Irreversible covalent inhibitors can have a beneficial pharmacokinetic/pharmacodynamics profile but are still often avoided due to the risk of indiscriminate covalent reactivity and resulting adverse effects. To overcome this potential liability, we introduced an alkyne moiety as a latent electrophile into small molecule inhibitors of Cathepsin K (CatK). Alkyne-based inhibitors do not show indiscriminate thiol reactivity, but potently inhibit CatK protease activity by formation of an irreversible covalent bond with the catalytic cysteine residue, which could be confirmed by crystal structure analysis. The rate of covalent bond formation (k_inact) does not correlate with electrophilicity of the alkyne moiety, indicative of a proximity-driven reactivity. Inhibition of CatK mediated bone resorption is validated in human osteoclasts. Together, this work illustrates the potential of alkynes as latent electrophiles in small molecule inhibitors, enabling the development of irreversible covalent inhibitors with an improved safety profile.

Covalent inhibition has become more accepted in the past two decades, as illustrated by the clinical approval of several irreversible inhibitors designed to covalently modify their target. Elucidation of the structure‐activity relationship and potency of such inhibitors requires a detailed kinetic evaluation. Here, we elucidate the relationship between the experimental read‐out and the underlying inhibitor binding kinetics. Interactive kinetic simulation scripts are employed to highlight the effects of in vitro enzyme activity assay conditions and inhibitor binding mode, thereby showcasing which assumptions and corrections are crucial. Four stepwise protocols to assess the biochemical potency of (ir)reversible covalent enzyme inhibitors targeting a nucleophilic active site residue are included, with accompanying data analysis tailored to the covalent binding mode. Together, this will serve as a guide to make an educated decision regarding the most suitable method to assess covalent inhibition potency. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol I: Progress curve analysis of substrate association competitionBasic Data Analysis Protocol 1A: Two‐step irreversible covalent inhibitionBasic Data Analysis Protocol 1B: One‐step irreversible covalent inhibitionBasic Data Analysis Protocol 1C: Two‐step reversible covalent inhibitionBasic Data Analysis Protocol 1D: Two‐step irreversible covalent inhibition with substrate depletionBasic Protocol II: Incubation time–dependent potency IC₅₀(t)Basic Data Analysis Protocol 2: Two‐step irreversible covalent inhibitionBasic Protocol III: Preincubation time–dependent inhibition without dilutionBasic Data Analysis Protocol 3: Preincubation time–dependent inhibition without dilutionBasic Data Analysis Protocol 3Ai: Two‐step irreversible covalent inhibitionAlternative Data Analysis Protocol 3Aii: Two‐step irreversible covalent inhibitionBasic Data Analysis Protocol 3Bi: One‐step irreversible covalent inhibitionAlternative Data Analysis Protocol 3Bii: One‐step irreversible covalent inhibitionBasic Data Analysis Protocol 3C: Two‐step reversible covalent inhibitionBasic Protocol IV: Preincubation time–dependent inhibition with dilution/competitionBasic Data Analysis Protocol 4: Preincubation time–dependent inhibition with dilutionBasic Data Analysis Protocol 4Ai: Two‐step irreversible covalent inhibitionAlternative Data Analysis Protocol 4Aii: Two‐step irreversible covalent inhibitionBasic Data Analysis Protocol 4Bi: One‐step irreversible covalent inhibitionAlternative Data Analysis Protocol 4Bii: One‐step irreversible covalent inhibition

Terminal unactivated alkynes are nowadays considered the golden standard for cysteine-reactive warheads in activity-based probes (ABPs) targeting cysteine deubiquitinating enzymes (DUBs). In this work, we study the versatility of the thiol–alkyne addition reaction in more depth. Contrary to previous findings with UCHL3, we now show that covalent adduct formation can progress with substituents on the terminal or internal alkyne position. Strikingly, acceptance of alkyne substituents is strictly DUB-specific as this is not conserved among members of the same subfamily. Covalent adduct formation with the catalytic cysteine residue was validated by gel analysis and mass spectrometry of intact ABP-treated USP16CD^WT and catalytically inactive mutant USP16CD^C205A. Bottom-up mass spectrometric analysis of the covalent adduct with a deuterated propargyl ABP provides mechanistic understanding of the in situ thiol–alkyne reaction, identifying the alkyne rather than an allenic intermediate as the reactive species. Furthermore, kinetic analysis revealed that introduction of (bulky/electron-donating) methyl substituents on the propargyl moiety decreases the rate of covalent adduct formation, thus providing a rational explanation for the commonly lower level of observed covalent adduct compared to unmodified alkynes. Altogether, our work extends the scope of possible propargyl derivatives in cysteine targeting ABPs from unmodified terminal alkynes to internal and substituted alkynes, which we anticipate will have great value in the development of ABPs with improved selectivity profiles.

Gram-positive bacterial infections present a major clinical challenge, with methicillin- and vancomycin-resistant strains continuing to be a cause for concern. In recent years, semisynthetic vancomycin derivatives have been developed to overcome this problem as exemplified by the clinically used telavancin, which exhibits increased antibacterial potency but has also raised toxicity concerns. Thus, glycopeptide antibiotics with enhanced antibacterial activities and improved safety profiles are still necessary. We describe the development of a class of highly potent semisynthetic glycopeptide antibiotics, the guanidino lipoglycopeptides, which contain a positively charged guanidino moiety bearing a variable lipid group. These glycopeptides exhibited enhanced in vitro activity against a panel of Gram-positive bacteria including clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant strains, showed minimal toxicity toward eukaryotic cells, and had a low propensity for resistance selection. Mechanistically, guanidino lipoglycopeptides engaged with bacterial cell wall precursor lipid II with a higher binding affinity than vancomycin. Binding to both wild-type d -Ala- d -Ala lipid II and the vancomycin-resistant d -Ala- d -Lac variant was confirmed, providing insight into the enhanced activity of guanidino lipoglycopeptides against vancomycin-resistant isolates. The in vivo efficacy of guanidino lipoglycopeptide EVG7 was evaluated in a S. aureus murine thigh infection model and a 7-day sepsis survival study, both of which demonstrated superiority to vancomycin. Moreover, the minimal to mild kidney effects at supratherapeutic doses of EVG7 indicate an improved therapeutic safety profile compared with vancomycin. These findings position guanidino lipoglycopeptides as candidates for further development as antibacterial agents for the treatment of clinically relevant multidrug-resistant Gram-positive infections.

In the past two decades, drug candidates with a covalent binding mode have gained the interest of medicinal chemists, as several covalent anticancer drugs have successfully reached the clinic. As a covalent binding mode changes the relevant parameters to rank inhibitor potency and investigate structure-activity relationship (SAR), it is important to gather experimental evidence on the existence of a covalent protein–drug adduct. In this work, we review established methods and technologies for the direct detection of a covalent protein–drug adduct, illustrated with examples from (recent) drug development endeavors. These technologies include subjecting covalent drug candidates to mass spectrometric (MS) analysis, protein crystallography, or monitoring intrinsic spectroscopic properties of the ligand upon covalent adduct formation. Alternatively, chemical modification of the covalent ligand is required to detect covalent adducts by NMR analysis or activity-based protein profiling (ABPP). Some techniques are more informative than others and can also elucidate the modified amino acid residue or bond layout. We will discuss the compatibility of these techniques with reversible covalent binding modes and the possibilities to evaluate reversibility or obtain kinetic parameters. Finally, we expand upon current challenges and future applications. Overall, these analytical techniques present an integral part of covalent drug development in this exciting new era of drug discovery.

Oral vancomycin has a long history as the first-line treatment for Clostridioides difficile infection (CDI), but its use is associated with high relapse rates. Antibiotics that more selectively target C. difficile while sparing protective commensal gut bacteria, have the potential to prevent recurrent CDI (rCDI). Here, we investigate the experimental glycopeptide antibiotic, EVG7, in the context of rCDI. In vitro susceptibility assays reveal that clinical C. difficile isolates are up to 16-times more sensitive to EVG7 (MIC = 0.063–0.25 mg/L) compared to vancomycin (MIC = 0.5–2 mg/L). In a validated mouse model of rCDI in male mice, low dose oral EVG7 (0.04 mg/mL in drinking water) more effectively treats primary CDI and prevents recurrence, outperforming a 10-fold higher dose of vancomycin. Subsequent microbiome analysis and in vitro susceptibility testing reveal that EVG7 preserves Lachnospiraceae, a family of commensal bacteria associated with protection against C. difficile colonization.