DrugMap: A quantitative pan-cancer analysis of cysteine ligandability

Mariko Takahashi, Harrison B. Chong,Siwen Zhang, Tzu-Yi Yang,Matthew J. Lazarov,Stefan Harry,Michelle Maynard, Brendan Hilbert,Ryan D. White,Heather E. Murrey, Chih-Chiang Tsou, Kira Vordermark, Jonathan Assaad, Magdy Gohar, Benedikt R. Dürr et al.

Cell, 2024
DOI: https://doi.org/10.1016/j.cell.2024.03.027

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed “DrugMap,” an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.

Thiophene-fused γ-lactams inhibit the SARS-CoV-2 main protease via reversible covalent acylation

Gayatri Gayatri , Lennart Brewitz , Lewis Ibbotson , Eidarus Saleh , Shyam Basak , Hani Choudhry and Christopher J Schofield

Chem. Sci., 2024

DOI: 10.1039/D4SC01027B (Edge Article) 

Enzyme inhibitors working by O-acylation of nucleophilic serine residues are of immense medicinal importance, as exemplified by the β-lactam antibiotics. By contrast, inhibition of nucleophilic cysteine enzymes by S-acylation has not been widely exploited for medicinal applications. The SARS-CoV-2 main protease (Mpro) is a nucleophilic cysteine protease and a validated therapeutic target for COVID-19 treatment using small-molecule inhibitors. The clinically used Mpro inhibitors nirmatrelvir and simnotrelvir work via reversible covalent reaction of their electrophilic nitrile with the Mpro nucleophilic cysteine (Cys145). We report combined structure activity relationship and mass spectrometric studies revealing that appropriately functionalized γ-lactams can potently inhibit Mpro by reversible covalent reaction with Cys145 of Mpro. The results suggest that γ-lactams have potential as electrophilic warheads for development of covalently reacting small-molecule inhibitors of Mpro and, by implication, other nucleophilic cysteine enzymes.

Targeting KRAS Diversity: Covalent Modulation of G12X and Beyond in Cancer Therapy

Tonia Kirschner, Matthias P. Müller, and Daniel Rauh

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.3c02403

The GTPase KRAS acts as a switch in cellular signaling, transitioning between inactive GDP-bound and active GTP-bound states. In about 20% of human cancers, oncogenic RAS mutations disrupt this balance, favoring the active form and promoting proliferative signaling, thus rendering KRAS an appealing target for precision medicine in oncology. In 2013, Shokat and co-workers achieved a groundbreaking feat by covalently targeting a previously undiscovered allosteric pocket (switch II pocket (SWIIP)) of KRASG12C. This breakthrough led to the development and approval of sotorasib (AMG510) and adagrasib (MRTX849), revolutionizing the treatment of KRASG12C-dependent lung cancer. Recent achievements in targeting various KRASG12X mutants, using SWIIP as a key binding pocket, are discussed. Insights from successful KRASG12C targeting informed the design of molecules addressing other mutations, often in a covalent manner. These findings offer promise for innovative approaches in addressing commonly occurring KRAS mutations such as G12D, G12V, G12A, G12S, and G12R in various cancers.

Vinylpyridine as a Tunable Covalent Warhead Targeting C797 in EGFR

Nils Pemberton, Nina Compagne, Argyrides Argyrou, Emma Evertsson, Anders Gunnarsson, Jason G. Kettle, Jonathan P. Orme, and Richard A. Ward

ACS Medicinal Chemistry Letters 2024

DOI: 10.1021/acsmedchemlett.3c00425

To further facilitate the discovery of cysteine reactive covalent inhibitors, there is a need to develop new reactive groups beyond the traditional acrylamide-type warheads. Herein we describe the design and synthesis of covalent EGFR inhibitors that use vinylpyridine as the reactive group. Structure-based design identified the quinazoline-containing vinylpyridine 6 as a starting point. Further modifications focused on reducing reactivity resulted in substituted vinyl compound 12, which shows high EGFR potency and good kinase selectivity, as well as significantly reduced reactivity compared to the starting compound 6, confirming that vinylpyridines can be applied as an alternative cysteine reactive warhead with tunable reactivity.

Fitting of kinact and KI Values from Endpoint Pre-incubation IC50 Data

Lavleen K. Mader and Jeffrey W. Keillor

ACS Medicinal Chemistry Letters 2024

DOI: 10.1021/acsmedchemlett.4c00054

Experiments comprising a “pre-incubation” phase, where enzyme is incubated with inhibitor prior to the addition of assay substrate, are commonly used to evaluate covalent inhibitors, often via discontinuous or “endpoint” IC50 assays. However, due to the lack of mathematical tools to describe its biphasic time-dependent nature, this experiment has thus far been unable to provide kinact and KI values. Herein we report EPIC-Fit, a new method to determine kinact and KI values from global fitting of Endpoint Pre-incubation IC50 data that can be implemented using Microsoft Excel. Experimental characterization of a known tissue transglutaminase inhibitor, AA9, using EPIC-Fit provided kinact and KI values with strong correlations to the values determined by other, previously established methods of evaluation. This unprecedented method serves to finally include time-dependent pre-incubation endpoint assays in the medicinal chemist’s toolbox for rigorous characterization of irreversible inhibitors.

Proteomic Ligandability Maps of Spirocycle Acrylamide Stereoprobes Identify Covalent ERCC3 Degraders

Zhonglin Liu, Jarrett R. Remsberg, Haoxin Li, Evert Njomen, Kristen E. DeMeester, Yongfeng Tao, Guoqin Xia, Rachel E. Hayward, Minjin Yoo, Tracey Nguyen, Gabriel M. Simon, Stuart L. Schreiber, Bruno Melillo, and Benjamin F. Cravatt

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.3c13448

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Here, we describe a set of stereo- and regiochemically defined spirocycle acrylamides and the analysis of these electrophilic “stereoprobes” in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamide stereoprobes, the spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound termed ZL-12A promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL-12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation but instead causes collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another’s protein degradation profiles. Finally, we provide evidence that the antihypertension drug spironolactone─previously found to promote ERCC3 degradation through an enigmatic mechanism─also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.

Reversible Covalent Inhibition─Desired Covalent Adduct Formation by Mass Action

Disha Patel, Zil E Huma, and Dustin Duncan

ACS Chemical Biology 2024

DOI: 10.1021/acschembio.3c00805

Covalent inhibition has seen a resurgence in the last several years. Although long-plagued by concerns of off-target effects due to nonspecific reactions leading to covalent adducts, there has been success in developing covalent inhibitors, especially within the field of anticancer therapy. Covalent inhibitors can have an advantage over noncovalent inhibitors since the formation of a covalent adduct may serve as an additional mode of selectivity due to the intrinsic reactivity of the target protein that is absent in many other proteins. Unfortunately, many covalent inhibitors form irreversible adducts with off-target proteins, which can lead to considerable side-effects. By designing the inhibitor to form reversible covalent adducts, one can leverage competing on/off kinetics in complex formation by taking advantage of the law of mass action. Although covalent adducts do form with off-target proteins, the reversible nature of inhibition prevents accumulation of the off-target adduct, thus limiting side-effects. In this perspective, we outline important characteristics of reversible covalent inhibitors, including examples and a guide for inhibitor development.