Discovery of RRx-001, a Myc and CD47 Downregulating Small Molecule with Tumor Targeted Cytotoxicity and Healthy Tissue Cytoprotective Properties in Clinical Development

 Bryan Oronsky, XiaoNing Guo, XiaoHui Wang, Pedro Cabrales, David Sher, Lou Cannizzo, Bob Wardle, Nacer Abrouk, Michelle Lybeck, Scott Caroen, Arnold Oronsky, and Tony R. Reid

Journal of Medicinal Chemistry 2021

DOI: 10.1021/acs.jmedchem.1c00599

After extensive screening of aerospace compounds in an effort to source a novel anticancer agent, RRx-001, a first-in-class dinitroazetidine small molecule, was selected for advancement into preclinical and clinical development. RRx-001 is a minimally toxic small molecule with a distinct chemical structure and mechanism of action. The paradox of RRx-001 is that it mediates both antitumor cytotoxicity and normal tissue protection. The question of exactly how RRx-001 does this, and by means of what mechanism(s), depending on the route of delivery, intravenous or intratumoral, are explored. RRx-001 is currently in phase 2 and 3 clinical trials for the treatment of multiple solid tumor malignancies and as a supportive care drug.

Identification of a Covalent Importin-5 Inhibitor, Goyazensolide, from a Collective Synthesis of Furanoheliangolides

Weilong Liu, Rémi Patouret, Sofia Barluenga, Michael Plank, Robbie Loewith, and Nicolas Winssinger

ACS Central Science 2021

DOI: 10.1021/acscentsci.1c00056

Sesquiterpenes are a rich source of covalent inhibitors with a long history in traditional medicine and include several important therapeutics and tool compounds. Herein, we report the total synthesis of 16 sesquiterpene lactones via a build/couple/pair strategy, including goyasensolide. Using an alkyne-tagged cellular probe and proteomics analysis, we discovered that goyazensolide selectively targets the oncoprotein importin-5 (IPO5) for covalent engagement. We further demonstrate that goyazensolide inhibits the translocation of RASAL-2, a cargo of IPO5, into the nucleus and perturbs the binding between IPO5 and two specific viral nuclear localization sequences.

Selective Inhibition of DNA Polymerase β by a Covalent Inhibitor

 Shelby C. Yuhas, Daniel J. Laverty, Huijin Lee, Ananya Majumdar, and Marc M. Greenberg

Journal of the American Chemical Society 2021

DOI: 10.1021/jacs.1c02453

DNA polymerase β (Pol β) plays a vital role in DNA repair and has been closely linked to cancer. Selective inhibitors of this enzyme are lacking. Inspired by DNA lesions produced by antitumor agents that inactivate Pol β, we have undertaken the development of covalent small-molecule inhibitors of this enzyme. Using a two-stage process involving chemically synthesized libraries, we identified a potent irreversible inhibitor (14) of Pol β (KI = 1.8 ± 0.45 μM, kinact = (7.0 ± 1.0) × 10–3 s–1). Inhibitor 14 selectively inactivates Pol β over other DNA polymerases. LC-MS/MS analysis of trypsin digests of Pol β treated with 14 identified two lysines within the polymerase binding site that are covalently modified, one of which was previously determined to play a role in DNA binding. Fluorescence anisotropy experiments show that pretreatment of Pol β with 14 prevents DNA binding. Experiments using a pro-inhibitor (pro-14) in wild type mouse embryonic fibroblasts (MEFs) indicate that the inhibitor (5 μM) is itself not cytotoxic but works synergistically with the DNA alkylating agent, methylmethanesulfonate (MMS), to kill cells. Moreover, experiments in Pol β null MEFs indicate that pro-14 is selective for the target enzyme. Finally, pro-14 also works synergistically with MMS and bleomycin to kill HeLa cells. The results suggest that pro-14 is a potentially useful tool in studies of the role of Pol β in disease.

Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain

Patrick Johe, Sascha Jung, Erik Endres, Christian Kersten, Collin Zimmer, Weixiang Ye, Carsten Sönnichsen, Ute A. Hellmich, Christoph Sotriffer, Tanja Schirmeister, and Hannes Neuweiler

ACS Chem. Biol. 2021, 16, 4, 661–670

https://doi.org/10.1021/acschembio.0c00911

Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.

 

Irreversible inhibition of BoNT/A protease: proximity-driven reactivity contingent upon a bifunctional approach

Lewis D. Turner, Alexander L. Nielsen, Lucy Lin, Sabine Pellettc Takashi Sugane, Margaret E. Olson, Eric A. Johnson, and Kim D. Janda

RSC Med. Chem., 2021

https://doi.org/10.1039/D1MD00089F

Botulinum neurotoxin A (BoNT/A) is categorized as a Tier 1 bioterrorism agent and persists within muscle neurons for months, causing paralysis. A readily available treatment that abrogates BoNT/A's toxicity and longevity is a necessity in the event of a widespread BoNT/A attack and for clinical treatment of botulism, yet remains an unmet need. Herein, we describe a comprehensive warhead screening campaign of bifunctional hydroxamate-based inhibitors for the irreversible inhibition of the BoNT/A light chain (LC). Using the 2,4-dichlorocinnamic hydroxamic acid (DCHA) metal-binding pharmacophore modified with a pendent warhead, a total of 37 compounds, possessing 13 distinct warhead types, were synthesized and evaluated for time-dependent inhibition against the BoNT/A LC. Iodoacetamides, maleimides, and an epoxide were found to exhibit time-dependent inhibition and their kGSH measured as a description of reactivity. The epoxide exhibited superior time-dependent inhibition over the iodoacetamides, despite reacting with glutathione (GSH) 51-fold slower. The proximity-driven covalent bond achieved with the epoxide inhibitor was contingent upon the vital hydroxamate–Zn2+ anchor in placing the warhead in an optimal position for reaction with Cys165. Monofunctional control compounds exemplified the necessity of the bifunctional approach, and Cys165 modification was confirmed through high-resolution mass spectrometry (HRMS) and ablation of time-dependent inhibitory activity against a C165A variant. Compounds were also evaluated against BoNT/A-intoxicated motor neuron cells, and their cell toxicity, serum stability, and selectivity against matrix metalloproteinases (MMPs) were characterized. The bifunctional approach allows the use of less intrinsically reactive electrophiles to intercept Cys165, thus expanding the toolbox of potential warheads for selective irreversible BoNT/A LC inhibition. We envision that this dual-targeted strategy is amenable to other metalloproteases that also possess non-catalytic cysteines proximal to the active-site metal center.

Refinement of Covalent EGFR Inhibitor AZD9291 to Eliminate Off-target Activity

Elise Bouffard, Balyn W Zaro, Melissa MD Dix, Benjamin Cravatt, Chi-HueyWong

Tetrahedron Letters, 2021

https://doi.org/10.1016/j.tetlet.2021.153178

Non-small-cell lung cancer (NSCLC) is a major disease that accounts for 85% of all lung cancer cases which claimed around 1.8 billion lives worldwide in 2020. Tyrosine kinase inhibitors (TKIs) that target EGFR have been used for the treatment of NSCLC, but often develop drug resistance, and the covalent inhibitor AZD9291 has been developed to tackle the problem of drug resistance mediated by the T790M EGFR mutation; however, there is a side effect of hyperglycemia that may be due to off-target activity. This study examines analogues of AZD9291 by chemical proteomics, identifying analogues that maintain T790M-EGFR engagement while showing reduced cross-reactivity with off-targets.

Exploring the Versatility of the Covalent Thiol–Alkyne Reaction with Substituted Propargyl Warheads: A Deciding Role for the Cysteine Protease

Elma Mons, Robbert Q. Kim, Bjorn R. van Doodewaerd, Peter A. van Veelen, Monique P. C. Mulder, and Huib Ovaa

J. Am. Chem. Soc. 2021, 143, 17, 6423–6433

https://doi.org/10.1021/jacs.0c10513

Terminal unactivated alkynes are nowadays considered the golden standard for cysteine-reactive warheads in activity-based probes (ABPs) targeting cysteine deubiquitinating enzymes (DUBs). In this work, we study the versatility of the thiol–alkyne addition reaction in more depth. Contrary to previous findings with UCHL3, we now show that covalent adduct formation can progress with substituents on the terminal or internal alkyne position. Strikingly, acceptance of alkyne substituents is strictly DUB-specific as this is not conserved among members of the same subfamily. Covalent adduct formation with the catalytic cysteine residue was validated by gel analysis and mass spectrometry of intact ABP-treated USP16CDWT and catalytically inactive mutant USP16CDC205A. Bottom-up mass spectrometric analysis of the covalent adduct with a deuterated propargyl ABP provides mechanistic understanding of the in situ thiol–alkyne reaction, identifying the alkyne rather than an allenic intermediate as the reactive species. Furthermore, kinetic analysis revealed that introduction of (bulky/electron-donating) methyl substituents on the propargyl moiety decreases the rate of covalent adduct formation, thus providing a rational explanation for the commonly lower level of observed covalent adduct compared to unmodified alkynes. Altogether, our work extends the scope of possible propargyl derivatives in cysteine targeting ABPs from unmodified terminal alkynes to internal and substituted alkynes, which we anticipate will have great value in the development of ABPs with improved selectivity profiles.