Comprehensive Exploration of Isocitrate Dehydrogenase (IDH) Mutations: Tumorigenesis, Drug Discovery, and Covalent Inhibitor Advances

Conghao Gai, Hairong Zeng ,  Haoming Xu, Xiaoyun Chai, Yan Zou, Chunlin Zhuang, Guangbo Ge, Qingjie Zhao 

European Journal of Medicinal Chemistry, 2024

https://doi.org/10.1016/j.ejmech.2024.117041

Isocitrate dehydrogenase (IDH) is an enzyme that catalyses the oxidative decarboxylation of isocitrate, producing α-ketoglutarate (α-KG) relative to the hydroxylation of substrates. However, IDH mutants can further reduce α-KG to 2-hydroxyglutarate (2-HG) which competitively inhibits α-KG dependent enzymes, leading to the downregulation of normal hydroxylation pathways. Good IDH mutant inhibitors can effectively reduce the level of 2-HG and therefore disturb cellular malignant transformation. In this review, we introduce the biological functions of IDH, describe the tumorigenesis mechanisms of IDH variants, and review the structure-based drug discovery of clinical inhibitors during 2012-2024. We also find successful applications of covalent strategy in the development of irreversible IDH inhibitors. Biological screening methods are also collected in this paper, which may help researchers to rapidly construct workflows for drug discovery and development.

Scalable Thiol Reactivity Profiling Identifies Azetidinyl Oxadiazoles as Cysteine-Targeting Electrophiles

Fereshte Ghorbani, Shaochen You, Gennadii A. Grabovyi, Mannkyu Hong, Garrett Lindsey, Arnab K. Chatterjee, and Michael J. Bollong

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c05711

Cysteine reactive groups are a mainstay in the design of covalent drugs and probe molecules, yet only a handful of electrophiles are routinely used to target this amino acid. Here, we report the development of scalable thiol reactivity (STRP), a method which enables the facile interrogation of large chemical libraries for intrinsic reactivity with cysteine. High throughput screening using STRP identified the azetidinyl oxadiazole as a moiety that selectively reacts with cysteine through a ring opening-based mechanism, capable of covalently engaging cysteine residues broadly across the human proteome. We show the utility of this reactive group with the discovery of an azetidinyl oxadiazole containing a small molecule that augments the catalytic activity of the deubiquitinase UCHL1 in vitro and in cells by covalently modifying a cysteine distal to its enzymatic active site. This study adds a novel cysteine targeting group to the electrophilic lexicon and provides robust methodology to rapidly surveil libraries for reactivity with cysteine.

An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor

Sijie Wang, Emily C. Woods, Jeyun Jo, Jiyun Zhu, Althea Hansel-Harris, Matthew Holcomb, Nichole J. Pedowitz, Tulsi Upadhyay, John Bennett, Matthias Fellner, Ki Wan Park, Anna Zhang, Tulio A. Valdez, Stefano Forli, Alix I Chan, Christian N. Cunningham, Matthew Bogyo

bioRxiv 2024.11.06.622387; 

doi: https://doi.org/10.1101/2024.11.06.622387

Staphylococcus aureus (S. aureus) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in Staphylococcus bacteria, they are valuable targets for diagnostics and therapeutics. Here we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue towards the desired target. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in live S. aureus bacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.

Mutant-selective AKT inhibition through lysine targeting and neo-zinc chelation

Gregory B. Craven, Hang Chu, Jessica D. Sun, Jordan D. Carelli, Brittany Coyne, Hao Chen, Ying Chen, Xiaolei Ma, Subhamoy Das, Wayne Kong, Adam D. Zajdlik, Kin S. Yang, Solomon H. Reisberg, Peter A. Thompson, J. Russell Lipford & Jack Taunton 

Nature, 2024

https://doi.org/10.1038/s41586-024-08176-4

Somatic alterations in the oncogenic kinase AKT1 have been identified in a broad spectrum of solid tumours. The most common AKT1 alteration replaces Glu17 with Lys (E17K) in the regulatory pleckstrin homology domain1, resulting in constitutive membrane localization and activation of oncogenic signalling. In clinical studies, pan-AKT inhibitors have been found to cause dose-limiting hyperglycaemia, which has motivated the search for mutant-selective inhibitors. We exploited the E17K mutation to design allosteric, lysine-targeted salicylaldehyde inhibitors with selectivity for AKT1 (E17K) over wild-type AKT paralogues, a major challenge given the presence of three conserved lysines near the allosteric site. Crystallographic analysis of the covalent inhibitor complex unexpectedly revealed an adventitious tetrahedral zinc ion that coordinates two proximal cysteines in the kinase activation loop while simultaneously engaging the E17K–imine conjugate. The salicylaldimine complex with AKT1 (E17K), but not that with wild-type AKT1, recruits endogenous Zn2+ in cells, resulting in sustained inhibition. A salicylaldehyde-based inhibitor was efficacious in AKT1 (E17K) tumour xenograft models at doses that did not induce hyperglycaemia. Our study demonstrates the potential to achieve exquisite residence-time-based selectivity for AKT1 (E17K) by targeting the mutant lysine together with Zn2+ chelation by the resulting salicylaldimine adduct.

Target Ligand Separation and Identification of Isoforsythiaside as a Histone Lysine-Specific Demethylase 1 Covalent Inhibitor Against Breast Cancer Metastasis

Mengzhen Gu, Xiaoqing Xu, Xiaoping Wang, Yun Wang, Yu Zhao, Xiaoxian Hu, Lu Zhu, Zhenzhong Deng, and Chao Han

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c02277

Histone lysine-specific demethylase 1 (LSD1) is hyperactive in breast cancer, which is associated with the metastasis of the tumor. Current irreversible LSD1 inhibitors are all synthesized by covalently binding to the flavin adenine dinucleotide cofactor, which often have side effects due to the high affinity for a variety of targets. Here, we identified isoforsythiaside (IFA), a natural phenylpropanoid glycoside isolated from Forsythia suspensa, as a novel covalent inhibitor of LSD1. The target ligand fishing technique and LC–MS/MS analysis identified that IFA could covalently bind to the Ser817 residue of LSD1 by α,β-unsaturated ketone moiety to block the amine oxidase-like domain of LSD1. Moreover, RBMS3/Twist1/MMP2, the downstream signaling pathway of LSD1, was activated after IFA treatment to inhibit the metastasis of MDA-MB-231 cells in vitro and in vivo. This study provided novel molecular templates for development of LSD1 covalence-binding inhibitor and laid a foundation for developing agents against breast carcinoma metastasis for targeting LSD1.

Covalency in PROTACs: Mechanisms and applications [@RPNowak]

Thomas M. Geiger, Radosław P. Nowak

Annual Reports in Medicinal Chemistry, 2024

https://doi.org/10.1016/bs.armc.2024.10.001

Proteolysis targeting chimeras (PROTACs) are hetero-bifunctional molecules that remove disease-causing proteins through the means of targeted protein degradation (TPD). Since their proof-of-concept over 20 years ago, PROTACs emerged as new modality in drug discovery and chemical biology. Historically, the vast majority of PROTACs use reversible-binding recruiters for both target and E3 ligase. However, in recent years more covalent PROTACs have been developed to harness the advantages of covalency such as unlocking the “undruggable” proteome to expand the repertoire of addressable targets and recruitable E3 ligases. Here, we review recent advances in covalent PROTACs, discuss their distinct mechanism of action and outline the key differences of this approach.

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.