glyco)amino acids mixtures to resin-bound amino acid was determined, along with developing an efficient protocol for on resin deprotection of O-acetyl groups. Enzyme-linked lectin assay was used to screen PS-SGCL against two plant lectins, Glycine max soybean agglutinin and Vicia villosa. The results revealed a carbohydrate density-dependent affinity trend and site-specific glycosylation requirements for high affinity binding to these lectins. Hence, PS-SGCLs provide a platform to systematically elucidate MUC1-lectin binding specificities, which in the long term may provide a rational design for novel inhibitors of MUC1-lectin interactions involved in tumor spread and glycopeptide-based cancer vaccines.
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Affiliation(s)
- YashoNandini Singh
- From the Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science , Florida Atlantic University , 777 Glades Road , Boca Raton , Florida 33431 , United States
| | - Maria C Rodriguez Benavente
- From the Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science , Florida Atlantic University , 777 Glades Road , Boca Raton , Florida 33431 , United States
| | - Mohammed H Al-Huniti
- From the Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science , Florida Atlantic University , 777 Glades Road , Boca Raton , Florida 33431 , United States
| | - Donella Beckwith
- From the Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science , Florida Atlantic University , 777 Glades Road , Boca Raton , Florida 33431 , United States
| | - Ramya Ayyalasomayajula
- From the Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science , Florida Atlantic University , 777 Glades Road , Boca Raton , Florida 33431 , United States
| | - Eric Patino
- From the Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science , Florida Atlantic University , 777 Glades Road , Boca Raton , Florida 33431 , United States
| | - William S Miranda
- From the Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science , Florida Atlantic University , 777 Glades Road , Boca Raton , Florida 33431 , United States
| | - Alex Wade
- From the Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science , Florida Atlantic University , 777 Glades Road , Boca Raton , Florida 33431 , United States
| | - Maré Cudic
- From the Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science , Florida Atlantic University , 777 Glades Road , Boca Raton , Florida 33431 , United States
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48
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Abstract
Traceless solid-phase synthesis represents an ultimate sophisticated synthetic strategy on insoluble supports. Compounds synthesized on solid supports can be released without a trace of the linker that was used to tether the intermediates during the synthesis. Thus, the target products are composed only of the components (atoms, functional groups) inherent to the target core structure. A wide variety of synthetic strategies have been developed to prepare products in a traceless manner, and this review is dedicated to all aspects of traceless solid-phase organic synthesis. Importantly, the synthesis does not need to be carried out on a linker designed for traceless synthesis; most of the synthetic approaches described herein were developed using standard, commercially available linkers (originally devised for solid-phase peptide synthesis). The type of structure prepared in a traceless fashion is not restricted. The individual synthetic approaches are divided into eight sections, each devoted to a different methodology for traceless synthesis. Each section consists of a brief outline of the synthetic strategy followed by a description of individual reported syntheses.
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Affiliation(s)
- Naděžda Cankařová
- Department of Organic Chemistry, Faculty of Science , Palacky University , 17. Listopadu 12 , Olomouc , 771 46 , Czech Republic
| | - Eva Schütznerová
- Department of Organic Chemistry, Faculty of Science , Palacky University , 17. Listopadu 12 , Olomouc , 771 46 , Czech Republic
| | - Viktor Krchňák
- Department of Organic Chemistry, Faculty of Science , Palacky University , 17. Listopadu 12 , Olomouc , 771 46 , Czech Republic.,Department of Chemistry and Biochemistry , University of Notre Dame , 251 Nieuwland Science Center , Notre Dame , Indiana 46556 , United States
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49
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Synthesis, solar cell application, and biological study of vinyl substituted isophorone derivatives. RESEARCH ON CHEMICAL INTERMEDIATES 2019. [DOI: 10.1007/s11164-019-03924-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
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50
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Pei D, Kubi GA. Developments with bead-based screening for novel drug discovery. Expert Opin Drug Discov 2019; 14:1097-1102. [PMID: 31335229 PMCID: PMC7301614 DOI: 10.1080/17460441.2019.1647164] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2019] [Accepted: 07/19/2019] [Indexed: 01/24/2023]
Abstract
Introduction: Combinatorial chemistry provides a cost-effective method for rapid discovery of drug hits/leads. The one-bead-one-compound (OBOC) library method is in principle ideally suited for this application, because it permits a large number of structurally diverse compounds to be rapidly synthesized and simultaneously screened for binding to a target of interest. However, application of OBOC libraries in drug discovery has encountered significant technical challenges. Areas covered: This Special Report covers the challenges associated with first-generation OBOC libraries (difficulty in structural identification of non-peptidic hits, screening biases and high false positive rates, and poor scalability). It also covers the many strategies developed over the past two decades to overcome these challenges. Expert opinion: With most of the technical challenges now overcome and the advent of powerful intracellular delivery technologies, OBOC libraries of metabolically stable and conformationally rigidified molecules (macrocyclic peptides and peptidomimetics, rigidified acyclic oligomers, and D-peptides) can be routinely synthesized and screened to discover initial hits against previously undruggable targets such as intracellular protein-protein interactions. On the other hand, further developments are still needed to expand the utility of the OBOC method to non-peptidic chemical scaffolds.
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Affiliation(s)
- Dehua Pei
- Department of Chemistry and Biochemistry, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA
| | - George Appiah Kubi
- Department of Chemistry and Biochemistry, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA
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