Tuesday, December 15, 2020

Proton-Coupled Conformational Activation of SARS Coronavirus Main Proteases and Opportunity for Designing Small-Molecule Broad-Spectrum Targeted Covalent Inhibitors

Neha Verma, Jack A. Henderson, and Jana Shen

J. Am. Chem. Soc., 2020


The SARS coronavirus 2 (SARS-CoV-2) main protease (Mpro) is an attractive broad-spectrum antiviral drug target. Despite the enormous progress in structure elucidation, the Mpro’s structure–function relationship remains poorly understood. Recently, a peptidomimetic inhibitor has entered clinical trial; however, small-molecule orally available antiviral drugs have yet to be developed. Intrigued by a long-standing controversy regarding the existence of an inactive state, we explored the proton-coupled dynamics of the Mpros of SARS-CoV-2 and the closely related SARS-CoV using a newly developed continuous constant pH molecular dynamics (MD) method and microsecond fixed-charge all-atom MD simulations. Our data supports a general base mechanism for Mpro’s proteolytic function. The simulations revealed that protonation of His172 alters a conserved interaction network that upholds the oxyanion loop, leading to a partial collapse of the conserved S1 pocket, consistent with the first and controversial crystal structure of SARS-CoV Mpro determined at pH 6. Interestingly, a natural flavonoid binds SARS-CoV-2 Mpro in the close proximity to a conserved cysteine (Cys44), which is hyper-reactive according to the CpHMD titration. This finding offers an exciting new opportunity for small-molecule targeted covalent inhibitor design. Our work represents a first step toward the mechanistic understanding of the proton-coupled structure–dynamics–function relationship of CoV Mpros; the proposed strategy of designing small-molecule covalent inhibitors may help accelerate the development of orally available broad-spectrum antiviral drugs to stop the current pandemic and prevent future outbreaks.

Sunday, December 6, 2020

10 years into the resurgence of covalent drugs

Elena De Vita

Future Medicinal Chemistry 2020


In the first decade of targeted covalent inhibition, scientists have successfully reversed the previous trend that had impeded the use of covalent inhibition in drug development. Successes in the clinic, mainly in the field of kinase inhibitors, are existing proof that safe covalent inhibitors can be designed and employed to develop effective treatments. The case of KRASG12C covalent inhibitors entering clinical trials in 2019 has been among the hottest topics discussed in drug discovery, raising expectations for the future of the field. In this perspective, an overview of the milestones hit with targeted covalent inhibitors, as well as the promise and the needs of current research, are presented. While recent results have confirmed the potential that was foreseen, many questions remain unexplored in this branch of precision medicine.

Friday, November 27, 2020

Targeted Covalent Inhibitors for the Treatment of Malaria?

Shashank Kulkarni, Klaus Urbahns, and Thomas Spangenberg
ACS Infectious Diseases 2020 6 (11), 2815-2817
DOI: 10.1021/acsinfecdis.0c00684

Malaria is a vector-borne disease caused by protozoan parasites of the genus Plasmodium. According to the World Health Organization, it is one of the most serious infectious diseases threatening more than 3 billion people worldwide. In recent years, targeted covalent inhibitors (TCIs) have gained a lot of attention and several TCI-based drugs have been approved across different therapeutic areas. For malaria, surprisingly, this approach has not been explored in depth even though lot of advancements have been made in understanding the biology of the parasite. Herein, we present our views on exploring TCIs as a new class of antimalarial agents.

Sunday, November 8, 2020

Development and Application of a Chemical Probe Based on a Neuroprotective Flavonoid Hybrid for Target Identification Using Activity-Based Protein Profiling

Sandra Gunesch, David Soriano-Castell, Stephanie Lamer, Andreas Schlosser, Pamela Maher, and Michael Decker

ACS Chemical Neuroscience 2020

DOI: 10.1021/acschemneuro.0c00589

Alzheimer’s disease (AD) is the most common form of dementia, and up to now, there are no disease-modifying drugs available. Natural product hybrids based on the flavonoid taxifolin and phenolic acids have shown a promising pleiotropic neuroprotective profile in cell culture assays and even disease-modifying effects in vivo. However, the detailed mechanisms of action remain unclear. To elucidate the distinct intracellular targets of 7-O-esters of taxifolin, we present in this work the development and application of a chemical probe, 7-O-cinnamoyltaxifolin-alkyne, for target identification using activity-based protein profiling. 7-O-Cinnamoyltaxifolin-alkyne remained neuroprotective in all cell culture assays. Western blot analysis showed a comparable influence on the same intracellular pathways as that of the lead compound 7-O-cinnamoyltaxifolin, thereby confirming its suitability as a probe for target identification experiments. Affinity pulldown and MS analysis revealed adenine nucleotide translocase 1 (ANT-1) and sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) as intracellular interaction partners of 7-O-cinnamoyltaxifolin-alkyne and thus of 7-O-esters of taxifolin.

An Untargeted Approach for Revealing Electrophilic Metabolites

Yan Yu, Henry H. Le, Brian J. Curtis, Chester J. J. Wrobel, Bingsen Zhang, Danielle N. Maxwell, Judy Y. Pan, and Frank C. Schroeder

ACS Chemical Biology 2020

DOI: 10.1021/acschembio.0c00706

Reactive electrophilic intermediates such as coenzyme A esters play central roles in metabolism but are difficult to detect with conventional strategies. Here, we introduce hydroxylamine-based stable isotope labeling to convert reactive electrophilic intermediates into stable derivatives that are easily detectable via LC–MS. In the model system Caenorhabditis elegans, parallel treatment with 14NH2OH and 15NH2OH revealed >1000 labeled metabolites, e.g., derived from peptide, fatty acid, and ascaroside pheromone biosyntheses. Results from NH2OH treatment of a pheromone biosynthesis mutant, acox-1.1, suggested upregulation of thioesterase activity, which was confirmed by gene expression analysis. The upregulated thioesterase contributes to the biosynthesis of a specific subset of ascarosides, determining the balance of dispersal and attractive signals. These results demonstrate the utility of NH2OH labeling for investigating complex biosynthetic networks. Initial results with Aspergillus and human cell lines indicate applicability toward uncovering reactive metabolomes in diverse living systems.

Saturday, November 7, 2020

Complex Crystal Structures of EGFR with Third-Generation Kinase Inhibitors and Simultaneously Bound Allosteric Ligands

ACS Med. Chem. Lett. 2020

doi: https://doi.org/10.1021/acsmedchemlett.0c00472

Osimertinib is a third-generation tyrosine kinase inhibitor (TKI) and currently the gold-standard for the treatment of patients suffering from non-small cell lung cancer (NSCLC) harboring T790M-mutated epidermal growth factor receptor (EGFR). The outcome of the treatment, however, is limited by the emergence of the C797S resistance mutation. Allosteric inhibitors have a different mode of action and were developed to overcome this limitation. However, most of these innovative molecules are not effective as a single agent. Recently, mutated EGFR was successfully addressed with osimertinib combined with the allosteric inhibitor JBJ-04-125-02, but surprisingly, structural insights into their binding mode were lacking. Here, we present the first complex crystal structures of mutant EGFR in complex with third-generation inhibitors such as osimertinib and mavelertinib in the presence of simultaneously bound allosteric inhibitors. These structures highlight the possibility of further combinations targeting EGFR and lay the foundation for hybrid inhibitors as next-generation TKIs.

Sunday, November 1, 2020

Chemical Control of Quorum Sensing in E. coli: Identification of Small Molecule Modulators of SdiA and Mechanistic Characterization of a Covalent Inhibitor

Matthew J. Styles, Stephen A. Early, Trisha Tucholski, Korbin H. J. West, Ying Ge, and Helen E. Blackwell

ACS Infectious Diseases 2020

DOI: 10.1021/acsinfecdis.0c00654

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of severe diarrheal disease in humans. Cattle are the natural reservoir of EHEC, and approximately 75% of EHEC infections in humans stem from bovine products. Many common bacterial pathogens, including EHEC, rely on chemical communication systems, such as quorum sensing (QS), to regulate virulence and facilitate host colonization. EHEC uses SdiA from E. coli (SdiAEC), an orphan LuxR-type receptor, to sense N-acyl l-homoserine lactone (AHL) QS signals produced by other members of the bovine enteric microbiome. SdiAEC regulates two phenotypes critical for colonizing cattle: acid resistance and the formation of attaching and effacing lesions. Despite the importance of SdiAEC, there is very little known about its selectivity for different AHL signals, and no chemical inhibitors that act specifically on SdiAEC have been reported. Such compounds would represent valuable tools to study the roles of QS in EHEC virulence. To identify chemical modulators of SdiAEC and delineate the structure–activity relationships (SARs) for AHL activity in this receptor, we report herein the screening of a focused library composed largely of AHLs and AHL analogues in an SdiAEC reporter assay. We describe the identity and SARs of potent modulators of SdiAEC activity, examine the promiscuity of SdiAEC, characterize the mechanism of a covalent inhibitor, and provide phenotypic assay data to support that these compounds can control SdiAEC-dependent acid resistance in E. coli. These SdiAEC modulators could be used to advance the study of LuxR-type receptor/ligand interactions, the biological roles of orphan LuxR-type receptors, and potential QS-based therapeutic approaches.

Saturday, October 24, 2020

Systematic Study of the Glutathione (GSH) Reactivity of N-Phenylacrylamides: 2. Effects of Acrylamide Substitution

Adam Birkholz, David J. Kopecky, Laurie P. Volak, Michael D. Bartberger, Yuping Chen, Christopher M. Tegley, Tara Arvedson, John D. McCarter, Christopher Fotsch, and Victor J. Cee

Journal of Medicinal Chemistry 2020 63 (20), 11602-11614
DOI: 10.1021/acs.jmedchem.0c00749

A comprehensive understanding of structure–reactivity relationships is critical to the design and optimization of cysteine-targeted covalent inhibitors. Herein, we report glutathione (GSH) reaction rates for N-phenyl acrylamides with varied substitutions at the α- and β-positions of the acrylamide moiety. We find that the GSH reaction rates can generally be understood in terms of the electron donating or withdrawing ability of the substituent. When installed at the β-position, aminomethyl substituents with amine pKa’s > 7 accelerate, while those with pKa’s < 7 slow the rate of GSH addition at pH 7.4, relative to a hydrogen substituent. Although a computational model was able to only approximately capture experimental reactivity trends, our calculations do not support a frequently invoked mechanism of concerted amine/thiol proton transfer and C–S bond formation and instead suggest that protonated aminomethyl functions as an electron-withdrawing group to reduce the barrier for thiolate addition to the acrylamide.

Itaconate is a covalent inhibitor of the Mycobacterium tuberculosis isocitrate lyase

Brooke X. C. Kwai, Annabelle J. Collins, Martin J. Middleditch, Jonathan Sperry,   Ghader Bashiri and  Ivanhoe K. H. Leung 

RSC Med. Chem., 2020

DOI: https://doi.org/10.1039/D0MD00301H

Itaconate is a mammalian antimicrobial metabolite that inhibits the isocitrate lyases (ICLs) of Mycobacterium tuberculosis. Herein, we report that ICLs form a covalent adduct with itaconate through their catalytic cysteine residue. These results reveal atomic details of itaconate inhibition and provide insights into the catalytic mechanism of ICLs.

Friday, October 23, 2020

CovalentInDB: a comprehensive database facilitating the discovery of covalent inhibitors

Hongyan Du, Junbo Gao, Gaoqi Weng, Junjie Ding, Xin Chai, Jinping Pang, Yu Kang, Dan Li, Dongsheng Cao, Tingjun Hou

Nucleic Acids Research, 2020, gkaa876, 

DOI: https://doi.org/10.1093/nar/gkaa876

Inhibitors that form covalent bonds with their targets have traditionally been considered highly adventurous due to their potential off-target effects and toxicity concerns. However, with the clinical validation and approval of many covalent inhibitors during the past decade, design and discovery of novel covalent inhibitors have attracted increasing attention. A large amount of scattered experimental data for covalent inhibitors have been reported, but a resource by integrating the experimental information for covalent inhibitor discovery is still lacking. In this study, we presented Covalent Inhibitor Database (CovalentInDB), the largest online database that provides the structural information and experimental data for covalent inhibitors. CovalentInDB contains 4511 covalent inhibitors (including 68 approved drugs) with 57 different reactive warheads for 280 protein targets. The crystal structures of some of the proteins bound with a covalent inhibitor are provided to visualize the protein–ligand interactions around the binding site. Each covalent inhibitor is annotated with the structure, warhead, experimental bioactivity, physicochemical properties, etc. Moreover, CovalentInDB provides the covalent reaction mechanism and the corresponding experimental verification methods for each inhibitor towards its target. High-quality datasets are downloadable for users to evaluate and develop computational methods for covalent drug design. CovalentInDB is freely accessible at http://cadd.zju.edu.cn/cidb/.

Wednesday, October 21, 2020

Triple, Mutually Orthogonal Bioorthogonal Pairs through the Design of Electronically Activated Sulfamate-Containing Cycloalkynes

Yun Hu, Jessica M. Roberts, Henry R. Kilgore, Amirah S. Mat Lani, Ronald T. Raines, and Jennifer M. Schomaker

Journal of the American Chemical Society 2020

DOI: 10.1021/jacs.0c06725

Interest in mutually exclusive pairs of bioorthogonal labeling reagents continues to drive the design of new compounds that are capable of fast and predictable reactions. The ability to easily modify S-, N-, and O-containing cyclooctynes (SNO-OCTs) enables electronic tuning of various SNO-OCTs to influence their cycloaddition rates with Type I–III dipoles. As opposed to optimizations based on just one specific dipole class, the electrophilicity of the alkynes in SNO-OCTs can be manipulated to achieve divergent reactivities and furnish mutually orthogonal dual ligation systems. Significant reaction rate enhancements of a difluorinated SNO-OCT derivative, as compared to the parent scaffold, were noted, with the second-order rate constant in cycloadditions with diazoacetamides exceeding 5.13 M–1 s–1. Computational and experimental studies were employed to inform the design of triple ligation systems that encompass three orthogonal reactivities. Finally, polar SNO-OCTs are rapidly internalized by mammalian cells and remain functional in the cytosol for live-cell labeling, highlighting their potential for diverse in vitro and in vivo applications.

Thursday, October 15, 2020

Assessment of tractable cysteines by covalent fragments screening

Petri, L., Ábrányi-Balogh, P., Imre, T., Pálfy, G., Perczel, A., Knez, D., Hrast, M., Gobec, M., Sosič, I., Nyíri, K., Vértessy, B..G., Jänsch, N., Desczyk, C., Meyer-Almes, F., Ogris, I., Grdadolnik, S..G., Iacovino, L..G., Binda, C., Gobec, S. and Keserű, G..M. 

ChemBioChem. 2020


Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Here, we present a toolbox of fragments containing a 3,5‐bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine‐containing enzymes.

Tuesday, October 13, 2020

Bicyclobutane Carboxylic Amide as a Cysteine-Directed Strained Electrophile for Selective Targeting of Proteins

Keisuke Tokunaga, Mami Sato, Keiko Kuwata, Chizuru Miura, Hirokazu Fuchida, Naoya Matsunaga, Satoru Koyanagi, Shigehiro Ohdo, Naoya Shindo, and Akio Ojida

Journal of the American Chemical Society 2020

DOI: 10.1021/jacs.0c07490

Expanding the repertoire of electrophiles with unique reactivity features would facilitate the development of covalent inhibitors with desirable reactivity profiles. We herein introduce bicyclo[1.1.0]butane (BCB) carboxylic amide as a new class of thiol-reactive electrophiles for selective and irreversible inhibition of targeted proteins. We first streamlined the synthetic routes to generate a variety of BCB amides. The strain-driven nucleophilic addition to BCB amides proceeded chemoselectively with cysteine thiols under neutral aqueous conditions, the rate of which was significantly slower than that of acrylamide. This reactivity profile of BCB amide was successfully exploited to develop covalent ligands targeting Bruton’s tyrosine kinase (BTK). By tuning BCB amide reactivity and optimizing its disposition on the ligand, we obtained a selective covalent inhibitor of BTK. The in-gel activity-based protein profiling and mass spectrometry-based chemical proteomics revealed that the selected BCB amide had a higher target selectivity for BTK in human cells than did a Michael acceptor probe. Further chemical proteomic study revealed that BTK probes bearing different classes of electrophiles exhibited distinct off-target profiles. This result suggests that incorporation of BCB amide as a cysteine-directed electrophile could expand the capability to develop covalent inhibitors with the desired proteome reactivity profile.

Monday, October 12, 2020

Discovery of a Selective, Covalent IRAK1 Inhibitor with Antiproliferative Activity in MYD88 Mutated B-Cell Lymphoma

John M. Hatcher, Guang Yang, Li Wang, Scott B. Ficarro, Sara Buhrlage, Hao Wu, Jarrod A. Marto, Steven P. Treon, and Nathanael S. Gray

ACS Med. Chem. Lett. 2020


Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating the innate immune response against foreign pathogens. Additionally, dysregulation of IRAK1 signaling plays a role in neoplastic disorders. For example, IRAK1 was shown to be important for survival and proliferation in many B-cell lymphomas, including Waldenström’s macroglobulinemia (WM) and ABC subtype Diffused Large B-cell Lymphoma (DLBCL) cells. Here, we report the discovery of a highly potent and selective covalent inhibitor of IRAK1, JH-X-119-01. Intact protein MS labeling studies confirmed that JH-X-119-01 irreversibly labels IRAK1 at C302. This compound exhibited cytotoxic activity at single digit micromolar concentrations in a panel of WM, DLBCL, and lymphoma cell lines expressing MYD88. Cotreatment of JH-X-119-01 with the BTK inhibitor ibrutinib resulted in synergistic killing effects in these systems. Taken together, JH-X-119-01 represents a highly selective probe of IRAK1 for further development.

Sunday, October 11, 2020

Discovery of CC-90011: A Potent and Selective Reversible Inhibitor of Lysine Specific Demethylase 1 (LSD1)

Toufike Kanouni, Christophe Severin, Robert W. Cho, Natalie Y.-Y. Yuen, Jiangchun Xu, Lihong Shi, Chon Lai, Joselyn R. Del Rosario, Ryan K. Stansfield, Lee N. Lawton, David Hosfield, Shawn O’Connell, Matt M. Kreilein, Paula Tavares-Greco, Zhe Nie, Stephen W. Kaldor, James M. Veal, Jeffrey A. Stafford, and Young K. Chen
Journal of Medicinal Chemistry 2020

DOI: 10.1021/acs.jmedchem.0c00978

Histone demethylase LSDl (KDMlA) belongs to the flavin adenine dinucleotide (FAD) dependent family of monoamine oxidases and is vital in regulation of mammalian biology. Dysregulation and overexpression of LSD1 are hallmarks of a number of human diseases, particularly cancers that are characterized as morphologically poorly differentiated. As such, inhibitors of LSD1 have potential to be beneficial as a cancer therapy. The most clinically advanced inhibitors of LSDl are covalent inhibitors derived from tranylcypromine (TCP). Herein, we report the discovery of a novel series of reversible and selective LSDl inhibitors. Exploration of structure–activity relationships (SARs) and optimization of ADME properties resulted in the identification of clinical candidate CC-90011. CC-90011 exhibits potent on-target induction of cellular differentiation in acute myeloid leukemia (AML) and small cell lung cancer (SCLC) cell lines, and antitumor efficacy in patient-derived xenograft (PDX) SCLC models. CC-90011 is currently in phase 2 trials in patients with first line, extensive stage SCLC (ClinicalTrials.gov identifier: NCT03850067).

Irreversible TrxR1 inhibitors block STAT3 activity and induce cancer cell death

S. Busker1, W. Qian, M. Haraldsson, B. Espinosa, L. Johansson,S. Attarha, I. Kolosenko, J. Liu6, M. Dagnell, D. Grandér, E. S. J. Arnér, K. Pokrovskaja Tamm, and B. D. G. Page

Science Advances  20 Mar 2020:

Vol. 6, no. 12, eaax7945

DOI: 10.1126/sciadv.aax7945

Because of its key role in cancer development and progression, STAT3 has become an attractive target for developing new cancer therapeutics. While several STAT3 inhibitors have progressed to advanced stages of development, their underlying biology and mechanisms of action are often more complex than would be expected from specific binding to STAT3. Here, we have identified and optimized a series of compounds that block STAT3-dependent luciferase expression with nanomolar potency. Unexpectedly, our lead compounds did not bind to cellular STAT3 but to another prominent anticancer drug target, TrxR1. We further identified that TrxR1 inhibition induced Prx2 and STAT3 oxidation, which subsequently blocked STAT3-dependent transcription. Moreover, previously identified inhibitors of STAT3 were also found to inhibit TrxR1, and likewise, established TrxR1 inhibitors block STAT3-dependent transcriptional activity. These results provide new insights into the complexities of STAT3 redox regulation while highlighting a novel mechanism to block aberrant STAT3 signaling in cancer cells.

Friday, October 9, 2020

Hydrogen Peroxide Inducible JAK3 Covalent Inhibitor: Prodrug for the Treatment of RA with Enhanced Safety Profile

Qichao Bao, Liangying Zhang, Nan Wang, Brian Gabet, Weikang Yang, Xingyang Gao, Qidong You, and Zhengyu Jiang

ACS Medicinal Chemistry Letters 2020

DOI: 10.1021/acsmedchemlett.0c00323

Selective inhibition of Janus kinases (JAKs) is an arising strategy in drug discovery. Covalent inhibitors targeting a unique cysteine in JAK3 exhibit ultraselectivity among JAK family members. However, safety and tissue specific concerns still remain. A prodrug of a known JAK3 covalent inhibitor sensitive to H2O2 was designed and synthesized and its therapeutic effect was evaluated in the CIA (collagen-induced arthritis) mice model of RA (rheumatoid arthritis). The prodrug strategy relied on the introduction of a hydrogen peroxide-sensitive borate trigger group to avoid random covalent binding to thiol functionalities in biomacromolecules. The results show that the prodrug can be activated and released under pathophysiological concentration of H2O2. In addition, the prodrug demonstrated stability to the physiological environment. In comparison to the parent compound, the prodrug showed a similar therapeutic effect in the CIA model but notably exhibited lower toxicity and a larger therapeutic window.

Wednesday, October 7, 2020

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

Alice Douangamath, Daren Fearon, Paul Gehrtz, Tobias Krojer, Petra Lukacik, C. David Owen, Efrat Resnick, Claire Strain-Damerell, Anthony Aimon, Péter Ábrányi-Balogh, José Brandão-Neto, Anna Carbery, Gemma Davison, Alexandre Dias, Thomas D. Downes, Louise Dunnett, Michael Fairhead, James D. Firth, S. Paul Jones, Aaron Keeley, György M. Keserü, Hanna F. Klein, Mathew P. Martin, Martin E. M. Noble, Peter O’Brien, Ailsa Powell, Rambabu N. Reddi, Rachael Skyner, Matthew Snee, Michael J. Waring, Conor Wild, Nir London, Frank von Delft & Martin A. Walsh -

Nat Commun 11, 5047 (2020).


COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

Tuesday, October 6, 2020

Approaches to Mitigate the Risk of Serious Adverse Reactions in Covalent Drug Design

Thomas A. Baillie (2020) Approaches to Mitigate the Risk of Serious Adverse Reactions in Covalent Drug Design, Expert Opinion on Drug Discovery 

DOI: 10.1080/17460441.2021.1832079

A review is provided of the current status of the covalent drug approach, emphasizing the unique benefits and attendant risks associated with reversible and irreversible binders. Areas of application beyond inhibition of tyrosine kinases are presented, and design considerations to de-risk covalent inhibitors with respect to undesirable off-target effects are discussed.

Wednesday, September 23, 2020

KRAS G12C Inhibition with Sotorasib in Advanced Solid Tumors

David S. Hong, M.D., Marwan G. Fakih, M.D., John H. Strickler, M.D., Jayesh Desai, M.D., Gregory A. Durm, M.D., Geoffrey I. Shapiro, M.D., Ph.D., Gerald S. Falchook, M.D., Timothy J. Price, M.B., B.S., D.Hlth.Sc., Adrian Sacher, M.D., M.M.Sc., Crystal S. Denlinger, M.D., Yung-Jue Bang, M.D., Ph.D., Grace K. Dy, M.D., John C. Krauss, M.D., Yasutoshi Kuboki, M.D., James C. Kuo, M.D., Andrew L. Coveler, M.D., Keunchil Park, M.D., Ph.D., Tae Won Kim, M.D., Ph.D., Fabrice Barlesi, M.D., Ph.D., Pamela N. Munster, M.D., Suresh S. Ramalingam, M.D., Timothy F. Burns, M.D., Ph.D., Funda Meric-Bernstam, M.D., Haby Henary, M.D., Jude Ngang, Pharm.D., Gataree Ngarmchamnanrith, M.D., June Kim, Ph.D., Brett E. Houk, Ph.D., Jude Canon, Ph.D., J. Russell Lipford, Ph.D., Gregory Friberg, M.D., Piro Lito, M.D., Ph.D., Ramaswamy Govindan, M.D., and Bob T. Li, M.D., M.P.H

N. Engl. J. Med. 2020; 383:1207-1217

DOI: 10.1056/NEJMoa1917239 



No therapies for targeting KRAS mutations in cancer have been approved. The KRASp.G12C mutation occurs in 13% of non–small-cell lung cancers (NSCLCs) and in 1 to 3% of colorectal cancers and other cancers. Sotorasib is a small molecule that selectively and irreversibly targets KRASG12C.


We conducted a phase 1 trial of sotorasib in patients with advanced solid tumors harboring the KRAS p.G12C mutation. Patients received sotorasib orally once daily. The primary end point was safety. Key secondary end points were pharmacokinetics and objective response, as assessed according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1.


A total of 129 patients (59 with NSCLC, 42 with colorectal cancer, and 28 with other tumors) were included in dose escalation and expansion cohorts. Patients had received a median of 3 (range, 0 to 11) previous lines of anticancer therapies for metastatic disease. No dose-limiting toxic effects or treatment-related deaths were observed. A total of 73 patients (56.6%) had treatment-related adverse events; 15 patients (11.6%) had grade 3 or 4 events. In the subgroup with NSCLC, 32.2% (19 patients) had a confirmed objective response (complete or partial response) and 88.1% (52 patients) had disease control (objective response or stable disease); the median progression-free survival was 6.3 months (range, 0.0+ to 14.9 [with + indicating that the value includes patient data that were censored at data cutoff]). In the subgroup with colorectal cancer, 7.1% (3 patients) had a confirmed response, and 73.8% (31 patients) had disease control; the median progression-free survival was 4.0 months (range, 0.0+ to 11.1+). Responses were also observed in patients with pancreatic, endometrial, and appendiceal cancers and melanoma.


Sotorasib showed encouraging anticancer activity in patients with heavily pretreated advanced solid tumors harboring the KRAS p.G12C mutation. Grade 3 or 4 treatment-related toxic effects occurred in 11.6% of the patients. (Funded by Amgen and others; CodeBreaK100 ClinicalTrials.gov number, NCT03600883. opens in new tab.)

Site-Specific Bioconjugation through Enzyme-Catalyzed Tyrosine–Cysteine Bond Formation

Marco J. Lobba, Marco J. Lobba, Christof Fellmann*, Alan M. Marmelstein, Johnathan C. Maza, Elijah N. Kissman, Stephanie A. Robinson, Brett T. Staahl, Cole Urnes, Rachel J. Lew, Casey S. Mogilevsky, Jennifer A. Doudna*, and Matthew B. Francis*

ACS Cent. Sci. 2020, 6, 9, 1564–1571


The synthesis of protein–protein and protein–peptide conjugates is an important capability for producing vaccines, immunotherapeutics, and targeted delivery agents. Herein we show that the enzyme tyrosinase is capable of oxidizing exposed tyrosine residues into o-quinones that react rapidly with cysteine residues on target proteins. This coupling reaction occurs under mild aerobic conditions and has the rare ability to join full-size proteins in under 2 h. The utility of the approach is demonstrated for the attachment of cationic peptides to enhance the cellular delivery of CRISPR-Cas9 20-fold and for the coupling of reporter proteins to a cancer-targeting antibody fragment without loss of its cell-specific binding ability. The broad applicability of this technique provides a new building block approach for the synthesis of protein chimeras.

Bardoxolone conjugation enables targeted protein degradation of BRD4

Bingqi Tong, Mai Luo, Yi Xie, Jessica N. Spradlin, John A. Tallarico, Jeffrey M. McKenna, Markus Schirle, Thomas J. Maimone & Daniel K. Nomura

Sci Rep 10, 15543 (2020).


Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.

Monday, September 21, 2020

Discovery of Roblitinib (FGF401) as a Reversible-Covalent Inhibitor of the Kinase Activity of Fibroblast Growth Factor Receptor 4

Robin A. Fairhurst, Thomas Knoepfel, Nicole Buschmann, Catherine Leblanc, Robert Mah, Milen Todorov, Pierre Nimsgern, Sebastien Ripoche, Michel Niklaus, Nicolas Warin, Van Huy Luu, Mario Madoerin, Jasmin Wirth, Diana Graus-Porta, Andreas Weiss, Michael Kiffe, Markus Wartmann, Jacqueline Kinyamu-Akunda, Dario Sterker, Christelle Stamm, Flavia Adler, Alexandra Buhles, Heiko Schadt, Philippe Couttet, Jutta Blank, Inga Galuba, Joerg Trappe, Johannes Voshol, Nils Ostermann, Chao Zou, Joerg Berghausen, Alberto Del Rio Espinola, Wolfgang Jahnke, and Pascal Furet

Journal of Medicinal Chemistry 2020

DOI: 10.1021/acs.jmedchem.0c01019

FGF19 signaling through the FGFR4/β-klotho receptor complex has been shown to be a key driver of growth and survival in a subset of hepatocellular carcinomas making selective FGFR4 inhibition an attractive treatment opportunity. A kinome-wide sequence alignment highlighted a poorly-conserved cysteine residue within the FGFR4 ATP-binding site at position 552, two positions beyond the gate-keeper residue. Several strategies for targeting this cysteine to identify FGFR4 selective inhibitor starting points are summarised which made use of both rationale and unbiased screening approaches. The optimisation of a 2-formylquinoline amide hit series is described in which the aldehyde makes a hemithioacetal reversible-covalent interaction with cysteine 552. Key challenges addressed during the optimisation are improving the FGFR4 potency, metabolic stability and solubility leading ultimately to the highly-selective first-in-class clinical candidate roblitinib.

Thursday, September 17, 2020

An electrophilic warhead library for mapping the reactivity and accessibility of tractable cysteines in protein kinases Author links open overlay panel

László Petria, Attila Egyeda, Dávid Bajusza, Tímea Imreb, Anasztázia Hetényic, Tamás Martinekc, Péter Ábrányi-Balogha, György M. Keserűa

European Journal of Medicinal Chemistry, 2020


Targeted covalent inhibitors represent a viable strategy to block protein kinases involved in different disease pathologies. Although a number of computational protocols have been published for identifying druggable cysteines, experimental approaches are limited for mapping the reactivity and accessibility of these residues. Here, we present a ligand based approach using a toolbox of fragment-sized molecules with identical scaffold but equipped with diverse covalent warheads. Our library represents a unique opportunity for the efficient integration of warhead-optimization and target-validation into the covalent drug development process. Screening this probe kit against multiple kinases could experimentally characterize the accessibility and reactivity of the targeted cysteines and helped to identify suitable warheads for designed covalent inhibitors. The usefulness of this approach has been confirmed retrospectively on Janus kinase 3 (JAK3). Furthermore, representing a prospective validation, we identified Maternal embryonic leucine zipper kinase (MELK), as a tractable covalent target. Covalently labelling and biochemical inhibition of MELK would suggest an alternative covalent strategy for MELK inhibitor programs.

Tuesday, September 1, 2020

Using sulfuramidimidoyl fluorides that undergo sulfur(VI) fluoride exchange for inverse drug discovery

Gabriel J. Brighty, Rachel C. Botham, Suhua Li, Luke Nelson, David E. Mortenson, Gencheng Li, Christophe Morisseau, Hua Wang, Bruce D. Hammock, K. Barry Sharpless & Jeffery W. Kelly 

Nat. Chem.2020


 candidates that form covalent linkages with their target proteins have been underexplored compared with the conventional counterparts that modulate biological function by reversibly binding to proteins, in part due to concerns about off-target reactivity. However, toxicity linked to off-target reactivity can be minimized by using latent electrophiles that only become activated towards covalent bond formation on binding a specific protein. Here we study sulfuramidimidoyl fluorides, a class of weak electrophiles that undergo sulfur(VI) fluoride exchange chemistry. We show that equilibrium binding of a sulfuramidimidoyl fluoride to a protein can allow nucleophilic attack by a specific amino acid side chain, which leads to conjugate formation. We incubated small molecules, each bearing a sulfuramidimidoyl fluoride electrophile, with human cell lysate, and the protein conjugates formed were identified by affinity chromatography–mass spectrometry. This inverse drug discovery approach identified a compound that covalently binds to and irreversibly inhibits the activity of poly(ADP-ribose) polymerase 1, an important anticancer target in living cells.

Friday, August 28, 2020

Physical and Functional Analysis of the Putative Rpn13 Inhibitor RA190

Paige Dickson, Daniel Abegg, Ekaterina Vinogradova, Junichiro Takaya, Hongchan An, Scott Simanski, Benjamin F. Cravatt, Alexander Adibekian, Thomas Kodadek

Cell Chem. Biol., 2020

DOI: https://doi.org/10.1016/j.chembiol.2020.08.007

Rpn13 is one of several ubiquitin receptors in the 26S proteasome. Cys88 of Rpn13 has been proposed to be the principal target of RA190, an electrophilic small molecule with interesting anti-cancer activities. Here, we examine the claim that RA190 mediates its cytotoxic effects through engagement with Rpn13. We find no evidence that this is the case. In vitro, RA190 is has no measurable effect on any of the known interactions of Rpn13. In cellulo, we see no physical engagement of Rpn13 by RA190, either on C88 or any other residue. However, chemical proteomics experiments in two different cell lines reveal that dozens of other proteins are heavily engaged by RA190. Finally, increasing or reducing the level of Rpn13 in HeLa and melanoma cells had no effect on the sensitivity of HeLa or melanoma cells to RA190. We conclude that Rpn13 is not the physiologically relevant target of RA190.

Thursday, August 27, 2020

Selective N-Terminal Cysteine Protein Modification with Cyclopropenones [@gbernardes_chem]

Istrate, A.; Navo, C. D.; Sousa, B. B.; Marques, M. C.; Deery, M.; Bond, A.; Corzana, F.; Jiménez-Osés, G.; Bernardes, G.

ChemRxiv. 2020

Protein conjugates are valuable tools to create therapeutics, such as antibody-drug conjugates, or to study biological processes. Despite a number of protein conjugation strategies having been developed over recent years, the ability to modify one specific amino acid on a protein in the presence of other side chains with similar reactivity remains a challenge. We used the reaction between a monosubstituted cyclopropenone (CPO) probe and the 1,2-aminothiol of an N-terminal cysteine to give a stable 1,4-thiazepa-5-none linkage under mild, biocompatible conditions. The method relies on the ability of cyclopropenones to ring-open after sequential sulfhydryl and α-amine conjugation to be truly specific for N-terminal cysteine. We show that our CPO probes selectively label N-terminal cysteine containing peptides and proteins even in the presence of internal, solvent-exposed cysteines, which can be subsequently modified by using conventional cysteine modification reagents. The ability to distinguish and specifically target N-terminal cysteine residues on a protein will facilitate the construction of elaborate multi-labelled bioconjugates.

Friday, August 21, 2020

An Irreversible Inhibitor to Probe the Role of Streptococcus pyogenes Cysteine Protease SpeB in Evasion of Host Complement Defenses

Jordan L. Woehl, Seiya Kitamura, Nicholas Dillon, Zhen Han, Landon J. Edgar, Victor Nizet, and Dennis W. Wolan
ACS Chemical Biology 2020 15 (8), 2060-2069

DOI: 10.1021/acschembio.0c00191

Members of the CA class of cysteine proteases have multifaceted roles in physiology and virulence for many bacteria. Streptococcal pyrogenic exotoxin B (SpeB) is secreted by Streptococcus pyogenes and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we developed and characterized a clickable inhibitor, 2S-alkyne, based on X-ray crystallographic analysis and structure–activity relationships. Our SpeB probe showed irreversible enzyme inhibition in biochemical assays and labeled endogenous SpeB in cultured S. pyogenes supernatants. Importantly, application of 2S-alkyne decreased S. pyogenes survival in the presence of human neutrophils and supports the role of SpeB-mediated proteolysis as a mechanism to limit complement-mediated host defense. We posit that our SpeB inhibitor will be a useful chemical tool to regulate, label, and quantitate secreted cysteine proteases with SpeB-like activity in complex biological samples and a lead candidate for new therapeutics designed to sensitize S. pyogenes to host immune clearance.

Monday, August 10, 2020

The Missing Link between (Un)druggable and Degradable KRAS

Elena De Vita, Maria Maneiro, and Edward W. Tate

ACS Central Science Article ASAP

DOI: 10.1021/acscentsci.0c00920

More than four decades since their first identification, small monomeric guanosine triphosphatases (GTPases) remain among the most studied oncogenic proteins. Three GTPases in the rat sarcoma viral oncogene (RAS) family, KRAS, NRAS, and HRAS, are activated by “gain-of-function” mutations in up to 25% of all cancers, driving proliferative signaling to support tumor growth.(1) Their flat topology and lack of a clear ligand binding site (with the exception of the undruggable GTP pocket) has given rise to a wide range of indirect strategies to target RAS proteins with varying degrees of success (Figure 1A).(1) Mutant KRAS remains among the hottest targets in oncology, and concerted efforts to target oncogenic KRASG12C culminated in 2019–2020 with the discovery of allosteric covalent inhibitors that attack the nucleophilic cysteine residue, which is present uniquely in this specific KRAS mutant. The work of numerous academic and industry teams ultimately delivered four KRASG12C covalent inhibitors currently in clinical trials for cancer.(2) Among these, Mirati Therapeutics’ drug candidate MRTX849 (Figure 1A) has shown promising results and tolerability in patients affected by nonsmall cell lung cancer (NSCLC) and colorectal cancer (CRC) driven by KRASG12C mutant.(3) Nevertheless, the search for improved strategies continues, and in the current issue, the Crews laboratory has used MRTX849 as a warhead in the first cell-active PROteolysis TArgeting Chimera (PROTAC) against KRASG12C.(4) This work elegantly illustrates the differentiating opportunities and rational design challenges of covalent PROTACs, and delivers striking insights into the trade-offs of inhibition versus degradation.

Wednesday, July 29, 2020

Multiparameter kinetic analysis for covalent fragment optimization using quantitative irreversible tethering (qIT)

Craven, G..B., Affron, D..P., Kösel, T., Wong, T.L..M., Jukes, Z..H., Liu, C., Morgan, R..M.L., Armstrong, A. and Mann, D..J. 
ChemBioChem, 2020 

Covalent fragments are increasingly being implemented to develop chemical probes but the complex relationship between fragment structure and binding kinetics makes optimization uniquely challenging. We describe a new technique in covalent probe discovery that enables data driven optimization of covalent fragment potency and selectivity. This platform extends beyond the existing methods for covalent fragment hit identification by facilitating rapid multiparameter kinetic analysis of covalent structure‐activity relationships through simultaneous determination of Ki, kinact and intrinsic reactivity. We apply this approach to develop novel probes against electrophile sensitive kinases and showcase how multiparameter kinetic analysis enabled a successful fragment merging strategy.

Tuesday, July 28, 2020

Strategies for Tuning the Selectivity of Chemical Probes that Target Serine Hydrolases

Franco Faucher, John M. Bennett, Matthew Bogyo, Scott Lovell
Cell Chemical Biology, 2020
DOI: https://doi.org/10.1016/j.chembiol.2020.07.008

Serine hydrolases comprise a large family of enzymes that have diverse roles in key cellular processes, such as lipid metabolism, cell signaling, and regulation of post-translation modifications of proteins. They are also therapeutic targets for multiple human pathologies, including viral infection, diabetes, hypertension, and Alzheimer disease; however, few have well-defined substrates and biological functions. Activity-based probes (ABPs) have been used as effective tools to both profile activity and screen for selective inhibitors of serine hydrolases. One broad-spectrum ABP containing a fluorophosphonate electrophile has been used extensively to advance our understanding of diverse serine hydrolases. Due to the success of this single reagent, several robust chemistries have been developed to further diversify and tune the selectivity of ABPs used to target serine hydrolases. In this review, we highlight approaches to identify selective serine hydrolase ABPs and suggest new synthetic methodologies that could be applied to further advance probe development.

Wednesday, July 22, 2020

The Chemical Biology of Reversible Lysine Post-translational Modifications

Zhipeng A. Wang, Philip A. Cole

Cell Chem. Biol. 2020

Lysine (Lys) residues in proteins undergo a wide range of reversible post-translational modifications (PTMs), which can regulate enzyme activities, chromatin structure, protein-protein interactions, protein stability, and cellular localization. Here we discuss the “writers,” “erasers,” and “readers” of some of the common protein Lys PTMs and summarize examples of their major biological impacts. We also review chemical biology approaches, from small-molecule probes to protein chemistry technologies, that have helped to delineate Lys PTM functions and show promise for a diverse set of biomedical applications.

Monday, July 20, 2020

Site-directed ligand discovery

Daniel A. Erlanson, Andrew C. Braisted, Darren R. Raphael, Mike Randal, Robert M. Stroud, Eric M. Gordon, and James A. Wells

PNAS  2000 97 (17) 9367-9372;

We report a strategy (called “tethering”) to discover low molecular weight ligands (≈250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules (≈1,200 compounds) at concentrations typically used in drug screening (10 to 200 μM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (Ki≈1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.

An irreversible inhibitor to probe the role of Streptococcus pyogenes cysteine protease SpeB in evasion of host complement defenses

Jordan L. Woehl, Seiya Kitamura, Nicholas Dillon, Zhen Han, Landon J. Edgar, Victor Nizet, and Dennis W. Wolan

ACS Chemical Biology 2020

Members of the CA class of cysteine proteases have multifaceted roles in physiology and virulence for many bacteria. Streptococcal pyrogenic exotoxin B (SpeB) is secreted by Streptococcus pyogenes and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we develop and characterize a clickable inhibitor, 2S-alkyne, based on x-ray crystallographic analysis and structure-activity relationships. Our SpeB probe showed irreversible enzyme inhibition in biochemical assays and labeled endogenous SpeB in cultured S. pyogenes supernatants. Importantly, application of 2S-alkyne decreased S. pyogenes survival in the presence of human neutrophils and supports the role of SpeB-mediated proteolysis as a mechanism to limit complement-mediated host defense. We posit that our SpeB inhibitor will be a useful chemical tool to regulate, label, and quantitate secreted cysteine proteases with SpeB-like activity in complex biological samples, and a lead candidate for new therapeutics designed to sensitize S. pyogenes to host immune clearance.

Monday, July 13, 2020

Chemoproteomics-Enabled Ligand Screening Yields Covalent RNF114-Based Degraders that Mimic Natural Product Function

Mai Luo, Jessica N Spradlin, Scott M Brittain, Jeffery M McKenna, John A Tallarico, Markus Schirle, Thomas J Maimone, Daniel K Nomura

bioRxiv 2020.07.12.198150; 

The translation of natural product function to fully synthetic small molecules has remained an important process in medicinal chemistry for decades resulting in numerous FDA-approved medicines. 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 (TPD) -- a powerful therapeutic modality within modern day drug discovery. Using activity-based protein profiling-enabled covalent ligand screening approaches, we herein 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.

Thursday, July 9, 2020

Targeted Degradation of Oncogenic KRASG12C by VHL-Recruiting PROTACs

Michael J. Bond, Ling Chu, Dhanusha A. Nalawansha, Ke Li, and Craig M. Crews
ACS Central Science 2020

KRAS is mutated in ∼20% of human cancers and is one of the most sought-after targets for pharmacological modulation, despite having historically been considered “undruggable.” The discovery of potent covalent inhibitors of the KRASG12C mutant in recent years has sparked a new wave of interest in small molecules targeting KRAS. While these inhibitors have shown promise in the clinic, we wanted to explore PROTAC-mediated degradation as a complementary strategy to modulate mutant KRAS. Herein, we report the development of LC-2, the first PROTAC capable of degrading endogenous KRASG12C. LC-2 covalently binds KRASG12C with a MRTX849 warhead and recruits the E3 ligase VHL, inducing rapid and sustained KRASG12C degradation leading to suppression of MAPK signaling in both homozygous and heterozygous KRASG12C cell lines. LC-2 demonstrates that PROTAC-mediated degradation is a viable option for attenuating oncogenic KRAS levels and downstream signaling in cancer cells.

Monday, July 6, 2020

Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

Chunlong Ma, Michael Dominic Sacco, Brett Hurst, Julia Alma Townsend, Yanmei Hu, Tommy Szeto, Xiujun Zhang, Bart Tarbet, Michael Thomas Marty, Yu Chen & Jun Wang 

Cell Research, 2020
doi: https://doi.org/10.1038/s41422-020-0356-z

A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

Thursday, July 2, 2020

Covalent Kinase Inhibitors: An Overview

Gehringer M. (2020) Covalent Kinase Inhibitors: An Overview. In: Topics in Medicinal Chemistry. Springer, Berlin, Heidelberg


Covalent targeting has experienced a revival in the last decade, especially in the area of protein kinase inhibitor development. Generally, covalent inhibitors make use of an electrophilic moiety often termed “warhead” to react with a nucleophilic amino acid, most frequently a cysteine. High efficacy and excellent selectivity in the kinome have been achieved by addressing poorly conserved, non-catalytic cysteine residues with so-called targeted covalent inhibitors (TCIs). Despite the challenges associated with covalent modifiers, application of the TCI approach for the discovery of new treatments has been very successful with six covalent kinase inhibitors having gained approval in the last few years. A multitude of reactive chemical probes and tool compounds has further been developed. Beside cysteine, other nucleophilic amino acids including tyrosine and lysine have also been addressed with suitable electrophiles and covalent-reversible chemistry has recently complemented our toolbox for designing covalent kinase inhibitors. Covalent ligands have also been used in the framework of chemical-genetics approaches or to tackle allosteric pockets, which are often difficult to address.

This chapter aims at providing a general introduction to covalent kinase inhibitors and an overview of the current state of research highlighting major targeting strategies, developments, and advances in this field. More detailed information on certain targets and approaches can be found in dedicated chapters of this book.

Wednesday, June 24, 2020

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.

Tuesday, June 23, 2020

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

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.

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...