Covalent Inhibitors: To Infinity and Beyond,Journal of Medicinal Chemistry

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Covalent Inhibitors: To Infinity and Beyond
Journal of Medicinal Chemistry ( IF 6.8 ) Pub Date : 2024-06-24 , DOI: 10.1021/acs.jmedchem.4c01308
Yasir S Raouf ₁

Affiliation

A future perspective on the “history of drug discovery” would more than likely characterize the 20th century (1901–1999) as one of the most significant collections of decades with respect to medicinal chemistry, and targeted molecular recognition. (1,2) A closer look into this time period would then quickly highlight several clinical milestones involving the use of covalent therapeutics – starting with the discovery of acetylsalicylic acid (Aspirin). Several years after its clinical use, this simple nonsteroidal anti-inflammatory drug (NSAID) was found to irreversibly acetylate Ser529 within the substrate-binding channel of cyclooxygenase 1 (COX-1), inhibiting the synthesis of pro-inflammatory prostaglandins (PGE₂), and thromboxanes (TXA₂). At the time, this unique nonreversible mechanism separated this molecule from fellow NSAIDs (e.g., ibuprofen, diclofenac) which engaged their target via standard reversible interactions. Following aspirin, several covalent molecules advanced to find widespread clinical use against a variety of human diseases. (3) Notable examples include nature-derived β-lactams (e.g., penicillin) as potent antibiotics via the covalent acylation of penicillin binding proteins (PBPs) involved in bacterial cell wall synthesis; proton-pump inhibitor omeprazole, a pro-drug which is metabolized in vivo into a reactive pyridinium sulfenamide prone to form covalent disulfide bonds with medicinal use against gastric reflux; pyrimidine nucleoside 5-fluorouracil (5-FU) which is a potent covalent inhibitor of thymidylate synthase (TS) and broad-use anticancer therapy; as well as bortezomib, a boronic acid dipeptide, which through the covalent modification of catalytic Thr residues of the 26S proteosome exerts antineoplastic effects in several human cancers (e.g., multiple myeloma, lymphoma). (4) In each case, these molecules utilize a reversible core scaffold appended with a partially positive electrophilic center designed to engage nucleophilic residues within disease-relevant target proteins (e.g., Cys, Ser, His). In a two-step mechanism, an initial molecular recognition event (K_i) is swiftly followed by covalent target inactivation (k_inact) to form a binary protein-inhibitor complex. The overall efficiency of this process depends on the rate of covalent modification, as described by k_inact/K_i (M^–1 s^–1). This second-order rate constant provides quantitative insights into the individual contributions of both thermodynamic affinity and kinetic electrophile reactivity to the covalent binding event. As a true measure of covalent inhibitor potency, several research articles have highlighted the benefits of this valuable metric to guide structure–activity relationships within covalent discovery programs, as compared to more standard practices focused on % inhibition, and IC₅₀ measurements. (5) While classic reversible inhibitors are entirely governed by standard binding kinetics, irreversible inactivation (k_inact) dissociates covalent molecules from this equilibrium, producing a time-dependent profile, with greater emphasis on kinetic reactivity, residence times, exposure, and target occupancy. (4,5) Despite these clinical milestones, the ability of an irreversible inhibitor to permanently modify cellular proteins, initially created an overwhelming sense of caution around this class of molecules, which lasted for many years. At the time, the primary motives against using a covalent binder involved safety concerns due to nonspecific reactivity, unpredictable polypharmacology, immunogenicity, and the potential for severe idiosyncratic toxicities. This hesitance was likely exacerbated by a poor understanding of covalent drug-target pharmacology, a partially characterized proteome, limited electrophile scope, and recurring research citing toxic metabolites derived from contemporary covalent therapeutics. (6) A commonly cited example is the nonopioid analgesic acetaminophen (Tylenol), now known to be metabolized in vivo to form a nonspecific alkylating agent (e.g., NAPQI), which is hepatotoxic at sufficiently elevated doses. Fortunately, as researchers continued to study this class of molecules in greater detail, the last 30 years has seen a dramatic resurgence of covalent inhibitors into a viable approach for clinical drug development with unique benefits, compared to a standard reversible molecule. (1) As a drug discovery strategy, the irreversible (or long-lived) inhibition profile of a covalent binder produces several unique advantages. Permanent target inactivation (with optimized structural design) enables exquisite potencies, with as low as pM-nM binding affinities. Covalent bond formation also fortifies the inhibitor binding event, resulting in minimal effects in the presence of competing cellular substrates, which is dramatically different from a reversible inhibitor that may be consistently competing with debilitating substrate concentrations (e.g., ATP, at 5–10 mM). In addition, irreversible target engagement (beyond the rate of protein resynthesis) essentially decouples inhibitor pharmacodynamics (PD) from pharmacokinetics (PK) allowing for persistent in vivo activity beyond metabolic clearance, which extends the systemic half-life of a covalent molecule. Ultimately, strong potencies, and prolonged cellular efficacy allows these special molecules to be possibly administered in smaller (e.g., <10–20 mg), and less frequent (e.g., > 12 h) drug doses, which can significantly improve patient compliance, limit the occurrence of unpredictable toxicities, and in most cases reduce both production, and treatment costs. As for target scope, given that covalent bond formation represents a significant driving force for the observed potency, these nonreversible inhibitors allow medicinal chemists to explore relatively challenging binding pockets (e.g., flat, solvated, hydrophilic) when compared to reversible binders that rely entirely on through-space intermolecular forces for target engagement. (6) Finally, with an increasingly narrowing intellectual property space for targeted small molecules, a covalent strategy may represent an entirely new strategy for disease relevant molecular targets, providing a novel and strong patent position for future clinical development. In recent years, important progress across several research lines around covalent inhibitor design, electrophilic mechanisms, kinetic reactivity, in vivo pharmacology, and required clinical benchmarks has largely resolved most (if not all) of the historical hesitance surrounding these reactive molecules. Important highlights include; the application of weakly reactive electrophiles (e.g., acrylamide), which largely eliminated fears of nonspecific or uncontrolled potency; the extensive characterization of the targetable proteome, enabling the design of highly selective covalent inhibitors with predictable pharmacology (i.e., nontoxic); as well as a much better understanding of the required preclinical/clinical target product profiles for a successful covalent drug candidate, with respect to in vivo efficacy, safety, and in-human dosing regimens. Also, while covalent inhibitors are almost always thought to act via irreversible mechanisms, there have been extensive progress in recent decades on reversible covalent chemistries. While historically serendipitous, advances in cocrystallographic studies, and detailed kinetic analyses have characterized several reversible warheads in recent years, allowing for a more targeted, and deliberate approach toward more tempered covalent inhibitors. (7) Common examples include nitriles, aldehydes, boronic acids, α-ketoamides, and more recently α-cyanoacrylamides. Despite all of these advances, there still remains a few key points to consider when developing a covalent small molecule, mainly with respect to target selection. First, to enable covalent bond formation, the target of interest must have a suitably reactive and mechanistically compatible nucleophile within an accessible distance of a functional protein binding site (e.g., 5–10 Å). Second, the observed cellular efficacy of a covalent inhibitor largely dependent on the rate of cellular degradation, and protein turn-over. These small molecules are typically most useful against targets with slow rates of resynthesis (e.g., 6–24 h) to enable persistent cellular efficacy. Note, while this is a general rule, a few exceptions have been reported in literature. Altogether, the ever-improving knowledge base around the entire process of covalent drug discovery has enabled extensive progress in a short period of time. This recent resurgence can be quickly verified through simple surveys of literature databases. For example, a quick search on www.pubmed.gov would return >1,000 research papers related to the term “covalent inhibitors” between 2023 and 2024, compared to a respectable ∼250 articles at the turn of the century. In addition, patent filings focused on covalent binding scaffolds have also seen a steady increase year over year for the past 20 years, with a persistent focus on oncology. In the clinic, as of 2024, >55 covalent drugs have received regulatory approval, with >15 approvals in the past decade alone, and estimated annual market cap in excess of US$50 billion. (5) As a whole, covalent inhibitors have found clinical success against a diverse set of diseases. This ranges from various types of human cancers (e.g., afatinib, ibrutinib, osimertinib, dacomitinib, acalabrutinib), sickle cell disease (e.g., voxelotor), viral infections (e.g., nirmatrelvir, telaprevir, boceprevir), hyperglycemia (e.g., saxagliptin), bacterial infections (e.g., fosfomycin), heart disease (e.g., clopidogrel), and so much more. Covalent programs can be found in almost all corners of the pharmaceutical sector. Whether in big pharma (e.g., Pfizer, Merck, Abbvie, Amgen, AstraZeneca, etc.) or covalent biotech specialists (e.g., Pharmacyclics, Mirati therapeutics, Matchpoint therapeutics, Ankaa therapeutics, etc.). At this stage, it is without a doubt that covalent inhibitors have ended their period of resurgence, and have firmly cemented themselves within mainstream drug discovery, or as some reports have said, going “from fringe to trendy”. All current indicators point to a rapidly developing, and exciting future, with a diverse set of biological, and clinical applications against a growing list of drug targets. This ability to target the most challenging pockets, and inhibit proteins long thought be undruggable is highlighted in recent reports against PI3Kα, WRN helicase, and K-RAS mutants. Phosphoinositide 3-kinase α (PI3Kα, p110α/p85) is well-studied lipid kinase, with pervasive roles in cell proliferation, motility, survival and adhesion. PIK3CA which codes for p110α (catalytic subunit of PI3Kα) represents one of the most commonly mutated genes in solid human cancers (e.g., ∼40% in HR⁺/HER2– breast cancer), which ultimately spurred extensive research into this kinase as a tractable oncology drug target. While PI3Kα has a rich clinical landscape, with the recent approvals of Novartis’s thiazole alpelisib (BYL719, Piqray) in combination with fulvestrant in various types of PIK3CA-active breast cancers, and Bayer’s pyrimidine copanlisib (Aliqopa) for relapsed follicular lymphoma, as well as > 50 clinical candidates currently under study (across several indications), most of these still suffer from poor isoform selectivity, and dose-limiting clinical toxicities. CNX-1351, a morpholine-substituted thieno[3,2-d]pyrimidine was the first covalent PI3Kα inhibitor reported by Nacht et al. (8) several years ago. While most covalent binders target nearby nucleophiles, CNX-1351 captures the relatively distal Cys862 (d ∼ 11 Å) with a β, β-dimethylenone electrophile. While enones are typically considered highly reactive warheads, β-carbon methylation is a common strategy, using sterics to reduce electrophilicity (k_inact = 6.57 × 10⁴ s^–1, K_i = 38.0 nM, SKOV3 IC₅₀ = 165 nM). Since this discovery, Borsari et al. (9) recently reported several second gen. covalent PI3Kα binders employing more tempered N-acrylamide electrophiles, linked to the structure of PQR514 – a reversible preclinical inhibitor of PI3Kα (IC₅₀ = 2.20 nM). While these molecules represent powerful chemical probes to interrogate PI3Kα pharmacology, the cited articles lay out tractable discovery strategies to develop a future covalent inhibitor of PI3Kα-implicated human diseases. In the past 10 years, it is rather unlikely to find a story in drug discovery as elegant (and thrilling) as the clinical development of covalent KRAS^G12X inhibitors. Ras proteins are a set of structurally related GTPases, with master regulatory influence on cytosolic signal transduction and cell-wide control of proliferation, differentiation, division, and survival. The human Ras genes (HRAS, NRAS, KRAS) are some of the most extensively studied oncogenes, found in ∼ 20–30% in all human cancers. (10) Of the 3 homologues, Kirsten rat sarcoma virus (KRAS) takes center stage as the most frequently mutated human oncogene, with high incidence rates in various aggressive human cancers, including pancreatic ductal adenocarcinoma (PDAC, 80–90%) non-small cell lung cancer (NSCLC, 30%), and colorectal cancer (CRC, 25%). (11) KRAS mutations tend to involve single-base missense mutants, primarily at G12X, G13X, and Q61X. codons (e.g., G12C, G12D, G12V, G13C, G13D, Q61H, Q61R,) and are typically observed to cause poor prognoses, and attenuated response to standards of care (Soc). While oncogene mutation frequencies vary as a function of cancer type, several subtypes have amassed significant interest in recent years, such as the KRAS^G12C mutant – implicated in NSCLC. Despite these roles in human cancers, KRAS proteins have actively resisted drug discovery efforts for over 30 years. While KRAS is an enzyme, it lacks a traditionally ligandable binding pocket, and reversible small molecules must outcompete strikingly high-affinity endogenous substrates, (e.g., pM) found at millimolar concentrations (e.g., GTP, GDP). (5) In fact, for several decades, KRAS was regarded as the archetype undruggable protein. The “eureka” moment arrived in 2013 out of the Shokat group (University of California, San Francisco), where an electrophile-guided tethering strategy of a 480 analog disulfide fragment library successfully identified a mutant-selective covalent KRAS^G12C inhibitor, which surprisingly binds a cryptic allosteric pocket (i.e., only apparent in the bound state) in the switch II region. (12) Note, this strategy was quite unique in the covalent space, as irreversible inhibitors are traditionally identified using the insertion of a reactive electrophile onto an already established reversible core. Ultimately, this landmark paper set a remarkable precedence in Ras drug discovery, and spurred an intense search toward a tractable covalent inhibitor of KRAS^G12C. Building on the preclinical advances of the ARS series (e.g., ARS-853, ARS-1620), the first clinical breakthrough involved the disclosure of AMG510 (sotorasib) out of Amgen. This atropisomer with a quinazoline core, and an acrylamide warhead appended off a 2-methylpiperazine handle which exploited a second cryptic pocket, first entered phase I clinical trials in 2018 (NCT03600883), and achieved regulatory approval in 2021 for adults with KRAS^G12C-mutated advanced or metastatic NSCLC (Lumakras). This first milestone was quickly followed by a second FDA approval in 2022 for MRTX849 (adagrasib, Krazati), a tetrahydropyridopyrimidine out of Mirati therapeutics for the same indication. Interestingly, adagrasib utilizes a substituted 2-fluoroacrylamide electrophile, with improved whole-blood stability relative to its nonfluorinated counterpart. All things considered, the molecular progression of KRAS proteins from “undruggable” to two FDA approvals in < 10 years (and many more candidates in the pipeline) represents an astounding success story in covalent drug discovery. WRN protein is a nuclear RecQ DNA helicase (with 3′–5′ exonuclease activity) which has been recently cited to involve synthetic lethal liabilities in human cancers with specific microsatellite instabilities (MSI). Studies have shown the inhibition of WRN helicase activity selectively promotes double-standard DNA breaks (and ultimately cell death) in MSI tumor cells. While the WRN gene was discovered in 1996, a recently growing understanding of WRN biology, upstream signal transduction (with complex downstream effects), poorly understood implications in disease, lack of sufficient structural data, and logistical challenges in inhibitor screening have historically positioned WRN as a challenging (and at times undruggable) protein target. Interestingly, as recently as May 2024, a mass spectrometry-based chemoproteomic screen out of Vividion therapeutics (acquired by Bayer AG in 2021) in collaboration with Roche identified VVD-109063 – an ATP-competitive covalent fragment, which utilizes a vinyl sulfone electrophile to engage Cys727 in an allosteric pocket within the ATPase domain (i.e., helicase core) of WRN. (13) Structural optimization of this novel series ultimately led to difluoromethyl-pyrimidine VVD-133214, a clinical candidate which through the covalent inhibition of WRN protein results in promising in vivo efficacy in MSI-H cancers, through the induction of dsDNA breaks, DNA repair responses, G2 cell cycle arrest, and subsequently cell death. These 3 examples elegantly demonstrate the power of a covalent binder, against highly challenging protein targets. One of the most important developments in recent memory is the discovery, design, optimization, and widespread adoption of weakly reactive (e.g., acrylamide), nucleophile specific (e.g., sulfonyl fluoride), and reversibly covalent (e.g., 2-cyanoacrylamide) electrophilic warheads. This was largely spurred by structural advances in tuning electrophile reactivity through the rational manipulation of both sterics, and electronics. Overall, while the field remains largely dominated by α,β-unsaturated warheads such as acrylamides, propiolamides, and butynamides, the past few years have seen several new electrophiles beyond the canonical Michael acceptor. (14) These reports likely came about as researchers attempt to capture less reactive Cys, or explore other amino acid nucleophiles beyond thiols, or try to cover novel ground in the rich, and saturated patent space of covalent small molecule. Examples of these electrophiles include: allenamides, propiolonitriles, vinyl pyrimidines, S_NAr systems (e.g., sulfonyls, activated haloarenes), haloacetamides (e.g., -Cl, -Br), aziridines, cyanamides, ethynylthienopyrimidines, and more. At the same time, while Cys thiols still represent the most widely studied nucleophile in covalent literature, they are in fact quite rare (e.g., ∼1.5%) and interest in more abundant/less explored nucleophiles is steadily on the rise. These includes lysine (e.g., aldehydes, sulfonyl fluoride, activated ester, vinyl sulfone), aspartic acid (e.g., β-lactone), histidine (e.g., sulfonyl fluoride, fluoro-sulfates, chloroacetamides), serine, threonine, tyrosine (e.g., sulfonyl fluoride). Several of these advances are comprehensively summarized in a 2019 J. Med. Chem. review article by Gehringer and Laufer. (7) Interestingly, a recent paper out of Nathanael Gray’s lab (Stanford, USA) reports the use of inhibitors with dual-warheads (e.g., 2 reactive electrophiles), as a novel covalent targeting strategy to overcome the recurring clinical vulnerabilities of standard irreversible binders to mutation-based acquired resistance. (15) Molecular bidents ZNL-8162/ZNL-0056 are ATP-competitive and retain sufficient potency, even in the presence of single Cys mutants (due to a second compensating electrophile). The clinical tractability of this approach will be interesting to observe in the coming years. As the biophysical toolkit around covalent binders continues to develop, the importance of evaluating second order rate constant (k_inact/K_i) as a measure of covalent inhibitor potency is evident in recent literature. This is further enabled by recent progress in covalent-specific assay design, optimized kinetic treatments of covalent binding data, as well as the design and validation of robust time-dependent experimental protocols (whether commercial or adaptable for in-house laboratory use) for k_inact and K_i determination. Additionally, these calculations have been determined in both biochemical and whole-cell settings. Altogether, while IC₅₀ values remain in use as a global measure of inhibitor potency in almost all covalent discovery literature, its limitation in the context of irreversible molecules is commonly highlighted, and widely understood. Another avenue which saw the recent adoption of covalent molecular recognition is within targeted protein degradation (TPD). This novel drug discovery strategy involves the use of heterobifunctional small molecules (e.g., inhibitor-linker-ligase binder) to selectively recruit a protein of interest (POI) to a ubiquitin E3 ligase, resulting in POI ubiquitylation and subsequent proteasomal degradation. Through the formation of a catalytic ternary complex, these proteolysis targeting chimeras (PROTACs) offer a unique approach to selectively decimate the populations of a disease-implicated target protein. Although still a relatively recent endeavor, coupling a covalent inhibitor to a targeted degrader can provide for quite unique drug pharmacology. While it sacrifices the catalytic nature of canonical PROTACs, the irreversible molecule can improve target selectivity, allow for smaller/more drug-like target binding scaffolds, as well as enable the targeting of cryptic, allosteric, or nonfunctional pockets, which may be hard to bind with a standard reversible molecule. In conclusion, whether as chemical probes, targeted inhibitors, clinical therapeutics, or protein degraders, the past 5 years has clearly demonstrated that the all-powerful (and versatile) covalent bond will remain a mainstay in the toolbox of medicinal chemists, and at the forefront of drug discovery for generations to come. The author would like to thank United Arab Emirates University (UAEU) for supporting this manuscript (Grant #12S156). 5-fluorouracil colorectal cancer cyclooxygenase 1 Kirsten rat sarcoma virus microsatellite instabilities non-small cell lung cancer nonsteroidal anti-inflammatory drug pancreatic ductal adenocarcinoma penicillin-binding protein pharmacodynamics pharmacokinetics proteolysis targeting chimeras targeted protein degradation thymidylate synthase This article references 15 other publications. This article has not yet been cited by other publications. This article references 15 other publications.

更新日期：2024-06-24

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