A Practical Guide for the Assay-Dependent Characterisation of Irreversible Inhibitors

Lavleen Mader,   Jessica Borean  and  Jeffrey W Keillor

RSC Med. Chem. 2024 

DOI 10.1039/D4MD00707G

Irreversible targeted covalent inhibitors, in the past regarded as inappropriately reactive and toxic, have seen a recent resurgence in clinical interest. This paradigm shift is attributed to the exploitation of the two-step mechanism, in which a high affinity and selectivity (i.e., low KI) scaffold binds the target and only then does a pendant low intrinsic reactivity warhead react with the target (moderate kinact). This highlights the importance of evaluating inhibitors by deriving both their KI and kinact values. The development of methods to evaluate these inhibitors by accounting for their time-dependent nature has been crucial to the discovery of promising clinical candidates. Herein, we report all the practical kinetic methods available to date to derive kinact and KI values. These methods include direct observation of covalent modification, continuous assay (Kitz & Wilson) evaluation, and discontinuous incubation and pre-incubation time-dependent IC50 assays. We also provide practical guidelines and examples for performing these assays, comparison of their utility, and perspectives for their extended applications. This review aims to provide clarity about the use of these methods for reporting complete inhibitor kinetic profiles, guiding irreversible drug development towards increased target affinity and selectivity, while modulating in-vivo stability and on-target reactivity.

Pan-Transcriptional Enhanced Associated Domain Palmitoylation Pocket Covalent Inhibitor

Jinhyuk Kim, Hadong Kim, Jongwan Kim, Seon Yeon Cho, Sungho Moon, Youngki Yoo, Hanseong Kim, Jin Kwan Kim, Hyejin Jeon, Wan Namkung, Gyoonhee Han, and Kyoung Tai No

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c01393

In the Hippo signaling pathway, the palmitoylated transcriptional enhanced associated domain (TEAD) protein interacts with the coactivator Yes-associated protein/PDZ-binding motif, leading to transcriptional upregulation of oncogenes such as Ctgf and Cyr61. Consequently, targeting the palmitoylation sites of TEAD has emerged as a promising strategy for treating TEAD-dependent cancers. Compound 1 was identified using a structure-based drug design approach, leveraging the molecular insights gained from the known TEAD palmitoylation site inhibitor, K-975. Optimization of the initial hit compound resulted in the development of compound 3, a covalent pan-TEAD inhibitor characterized by high potency and oral bioavailability.

Total Synthesis of Tagitinins, Goyazensolide and RelatedFuranoheliangolides and their Covalent Interaction withImportin-5 (IPO5)

W Liu, R Patouret, E Peev, S Barluenga, N Winssinger

Helvetica Chimica Acta, 2024 
https://doi.org/10.1002/hlca.202400122

Herein, we detail an extension of our research on the synthesis of a small library of furanoheliangolides and the characterization of the covalent interaction between goyazensolide and IPO5. Using a build‐couple‐pair strategy, we assembled a small library of germacrene‐type lactones and diversified them into eight groups of structurally different analogues. The germacrene lactones were synthesized using Sonogashira coupling and Barbier‐type macrocyclization, while the furanoheliangolides were further elaborated through gold‐catalyzed transannulation followed by esterification. This synthetic approach enabled the generation of a goyazensolide alkyne‐tagged cellular probe, which was used to identify the selective binding between goyazensolide and the oncoprotein importin‐5 (IPO5). Mass spectrometry analysis of the proteolytic digest from the reaction between the goyazensolide probe and a recombinant IPO5 indicated a covalent engagement at Cys560 of IPO5, which was confirmed by site‐directed mutagenesis.

Slow-Binding and Covalent HDAC Inhibition: A New Paradigm?

Yasir S. Raouf and Carlos Moreno-Yruela

JACS Au, 2024

DOI: 10.1021/jacsau.4c00828

The dysregulated post-translational modification of proteins is an established hallmark of human disease. Through Zn2+-dependent hydrolysis of acyl-lysine modifications, histone deacetylases (HDACs) are key regulators of disease-implicated signaling pathways and tractable drug targets in the clinic. Early targeting of this family of 11 enzymes (HDAC1–11) afforded a first generation of broadly acting inhibitors with medicinal applications in oncology, specifically in cutaneous and peripheral T-cell lymphomas and in multiple myeloma. However, first-generation HDAC inhibitors are often associated with weak-to-modest patient benefits, dose-limited efficacies, pharmacokinetic liabilities, and recurring clinical toxicities. Alternative inhibitor design to target single enzymes and avoid toxic Zn2+-binding moieties have not overcome these limitations. Instead, recent literature has seen a shift toward noncanonical mechanistic approaches focused on slow-binding and covalent inhibition. Such compounds hold the potential of improving the pharmacokinetic and pharmacodynamic profiles of HDAC inhibitors through the extension of the drug–target residence time. This perspective aims to capture this emerging paradigm and discuss its potential to improve the preclinical/clinical outlook of HDAC inhibitors in the coming years.

Discovery and development of Krazati (adagrasib/MRTX849), a potent, selective, orally bioavailable, covalent KRASG12C(OFF) inhibitor

Adrian L. Gill, Mathew A. Marx

RAS Drug Discovery Past, Present and Future 2025, 205-227

https://doi.org/10.1016/B978-0-443-21861-3.00017-6

Krazati (adagrasib/MRTX849) is a potent, selective, and covalent KRASG12C inhibitor, representing a significant breakthrough in directly targeting KRAS. Adagrasib demonstrates favorable drug-like properties, selectively modifying mutant cysteine 12 in GDP-bound KRASG12C, leading to the inhibition of KRAS-dependent signaling both in vitro and in vivo, and tumor regression in many KRASG12C-positive cell lines and patient-derived xenograft models across various tumor types. Objective responses have been observed in clinical trials, particularly in lung and colon adenocarcinoma patients with KRASG12C mutations. Comprehensive pharmacodynamic and pharmacogenomic profiling in both sensitive and partially resistant nonclinical models has shed light on the mechanisms limiting adagrasib antitumor activity. These resistance mechanisms include contributing factors related to KRAS nucleotide cycling, feedback reactivation pathways through activation of receptor tyrosine kinases, instances where tumors bypass KRAS dependence, and genetic dysregulation of the cell cycle. The ongoing characterization of adagrasib's activity, along with insights into response and resistance mechanisms, provides valuable understanding of KRAS dependence and opened a long-awaited opportunity to selectively target KRASG12C in patients. Furthermore, the identification of effective preclinical combinations, such as adagrasib with agents targeting RTKs, SHP2, mTOR, or the cell cycle, demonstrates enhanced responses and marked tumor regression. These findings contribute to the rational development of this class of agents, holding promise for improving therapeutic outcomes in KRASG12C-mutant human cancers.

Tuning isatoic anhydrides’ lysine ligation chemistry for bioconjugation and drug delivery

Tiwari, Sona, Senthil, Sathyapriya, Khanna, Shweta, Duraisamy, Santhosh, Vechalapu, Sai Kumari, Mallojjala, Sharath Chandra, Allimuthu, Dharmaraja,

Cell Reports Physical Science, 2024

DOI: https://doi.org/10.1016/j.xcrp.2024.102260

The discovery of new chemical entities for the selective modification of protein lysines is a recent interest in the development of unique covalent chemical probes. Isatoic anhydride (benzoxauracil), possessing aminophilic reactivity, was employed for the profiling of ligandable lysines in the cellular proteome. Our reactivity evaluation of benzoxauracil with proteins using mass spectral peptide mapping revealed a biased reactivity profile with nearly all the nucleophilic amino acids. The chemoselective reactivity of electrophilic tags is a key determinant of their idiosyncratic reactions. We applied the hard-soft-acid-base (HSAB) principle for tuning isatoic anhydride’s reactivity through systematic chemical modifications for lysine-dominant reactivity. We demonstrated the employability of ring-opening chemistry in isatoic anhydride as a drug delivery modality for the release of a small molecule and doxorubicin in cancer cells. Broadly, the tunable reactivity of isatoic anhydride could be leveraged for developing lysine-selective probes and drug delivery cargos.

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

Hongyan Du, Xujun Zhang, Zhenxing Wu, Odin Zhang, Shukai Gu, Mingyang Wang, Feng Zhu, Dan Li, Tingjun Hou, Peichen Pan 

Nucleic Acids Research, 2024, gkae946

 https://doi.org/10.1093/nar/gkae946

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/.