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.

Rational Design of a Highly Reactive Dicysteine Peptide Tag For Fluorogenic Protein Labelling [@theKeillors]

Miroslava Strmiskova,  Kelvin Tsao  and  Jeffrey W Keillor
Org. Biomol. Chem., 2018
doi: 10.1039/C8OB01417E

Rationally designed libraries of a short helical peptide sequence containing two cysteine residues were screened kinetically for their reactivity towards complementary dimaleimide fluorogens. This screening revealed variant sequences whose reactivity has been increased by an order of magnitude relative to the original sequence. The most reactive engineered sequences feature mutant residues bearing positive charges, suggesting the pKa values of the adjacent thiol groups have been significantly lowered, through electrostatic stabilization of the thiolate ionization state. pH-rate profiles measured for several mutant sequences support this mechanism of rate enhancement. The practical utility of the enhanced reactivity of the final engineered dicysteine tag (‘dC10*’) was then demonstrated in the fluorogenic intracellular labelling of a specific protein in living cells.

Target Identification of Bioactive Covalently Acting Natural Products

Nomura D.K., Maimone T.J.

Current Topics in Microbiology and Immunology, 2018
doi: 10.1007/82_2018_121

There are countless natural products that have been isolated from microbes, plants, and other living organisms that have been shown to possess therapeutic activities such as antimicrobial, anticancer, or anti-inflammatory effects. However, developing these bioactive natural products into drugs has remained challenging in part because of their difficulty in isolation, synthesis, mechanistic understanding, and off-target effects. Among the large pool of bioactive natural products lies classes of compounds that contain potential reactive electrophilic centers that can covalently react with nucleophilic amino acid hotspots on proteins and other biological molecules to modulate their biological action. Covalently acting natural products are more amenable to rapid target identification and mapping of specific druggable hotspots within proteins using activity-based protein profiling (ABPP)-based chemoproteomic strategies. In addition, the granular biochemical insights afforded by knowing specific sites of protein modifications of covalently acting natural products enable the pharmacological interrogation of these sites with more synthetically tractable covalently acting small molecules whose structures are more easily tuned. Both discovering binding pockets and targets hit by natural products and exploiting druggable modalities targeted by natural products with simpler molecules may overcome some of the challenges faced with translating natural products into drugs.

A green BODIPY‐based, super‐fluorogenic, protein‐specific labelling agent

Yingche Chen, Kelvin Tsao, Sydney L. Acton, Jeffrey W. Keillor

Angewandte Chemie, 2018
doi: 10.1002/ange.201805482

We report the development of YC23, a novel green BODIPY‐based dimaleimide derivative that undergoes a Fluorogenic Addition Reaction (FlARe) with a genetically encodable peptide tag (dC10α) that can be fused to a protein of interest (POI). We also demonstrate the application of this reaction for the fluorogenic labelling of a specific POI in bacterial lysate and in living mammalian cells.

Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315

Clarissa G. Jakob, Anup K. Upadhyay, Pamela L. Donner, Emily Nicholl, Sadiya N. Addo, Wei Qiu, Christopher Ling, Sujatha M. Gopalakrishnan, Maricel Torrent, Steven P. Cepa, Jason Shanley, Alexander R. Shoemaker, Chaohong C. Sun∥, Anil Vasudevan, Kevin R. Woller, J. Brad Shotwell, Bailin Shaw, Zhiguo Bian, and Jessica E. Hutti

J. Med. Chem, 2018 61 (15), 6647–6657

DOI: 10.1021/acs.jmedchem.8b00305

IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsaturated enone 1 makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design.

Precedence and Promise of Covalent Inhibitors of EGFR and KRAS for Patients with Non-Small-Cell Lung Cancer

Hengmiao Cheng  and Simon Planken

ACS Med. Chem. Lett., 2018
DOI: 10.1021/acsmedchemlett.8b00311

Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogenic mutations are leading causes for lung cancer. Extensive drug discovery efforts targeting EGFR have led to the discovery and FDA approval of both reversible and covalent inhibitors. Second and third generation covalent inhibitors for EGFR have also been described, with the latter targeting specific emerging mutations. After decades of extensive effort, KRAS is widely regarded as an intractable therapeutic target; however, recent publications suggest covalent inhibition is a promising strategy to deliver inhibitors of the KRASG12C mutation.