Exploring Covalent Modulators in Drug Discovery and Chemical Biology

In the call for papers for this Special Issue, we invited the scientific community to submit Articles that embraced the interface between chemistry and biology as it relates to covalent modulators of activity. The Articles captured in this Special Issue are exactly the breadth of science these editors had hoped for, with Articles that span the drug discovery continuum from technology development (in the case of new chemical biology techniques leveraging covalency and novel warheads) to the disclosure of covalent clinical candidates.

No special issue around covalent modulators would be complete without Articles that both help us gain understanding of the specific parameters that drive efficient covalent modification as well as the development of new chemistries and approaches utilizing covalency. An example of this is in the collaborative work between the Conway lab and GSK, a method enabling rapid in cell target validation utilizing a “mutate and conjugate” approach that combines both site-directed mutagenesis and screening of electrophilic fragments. The marriage of DNA-encoded library technology and the application toward covalent ligand discovery was captured in a Perspective by Dickson. Focusing on another early stage drug discovery technique exploiting the Minifrags concept (screening of ultralow molecular weight ligands), the joint teams of Meyer-Almes and Keseru outline a systematic approach utilizing the electrophilic reactivity of their fragments to map binding sites in HDAC8.

Novel Warheads

There is growing interest in developing new covalent electrophiles as a strategy to increase potency, prolong the duration of action, and improve selectivity, and this interest is reflected in this Special Issue. Novel electrophiles can expand the druggable proteome by targeting previously “undruggable” residues beyond cysteine, such as lysine, tyrosine, or serine. As an update to their 2019 contribution, Gehringer et al. provided a summary of Emerging and Re-emerging Covalent Warheads, providing a big picture view of the current trends across the medicinal chemistry and chemical biology community. Additionally, Ojida et al. provided a detailed report exploring the chemistry and reactivity of the dihaloacetamide functional group for reversible targeting of cysteine, while Baud et al. identified 2-sulfonylpyrimidine as an irreversible alternative to acrylamides. Pemberton and co-workers characterize the reactivity of vinylpyridines in the context of covalent EGFR inhibitors. Gehringer and colleagues demonstrated that a chloronitropyridine was able to generate isoform selective inhibitors of FGFR4, via an unconventional S_NAr with a cysteine in the hinge region that is not present in other FGFR family members. Separately, the team of Yoo and co-workers detail the discovery of a reactive scaffold, 4-chloropyrazolopyridine, which in chemoproteomic profiling was shown to engage a number of interesting cellular targets.

Non-cysteine Targeting

While several effective cysteine-targeting covalent drugs have been approved, the limited availability of druggable cysteine residues has driven interest in targeting alternative nucleophilic residues such as lysine, serine, and histidine. The work from Pellechia at the University of California Riverside reports the rational design of stapled peptides that covalently target His252 of hMcl-1, an antiapoptotic Bcl-2 protein. The team were originally seeking to target Lys234 but were drawn to the nucleophilic properties of the histidine imidazole and demonstrated that they were able to tune reactivity to either the histidine or lysine using stapling strategies.

Sulfur­(VI)-diazoles (SuDEX) were leveraged by the laboratories of Jones and Fischer from Dana-Farber to covalently staple the sensor loop histidine of the E3 ligase Cereblon. This work was driven by the need to improve the plasma stability of previous covalent inhibitors that contained either a triazole or sulfonyl fluoride electrophile and was ultimately successful at improving the pharmacokinetic profile.

Researchers have also explored serine in their efforts to develop selective monoacylglycerol lipase inhibitors based on an azetidin-2-one motif. They were able to confirm covalent binding via X-ray crystallography of three compounds in their library, and have profiled exemplar compounds for their potential off-target selectivity. In preliminary in vivo pharmacodynamic studies, the team was able to demonstrate an effect on monoacylglycerol lipase levels in the brain and believe that further work is warranted to develop the series further.

Serine was also targeted in the identification of inhibitors of plasma kallikrein using boronate-containing molecules. Stocks and colleagues used molecular docking of non-covalent compounds into the binding site of plasma kallikrein before serendipitously identifying Ser195 that was within the binding pocket. Following identification of an appropriate vector, they synthesized a library of compounds, arriving at compounds that exhibited time-dependent inhibition, and improved potency (into the picomolar range).

Cysteine Targeting

Cysteine-targeted covalent modulation is the most well-established strategy for covalent enzyme targeting in drug discovery, widely embraced in medicinal chemistry for its demonstrated efficacy, selectivity, and clinical success. We see in this Special Issue that groups are also taking inspiration from non-covalent binders in their optimization with the goal of increasing potency and selectivity versus a protein family. For example, selective inhibitors of nonreceptor tyrosine kinase c-Src have remained elusive. Through targeting a nonconserved cysteine and engineering an additional fluorine-mediated interaction (confirmed by crystallography), the team improved potency 75-fold against c-Src, demonstrated selectivity over 76 tyrosine kinases, and provided preliminary evidence of disruption of autophosphorylation in vitro.

Researchers from AstraZeneca reported their efforts to develop a ∼12,000-member compound library containing lead-like covalent compoundsspecifically curating the physicochemical properties including molecular weight, lipophilicity, ionization class, and hydrogen bond donors and acceptors. The team outlines the results of a screen against the antiapoptotic B-cell Lymphoma 2 (Bcl-2)-related protein A1 (Bfl-1) and hits that are primed for SAR optimization. In separate work on Bfl-1, an AstraZeneca team also disclosed their efforts to use DEL-screening to identify, and optimize a cyanoacrylamide-containing hit molecule, demonstrating a 10-fold improvement in potency.

The Flaherty group at Purdue University described a covalent fragment screen that identified a chloroacetohydrazide scaffold as a covalent inhibitor of Ubiquitin C-terminal hydrolase L1 (UCHL1), which was subsequently optimized to yield a selective, single-digit micromolar inhibitor with efficacy in cellular metastasis assays. Additionally, they obtained a ligand-bound crystal structure of the most potent compound with UCHL1 revealing its binding mode in preparation for future optimization efforts.

Gagnon and colleagues from the University of Quebec in Montreal identified HC-258, a covalent transcriptional enhanced associate domain (TEAD) inhibitor derived from flufenamic acid, which features an oxopentyl chain that mimics palmitic acid and an acrylamide moiety that covalently binds TEAD’s cysteine. It reduces expression of Hippo pathway target genes and inhibits breast cancer cell migration, with co-crystal structures, and HRMS analysis confirming covalent engagement within the TEAD palmitic acid pocket; HC-258 may also serve as a tool compound to further study the role of TEAD in cancer development.

Cathepsin S was explored by a group from the University of Mainz, with a specific focus on the pharmacodynamic and pharmacokinetic profile of the compounds due to the limited tissue distribution of the target. The most promising compounds consisted of an aldehyde or ketone electrophile, which was masked as the hydrazone to facilitate compound diversification without compromising the activity or selectivity of the compounds.

Selinexor, a selective inhibitor of nuclear export, acts as a slowly reversible covalent inhibitor of exportin-1. Researchers at Case Western Reserve University School of Medicine have shown that modifications to the electrophilic warhead can yield compounds with distinct pharmacological profilesincluding full and partial antagonists, as well as degraders. Although the mechanisms driving these divergent cellular responses remain unresolved, several hypotheses have been proposed, underscoring the need for further mechanistic investigation.

Retinoic acid receptor-related orphan receptor γ (RORγ) represents an interesting therapeutic target in inflammatory diseases, and has been identified as a key protein in castration-resistant prostate cancers. Researchers noted that there was a cysteine present in the binding pocket of crystal structures bound to noncovalent inhibitors that could be leveraged. Following structure-based optimization, the team of Wang and Wang, identified compounds that were able to suppress tumor growth in a mouse xenograft model with a good safety profile.

In a strategy analogous to alanine scanning, López and Pentelute developed electrophile scanning to identify reactivity hotspots by systematically substituting each residue in a peptide sequence with a protein-reactive modifier. Using the nonameric peptide antigen VL9 and its interaction with HLA-Ea complex associated with immune evasionthey identified specific sites for covalent conjugation that blocked recognition by the NK cell receptor CD94-NKG2A, showcasing the method’s potential for developing covalent peptide-based PPI inhibitors. A team from the Scripps Research Institute identified covalent modulators of the NLRP3 inflammasome assembly via an unbiased high-throughput screen and demonstrated interactions with multiple cysteines across various domains.

We see covalent modulation of targets being explored across the infectious disease space, including resistant Gram-positive bacteria such as Streptococcus pyogenes, and Staphylococcus aureus where researchers designed new covalent modulators from a previously published crystal structure of sortase A (a cysteine transpeptidase). Another example is of β-lactam antibiotics derived from cephamycin for Clostridioides difficile infection, supported by AlphaFold, and a crystal structure of sporulation-specific protein (CdSpoVD). Researchers have also investigated targeting Chlamydia trachomatis deubiquitinase ChlaDUB1which suppresses host cell apoptosiswith a second group employing a DFT-based approach to refine predictions of its in vitro enzymatic binding.

Inhibition of SARS-CoV-2 M^pro continues to receive significant attention from the community for its potential as broad spectrum coronavirus inhibitors. The Müller lab and colleagues focused their efforts on the benzoic acid chloropyridyl ester scaffold, identifying inhibitors in the <100 nM range, highlighting the importance of an ortho-fluorine to both activate the electrophile and stabilize the enzyme–inhibitor complex via a water-mediated network. Furthermore, they demonstrated the ability to tune the GSH reactivity of the electrophile, and that it did not correlate with M^pro activity. Others in this Special Issue have explored human cathepsin L as a potential broad-spectrum RNA virus target given that it is less prone to mutation over time, than M^pro.

Technical approach

There are several Perspectives and Reviews providing signposts for practitioners in this field. One focuses on providing “quality criteria for covalent and degrader probes”this is an important step toward driving a consistent way of capturing high-quality tool compounds across two important modalities. Another Perspective comes from Duncan and co-workers, concentrating on the important aspects of reversible covalent inhibitors, particularly to address off-target effects sometimes seen due to the reactivity of the electrophilic motif.

As interest in covalent drugs continues to grow, so too does the need for thoughtful clinical translation, driving the development of standardized guidelines and benchmarks to ensure reproducibility, clarity, and rigor in the field. Heppner and colleagues contributed two editorials to the collection, the first focused on “demystifying functional parameters for irreversible enzyme inhibitors,” while the second developed a best practices guide with respect to collecting kinetic inhibition values, and the impacts that can be seen with variations in liquid handling and assay reagents. Hartung and colleagues also sought to define a full set of criteria for the characterization of covalent, irreversible inhibitors as well as heterobifunctional degraders and molecular glue degraders in their editorial. Specifically, this includes modifications in potency and selectivity criteria compared to those for reversible inhibitors. Finally, researchers from Novartis Biomedical Research analyzed 30 covalent drugs to provide a quantitative framework to guide method selection for intravenous pharmacokinetic studies in humans.

In a Technical Note, Keillor and Medor highlight the method EPIC-Fita solution toward the inclusion of time-dependent preincubation assays in data analysis.

Clinical

As a community of drug hunters, the ultimate success of the technology is delivery of a clinical candidate derived from that discovery. Tyhonas and co-workers published the discovery of KIN-3248, an acrylamide-bearing irreversible inhibitor of FGFR. A contribution from Biogen from Hopkins et al. describes the development of their brain penetrant covalent BTK inhibitor BIIB129 that adopts a unique binding mode that is responsible for broad kinome selectivity. Kettle and co-workers from AstraZeneca outline the evolution of their initial covalent candidate AZD4625 to AZD4747 as a KRAS^G12C with high CNS exposure. A second Perspective discusses the approval of capivasertib, an ATP-competitive Akt inhibitor, as well as highlighting some of the challenges that still remain in terms of optimizing selectivity and enhancing therapeutic efficacy.

This Special Issue is a testament to the dynamic and rapidly evolving field of covalent modulators in medicinal chemistry and drug discovery. The collection of Articles showcases the breadth and depth of research at the interface of chemistry and biologyfrom foundational technology development and novel electrophile design to innovative targeting strategies and the advancement of clinical candidates. Collectively, these contributions reflect the collaborative spirit of our community, highlighting both the relevance of covalent approaches and their expanding potential beyond traditional cysteine targeting. As the field continues to mature, we anticipate that the insights and methodologies shared in this Special Issue will not only inspire future innovations but also help guide the translation of covalent modulators into effective therapeutics.

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Both authors contributed equally.

Views expressed in this editorial are those of the authors and not necessarily the views of the ACS.