bioTCIs: Middle-to-Macro Biomolecular Targeted Covalent Inhibitors Possessing Both Semi-Permanent Drug Action and Stringent Target Specificity as Potential Antibody Replacements.

Monoclonal antibody therapies targeting immuno-modulatory targets such as checkpoint proteins, chemokines, and cytokines have made significant impact in several areas, including cancer, inflammatory disease, and infection. However, antibodies are complex biologics with well-known limitations, including high cost for development and production, immunogenicity, a limited shelf-life because of aggregation, denaturation, and fragmentation of the large protein. Drug modalities such as peptides and nucleic acid aptamers showing high-affinity and highly selective interaction with the target protein have been proposed alternatives to therapeutic antibodies. The fundamental limitation of short in vivo half-life has prevented the wide acceptance of these alternatives. Covalent drugs, also known as targeted covalent inhibitors (TCIs), form permanent bonds to target proteins and, in theory, eternally exert the drug action, circumventing the pharmacokinetic limitation of other antibody alternatives. The TCI drug platform, too, has been slow in gaining acceptance because of its potential prolonged side-effect from off-target covalent binding. To avoid the potential risks of irreversible adverse drug effects from off-target conjugation, the TCI modality is broadening from the conventional small molecules to larger biomolecules possessing desirable properties (e.g., hydrolysis resistance, drug-action reversal, unique pharmacokinetics, stringent target specificity, and inhibition of protein-protein interactions). Here, we review the historical development of the TCI made of bio-oligomers/polymers (i.e., peptide-, protein-, or nucleic-acid-type) obtained by rational design and combinatorial screening. The structural optimization of the reactive warheads and incorporation into the targeted biomolecules enabling a highly selective covalent interaction between the TCI and the target protein is discussed. Through this review, we hope to highlight the middle to macro-molecular TCI platform as a realistic replacement for the antibody.

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.

Discovery, synthesis and mechanism study of 2,3,5-substituted [1,2,4]-thiadiazoles as covalent inhibitors targeting 3C-Like protease of SARS-CoV-2.

The 3C-like protease (3CLpro) is essential for the replication and transcription of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it a promising target for the treatment of corona virus disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to be able to inhibit 3CLpro as non-peptidomimetic covalent binders at submicromolar levels, with IC50 values ranging from 0.118 to 0.582 µM. Interestingly, these compounds were also shown to inhibit PLpro with the same level of IC50 values, but had negligible effect on proteases such as chymotrypsin, cathepsin B, and cathepsin L. Subsequently, the antiviral abilities of these compounds were evaluated in cell-based assays, and compound 6g showed potent antiviral activity with an EC50 value of 7.249 µM. It was proposed that these compounds covalently bind to the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. To understand this covalent-binding reaction, we chose compound 6a, one of the identified hit compounds, as a representative to investigate the reaction mechanism in detail by combing several computational predictions and experimental validation. The process of ring-opening metathesis was theoretically studied using quantum chemistry calculations according to the transition state theory. Our study revealed that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CLpro as a potential warhead. Moreover, 6a was a known GPCR modulator, and our study is also a successful computational method-based drug-repurposing study.

Structural Basis for the Inhibition of Coronaviral Main Proteases by a Benzothiazole-Based Inhibitor.

The ongoing spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused hundreds of millions of cases and millions of victims worldwide with serious consequences to global health and economies. Although many vaccines protecting against SARS-CoV-2 are currently available, constantly emerging new variants necessitate the development of alternative strategies for prevention and treatment of COVID-19. Inhibitors that target the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation, represent a key class of antivirals. Here, we showed that a peptidomimetic compound with benzothiazolyl ketone as warhead, YH-53, is an effective inhibitor of SARS-CoV-2, SARS-CoV, and MERS-CoV Mpros. Crystal structures of Mpros from SARS-CoV-2, SARS-CoV, and MERS-CoV bound to the inhibitor YH-53 revealed a unique ligand-binding site, which provides new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures defined the key molecular determinants required for inhibition and illustrate the binding mode of Mpros from other coronaviruses. In consideration of the important role of Mpro in developing antivirals against coronaviruses, insights derived from this study should add to the design of pan-coronaviral Mpro inhibitors that are safer and more effective.

Covalent Reversible Inhibitors of Cysteine Proteases Containing the Nitrile Warhead: Recent Advancement in the Field of Viral and Parasitic Diseases.

In the field of drug discovery, the nitrile group is well represented among drugs and biologically active compounds. It can form both non-covalent and covalent interactions with diverse biological targets, and it is amenable as an electrophilic warhead for covalent inhibition. The main advantage of the nitrile group as a warhead is mainly due to its milder electrophilic character relative to other more reactive groups (e.g., -CHO), reducing the possibility of unwanted reactions that would hinder the development of safe drugs, coupled to the ease of installation through different synthetic approaches. The covalent inhibition is a well-assessed design approach for serine, threonine, and cysteine protease inhibitors. The mechanism of hydrolysis of these enzymes involves the formation of a covalent acyl intermediate, and this mechanism can be exploited by introducing electrophilic warheads in order to mimic this covalent intermediate. Due to the relevant role played by the cysteine protease in the survival and replication of infective agents, spanning from viruses to protozoan parasites, we will review the most relevant and recent examples of protease inhibitors presenting a nitrile group that have been introduced to form or to facilitate the formation of a covalent bond with the catalytic cysteine active site residue.

Discovery of Novel Epoxyketone Peptides as Lipase Inhibitors.

Obesity is the most common nutritional disorder in the developed world and is associated with important comorbidities. Pancreatic lipase (PL) inhibitors play a key role in the metabolism of human fat. A series of novel epoxyketones peptide derivatives were investigated for their pancreatic lipase inhibitory activity. The epoxyketone moiety is a well-known reactive electrophile group that has been used as part of proteasome inhibitors in cancer therapy, and it is widely believed that these are very selective for targeting the proteasome active site. Here we investigated various peptide derivatives with an epoxide warhead for their anti-lipase activity. The assessment of these novel epoxyketones was performed by an in-house method that we developed for rapid screening and identification of lipase inhibitors using GC-FID. Herein, we present a novel anti-lipase pharmacophore based on epoxyketone peptide derivatives that showed potent anti-lipase activity. Many of these derivatives had comparable or more potent activity than the clinically used lipase inhibitors such as orlistat. In addition, the lipase appears to be inhibited by a wide range of epoxyketone analogues regardless of the configuration of the epoxide in the epoxyketone moiety. The presented data in this study shows the first example of the use of epoxyketone peptides as novel lipase inhibitors.