Covalent modification of biomolecules through maleimide-based labeling strategies

Cyrille Sabot, Pierre-Yves Renard, Kevin Renault, and Jean Wilfried Fredy

Bioconjugate Chem., 2018
DOI: 10.1021/acs.bioconjchem.8b00252

Since their first use in bioconjugation more than 50 years ago, maleimides have become privileged chemical partners for the site selective modification of proteins through thio-Michael addition of biothiols and, to a lesser extent, via Diels‒Alder (DA) reactions in combination with biocompatible dienes. Prominent examples include immunotoxins and marketed maleimide-based antibody-drug conjugates (ADCs) such as Adcetris® used in cancer therapies. Among the keys to success is the availability of several maleimides N-functionalized by fluorophores, affinity tags, spin labels, pharmacophores, as well as their unique reactivity features in terms of selectivity or kinetics. However, maleimide conjugate reactions have long been thought to be irreversible, and it is only recently that systematic studies regarding their reversibility and stability towards hydrolysis have been reported. This review provides an overview of the diversity of applications of maleimides in bioconjugation, highlighting their strengths and weaknesses, which are being circumvented by recent strategies. Finally, the fluorescence quenching ability of maleimides was leveraged in the preparation of fluorogenic probes mainly involved in the specific detection of thiol analytes. A summary of reported structures, their photophysical features and relative efficiency are discussed in the last part of the review.

Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide

Tang Li, René Maltais, Donald Poirier, and Sheng-Xiang Lin


J. Phys. Chem. Lett., 2018, 9, 5275–5280

DOI: 10.1021/acs.jpclett.8b02225

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) plays a pivotal role in the progression of estrogen-related diseases because of its involvement in the biosynthesis of estradiol (E2), constituting a valuable therapeutic target for endocrine treatment. In the present study, we successfully cocrystallized the enzyme with the reversible inhibitor 2-methoxy-16β-(m-carbamoylbenzyl)-E2 (2-MeO-CC-156) as well as the enzyme with the irreversible inhibitor 3-(2-bromoethyl)-16β-(m-carbamoylbenzyl)-17β-hydroxy-1,3,5(10)-estratriene (PBRM). The structures of ternary complexes of 17β-HSD1–2-MeO-CC-156–NADP+ and 17β-HSD1–PBRM–NADP+ comparatively show the formation of a covalent bond between His221 and the bromoethyl side chain of the inhibitor in the PBRM structure. A dynamic process including beneficial molecular interactions that favor the specific binding of a low-reactivity inhibitor and subsequent N-alkylation event through the participation of His221 in the enzyme catalytic site clearly demonstrates the covalent bond formation. This finding opens the door to a new design of alkyl halide-based specific covalent inhibitors as potential therapeutic agents for different enzymes, contributing to the development of highly efficient inhibitors.

Discovery and Optimization of Inhibitors of the Parkinson’s Disease Associated Protein DJ-1

ACS Chem. Biol., 2018

DOI: 10.1021/acschembio.8b00701

DJ-1 is a Parkinson’s disease associated protein endowed with enzymatic, redox sensing, regulatory, chaperoning, and neuroprotective activities. Although DJ-1 has been vigorously studied for the past decade and a half, its exact role in the progression of the disease remains uncertain. In addition, little is known about the spatiotemporal regulation of DJ-1, or the biochemical basis explaining its numerous biological functions. Progress has been hampered by the lack of inhibitors with precisely known mechanisms of action. Herein, we have employed biophysical methodologies and X-ray crystallography to identify and to optimize a family of compounds inactivating the critical Cys106 residue of human DJ-1. We demonstrate these compounds are potent inhibitors of various activities of DJ-1 in vitro and in cell-based assays. This study reports a new family of DJ-1 inhibitors with a defined mechanism of action, and contributes toward the understanding of the biological function of DJ-1.

Design and Characterization of Novel Covalent Bromodomain and Extra-terminal Domain (BET) Inhibitors Targeting a Methionine

Olesya Kharenko, Reena G Patel, S. David Brown, Cyrus Calosing, Andre White, Damodharan Lakshminarasimhan, Robert K Suto, Bryan C Duffy, Douglas B. Kitchen, Kevin G McLure, Henrik C. Hansen, Edward H van der Horst, and Peter R. Young

J. Med. Chem., 2018
DOI: 10.1021/acs.jmedchem.8b00666

BET proteins are key epigenetic regulators that alter transcription by binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small molecule bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacology. Here we describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented non-covalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry and an X-ray crystal structure, we demonstrate that these inhibitors selectively form a covalent bond with Met149 in BRD4(BD1) but not other bromodomains, and provide durable transcriptional and anti-proliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.

Covalent Modification of Biomolecules through Maleimide-Based Labeling Strategies

Kévin Renault, Jean Wilfried Fredy, Pierre-Yves Renard, and Cyrille Sabot

Bioconjugate Chem., 2018, 29 (8),  2497–2513
DOI: 10.1021/acs.bioconjchem.8b00252

Since their first use in bioconjugation more than 50 years ago, maleimides have become privileged chemical partners for the site-selective modification of proteins via thio-Michael addition of biothiols and, to a lesser extent, via Diels–Alder (DA) reactions with biocompatible dienes. Prominent examples include immunotoxins and marketed maleimide-based antibody–drug conjugates (ADCs) such as Adcetris, which are used in cancer therapies. Among the key factors in the success of these groups is the availability of several maleimides that can be N-functionalized by fluorophores, affinity tags, spin labels, and pharmacophores, as well as their unique reactivities in terms of selectivity and kinetics. However, maleimide conjugate reactions have long been considered irreversible, and only recently have systematic studies regarding their reversibility and stability toward hydrolysis been reported. This review provides an overview of the diverse applications for maleimides in bioconjugation, highlighting their strengths and weaknesses, which are being overcome by recent strategies. Finally, the fluorescence quenching ability of maleimides was leveraged for the preparation of fluorogenic probes, which are mainly used for the specific detection of thiol analytes. A summary of the reported structures, their photophysical features, and their relative efficiencies is discussed in the last part of the review.

How Reactive are Druggable Cysteines in Protein Kinases? [@RowleyGroup]

Ernest Awoonor-Williams and Christopher N. Rowley

J. Chem. Inf. Model., 2018
DOI: 10.1021/acs.jcim.8b00454

Targeted covalent inhibitors (TCIs) have been successfully developed as high-affinity and selective inhibitors of enzymes of the protein kinase family. These drugs typically act by undergoing an electrophilic addition with an active-site cysteine residue, so design of a TCI begins with the identification of a “druggable” cysteine. These electrophilic additions generally require deprotonation of the thiol to form a reactive anionic thiolate, so the acidity of the residue is a critical factor. Few experimental measurements of the pKa’s of druggable cysteines have been reported, so computational prediction could prove to be very important in selecting reactive cysteine targets. Here we report the computed pKa’s of druggable cysteines in selected protein kinases that are of clinical relevance for targeted therapies. The pKa’s of the cysteines were calculated using advanced computational methods based on all-atom replica-exchange thermodynamic integration molecular dynamics simulations in explicit solvent. We found that the acidities of druggable cysteines within protein kinases are diverse and elevated, indicating enormous differences in their reactivity. Constant-pH molecular dynamics simulations were also performed on selected protein kinases, and the results confirmed this varied range in the acidities of druggable cysteines. Many of these active-site cysteines have low exposure to solvent molecules, elevating their pKa values. Electrostatic interactions with nearby anionic residues also elevate the pKa’s of cysteine residues in the active site. The results suggest that some cysteine residues within kinase binding sites will be slow to react with a TCI because of their low acidity. Several oncogenic kinase mutations were also modeled and found to have pKa’s similar to that of the wild-type kinase.

Applications of Reactive Cysteine Profiling

Keriann M. Backus

Current Topics in Microbiology and Immunology book series, 2018
doi: 10.1007/82_2018_120

Cysteine thiols are involved in a diverse set of biological transformations, including nucleophilic and redox catalysis, metal coordination and formation of both dynamic and structural disulfides. Often posttranslationally modified, cysteines are also frequently alkylated by electrophilic compounds, including electrophilic metabolites, drugs, and natural products, and are attractive sites for covalent probe and drug development. Quantitative proteomics combined with activity-based protein profiling has been applied to annotate cysteine reactivity, susceptibility to posttranslational modifications, and accessibility to chemical probes, uncovering thousands of functional and small-molecule targetable cysteines across a diverse set of proteins, proteome-wide in an unbiased manner. Reactive cysteines have been targeted by high-throughput screening and fragment-based ligand discovery efforts. New cysteine-reactive electrophiles and compound libraries have been synthesized to enable inhibitor discovery broadly and to minimize nonspecific toxicity and off-target activity of compounds. With the recent blockbuster success of several covalent inhibitors, and the development of new chemical proteomic strategies to broadly identify reactive, ligandable and posttranslationally modified cysteines, cysteine profiling is poised to enable the development of new potent and selective chemical probes and even, in some cases, new drugs.