Development Of Covalent Drugs Research Articles

CovalentInDB 2.0: an updated comprehensive database for structure-based and ligand-based covalent inhibitor design and screening.

The rational design of targeted covalent inhibitors (TCIs) has emerged as a powerful strategy in drug discovery, known for its ability to achieve strong binding affinity and prolonged target engagement. However, the development of covalent drugs is often challenged by the need to optimize both covalent warhead and non-covalent interactions, alongside the limitations of existing compound libraries. To address these challenges, we present CovalentInDB 2.0, an updated online database designed to support covalent drug discovery. This updated version includes 8303 inhibitors and 368 targets, supplemented by 3445 newly added cocrystal structures, providing detailed analyses of non-covalent interactions. Furthermore, we have employed an AI-based model to profile the ligandability of 144 864 cysteines across the human proteome. CovalentInDB 2.0 also features the largest covalent virtual screening library with 2 030 192 commercially available compounds and a natural product library with 105 901 molecules, crucial for covalent drug screening and discovery. To enhance the utility of these compounds, we performed structural similarity analysis and drug-likeness predictions. Additionally, a new user data upload feature enables efficient data contribution and continuous updates. CovalentInDB 2.0 is freely accessible at http://cadd.zju.edu.cn/cidb/.

Precious Cargo: The Role of Polymeric Nanoparticles in the Delivery of Covalent Drugs

Covalent drugs can offer significant advantages over non-covalent drugs in terms of pharmacodynamics (i.e., target-binding properties). However, the development of covalent drugs is sometimes hampered by pharmacokinetic limitations (e.g., low bioavailability, rapid metabolism and toxicity due to off-target binding). Polymeric nanoparticles offer a potential solution to these limitations. Delivering covalent drugs via polymeric nanoparticles provides myriad benefits in terms of drug solubility, permeability, lifetime, selectivity, controlled release and the opportunity for synergistic administration alongside other drugs. In this short review, we examine each of these benefits in turn, illustrated through multiple case studies.

Two-Step Covalent Docking with Attracting Cavities.

Due to their various advantages, interest in the development of covalent drugs has been renewed in the past few years. It is therefore important to accurately describe and predict their interactions with biological targets by computer-aided drug design tools such as docking algorithms. Here, we report a covalent docking procedure for our in-house docking code Attracting Cavities (AC), which mimics the two-step mechanism of covalent ligand binding. Ligand binding to the protein cavity is driven by nonbonded interactions, followed by the formation of a covalent bond between the ligand and the protein through a chemical reaction. To test the performance of this method, we developed a diverse, high-quality, openly accessible re-docking benchmark set of 95 covalent complexes bound by 8 chemical reactions to 5 different reactive amino acids. Combination with structures from previous studies resulted in a set of 304 complexes, on which AC obtained a success rate (rmsd ≤ 2 Å) of 78%, outperforming two state-of-the-art covalent docking codes, genetic optimization for ligand docking (GOLD (66%)) and AutoDock (AD (35%)). Using a more stringent success criterion (rmsd ≤ 1.5 Å), AC reached a success rate of 71 vs 55% for GOLD and 26% for AD. We additionally assessed the cross-docking performance of AC on a set of 76 covalent complexes of the SARS-CoV-2 main protease. On this challenging test set of mainly small and highly solvent-exposed ligands, AC yielded success rates of 58 and 28% for re-docking and cross-docking, respectively, compared to 45 and 17% for GOLD.

AzidoTMT Enables Direct Enrichment and Highly Multiplexed Quantitation of Proteome-Wide Functional Residues.

Recent advances in targeted covalent inhibitors have aroused significant interest for their potential in drug development for difficult therapeutic targets. Proteome-wide profiling of functional residues is an integral step of covalent drug discovery aimed at defining actionable sites and evaluating compound selectivity in cells. A classical workflow for this purpose is called IsoTOP-ABPP, which employs an activity-based probe and two isotopically labeled azide-TEV-biotin tags to mark, enrich, and quantify proteome from two samples. Here we report a novel isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP, that significantly expands multiplexing power as compared to the original isoTOP-ABPP. We demonstrate its application in identifying cysteine on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620. However, changes in some of these hits can be explained by modulation at the protein and post-translational levels. Thus, it would be crucial to interrogate site-level bona fide changes in concurrence to proteome-level changes for corroboration. In addition, we perform a multiplexed covalent fragment screening using four acrylamide-based compounds as a proof-of-concept. This study identifies a diverse set of liganded cysteine residues in a compound-dependent manner with an average hit rate of 0.07% in intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds to demonstrate that the AT-MAPP assay is flexible for noncysteine functional residues such as tyrosine and lysine. Overall, we envision that 11plex-AzidoTMT will be a useful addition to the current toolbox for activity-based protein profiling and covalent drug development.

Recent Advances in Covalent Drug Discovery.

In spite of the increasing number of biologics license applications, the development of covalent inhibitors is still a growing field within drug discovery. The successful approval of some covalent protein kinase inhibitors, such as ibrutinib (BTK covalent inhibitor) and dacomitinib (EGFR covalent inhibitor), and the very recent discovery of covalent inhibitors for viral proteases, such as boceprevir, narlaprevir, and nirmatrelvir, represent a new milestone in covalent drug development. Generally, the formation of covalent bonds that target proteins can offer drugs diverse advantages in terms of target selectivity, drug resistance, and administration concentration. The most important factor for covalent inhibitors is the electrophile (warhead), which dictates selectivity, reactivity, and the type of protein binding (i.e., reversible or irreversible) and can be modified/optimized through rational designs. Furthermore, covalent inhibitors are becoming more and more common in proteolysis, targeting chimeras (PROTACs) for degrading proteins, including those that are currently considered to be 'undruggable'. The aim of this review is to highlight the current state of covalent inhibitor development, including a short historical overview and some examples of applications of PROTAC technologies and treatment of the SARS-CoV-2 virus.

CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

Hitting KRAS When It's Down.

KRAS, one of the most prevalent oncogenes and sought-after anticancer targets, has eluded chemists for decades until an irreversible covalent strategy targeting a specific mutation (G12C) paved the way for the first KRAS inhibitors to reach the clinic. MRTX849 is one such clinical candidate with promising initial results in patients harboring the mutation. The impressive optimization story of MRTX849 highlights challenges and solutions in the development of covalent drugs, including the use of an α-fluoroacrylamide electrophile.

Toward Atomistic Modeling of Irreversible Covalent Inhibitor Binding Kinetics.

Covalent inhibitors have emerged as an important drug class in recent years, largely due to their many unique advantages as compared to noncovalent inhibitors, including longer duration of action, lower prolonged systemic exposure, higher potency, and selectivity. However, the potential off-target toxicity of covalent inhibitors, particularly of irreversible covalent inhibitors, represents a great challenge in covalent drug development. Therefore, accurate calculation of protein covalent inhibitor reaction kinetics to guide the design of selective inhibitors would greatly benefit covalent drug discovery efforts. In the present paper, we present a computational method to calculate the relative reaction kinetics between congeneric irreversible covalent inhibitors and their protein receptors. The method combines density functional theory calculations of the transition state barrier height of the rate-limiting step for reaction between the warhead of the inhibitor and a single protein residue, and molecular-mechanics-based free energy calculations to account for the interactions between the ligand in the transition state and the protein environment. The method was tested on four pharmaceutically interesting irreversible covalent binding systems involving 28 ligands; the mean unsigned error (MUE) of the relative reaction rate for all pairs of ligands between the predictions and experimental results for these tested systems is 0.79 log unit. This is to our knowledge the first time where the reaction kinetics of protein irreversible covalent inhibition have been directly calculated with physics-based free energy calculation methods and transition state theory. We anticipate the outstanding accuracy demonstrated here across a broad range of target classes will have a strong impact on the design of selective covalent inhibitors.

Distinguishing the optimal binding mechanism of an E3 ubiquitin ligase: Covalent versus noncovalent inhibition.

The development of covalent drugs, specifically in cancer therapeutics, has recently sparked interest among the pharmaceutical research community. While representing a significant fraction of the drugs in the market, very few have been deliberately designed to interact covalently with their biological target. One of the enzymes that have been both covalently and noncovalently targeted is the Neural Precursor Cell Expressed Developmentally Downregulated gene 4-1 (Nedd4-1). This enzyme has been found to have multiple physiological implications, including its involvement in cancer invasion. A critical gap still remains in the molecular understanding of the structural mechanism upon the covalent and noncovalent binding to Nedd4-1. In this study, we explore the most optimal binding mechanism in the inhibition of the catalytic site of the Nedd4-1. Our results exhibited a greater stability in the covalent complex compared with the noncovalent complex. This was supported by the secondary structure elements that were more dominant in the covalently inhibited complex. This complex disclosed an optimal free binding energy landscape, induced by the catalytic site energy contributions that showed to be more favorable. The insights demonstrating the above binding mechanism of Nedd4-1 establishes covalent inhibition as the preferred method of inhibition of the enzyme. This investigation aids in the understanding of the structural mechanism of Nedd4-1 inhibition and would assist in the design of more potent covalent inhibitors at the catalytic site of Nedd4-1.

Versatile Bioconjugation Chemistries of ortho-Boronyl Aryl Ketones and Aldehydes.

Biocompatible and bioorthogonal conjugation reactions have proven to be powerful tools in biological research and medicine. While the advent of bioorthogonal conjugation chemistries greatly expands our capacity to interrogate specific biomolecules in situ, biocompatible reactions that target endogenous reactive groups have given rise to a number of covalent drugs as well as a battery of powerful research tools. Despite the impressive progress, limitations do exist with the current conjugation chemistries. For example, most known bioorthogonal conjugations suffer from slow reaction rates and imperfect bioorthogonality. On the other hand, covalent drugs often display high toxicity due to off-target labeling and immunogenicity. These limitations demand continued pursuit of conjugation chemistries with optimal characteristics for biological applications. A spate of papers appearing in recent literature report the conjugation chemistries of 2-formyl and 2-acetyl phenylboronic acids (abbreviated as 2-FPBA and 2-APBA, respectively). These simple reactants are found to undergo fast conjugation with various nucleophiles under physiological conditions, showing great promise for biological applications. The versatile reactivity of 2-FPBA and 2-APBA manifests in dynamic conjugation with endogenous nucleophiles as well as conjugation with designer nucleophiles in a bioorthogonal manner. 2-FPBA/APBA conjugates with amines in biomolecules, such as lysine side chains and aminophospholipids, in a highly dynamic manner to give iminoboronates. In contrast to typical imines, iminoboronates enjoy much improved thermodynamic stability, yet are kinetically labile for hydrolysis due to imine activation bythe boronic acid. Dynamic conjugations as such presenta novel binding mechanism analogous to hydrogen bonding and electrostatic interactions. Implementation of this covalent binding mechanism has yielded reversible covalent probes of prevalent bacterial pathogens. It has also resulted in reversible covalent inhibitors of a therapeutically important protein Mcl-1. Such covalent probes/inhibitors with 2-FPBA/APBA warheads avoid permanent modification of their biological target, potentially able to mitigate off-target labeling and immunogenicity of covalent drugs. The dynamic conjugation of 2-FPBA/APBA has been recently extended to N-terminal cysteines, which can be selectively targeted via formation of a thiazolidino boronate (TzB) complex. The dynamic TzB formation expands the toolbox for site-specific protein labeling and the development of covalent drugs. On the front of bioorthogonal conjugation, 2-FPBA/APBA has been found to conjugate with α-nucleophiles under physiologic conditions with rate constant ( k2) over 1000 M-1 s-1, which overcomes the slow kinetics problems and rekindles the interest of using the conjugation of α-nucleophiles for biological studies. With fast kinetics being a shared feature, this family of conjugation chemistries gives remarkably diverse product structures depending on the choice of nucleophile. Importantly, both dynamic and irreversible conjugations have been developed, which we believe will enable a wide array of applications in biological research. In this Account, we collectively examine this rapidly expanding family of conjugation reactions, seeking to elucidate the unifying principles that would guidefurther development of novel conjugation reactions, as well as their applications in biology.

Using Protein-Confined Proximity To Determine Chemical Reactivity.

Chemical reactivity is essential for functional modification of biomolecules with small molecules and the development of covalent drugs. The reactivity between a chemical functional group of a small molecule and that of a large biomolecule cannot be reliably predicted from the reactivity of the corresponding functional groups separately installed on two small molecules, because the proximity effect on reactivity resulting from the binding of the small molecule to the biomolecule is challenging to achieve by mixing two small molecules. Here we present a new strategy to determine the chemical reactivity of two functional groups in the context of close proximity afforded by proteins. The functional groups to be tested were separately installed at the interface of two interacting proteins in the format of amino acid side chains via the expansion of the genetic code. Reaction of the two functional groups resulted in covalent cross-linking of interacting proteins, readily detectable by gel electrophoresis. Using this strategy, we evolved new synthetases to genetically encode Nε-fluoroacetyllysine (FAcK), an isosteric fluorine analogue of acetyllysine. We demonstrated that fluoroacetamide installed on FAcK, previously thought inert to biological functional groups, actually reacted with the thiol group of cysteine when in proximity. This strategy should be valuable for accurately evaluating chemical reactivity of small molecules toward large biomolecules, which will help avoid undesired side reactions of drugs and expand the repertoire of functional groups to covalently target biomolecules.

Celgene Gains Covalent Drug Pipeline and Platform via Avila Therapeutics Acquisition

Celgene has agreed to acquire Avila Therapeutics in a deal that is valued at up to US$925 M. With the acquisition, Celgene will gain AVL-292, an oral Bruton’s tyrosine kinase (Btk) inhibitor in Phase I development for haematological malignancies and autoimmune diseases, and Avila’s Avilomics™ platform for the discovery and development of targeted covalent drugs. The deal, which has been approved by the Boards of Directors of both companies, is expected to complete during the first quarter of 2012.