Substitution-Induced Mechanistic Switching in SNAr-Warheads for Cysteine Proteases.

The aim of this study was to investigate the transition from non-covalent reversible over covalent reversible to covalent irreversible inhibition of cysteine proteases by making delicate structural changes to the warhead scaffold. To this end, dipeptidic rhodesain inhibitors with different N-terminal electrophilic arenes as warheads relying on the SNAr mechanism were synthesized and investigated. Strong structure-activity relationships of the inhibition potency, the degree of covalency, and the reversibility of binding on the arene substitution pattern were found. The studies were complemented and substantiated by molecular docking and quantum-mechanical calculations of model systems. Furthermore, the improvement in the membrane permeability of peptide esters in comparison to their corresponding carboxylic acids was exemplified.

Activation-Free Sulfonyl Fluoride Probes for Fragment Screening.

SuFEx chemistry is based on the unique reactivity of the sulfonyl fluoride group with a range of nucleophiles. Accordingly, sulfonyl fluorides label multiple nucleophilic amino acid residues, making these reagents popular in both chemical biology and medicinal chemistry applications. The reactivity of sulfonyl fluorides nominates this warhead chemotype as a candidate for an external, activation-free general labelling tag. Here, we report the synthesis and characterization of a small sulfonyl fluoride library that yielded the 3-carboxybenzenesulfonyl fluoride warhead for tagging tractable targets at nucleophilic residues. Based on these results, we propose that coupling diverse fragments to this warhead would result in a library of sulfonyl fluoride bits (SuFBits), available for screening against protein targets. SuFBits will label the target if it binds to the core fragment, which facilitates the identification of weak fragments by mass spectrometry.

Discovery of a Potent Deubiquitinase (DUB) Small-Molecule Activity-Based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells.

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes.

The Design, Synthesis and Mechanism of Action of Paxlovid, a Protease Inhibitor Drug Combination for the Treatment of COVID-19.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has presented an enormous challenge to health care systems and medicine. As a result of global research efforts aimed at preventing and effectively treating SARS-CoV-2 infection, vaccines with fundamentally new mechanisms of action and some small-molecule antiviral drugs targeting key proteins in the viral cycle have been developed. The most effective small-molecule drug approved to date for the treatment of COVID-19 is PaxlovidTM, which is a combination of two protease inhibitors, nirmatrelvir and ritonavir. Nirmatrelvir is a reversible covalent peptidomimetic inhibitor of the main protease (Mpro) of SARS-CoV-2, which enzyme plays a crucial role in viral reproduction. In this combination, ritonavir serves as a pharmacokinetic enhancer, it irreversibly inhibits the cytochrome CYP3A4 enzyme responsible for the rapid metabolism of nirmatrelvir, thereby increasing the half-life and bioavailability of nirmatrelvir. In this tutorial review, we summarize the development and pharmaceutical chemistry aspects of Paxlovid, covering the evolution of protease inhibitors, the warhead design, synthesis and the mechanism of action of nirmatrelvir, as well as the synthesis of ritonavir and its CYP3A4 inhibition mechanism. The efficacy of Paxlovid to novel virus mutants is also overviewed.

Covalent drug discovery using sulfur(VI) fluoride exchange warheads.

INTRODUCTION: Covalent drug discovery has traditionally focused on targeting cysteine, but the amino acid is often absent in protein binding sites. This review makes the case to move beyond cysteine labeling using sulfur (VI) fluoride exchange (SuFEx) chemistry to expand the druggable proteome. AREAS COVERED: Recent advances in SuFEx medicinal chemistry and chemical biology are described, which have enabled the development of covalent chemical probes that site-selectively engage amino acid residues (including tyrosine, lysine, histidine, serine, and threonine) in binding pockets. Areas covered include chemoproteomic mapping of the targetable proteome, structure-based design of covalent inhibitors and molecular glues, metabolic stability profiling, and synthetic methodologies that have expedited the delivery of SuFEx modulators. EXPERT OPINION: Despite recent innovations in SuFEx medicinal chemistry, focused preclinical research is required to ensure the field moves from early chemical probe discovery to the delivery of transformational covalent drug candidates. The authors believe that covalent drug candidates designed to engage residues beyond cysteine using sulfonyl exchange warheads will likely enter clinical trials in the coming years.

The expanding repertoire of covalent warheads for drug discovery.

The reactive functionalities of drugs that engage in covalent interactions with the enzyme/receptor residue in either a reversible or an irreversible manner are called 'warheads'. Covalent warheads that were previously neglected because of safety concerns have recently gained center stage as a result of their various advantages over noncovalent drugs, including increased selectivity, increased residence time, and higher potency. With the approval of several covalent inhibitors over the past decade, research in this area has accelerated. Various strategies are being continuously developed to tune the characteristics of warheads to improve their potency and mitigate toxicity. Here, we review research progress in warhead discovery over the past 5 years to provide valuable insights for future drug discovery.

A covalent strategy to target intrinsically disordered proteins: Discovery of novel tau aggregation inhibitors.

Intrinsically disordered proteins (IDPs) play important roles in disease pathologies; however, their lack of defined stable 3D structures make traditional drug design strategies typically less effective against these targets. Based on promising results of targeted covalent inhibitors (TCIs) on challenging targets, we have developed a covalent design strategy targeting IDPs. As a model system we chose tau, an endogenous IDP of the central nervous system that is associated with severe neurodegenerative diseases via its aggregation. First, we mapped the tractability of available cysteines in tau and prioritized suitable warheads. Next, we introduced the selected vinylsulfone warhead to the non-covalent scaffolds of potential tau aggregation inhibitors. The designed covalent tau binders were synthesized and tested in aggregation models, and inhibited tau aggregation effectively. Our results revealed the usefulness of the covalent design strategy against therapeutically relevant IDP targets and provided promising candidates for the treatment of tauopathies.

Reactivity of Covalent Fragments and Their Role in Fragment Based Drug Discovery.

Fragment based drug discovery has long been used for the identification of new ligands and interest in targeted covalent inhibitors has continued to grow in recent years, with high profile drugs such as osimertinib and sotorasib gaining FDA approval. It is therefore unsurprising that covalent fragment-based approaches have become popular and have recently led to the identification of novel targets and binding sites, as well as ligands for targets previously thought to be 'undruggable'. Understanding the properties of such covalent fragments is important, and characterizing and/or predicting reactivity can be highly useful. This review aims to discuss the requirements for an electrophilic fragment library and the importance of differing warhead reactivity. Successful case studies from the world of drug discovery are then be examined.

Electrophilic warheads in covalent drug discovery: an overview.

INTRODUCTION: Covalent drugs have been used for more than hundred years, but gathered larger interest in the last two decades. There are currently over a 100 different electrophilic warheads used in covalent ligands, and there are several considerations tailoring their reactivity against the target of interest, which is still a challenging task. AREAS COVERED: This review aims to give an overview of electrophilic warheads used for protein labeling in chemical biology and medicinal chemistry. The warheads are discussed by targeted residues, mechanism and selectivity, and analyzed through three different datasets including our collection of warheads, the CovPDB database, and the FDA approved covalent drugs. Moreover, the authors summarize general practices that facilitate the selection of the appropriate warhead for the target of interest. EXPERT OPINION: In spite of the numerous electrophilic warheads, only a fraction of them is used in current drug discovery projects. Recent studies identified new tractable residues by applying a wider array of warhead chemistries. However, versatile, selective warheads are not available for all targetable amino acids, hence discovery of new warheads for these residues is needed. Broadening the toolbox of the warheads could result in novel inhibitors even for challenging targets developing with significant therapeutic potential.

Development and application of novel electrophilic warheads in target identification and drug discovery.

Nucleophilic amino acids play important roles in maintenance of protein structure and function, covalent modification of such amino acid residues by therapeutic agents is an efficient way to treat human diseases. Most of current clinical drugs are structurally limited to α,ß-unsaturated amide as an electrophilic warhead. To alleviate this issue, many novel electrophiles have been developed in recent years that can covalently bind to different amino acid residues and provides a unique way to interrogate proteins, including "undruggable" targets. With an activity-based protein profiling (ABPP) approach, the activity and functionality of a protein and its binding sites can be assessed. This facilitates an understanding of protein function, and contributes to the discovery of new druggable targets and lead compounds. Meanwhile, many novel inhibitors bearing new reactive warhead were developed and displayed remarkable pharmaceutical properties. In this perspective, we have reviewed the recent remarkable progress of novel electrophiles and their applications in target identification and drug discovery.

Covalent inhibitors of GAPDH: From unspecific warheads to selective compounds.

Targeting glycolysis is an attractive approach for the treatment of a wide range of pathologies, such as various tumors and parasitic infections. Due to its pivotal role in the glycolysis, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) represents a rate-limiting enzyme in those cells that mostly, or exclusively rely on this pathway for energy production. In this context, GAPDH inhibition can be a valuable approach for the development of anticancer and antiparasitic drugs. In addition to its glycolytic role, GAPDH possesses several moonlight functions, whose deregulation is involved in some pathological conditions. Covalent modification on different amino acids of GAPDH, in particular on cysteine residues, can lead to a modulation of the enzyme activity. The selectivity towards specific cysteine residues is essential to achieve a specific phenotypic effect. In this work we report an extensive overview of the latest advances on the numerous compounds able to inhibit GAPDH through the covalent binding to cysteine residues, ranging from endogenous metabolites and xenobiotics, which may serve as pharmacological tools to actual drug-like compounds with promising therapeutic perspectives. Furthermore, we focused on the potentialities of the different warheads, shedding light on the possibility to exploit a combination of a finely tuned electrophilic group with a well-designed recognition moiety. These findings can provide useful information for the rational design of novel covalent inhibitors of GAPDH, with the final goal to expand the current treatment options.

Novel disaccharide inhibitors for the bacterial galactosyltransferase LgtC: Design, synthesis via Heyns rearrangement, and biochemical evaluation.

Bacterial glycosyltransferases are potential targets for the development of novel antibiotics and anti-virulence agents. We report a novel inhibitor design for the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. Our design is based on the installation of an electrophilic warhead on the LgtC acceptor substrate and targeted at a non-catalytic cysteine residue in the LgtC active site. We have successfully synthesised two prototype inhibitors in four steps from lactulose. The key step in our synthesis is a Heyns rearrangement, during which we observed the formation of a hitherto unknown side product. While both lactosamine derivatives behaved as moderate inhibitors of LgtC, they also retained residual substrate activity. These results suggest that in contrast to our original design, these inhibitors do not act via a covalent mode of action, but are most likely non-covalent inhibitors.