Re-Evaluating the Mechanism of Action of α,β-Unsaturated Carbonyl DUB Inhibitors B-AP15 and VLX1570: A Paradigmatic Example of Unspecific Protein Crosslinking with Michael Acceptor Motif-Containing Drugs [@TateScience]

Ward, J.; Pinto-Fernández, A.; Cornelissen, L.; Bonham, S.; Saez, L. D.; Riant, O.; Huber, K.; Kessler, B. M.; Feron, O.; Tate, E. W

ChemRxiv. 2019

doi: 10.26434/chemrxiv.10028444.v1

Deubiquitinating enzymes are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors with a shared α,β-unsaturated carbonyl substructure motif. Initially taken forward into a phase I/II clinical trial for refractory multiple myeloma, VLX1570 has since been put on full clinical hold due to dose limiting toxicity. Through a proteomic approach, here we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight complexes. Activity-based proteome profiling identified CIAPIN1 as a sub-micromolar covalent target of VLX1570, and further analysis demonstrated that high molecular weight complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce non-specific protein aggregation through cross-linking, providing a molecular explanation for general cellular toxicity.

The KRASG12C Inhibitor, MRTX849, Provides Insight Toward Therapeutic Susceptibility of KRAS Mutant Cancers in Mouse Models and Patients

James G. Christensen, Jill Hallin, Lars D Engstrom, Lauren Hargis, Andrew Calinisan, Ruth Aranda, David M Briere, Niranjan Sudhakar, Vickie Bowcut, Brian R Baer, Joshua A Ballard, Michael R Burkard, Jay B Fell, John P Fischer, Guy P Vigers, Jenny Y Xue, Sole Gatto, Julio Fernandez-Banet, Adam Pavlicek, Karen Velastegui, Richard C Chao, Jeremy Barton, Mariaelena Pierobon, Elisa Baldelli, Emmanuel F Patricoin, Douglas P Cassidy, Matthew A Marx, Igor I Rybkin, Melissa L Johnson, Sai-Hong Ignatius Ou, Piro Lito, Kyriakos P. Papadopoulos, Pasi A Janne and Peter Olson

Cancer Discov.  2019 DOI: 10.1158/2159-8290.CD-19-1167
DOI: 10.1158/2159-8290.CD-19-1167

Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties, selectively modifies mutant cysteine 12 in GDP-bound KRASG12C and inhibits KRAS-dependent signaling. MRTX849 demonstrated pronounced tumor regression in 17 of 26 (65%) of KRASG12C-positive cell line- and patient-derived xenograft models from multiple tumor types and objective responses have been observed in KRASG12C-positive lung and colon adenocarcinoma patients. Comprehensive pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant non-clinical models identified mechanisms implicated in limiting anti-tumor activity including KRAS nucleotide cycling and pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included activation of RTKs, bypass of KRAS dependence, and genetic dysregulation of cell cycle. Combinations of MRTX849 with agents that target RTKs, mTOR, or cell cycle demonstrated enhanced response and marked tumor regression in several tumor models, including MRTX849-refractory models.

Recent Advances in Selective and Irreversible Covalent Ligand Development and Validation

Tinghu Zhang, John M. Hatcher, Mingxing Teng, Nathanael S. Gray, Milka Kostic

Cell Chemical Biology, 2019
doi: 10.1002/wcms.1446

Some of the most widely used drugs, such as aspirin and penicillin, are covalent drugs. Covalent binding can improve potency, selectivity, and duration of the effects, but the intrinsic reactivity represents a potential liability and may result in idiosyncratic toxicity. For decades, the cons were believed to outweigh the pros, and covalent targeting was deprioritized in drug discovery. Recently, several covalent inhibitors have been approved for cancer treatment, thus rebooting the field. In this review, we briefly reflect on the history of selective covalent targeting, and provide a comprehensive overview of emerging developments from a chemical biology stand-point. Our discussion will reflect on efforts to validate irreversible covalent ligands, expand the scope of targets, and discover new ligands and warheads. We conclude with a brief commentary of remaining limitations and emerging opportunities in selective covalent targeting.

Manumycin Polyketides Act as Molecular Glues Between UBR7 and P53 to Impair Breast Cancer Pathogenicity [@dannomura]

Yosuke Isobe, Mikiko Okumura, Ross White, Lynn M McGregor, Jeffrey M McKenna, John A Tallarico, Markus Schirle, Thomas J Maimone, Daniel K Nomura

BioRxiv, 2019
doi: https://www.biorxiv.org/content/10.1101/814285v1

Molecular glues are an intriguing therapeutic modality that harness small-molecules to induce interactions between proteins that typically do not interact, thus enabling the creation of novel protein functions not naturally encoded in biology. While molecular glues such as thalidomide and rapamycin have catalyzed drug discovery efforts, such molecules are rare and have often been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention of disease. Historically, natural products have proven to be important sources of molecular glues and we postulated that natural products bearing multiple electrophilic sites may be an unexplored source of such molecules, potentially through multi-covalent attachment. Using activity-based protein profiling (ABPP)-based chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells to impair breast cancer pathogenicity through engaging in molecular glue interactions with the neo-substrate tumor-suppressor TP53, leading to the activation of p53 transcriptional activity and cell death. Our results reveal a previously undiscovered anti-cancer mechanism of this natural product family and highlight the potential for combining chemoproteomics and multi-covalent natural products for the discovery and characterization of new molecular glues.

An activity-guided map of electrophile-cysteine interactions in primary human immune cells

Ekaterina Vinogradova, Daniel Lazar, Radu Suciu, Yujia Wang, Giulia Bianco, Yu Yamashita, Vincent Crowley, Dave Remillard, Kenneth Lum, Gabriel Simon, Esther Kemper, Michael Lazear, Sifei Yin, Megan Blewett, Melissa Dix, Nhan Nguyen, Maxim Shokhirev, Emily Chin, Luke Lairson, Stefano Forli, John Teijaro, Benjamin Cravatt
BioRxiv, 2019
doi: https://www.biorxiv.org/content/10.1101/808113v1

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving direct functional perturbation and/or ligand-induced degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells, underscoring the potential of electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

Synthesis of peptides with cysteine sulfinic acid via the cysteine methoxybenzyl sulfone

Urmey, AR, Zondlo, NJ.

Pept Sci. 2019; e24137.  
doi: https://doi.org/10.1002/pep2.24137

Cysteine sulfinic acid is a protein posttranslational modification that is formed under oxidative conditions and is regulated both enzymatically and nonenzymatically. Cysteine oxidation to the sulfinic acid has been observed broadly throughout the proteome and can induce activation or inhibition of function in proteins. Recently, wide‐scale, reversible regulation of the sulfinic acid state of cysteine within proteins was identified, posing new questions in cysteine sulfinic acid biology. Existing methods to synthesize peptides with cysteine sulfinic acid can suffer from low yield, due to the formation of side products in the disulfide, sulfenic acid, and/or sulfonic acid oxidation states. Herein, a method for the synthesis of peptides with cysteine sulfinic acids was developed, via protection of cysteine sulfinic acid as the methoxybenzyl (Mob) sulfone. Cysteine Mob sulfone was synthesized as an Fmoc amino acid in one step from the commercially available Mob‐protected Fmoc‐cysteine (Fmoc‐Cys(Mob)‐OH). This amino acid was directly incorporated into peptides via solid‐phase peptide synthesis. Alternatively, peptides were synthesized using Fmoc‐Cys(Mob)‐OH, followed by subsequent oxidation within peptides of the thioether to the Mob sulfone via H2O2 and catalytic niobium carbide. Deprotection of peptides under strongly acidic conditions (50% triflic acid, 45% trifluoroacetic acid, 5% water) generated peptides with cysteine sulfinic acid. This approach was applied to the synthesis of peptides containing cysteine sulfinic acid within diverse peptide sequence contexts.

Covalent‐Allosteric Inhibitors to Achieve Akt Isoform‐Selectivity

Quambusch, L. , Landel, I. , Depta, L. , Weisner, J. , Uhlenbrock, N. , Müller, M. P., Glanemann, F. , Althoff, K. , Siveke, J. T. and Rauh, D.

Angew. Chem. Int. Ed., 2019

doi: 10.1002/anie.201909857

Isoforms of protein kinase Akt (Akt1/2/3) are involved in a myriad of essential processes including cell proliferation, survival, and metabolism. However, their individual roles in health and disease have not been thoroughly evaluated. Thus, there is an urgent need for perturbation studies, preferably mediated by highly selective bioactive small molecules. Here, we present a structure‐guided approach for the design of structurally diverse and pharmacologically beneficial covalent‐allosteric modifiers which enabled an investigation of the isoform‐specific preferences and the important residues within the allosteric site of the different isoforms. The biochemical, cellular, and structural evaluations revealed interactions responsible for the selective binding profiles. The first set of isoform‐selective covalent‐allosteric Akt inhibitors that emerged from this approach showed a conclusive structure‐activity relationship and broke ground for further structure‐guided development of selective probes to delineate the isoform‐specific functions of Akt kinases.

https://www.rauh-lab.de/