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 

doi:10.1002/cbic.202000457

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.

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.

The Chemical Biology of Reversible Lysine Post-translational Modifications

Zhipeng A. Wang, Philip A. Cole

Cell Chem. Biol. 2020

DOI: https://doi.org/10.1016/j.chembiol.2020.07.002

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.

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;

doi: https://doi.org/10.1073/pnas.97.17.9367

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

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 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.

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; 

doi: https://doi.org/10.1101/2020.07.12.198150

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.

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

DOI: 10.1021/acscentsci.0c00411

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.

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.

Covalent Kinase Inhibitors: An Overview

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

https://doi.org/10.1007/7355_2020_103

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.