Friday, April 30, 2021

Deubiquitinase-Targeting Chimeras for Targeted Protein Stabilization

Nathaniel J Henning, Lydia Boike, Jessica N Spradlin, Carl C Ward, Bridget Belcher, Scott M Brittain, Matthew Hesse, Dustin Dovala, Lynn M McGregor, Jeffrey M McKenna, John A Tallarico, Markus Schirle, Daniel K Nomura

bioRxiv 2021.04.30.441959; doi:

Targeted protein degradation is a powerful therapeutic modality that uses heterobifunctional small-molecules to induce proximity between E3 ubiquitin ligases and target proteins to ubiquitinate and degrade specific proteins of interest. However, many proteins are ubiquitinated and degraded to drive disease pathology; in these cases targeted protein stabilization (TPS), rather than degradation, of the actively degraded target using a small-molecule would be therapeutically beneficial. Here, we present the Deubiquitinase-Targeting Chimera (DUBTAC) platform for TPS of specific proteins. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48 ubiquitin-specific deubiquitinase OTUB1. We then developed a heterobifunctional DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-CFTR. We demonstrated proof-of-concept of TPS by showing that this DUBTAC robustly stabilized ΔF508-CFTR in human cystic fibrosis bronchial epithelial cells in an OTUB1-dependent manner. Our study underscores the utility of chemoproteomics-enabled covalent ligand discovery approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.

Monday, April 26, 2021

Electrophilic Natural Products as Drug Discovery Tools

Paul Gehrtz, Nir London,

Trends in Pharmacological Sciences, 2021

Electrophilic natural products (ENPs) are a rich source of bioactive molecules with tremendous therapeutic potential. While their synthetic complexity may hinder their direct use as therapeutics, they represent tools for elucidation of suitable molecular targets and serve as inspiration for the design of simplified synthetic counterparts. Here, we review the recent use of various activity-based protein profiling methods to uncover molecular targets of ENPs. Beyond target identification, these examples also showcase further development of synthetic ligands from natural product starting points. Two examples demonstrate how ENPs can progress the emerging fields of targeted protein degradation and molecular glues. Though challenges still remain in the synthesis of ENP-based probes, and in their synthetic simplification, their potential for discovery of novel mechanisms of action makes it well worth the effort.

Friday, April 23, 2021

Structure-Activity Relationship Study of THZ531 Derivatives Enables the Discovery of BSJ-01-175 as a Dual CDK12/13 Covalent Inhibitor with Efficacy in Ewing Sarcoma

Baishan Jiang, Jie Jiang, Ines H. Kaltheuner, Amanda Balboni Iniguez, Kanchan Anand, Fleur M. Ferguson, Scott B. Ficarro, Bo Kyung Alex Seong, Ann Katrin Greifenberg, Sofia Dust, Nicholas P. Kwiatkowski, Jarrod A. Marto, Kimberly Stegmaier, Tinghu Zhang, Matthias Geyer, Nathanael S. Gray

European Journal of Medicinal Chemistry, 2021

Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.

Thursday, April 15, 2021

Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications [@DanNomura]

Nathaniel J Henning, Andrew G Manford, Jessica N Spradlin, Scott M Brittain, Jeffrey M McKenna, John A Tallarico, Markus Schirle, Michael Rape, Daniel K Nomura

bioRxiv 2021.04.15.439993;


Proteolysis Targeting Chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number of E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand, EN106, that targets FEM1B, an E3 ligase recently discovered as the critical component of the cellular response to reductive stress. By targeting Cys186 in FEM1B, EN106 disrupts recognition of the key reductive stress substrate of FEM1B, FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications, in which we demonstrate that a PROTAC linking EN106 to the BET Bromodomain inhibitor JQ1 leads to specific FEM1B- and proteasome-dependent degradation of BRD4 in cells. Our study showcases a covalent ligand that targets a natural E3 ligase-substrate binding site and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters that can be deployed for TPD applications.

Tuesday, April 13, 2021

Functionalized Scout Fragments for Site-Specific Covalent Ligand Discovery and Optimization

Vincent M. Crowley, Marvin Thielert, and Benjamin F. Cravatt

ACS Central Science 2021

DOI: 10.1021/acscentsci.0c01336

Covalent ligands are a versatile class of chemical probes and drugs that can target noncanonical sites on proteins and display differentiated pharmacodynamic properties. Chemical proteomic methods have been introduced that leverage electrophilic fragments to globally profile the covalent ligandability of nucleophilic residues, such as cysteine and lysine, in native biological systems. Further optimization of these initial ligandability events without resorting to the time-consuming process of individualized protein purification and functional assay development, however, presents a persistent technical challenge. Here, we show that broadly reactive electrophilic fragments, or “scouts”, can be converted into site-specific target engagement probes for screening small molecules against a wide array of proteins in convenient gel- and ELISA-based assay formats. We use these assays to expediently optimize a weak potency fragment hit into a sub-μM inhibitor that selectively engages an active-site cysteine in the retinaldehyde reductase AKR1B10. Our findings provide a road map to optimize covalent fragments into more advanced chemical probes without requiring protein purification or structural analysis.

Friday, April 9, 2021

Discovery of a Potent and Selective Covalent p300/CBP Inhibitor

Anthony Mastracchio, Chunqiu Lai, Enrico Digiammarino, Damien B. Ready, Loren M. Lasko, Kenneth D. Bromberg, William J. McClellan, Debra Montgomery, Vlasios Manaves, Bailin Shaw, Mikkel Algire, Melanie J. Patterson, Chaohong C. Sun, Saul Rosenberg, Albert Lai, and Michael R. Michaelides

ACS Medicinal Chemistry Letters 2021

DOI: 10.1021/acsmedchemlett.0c00654

Aberrant gene activation driven by the histone acetyltransferases p300 and CREB binding protein (CBP) has been linked to several diseases, including cancers. Because of this, many efforts have been aimed toward the targeting of the closely related paralogues, p300 and CBP, but these endeavors have been exclusively directed toward noncovalent inhibitors. X-ray crystallography of A-485 revealed that both p300 and CBP possess a cysteine (C1450) near the active site, thus rendering covalent inhibition an attractive chemical approach. Herein we report the development of compound 2, an acrylamide-based inhibitor of p300/CBP that forms a covalent adduct with C1450. We demonstrated using mass spectrometry that compound 2 selectively targets C1450, and we also validated covalent binding using kinetics experiments and cellular washout studies. The discovery of covalent inhibitor 2 gives us a unique tool for the study of p300/CBP biology.

Discovery of a Potent and Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase with Oral Anti-Inflammatory Activity

Mark S. Tichenor, John J. M. Wiener, Navin L. Rao, Charlotte Pooley Deckhut, J. Kent Barbay, Kevin D. Kreutter, Genesis M. Bacani, Jianmei Wei, Leon Chang, Heather E. Murrey, Weixue Wang, Kay Ahn, Michael Huber, Elizabeth Rex, Kevin J. Coe, JieJun Wu, Mark Seierstad, Scott D. Bembenek, Kristi A. Leonard, Alec D. Lebsack, Jennifer D. Venable, and James P. Edwards

ACS Medicinal Chemistry Letters 2021

DOI: 10.1021/acsmedchemlett.1c00044

Bruton’s tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that plays a critical role in the activation of B cells, macrophages, and osteoclasts. Given the key role of these cell types in the pathology of autoimmune disorders, BTK inhibitors have the potential to improve treatment outcomes in multiple diseases. Herein, we report the discovery and characterization of a novel potent and selective covalent 4-oxo-4,5-dihydro-3H-1-thia-3,5,8-triazaacenaphthylene-2-carboxamide BTK inhibitor chemotype. Compound 27 irreversibly inhibits BTK by targeting a noncatalytic cysteine residue (Cys481) for covalent bond formation. Compound 27 is characterized by selectivity for BTK, potent in vivo BTK occupancy that is sustained after it is cleared from systemic circulation, and dose-dependent efficacy at reducing joint inflammation in a rat collagen-induced arthritis model.

Covalent Probes for Aggregated Protein Imaging via Michael Addition

Wang Wan  Yanan Huang  Qiuxuan Xia  Yulong Bai  Yuwen Chen  Wenhan Jin  Mengdie Wang  Di Shen  Haochen Lyu  Yuqi Tang  Xuepeng Dong  Zhenming Gao  Qun Zhao  Lihua Zhang  Yu Liu

Angew. Chem. Int. Ed. 2021

Covalent chemical reactions to modify aggregated proteins are rare. Here, we reported covalent Michael addition can generally occur upon protein aggregation. Such reactivity was initially discovered by a bioinspired fluorescent color‐switch probe mimicking the photo‐conversion mechanism of Kaede fluorescent protein. This probe was dark with folded proteins but turned on red fluorescence (620 nm) when it non‐covalently bound to misfolded proteins. Supported by the biochemical and mass spectrometry results, the probe chemoselectively reacted with the reactive cysteines of aggregated proteins via covalent Michael addition and gradually switched to green fluorescence (515 nm) upon protein aggregation. Exploiting this Michael addition chemistry in the malachite green dye derivatives demonstrated its general applicability and chemical tunability, resulting in different fluorescence color‐switch responses. Our work may offer a new avenue to explore other chemical reactions upon protein aggregation and design covalent probes for imaging, chemical proteomics, and therapeutic purposes.

Linchpin empowers promiscuous electrophile to render site-selective modification of histidine and aspartic acid in proteins [@vishal_iiserb]

Dattatraya Gautam Rawale,   Kalyani Thakur,   Pranav Sreekumar,   Sajeev T. K.,   Ramesh A,   Srinivasa Rao Adusumalli,   Ram Kumar Mishra  and  Vishal Rai  

Chemical Science, 2021

The conservation of chemoselectivity becomes invalid for multiple electrophilic warheads during protein bioconjugation. Consequently, it leads to unpredictable heterogeneous labeling of proteins. Here, we report that a linchpin can create a unique chemical space to render site-selectivity for histidine and aspartic acid modification overcoming the pre-requisite of chemoselectivity.

Monday, April 5, 2021

A targeted covalent small molecule inhibitor of HIV-1 fusion

Guangyan Zhou Li He Kathy H Li Cassio Cardoso Santos Pedroso and Miriam Gochin

Chem. Commun., 2021

DOI: 10.1039/D1CC01013A 

We describe a low molecular weight covalent inhibitor targeting a conserved lysine residue within the hydrophobic pocket of HIV-1 glycoprotein-41. The inhibitor bound selectively to the hydrophobic pocket and exhibited an order of magnitude enhancement of anti-fusion activity against HIV-1 compared to its non-covalent counterpart. The findings represent a significant advance in the quest to obtain non-peptide fusion inhibitor.

DCAF11 Supports Targeted Protein Degradation by Electrophilic Proteolysis-Targeting Chimeras

Xiaoyu Zhang, Lena M. Luukkonen, Christie L. Eissler, Vincent M. Crowley, Yu Yamashita, Michael A. Schafroth, Shota Kikuchi, David S. Weinstein, Kent T. Symons, Brian E. Nordin, Joe L. Rodriguez, Thomas G. Wucherpfennig, Ludwig G. Bauer, Melissa M. Dix, Dean Stamos, Todd M. Kinsella, Gabriel M. Simon, Kristen A. Baltgalvis, and Benjamin F. Cravatt

Journal of the American Chemical Society Article 2021

DOI: 10.1021/jacs.1c00990

Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.

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...