Sunday, May 30, 2021

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.

Friday, May 28, 2021

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.

Monday, May 24, 2021

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.

Sunday, May 23, 2021

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

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.


Wednesday, May 19, 2021

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

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.

Monday, May 17, 2021

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

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.

Sunday, May 16, 2021

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

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.

Tuesday, May 11, 2021

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

Christian Dubiella, Benika J. Pinch, Kazuhiro Koikawa, Daniel Zaidman, Evon Poon, Theresa D. Manz, Behnam Nabet, Shuning He, Efrat Resnick, Adi Rogel, Ellen M. Langer, Colin J. Daniel, Hyuk-Soo Seo, Ying Chen, Guillaume Adelmant, Shabnam Sharifzadeh, Scott B. Ficarro, Yann Jamin, Barbara Martins da Costa, Mark W. Zimmerman, Xiaolan Lian, Shin Kibe, Shingo Kozono, Zainab M. Doctor, Christopher M. Browne, Annan Yang, Liat Stoler-Barak, Richa B. Shah, Nicholas E. Vangos, Ezekiel A. Geffken, Roni Oren, Eriko Koide, Samuel Sidi, Ziv Shulman, Chu Wang, Jarrod A. Marto, Sirano Dhe-Paganon, Thomas Look, Xiao Zhen Zhou, Kun Ping Lu, Rosalie C. Sears, Louis Chesler, Nathanael S. Gray & Nir London

Nat Chem Biol2021


The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1’s active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target. 

Friday, May 7, 2021

Covalent Flexible Peptide Docking in Rosetta

Barr Tivon, Ronen Gabizon, Bente A Somsen, Peter J Cossar, Christian Ottmann, Nir London


Electrophilic peptides that form an irreversible covalent bond with their target have great potential for binding targets that have been previously considered undruggable. However, the discovery of such peptides remains a challenge. Here, we present CovPepDock, a computational pipeline for peptide docking that incorporates covalent binding between the peptide and a receptor cysteine. We applied CovPepDock retrospectively to a dataset of 115 disulfide-bound peptides and a dataset of 54 electrophilic peptides, for which it produced a top-five scoring, near-native model, in 89% and 100% of the cases, respectively. In addition, we developed a protocol for designing electrophilic peptide binders based on known non-covalent binders or protein-protein interfaces. We identified 7,154 peptide candidates in the PDB for application of this protocol. As a proof-of-concept we validated the protocol on the non-covalent complex of 14-3-3σ and YAP1 phosphopeptide. The protocol identified seven highly potent and selective irreversible peptide binders. The predicted binding mode of one of the peptides was validated using X-ray crystallography. This case-study demonstrates the utility and impact of CovPepDock. It suggests that many new electrophilic peptide binders can be rapidly discovered, with significant potential as therapeutic molecules and chemical probes.

Thursday, May 6, 2021

A lysine–cysteine redox switch with an NOS bridge regulates enzyme function

Wensien, M., von Pappenheim, F.R., Funk, LM. et al.

Nature, 2021


Disulfide bonds between cysteine residues are important post-translational modifications in proteins that have critical roles for protein structure and stability, as redox-active catalytic groups in enzymes or allosteric redox switches that govern protein function1,2,3,4. In addition to forming disulfide bridges, cysteine residues are susceptible to oxidation by reactive oxygen species, and are thus central not only to the scavenging of these but also to cellular signalling and communication in biological as well as pathological contexts5,6. Oxidized cysteine species are highly reactive and may form covalent conjugates with, for example, tyrosines in the active sites of some redox enzymes7,8. However, to our knowledge, regulatory switches with covalent crosslinks other than disulfides have not previously been demonstrated. Here we report the discovery of a covalent crosslink between a cysteine and a lysine residue with a NOS bridge that serves as an allosteric redox switch in the transaldolase enzyme of Neisseria gonorrhoeae, the pathogen that causes gonorrhoea. X-ray structure analysis of the protein in the oxidized and reduced state reveals a loaded-spring mechanism that involves a structural relaxation upon redox activation, which is propagated from the allosteric redox switch at the protein surface to the active site in the protein interior. This relaxation leads to a reconfiguration of key catalytic residues and elicits an increase in enzymatic activity of several orders of magnitude. The redox switch is highly conserved in related transaldolases from other members of the Neisseriaceae; for example, it is present in the transaldolase of Neisseria meningitides (a pathogen that is the primary cause of meningitis and septicaemia in children). We surveyed the Protein Data Bank and found that the NOS bridge exists in diverse protein families across all domains of life (including Homo sapiens) and that it is often located at catalytic or regulatory hotspots. Our findings will inform strategies for the design of proteins and peptides, as well as the development of new classes of drugs and antibodies that target the lysine–cysteine redox switch9,10.

Monday, May 3, 2021

Discovery of a covalent inhibitor of heat shock protein 90 with antitumor activity that blocks the co-chaperone binding via C-terminal modification

Li Li, Nannan Chen, Dandan Xia, Shicheng Xu, Wei Dai, Yuanyuan Tong, Lei Wang, Zhengyu Jiang, Qidong You, Xiaoli Xu

Cell Chemical Biology, 2021

Heat shock protein (Hsp90), a critical molecular chaperone that regulates the maturation of a large number of oncogenic client proteins, plays an essential role in the growth of neoplastic cells. Herein, DDO-6600 is identified to covalent modification of Cys598 on Hsp90 from in silico study and is verified by a series of biological assays. We demonstrated that DDO-6600 covalently bound to Cys598 on the Hsp90 C terminus and exhibited antiproliferative activities against multiple tumor cells without inhibiting ATPase activity. Further studies showed that DDO-6600 disrupted the interaction between Hsp90 and Cdc37, which induced the degradation of kinase client proteins in multiple tumor cell lines, promoted apoptosis, and inhibited cell motility. Our findings offer mechanic insights into the covalent modification of Hsp90 and provide an alternative strategy for the development of Hsp90 covalent regulators or chemical probes to explore the therapeutical potential of Hsp90.

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...