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Pharmaceutical Research Nov 2015The list of ADCs in the clinic continues to grow, bolstered by the success of first two marketed ADCs: ADCETRIS® and Kadcyla®. Currently, there are 40 ADCs in various... (Review)
Review
The list of ADCs in the clinic continues to grow, bolstered by the success of first two marketed ADCs: ADCETRIS® and Kadcyla®. Currently, there are 40 ADCs in various phases of clinical development. However, only 34 of these have published their structures. Of the 34 disclosed structures, 24 of them use a linkage to the thiol of cysteines on the monoclonal antibody. The remaining 10 candidates utilize chemistry to surface lysines of the antibody. Due to the inherent heterogeneity of conjugation to the multiple lysines or cysteines found in mAbs, significant research efforts are now being directed toward the production of discrete, homogeneous ADC products, via site-specific conjugation. These site-specific conjugations may involve genetic engineering of the mAb to introduce discrete, available cysteines or non-natural amino acids with an orthogonally-reactive functional group handle such as an aldehyde, ketone, azido, or alkynyl tag. These site-specific approaches not only increase the homogeneity of ADCs but also enable novel bio-orthogonal chemistries that utilize reactive moieties other than thiol or amine. This broadens the diversity of linkers that can be utilized which will lead to better linker design in future generations of ADCs.
Topics: Antibodies, Monoclonal; Cysteine; Immunoconjugates; Lysine; Pharmaceutical Preparations; Protein Engineering
PubMed: 25759187
DOI: 10.1007/s11095-015-1657-7 -
Nature Chemical Biology Nov 2023Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive...
Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.
Topics: Humans; Cysteine; Proteomics; Gene Editing; Proteome; Neoplasms; Nuclear Proteins
PubMed: 37783940
DOI: 10.1038/s41589-023-01428-w -
Angewandte Chemie (International Ed. in... May 2018We report a site-selective cysteine-cyclooctyne conjugation reaction between a seven-residue peptide tag (DBCO-tag, Leu-Cys-Tyr-Pro-Trp-Val-Tyr) at the N or C terminus...
We report a site-selective cysteine-cyclooctyne conjugation reaction between a seven-residue peptide tag (DBCO-tag, Leu-Cys-Tyr-Pro-Trp-Val-Tyr) at the N or C terminus of a peptide or protein and various aza-dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO-tag increases the rate of the thiol-yne reaction 220-fold, thereby enabling selective conjugation of DBCO-tag to DBCO-linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO-tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. This study demonstrates that short peptide tags can aid in accelerating bond-forming reactions that are often slow to non-existent in water.
Topics: Cyclooctanes; Cysteine; Molecular Structure
PubMed: 29575377
DOI: 10.1002/anie.201800860 -
Current Opinion in Chemical Biology Dec 2022Protein S-glutathionylation serves a regulatory role in proteins and modulates distinct biological processes implicated in health and diseases. Despite challenges in... (Review)
Review
Protein S-glutathionylation serves a regulatory role in proteins and modulates distinct biological processes implicated in health and diseases. Despite challenges in analyzing the dynamic and reversible nature of S-glutathionylation, recent chemical and biological methods have significantly advanced the field of S-glutathionylation, culminating in selective identification and detection, structural motif analysis, and functional studies of S-glutathionylation. This review will highlight emerging studies of protein glutathionylation, beginning by introducing biochemical tools that enable mass spectrometric identification and live-cell imaging of S-glutathionylation. Next, it will spotlight recent examples of S-glutathionylation regulating physiology and inflammation. Lastly, we will feature two emerging lines of glutathionylation research in cryptic cysteine glutathionylation and protein C-glutathionylation.
Topics: Glutathione; Oxidation-Reduction; Cysteine; Proteins; Protein Processing, Post-Translational; Biology
PubMed: 36223700
DOI: 10.1016/j.cbpa.2022.102221 -
Cell Chemical Biology Jul 2023Covalent drug discovery has undergone a resurgence over the past two decades and reactive cysteine profiling has emerged in parallel as a platform for ligand discovery... (Meta-Analysis)
Meta-Analysis
Covalent drug discovery has undergone a resurgence over the past two decades and reactive cysteine profiling has emerged in parallel as a platform for ligand discovery through on- and off-target profiling; however, the scope of this approach has not been fully explored at the whole-proteome level. We combined AlphaFold2-predicted side-chain accessibilities for >95% of the human proteome with a meta-analysis of eighteen public cysteine profiling datasets, totaling 44,187 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further showed that hit elaboration and optimization drives increased bias against buried cysteine residues. Based on these data, we suggest that current profiling approaches cover a small proportion of potential ligandable cysteine residues and propose future directions for increasing coverage, focusing on high-priority residues and depth. All analysis and produced resources are freely available and extendable to other reactive amino acids.
Topics: Humans; Cysteine; Proteome; Amino Acids; Drug Discovery; Ligands
PubMed: 37451266
DOI: 10.1016/j.chembiol.2023.06.021 -
Angewandte Chemie (International Ed. in... Oct 2019Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic-peptide ligands... (Review)
Review
Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic-peptide ligands for therapeutic targets, phage-displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage-display technique in which its displayed peptides are cyclized through a proximity-driven Michael addition reaction between a cysteine and an amber-codon-encoded N -acryloyl-lysine (AcrK). Using a randomized 6-mer library in which peptides were cyclized at two ends through a cysteine-AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4- to 6-fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.
Topics: Cyclization; Cysteine; Genetic Code; Humans; Ligands; Lysine; Peptide Library; Peptides, Cyclic
PubMed: 31398275
DOI: 10.1002/anie.201908713 -
Current Opinion in Chemical Biology Jun 2019The recognition that only a small percentage of known human gene products are druggable using traditional modes of non-covalent ligand design, has led to a resurgence in... (Review)
Review
The recognition that only a small percentage of known human gene products are druggable using traditional modes of non-covalent ligand design, has led to a resurgence in targeted covalent inhibitors. Covalent inhibitors offer advantages over non-covalent inhibitors in engaging otherwise challenging targets. Reactive cysteine residues on proteins are a common target for covalent inhibitors, whereby the high nucleophilicity of the cysteine thiol under physiological conditions provides an ideal anchoring site for electrophilic small molecules. A chemical-proteomic platform, termed isoTOP-ABPP, allows for profiling cysteine reactivity in complex proteomes and is one of many techniques that can aid in two aspects of the covalent-inhibitor development process: (1) to identify functional cysteines that lead to modulation of protein activity through covalent modification; and, (2) to determine cellular targets and evaluate promiscuity of electrophilic fragments, small molecules, and natural products. Herein, we discuss recent advances in isoTOP-ABPP and potential applications of this technology in the drug-discovery pipeline.
Topics: Cysteine; Drug Discovery; High-Throughput Screening Assays; Humans
PubMed: 30897495
DOI: 10.1016/j.cbpa.2019.02.010 -
Journal of the American Chemical Society Nov 2022Efficient, site-specific, and bio-orthogonal conjugation of chemical functionalities to proteins is of great utility in fundamental research as well as industrial...
Efficient, site-specific, and bio-orthogonal conjugation of chemical functionalities to proteins is of great utility in fundamental research as well as industrial processes (e.g., the production of antibody-drug conjugates and immobilization of enzymes for biocatalysis). A popular approach involves reacting a free N-terminal cysteine with a variety of electrophilic reagents. However, current methods for generating proteins with N-terminal cysteines have significant limitations. Herein we report a novel, efficient, and convenient method for producing recombinant proteins with free N-terminal cysteines by genetically fusing a Met-Pro-Cys sequence to the N-terminus of a protein of interest and subjecting the recombinant protein to the sequential action of methionine and proline aminopeptidases. The resulting protein was site-specifically labeled at the N-terminus with fluorescein and a cyclic cell-penetrating peptide through native chemical ligation and a 2-cyanobenzothiazole moiety, respectively. In addition, the optimal recognition sequence of proline aminopeptidase was determined by screening a combinatorial peptide library and incorporated into the N-terminus of a protein of interest for most efficient N-terminal processing.
Topics: Cysteine; Aminopeptidases; Recombinant Proteins; Fluorescein; Peptide Library
PubMed: 36378906
DOI: 10.1021/jacs.2c10194 -
The Journal of Biological Chemistry Sep 2013Post-translational S-glutathionylation occurs through the reversible addition of a proximal donor of glutathione to thiolate anions of cysteines in target proteins,... (Review)
Review
Post-translational S-glutathionylation occurs through the reversible addition of a proximal donor of glutathione to thiolate anions of cysteines in target proteins, where the modification alters molecular mass, charge, and structure/function and/or prevents degradation from sulfhydryl overoxidation or proteolysis. Catalysis of both the forward (glutathione S-transferase P) and reverse (glutaredoxin) reactions creates a functional cycle that can also regulate certain protein functional clusters, including those involved in redox-dependent cell signaling events. For translational application, S-glutathionylated serum proteins may be useful as biomarkers in individuals (who may also have polymorphic expression of glutathione S-transferase P) exposed to agents that cause oxidative or nitrosative stress.
Topics: Animals; Cysteine; Gene Expression Regulation, Enzymologic; Glutaredoxins; Glutathione; Glutathione Transferase; Humans; Nitric Oxide; Nitrogen; Oxidation-Reduction; Oxidative Stress; Peroxidases; Protein Processing, Post-Translational; Reactive Oxygen Species; Serpins; Sulfhydryl Compounds
PubMed: 23861399
DOI: 10.1074/jbc.R113.461368 -
Frontiers in Endocrinology 2023Cysteine is one of the least abundant but most conserved amino acid residues in proteins, playing a role in their structure, metal binding, catalysis, and redox... (Review)
Review
Cysteine is one of the least abundant but most conserved amino acid residues in proteins, playing a role in their structure, metal binding, catalysis, and redox chemistry. Thiols present in cysteines can be modified by post-translational modifications like sulfenylation, acylation, or glutathionylation, regulating protein activity and function and serving as signals. Their modification depends on their position in the structure, surrounding amino acids, solvent accessibility, pH, etc. The most studied modifications are the redox modifications by reactive oxygen, nitrogen, and sulfur species, leading to reversible changes that serve as cell signals or irreversible changes indicating oxidative stress and cell damage. Selected antioxidants undergoing reversible oxidative modifications like peroxiredoxin-thioredoxin system are involved in a redox-relay signaling that can propagate to target proteins. Cysteine thiols can also be modified by acyl moieties' addition (derived from lipid metabolism), resulting in protein functional modification or changes in protein anchoring in the membrane. In this review, we update the current knowledge on cysteine modifications and their consequences in pancreatic β-cells. Because β-cells exhibit well-balanced redox homeostasis, the redox modifications of cysteines here serve primarily for signaling purposes. Similarly, lipid metabolism provides regulatory intermediates that have been shown to be necessary in addition to redox modifications for proper β-cell function and, in particular, for efficient insulin secretion. On the contrary, the excess of reactive oxygen, nitrogen, and sulfur species and the imbalance of lipids under pathological conditions cause irreversible changes and contribute to oxidative stress leading to cell failure and the development of type 2 diabetes.
Topics: Humans; Cysteine; Diabetes Mellitus, Type 2; Insulin-Secreting Cells; Sulfhydryl Compounds; Signal Transduction; Oxygen
PubMed: 37455926
DOI: 10.3389/fendo.2023.1221520