Sunday, August 7, 2022

Thiol Reactivity of N-Aryl α-Methylene-γ-lactams: Influence of the Guaianolide Structure [@KayBrummond]

 Daniel P. Dempe, Chong-Lei Ji, Peng Liu, and Kay M. Brummond

The Journal of Organic Chemistry, 2020

DOI: 10.1021/acs.joc.2c01530

The α-methylene-γ-lactam offers promise as a complementary warhead for the development of targeted covalent inhibitors. However, an understanding of the factors governing its electrophilic reactivity is needed to promote the development of lead compounds utilizing this motif. Herein we synthesize a series of N-aryl-substituted α-methylene-γ-lactams installed within the framework of a bioactive guaianolide analog. To determine the effects of the guaianolide structure on the electrophilic reactivity, these compounds were reacted with glutathione under biomimetic conditions, and the rate constants were measured. A linear free-energy relationship was observed with the Hammett parameter of the N-aryl group within the cis- or trans-annulated isomeric series of compounds. However, the trans-annulated compounds exhibited a ca. 10-fold increase in reactivity relative to both the cis-annulated compounds and the corresponding N-arylated 3-methylene-2-pyrrolidinones. Density functional theory calculations revealed that the reactivity of the trans-annulated stereoisomers is promoted by the partial release of the ring strain of the fused seven-membered ring in the thio-Michael addition transition state.

Thursday, July 21, 2022

Chemical acylation of an acquired serine suppresses oncogenic signaling of K-Ras(G12S) [@kevansf]

Zhang, Z., Guiley, K.Z. & Shokat, K.M. 

Nat Chem Biol, 2022

Drugs that directly impede the function of driver oncogenes offer exceptional efficacy and a therapeutic window. The recently approved mutant selective small-molecule cysteine-reactive covalent inhibitor of the G12C mutant of K-Ras, sotorasib, provides a case in point. KRAS is the most frequently mutated proto-oncogene in human cancer, yet despite success targeting the G12C allele, targeted therapy for other hotspot mutants of KRAS has not been described. Here we report the discovery of small molecules that covalently target a G12S somatic mutation in K-Ras and suppress its oncogenic signaling. We show that these molecules are active in cells expressing K-Ras(G12S) but spare the wild-type protein. Our results provide a path to targeting a second somatic mutation in the oncogene KRAS by overcoming the weak nucleophilicity of an acquired serine residue. The chemistry we describe may serve as a basis for the selective targeting of other unactivated serines.

Monday, July 11, 2022

Covalent Disruptor of YAP-TEAD Association Suppresses Defective Hippo Signaling

Mengyang Fan, Wenchao Lu, Jianwei Che, Nicholas Kwiatkowski, Yang Gao, Hyuk-Soo Seo, Scott B. Ficarro, Prafulla C. Gokhale, Yao Liu, Ezekiel A. Geffken, Jimit Lakhani, Kijun Song, Miljan Kuljanin, Wenzhi Ji, Jie Jiang, Zhixiang He, Jason Tse, Andrew S. Boghossian, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Joseph D. Mancias, Jarrod A. Marto, Sirano Dhe-Paganon, Tinghu Zhang, Nathanael S. Gray

bioRxiv 2022.05.10.491316


The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

Wednesday, June 29, 2022

Oncogenic KRAS G12C: Kinetic and Redox Characterization of Covalent Inhibition

Minh V. Huynh, Derek Parsonage, Tom E. Forshaw, Venkat R. Chirasani, G. Aaron Hobbs, Hanzhi Wu, Jingyun Lee, Cristina M. Furdui, Leslie B. Poole, Sharon L. Campbell

Journal of Biological Chemistry, 2022

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, Kd = 36.0 ± 0.7 μM), and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, as well as identifying the occurrence of G12C oxidation with currently unknown biological ramifications.

Sunday, June 26, 2022

Discovery and Characterization of a Novel Series of Chloropyrimidines as Covalent Inhibitors of the Kinase MSK1

Adrian Hall, Jan Abendroth, Madison J. Bolejack, Tom Ceska, Sylvie Dell’Aiera, Victoria Ellis, David Fox, Cyril François, Muigai M. Muruthi, Camille Prével, Karine Poullennec, Sergei Romanov, Anne Valade, Alain Vanbellinghen, Jason Yano, and Martine Geraerts

ACS Medicinal Chemistry Letters 2022

DOI: 10.1021/acsmedchemlett.2c00134

We describe the identification and characterization of a series of covalent inhibitors of the C-terminal kinase domain (CTKD) of MSK1. The initial hit was identified via a high-throughput screening and represents a rare example of a covalent inhibitor which acts via an SNAr reaction of a 2,5-dichloropyrimidine with a cysteine residue (Cys440). The covalent mechanism of action was supported by in vitro biochemical experiments and was confirmed by mass spectrometry. Ultimately, the displacement of the 2-chloro moiety was confirmed by crystallization of an inhibitor with the CTKD. We also disclose the crystal structures of three compounds from this series bound to the CTKD of MSK1, in addition to the crystal structures of two unrelated RSK2 covalent inhibitors bound to the CTKD of MSK1.

Sunday, June 19, 2022

Nucleophilic covalent ligand discovery for the cysteine redoxome

Fu, L.; Jung, Y.; Tian, C.; Ferreira, R.; He, F.; Yang, J.; Carroll, K. ChemRxiv 2022.

The convergence of reactive cysteine-targeted electrophilic fragments and chemoproteomics have dramatically accelerated the discovery of ligandable sites in the proteome. Our genome encodes 214,000 cysteine residues, at least 20% of which are estimated to be redox-active. Oxidation blunts sulfur reactivity toward electrophiles but opens the door to a new class of nucleophilic covalent ligands that target cysteinyl sulfenic acids, which are widespread post-translational modifications. Here we report a quantitative analysis of nucleophilic fragments screened against the human sulfenome. Ligands were discovered for >500 sulfenated cysteines in >400 proteins, including sites not targeted by electrophiles with the same scaffold. Among these were compounds that preferentially react with hepatoma-derived growth factor (HDGF)-related proteins (HRPs) one of which was able to block nuclear transport of this oncoprotein. Nucleophilic fragments provide a rich resource for chemical biology and drug discovery, where ligandability in the human proteome extends beyond protein thiols.

Sunday, June 5, 2022

Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents

 Alena Istrate, Michael B. Geeson, Claudio D. Navo, Barbara B. Sousa, Marta C. Marques, Ross J. Taylor, Toby Journeaux, Sebastian R. Oehler, Michael R. Mortensen, Michael J. Deery, Andrew D. Bond, Francisco Corzana, Gonzalo Jiménez-Osés, and Gonçalo J. L. Bernardes

Journal of the American Chemical Society 2022
DOI: 10.1021/jacs.2c02185

Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody–drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the β-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.

Thiol Reactivity of N-Aryl α-Methylene-γ-lactams: Influence of the Guaianolide Structure [@KayBrummond]

 Daniel P. Dempe, Chong-Lei Ji, Peng Liu, and Kay M. Brummond The Journal of Organic Chemistry, 2020 DOI: 10.1021/acs.joc.2c01530 The α-meth...