Thursday, January 28, 2021

Investigating β-Lactam Drug Targets in Mycobacterium tuberculosis Using Chemical Probes [@BeattyLab]

Samantha R. Levine and Kimberly E. Beatty

ACS Infect. Dis. 2021

Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), infects 10 million people a year. An estimated 25% of humans harbor latent TB infections, an asymptomatic form of the disease. In both active and latent infections, Mtb relies on cell wall peptidoglycan for viability. In the current work, we synthesized fluorescent analogues of β-lactam antibiotics to study two classes of enzymes that maintain Mtb’s peptidoglycan: penicillin-binding proteins (PBPs) and l,d-transpeptidases (LDTs). This set of activity-based probes included analogues of three classes of β-lactams: a monobactam (aztreonam-Cy5), a cephalosporin (cephalexin-Cy5), and a carbapenem (meropenem-Cy5). We used these probes to profile enzyme activity in protein gel-resolved lysates of Mtb. All three out-performed the commercial reagent Bocillin-FL, a penam. Meropenem-Cy5 was used to identify β-lactam targets by mass spectrometry, including PBPs, LDTs, and the β-lactamase BlaC. New probes were also used to compare PBP and LDT activity in two metabolic states: dormancy and active replication. We provide the first direct evidence that Mtb dynamically regulates the enzymes responsible for maintaining peptidoglycan in dormancy. Lastly, we profiled drug susceptibility in lysates and found that meropenem inhibits PBPs, LDTs, and BlaC.

Chemoproteomics-enabled discovery of covalent RNF114-based degraders that mimic natural product function [@DanNomura, @j_sprads]

Luo M, Spradlin JN,  Boike L, Tong B, Brittain SM, McKenna JM, Tallarico JA, Schirle M, Maimone TJ#, Nomura DK

Cell Chemical Biology, 2021


The translation of functionally active natural products into fully synthetic small-molecule mimetics has remained an important process in medicinal chemistry. We recently discovered that the terpene natural product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted protein degradation—a powerful therapeutic modality within modern-day drug discovery. Using activity-based protein profiling-enabled covalent ligand-screening approaches, here we report the discovery of fully synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and demonstrate their utility in degrading therapeutically relevant targets, such as BRD4 and BCR-ABL, in cells. The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a complex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great importance in drug discovery and chemical biology.

Tuesday, January 26, 2021

Targeting Cysteine Located Outside the Active Site: An Effective Strategy for Covalent ALKi Design

Guoyi Yan, Xinxin Zhong, Chunlan Pu, Lin Yue, Huifang Shan, Suke Lan, Meng Zhou, Xueyan Hou, Jie Yang, Deyu Li, Shilong Fan, and Rui Li
Journal of Medicinal Chemistry 2021

DOI: 10.1021/acs.jmedchem.0c01707

Potent inhibitors of ALK are highly desired because of the occurrence of drug resistance. We herein firstly report the development of a rationally designed inhibitor, Con B-1, which can covalently bind to Cys1259, a cysteine located outside the ALK active site by linking a warhead with Ceritinib through a 2,2′-Oxybis(ethylamine) linker. The in vitro and in vivo assays showed ConB-1 is a potent selective ALKi with low toxicity to normal cells. In addition, the molecule showed significant improvement of anticancer activities and potential antidrug resistant activity compared with Ceritinib, demonstrating the covalent inhibitor of ALK can be a promising drug candidate for the treatment of NSCLC. This work may provide a novel perspective on the design of covalent inhibitors.

Thursday, January 21, 2021

Chemoproteomic profiling of kinases in live cells using electrophilic sulfonyl triazole probes [@Hsu_Lab_UVa]

Tao Huang,  Seyyedmohsen Hosseinibarkooie, Adam L. Borne,  Mitchell E. Granade,  Jeffrey W. Brulet,   Thurl E. Harris,    Heather A. Ferris and  Ku-Lung Hsu 

Chemical Science, 2021

DOI: 10.1039/D0SC06623K

Sulfonyl-triazoles are a new class of electrophiles that mediate covalent reaction with tyrosine residues on proteins through sulfur-triazole exchange (SuTEx) chemistry. Recent studies demonstrate the broad utility and tunability of SuTEx chemistry for chemical proteomics and protein ligand discovery. Here, we present a strategy for mapping protein interaction networks of structurally complex binding elements using functionalized SuTEx probes. We show that the triazole leaving group (LG) can serve as a releasable linker for embedding hydrophobic fragments to direct molecular recognition while permitting efficient proteome-wide identification of binding sites in live cells. We synthesized a series of SuTEx probes functionalized with a lipid kinase fragment binder for discovery of ligandable tyrosines residing in catalytic and regulatory domains of protein and metabolic kinases in live cells. We performed competition studies with kinase inhibitors and substrates to demonstrate that probe binding is occurring in an activity-dependent manner. Our functional studies led to discovery of probe-modified sites within the C2 domain that were important for downregulation of protein kinase C-alpha in response to phorbol ester activation. Our proof of concept studies highlight the triazole LG of SuTEx probes as a traceless linker for locating protein binding sites targeted by complex recognition elements in live cells.

Wednesday, January 20, 2021

Multiplexed CuAAC Suzuki–Miyaura Labeling for Tandem Activity-Based Chemoproteomic Profiling [@Keribackus]

Jian Cao, Lisa M. Boatner, Heta S. Desai, Nikolas R. Burton, Ernest Armenta, Neil J. Chan, José O. Castellón, and Keriann M. Backus
Analytical Chemistry 2021

DOI: 10.1021/acs.analchem.0c04726

Mass-spectrometry-based chemoproteomics has enabled the rapid and proteome-wide discovery of functional and potentially ’druggable’ hotspots in proteins. While numerous transformations are now available, chemoproteomic studies still rely overwhelmingly on copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) or ’click’ chemistry. The absence of bio-orthogonal chemistries that are functionally equivalent and complementary to CuAAC for chemoproteomic applications has hindered the development of multiplexed chemoproteomic platforms capable of assaying multiple amino acid side chains in parallel. Here, we identify and optimize Suzuki–Miyaura cross-coupling conditions for activity-based protein profiling and mass-spectrometry-based chemoproteomics, including for target deconvolution and labeling site identification. Uniquely enabled by the observed orthogonality of palladium-catalyzed cross-coupling and CuAAC, we combine both reactions to achieve dual labeling. Multiplexed targeted deconvolution identified the protein targets of bifunctional cysteine- and lysine-reactive probes.

Tuesday, January 19, 2021

Development and biological applications of sulfur–triazole exchange (SuTEx) chemistry

Adam L. Borne,   Jeffrey W. Brule ,  Kun Yuan  and  Ku-Lung Hsu 

RSC Chem. Biol., 2021, Advance Article

Sulfur electrophiles constitute an important class of covalent small molecules that have found widespread applications in synthetic chemistry and chemical biology. Various electrophilic scaffolds, including sulfonyl fluorides and arylfluorosulfates as recent examples, have been applied for protein bioconjugation to probe ligand sites amenable for chemical proteomics and drug discovery. In this review, we describe the development of sulfonyl-triazoles as a new class of electrophiles for sulfur–triazole exchange (SuTEx) chemistry. SuTEx achieves covalent reaction with protein sites through irreversible modification of a residue with an adduct group (AG) upon departure of a leaving group (LG). A principal differentiator of SuTEx from other chemotypes is the selection of a triazole heterocycle as the LG, which introduces additional capabilities for tuning the sulfur electrophile. We describe the opportunities afforded by modifications to the LG and AG alone or in tandem to facilitate nucleophilic substitution reactions at the SO2 center in cell lysates and live cells. As a result of these features, SuTEx serves as an efficient platform for developing chemical probes with tunable bioactivity to study novel nucleophilic sites on established and poorly annotated protein targets. Here, we highlight a suite of biological applications for the SuTEx electrophile and discuss future goals for this enabling covalent chemistry.

Thursday, January 14, 2021

Targeting EGFR in glioblastoma with a novel brain-penetrant small molecule EGFR-TKI

Jing Ni, Yanzhi Yang, Qiwei Wang, Johann S. Bergholz, Tao Jiang, Thomas M. Roberts, Nathanael S. Gray, Jean J. Zhao


Epidermal growth factor receptor (EGFR) is mutated or amplified in a majority of glioblastoma (GBM), and its mutation and focal amplification correlate with a more aggressive disease course. However, EGFR-directed tyrosine kinase inhibitors (TKIs) tested to date have yielded minimal clinical benefit. Here, we report a novel covalent EGFR-TKI, CM93, as a potential specific drug to target adult GBMs with aberrant EGFR. CM93 has extraordinary brain-selective distribution, with a brain-to-plasma ratio greater than 20 (>2,000% brain penetration). While all currently approved EGFR-TKIs are subject to extensive efflux transporter activity, CM93 is not a substrate of efflux transporters (P-gp and BCRP). Pre-clinical efficacy studies showed that CM93 is more effective than other EGFR-TKIs in blocking the proliferation of GBM tumor cells from both patient-derived and cultured human GBM cell lines with EGFR amplification and/or EGFRvIII mutation. In addition, CM93 administered as a single agent was able to attenuate the growth of orthotopic U251-EGFRvIII xenografts and extend the survival of tumor-bearing mice in a dose-dependent manner. Moreover, CM93 inhibited EGFR phosphorylation in GBM tumors derived from a novel genetically-engineered mouse (GEM) model of GBM with EGFRvIII expression both in vitro and in vivo. CM93 also extended the survival of mice bearing orthotopic allografts of GBM. Notably, mice maintained stable body weight during treatments with increasing doses of CM93 up to 75 mg/kg per day. Together, these data suggest that CM93 is a potential EGFR-TKI well suited for the treatment of adult GBM with mutant EGFR.

Wednesday, January 13, 2021

Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries

Miljan Kuljanin, Dylan C. Mitchell, Devin K. Schweppe, Ajami S. Gikandi, David P. Nusinow, Nathan J. Bulloch, Ekaterina V. Vinogradova, David L. Wilson, Eric T. Kool, Joseph D. Mancias, Benjamin F. Cravatt & Steven P. Gygi

Nature Biotechnology, 2021

Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound. We applied it to identify proteome-wide targets of covalent inhibitors to mutant Kirsten rat sarcoma (KRAS)G12C and Bruton’s tyrosine kinase (BTK). In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines, which includes >20,000 cysteines from >6,000 proteins per line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.

Steric-Free Bioorthogonal Labeling of Acetylation Substrates Based on a Fluorine–Thiol Displacement Reaction

Zhigang Lyu, Yue Zhao, Zakey Yusuf Buuh, Nicole Gorman, Aaron R. Goldman, Md Shafiqul Islam, Hsin-Yao Tang, and Rongsheng E. Wang*

J. Am. Chem. Soc. 2021

We have developed a novel bioorthogonal reaction that can selectively displace fluorine substitutions alpha to amide bonds. This fluorine–thiol displacement reaction (FTDR) allows for fluorinated cofactors or precursors to be utilized as chemical reporters, hijacking acetyltransferase-mediated acetylation both in vitro and in live cells, which cannot be achieved with azide- or alkyne-based chemical reporters. The fluoroacetamide labels can be further converted to biotin or fluorophore tags using FTDR, enabling the general detection and imaging of acetyl substrates. This strategy may lead to a steric-free labeling platform for substrate proteins, expanding our chemical toolbox for functional annotation of post-translational modifications in a systematic manner.

Monday, January 11, 2021

Identification of highly selective covalent inhibitors by phage display [@mbogyo]

Shiyu Chen, Scott Lovell, Sumin Lee, Matthias Fellner, Peter D. Mace & Matthew Bogyo

Nature Biotechnology2020

Molecules that covalently bind macromolecular targets have found widespread applications as activity-based probes and as irreversibly binding drugs. However, the general reactivity of the electrophiles needed for covalent bond formation makes control of selectivity difficult. There is currently no rapid, unbiased screening method to identify new classes of covalent inhibitors from highly diverse pools of candidate molecules. Here we describe a phage display method to directly screen for ligands that bind to protein targets through covalent bond formation. This approach makes use of a reactive linker to form cyclic peptides on the phage surface while simultaneously introducing an electrophilic ‘warhead’ to covalently react with a nucleophile on the target. Using this approach, we identified cyclic peptides that irreversibly inhibited a cysteine protease and a serine hydrolase with nanomolar potency and exceptional specificity. This approach should enable rapid, unbiased screening to identify new classes of highly selective covalent inhibitors for diverse molecular targets.

Wednesday, January 6, 2021

A Covalent p97/VCP ATPase Inhibitor can overcome resistance to CB-5083 and NMS-873 in colorectal cancer cells,

Gang Zhang, Shan Li, Feng Wang, Amanda C. Jones, Alexander F.G. Goldberg, Benjamin Lin, Scott Virgil, Brian M. Stoltz, Raymond J. Deshaies, Tsui-Fen Chou,

European Journal of Medicinal Chemistry, 2021, 113148

We incorporated electrophiles into a PP-like compound 17 (4-amino-1-tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18 (N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent selectivity in an in vitro ATPase activity assay: IC50 of 0.6 μM, 300 μM, and 100 μM for wild type p97, yeast Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified and shown to increase IC50 values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with Cysteine decreased the IC50 by 10-fold. The molecular modeling suggested the hydrogen bonds and hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA. Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was inhibited by PPA with IC50 of 2.7 μM, 6.1 μM, and 3.4 μM, respectively. In addition, PPA is able to inhibit proliferation of two HCT116 cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future exploration to improve the potency of p97 inhibitors with different mechanisms of action.

Covalent Inhibition of Wild-Type HIV-1 Reverse Transcriptase Using a Fluorosulfate Warhead [@JorgensenWL]

Joseph A. Ippolito, Haichan Niu, Nicole Bertoletti, Zachary J. Carter, Shengyan Jin, Krasimir A. Spasov, José A. Cisneros, Margarita Valhondo, Kara J. Cutrona, Karen S. Anderson, and William L. Jorgensen

ACS Medicinal Chemistry Letters 2021

DOI: 10.1021/acsmedchemlett.0c00612

Covalent inhibitors of wild-type HIV-1 reverse transcriptase (CRTIs) are reported. Three compounds derived from catechol diether non-nucleoside inhibitors (NNRTIs) with addition of a fluorosulfate warhead are demonstrated to covalently modify Tyr181 of HIV-RT. X-ray crystal structures for complexes of the CRTIs with the enzyme are provided, which fully demonstrate the covalent attachment, and confirmation is provided by appropriate mass shifts in ESI-TOF mass spectra. The three CRTIs and six noncovalent analogues are found to be potent inhibitors with both IC50 values for in vitro inhibition of WT RT and EC50 values for cytopathic protection of HIV-1-infected human T-cells in the 5–320 nM range.

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...