Developing Covalent Protein Drugs via Proximity-Enabled Reactive Therapeutics

Qingke Li , Qu Chen, Paul C. Klauser, Mengyuan Li, Feng Zheng, Nanxi Wang, Xiaoying Li, Qianbing Zhang, Xuemei Fu, Qian Wang, Yang Xu, Lei Wang 

Cell, 2020

doi: 10.1016/j.cell.2020.05.028

Small molecule covalent drugs provide desirable therapeutic properties over noncovalent ones for treating challenging diseases. The potential of covalent protein drugs, however, remains unexplored due to protein’s inability to bind targets covalently. We report a proximity-enabled reactive therapeutics (PERx) approach to generate covalent protein drugs. Through genetic code expansion, a latent bioreactive amino acid fluorosulfate-L-tyrosine (FSY) was incorporated into human programmed cell death protein-1 (PD-1). Only when PD-1 interacts with PD-L1 did the FSY react with a proximal histidine of PD-L1 selectively, enabling irreversible binding of PD-1 to only PD-L1 in vitro and in vivo. When administrated in immune-humanized mice, the covalent PD-1(FSY) exhibited strikingly more potent antitumor effect over the noncovalent wild-type PD-1, attaining therapeutic efficacy equivalent or superior to anti-PD-L1 antibody. PERx should provide a general platform technology for converting various interacting proteins into covalent binders, achieving specific covalent protein targeting for biological studies and therapeutic capability unattainable with conventional noncovalent protein drugs.

Hydrazines as versatile chemical biology probes and drug-discovery tools for cofactor-dependent enzymes

Zongtao Lin, Xie Wang, Katelyn A. Bustin, Lin He, Radu M. Suciu, Nancy Schek, Mina Ahmadi, Kai Hu, Erika J. Olson, William H. Parsons, Eric S. Witze, Paul D. Morton, Ann M. Gregus, Matthew W. Buczynski, Megan L. Matthews

BioRXiv, 2020

doi: https://doi.org/10.1101/2020.06.17.154864

Known chemoproteomic probes generally use warheads that tag a single type of amino acid or modified form thereof to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electron-poor enzyme cofactors, transient intermediates and chemically-labile regulatory modifications, but probes for such species are underdeveloped. Here, we have innovated a versatile class of chemoproteomic probes for this less charted hemisphere of the proteome by using hydrazine as the common chemical warhead. Its electron-rich nature allows it to react by both polar and radicaloid mechanisms and to target multiple, pharmacologically important functional classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment can be blocked by active-site-directed inhibitors, and elaboration of the warhead supports connection of a target to a lead compound. The capacity of substituted hydrazines to profile, discover and inhibit diverse cofactor-dependent enzymes enables cell and tissue imaging and makes this platform useful for enzyme and drug discovery.

Manumycin polyketides act as molecular glues between UBR7 and P53 [@DanNomura]

Yosuke Isobe, Mikiko Okumura, Lynn M. McGregor, Scott M. Brittain, Michael D. Jones, Xiaoyou Liang, Ross White, William Forrester, Jeffrey M. McKenna, John A. Tallarico, Markus Schirle, Thomas J. Maimone & Daniel K. Nomura 

Nat Chem Biol, 2020

https://doi.org/10.1038/s41589-020-0557-2

Molecular glues are an intriguing therapeutic modality that harness small molecules to induce interactions between proteins that typically do not interact. However, such molecules are rare and have been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention. We postulated that natural products bearing one or more electrophilic sites may be an unexplored source of new molecular glues, potentially acting through multicovalent attachment. Using 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, and engage in molecular glue interactions with the neosubstrate tumor-suppressor TP53, leading to p53 transcriptional activation and cell death. Our results reveal an anticancer mechanism of this natural product family, and highlight the potential for combining chemoproteomics and multicovalent natural products for the discovery of new molecular glues.

In Vivo Imaging of the Tumor‐Associated Enzyme NCEH1 with a Covalent PET Probe

Chang, J.W., Bhuiyan, M., Tsai, H.‐M., Zhang, H.J., Li, G., Fathi, S., McCutcheon, D.C., Leoni, L., Freifelder, R., Chen, C.‐T. and Moellering, R.E. 

Angew. Chem. Int. Ed. 2020

doi:10.1002/anie.202004762

Herein, we report the development of an 18F‐labeled, activity‐based small‐molecule probe targeting the cancer‐associated serine hydrolase NCEH1. We undertook a focused medicinal chemistry campaign to simultaneously preserve potent and specific NCEH1 labeling in live cells and animals, while permitting facile 18F radionuclide incorporation required for PET imaging. The resulting molecule, [18F]JW199, labels active NCEH1 in live cells at nanomolar concentrations and greater than 1000‐fold selectivity relative to other serine hydrolases. [18F]JW199 displays rapid, NCEH1‐dependent accumulation in mouse tissues. Finally, we demonstrate that [18F]JW199 labels aggressive cancer tumor cells in vivo, which uncovered localized NCEH1 activity at the leading edge of triple‐negative breast cancer tumors, suggesting roles for NCEH1 in tumor aggressiveness and metastasis.

Mutant‐Selective Allosteric EGFR Degraders are Effective Against a Broad Range of Drug‐Resistant Mutations

Jaebong Jang  Ciric To  Dries J. H. De Clercq  Eunyoung Park  Charles M. Ponthier  Bo Hee Shin  Mierzhati Mushajiang  Radosław P. Nowak  Eric S. Fischer  Michael J. Eck  Pasi A. Jänne  Nathanael Schiander Gray

Angewante Chemie, 2020

https://doi.org/10.1002/ange.202003500

Targeting epidermal growth factor receptor (EGFR) through an allosteric mechanism provides a potential therapeutic strategy to overcome drug‐resistant EGFR mutations that emerge within the ATP binding site. Here, we develop an allosteric EGFR degrader, DDC‐01‐163, which can selectively inhibit the proliferation of L858R/T790 (L/T) mutant Ba/F3 cells while leaving wildtype EGFR Ba/F3 cells unaffected. DDC‐01‐163 is also effective against osimertinib‐resistant cells with L/T/C797S and L/T/L718Q EGFR mutations. When combined with an ATP‐site EGFR inhibitor, osimertinib, the anti‐proliferative activity of DDC‐01‐163 against L858R/T790M EGFR‐Ba/F3 cells is enhanced. Collectively, DDC‐01‐163 is a promising allosteric EGFR degrader with selective activity against various clinically relevant EGFR mutants as a single agent and when combined with an ATP‐site inhibitor. Our data suggests that targeted protein degradation is a promising drug development approach for mutant EGFR.

Sulfur(VI) Fluoride Exchange (SuFEx)-Enabled High-Throughput Medicinal Chemistry

Seiya Kitamura, Qinheng Zheng, Jordan L. Woehl, Angelo Solania, Emily Chen, Nicholas Dillon, Mitchell V. Hull, Miyako Kotaniguchi, John R. Cappiello, Shinichi Kitamura, Victor Nizet, K. Barry Sharpless, and Dennis W. Wolan

Journal of the American Chemical Society 2020
DOI: 10.1021/jacs.9b13652

Optimization of small-molecule probes or drugs is a synthetically lengthy, challenging, and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible sulfur(VI) fluoride exchange (SuFEx) click chemistry. A high-throughput screening hit benzyl (cyanomethyl)carbamate (Ki = 8 μM) against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN═S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products were directly screened to yield drug-like inhibitors with 480-fold higher potency (Ki = 18 nM). We showed that the improved molecule is active in a bacteria-host coculture. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.

Discovery of MFH290: A Potent and Highly Selective Covalent Inhibitor for Cyclin-Dependent Kinase 12/13

Yao Liu, Mingfeng Hao, Alan Leggett, Yang Gao, Scott B Ficarro, Jianwei Che, Zhixiang He, Calla Olson, Jarrod A. Marto, Nicholas Kwiatkowski, Tinghu Zhang, and Nathanael S Gray

J. Med. Chem. 2020
https://doi.org/10.1021/acs.jmedchem.9b01929

Genetic depletion of cyclin-dependent kinase 12 (CDK12) or selective inhibition of an analog-sensitive CDK12 reduces DNA damage repair gene expression, but selective inhibition of endogenous CDK12 is difficult. Here, we report the development of MFH290, a novel cysteine (Cys)-directed covalent inhibitor of CDK12/13. MFH290 forms a covalent bond with Cys-1039 of CDK12, exhibits excellent kinome selectivity, inhibits the phosphorylation of serine-2 in the C-terminal domain (CTD) of RNA-polymerase II (Pol II) and reduces the expression of key DNA damage repair genes. Importantly, these effects were demonstrated to be CDK12-dependent as mutation of Cys-1039 rendered the kinase refractory to MFH290 and restored Pol II CTD phosphorylation and DNA damage repair gene expression. Consistent with its effect on DNA damage repair gene expression, MFH290 augments the anti-proliferative effect of the PARP inhibitor, Olaparib.

Identification of a potent and selective covalent Pin1 inhibitor

Benika J. Pinch, Zainab M. Doctor, Behnam Nabet, Christopher M. Browne, Hyuk-Soo Seo, Mikaela L. Mohardt, Shingo Kozono, Xiaolan Lian, Theresa D. Manz, Yujin Chun, Shin Kibe, Daniel Zaidman, Dina Daitchman, Zoe C. Yeoh, Nicholas E. Vangos, Ezekiel A. Geffken, Li Tan, Scott B. Ficarro, Nir London, Jarrod A. Marto, Stephen Buratowski, Sirano Dhe-Paganon, Xiao Zhen Zhou, Kun Ping Lu & Nathanael S. Gray

Nature Chemical Biology, 2020
DOI https://doi.org/10.1038/s41589-020-0550-9

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.