A Comprehensive Guide for Assessing Covalent Inhibition in Enzymatic Assays Illustrated with Kinetic Simulations
Bibliographic record
Abstract
Abstract 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 competition Basic Data Analysis Protocol 1A: Two‐step irreversible covalent inhibition Basic Data Analysis Protocol 1B: One‐step irreversible covalent inhibition Basic Data Analysis Protocol 1C: Two‐step reversible covalent inhibition Basic Data Analysis Protocol 1D: Two‐step irreversible covalent inhibition with substrate depletion Basic Protocol II: Incubation time–dependent potency IC50(t) Basic Data Analysis Protocol 2: Two‐step irreversible covalent inhibition Basic Protocol III: Preincubation time–dependent inhibition without dilution Basic Data Analysis Protocol 3: Preincubation time–dependent inhibition without dilution Basic Data Analysis Protocol 3Ai: Two‐step irreversible covalent inhibition Alternative Data Analysis Protocol 3Aii: Two‐step irreversible covalent inhibition Basic Data Analysis Protocol 3Bi: One‐step irreversible covalent inhibition Alternative Data Analysis Protocol 3Bii: One‐step irreversible covalent inhibition Basic Data Analysis Protocol 3C: Two‐step reversible covalent inhibition Basic Protocol IV: Preincubation time–dependent inhibition with dilution/competition Basic Data Analysis Protocol 4: Preincubation time–dependent inhibition with dilution Basic Data Analysis Protocol 4Ai: Two‐step irreversible covalent inhibition Alternative Data Analysis Protocol 4Aii: Two‐step irreversible covalent inhibition Basic Data Analysis Protocol 4Bi: One‐step irreversible covalent inhibition Alternative Data Analysis Protocol 4Bii: One‐step irreversible covalent inhibition
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.004 | 0.008 |
| Meta-epidemiology (narrow) | 0.003 | 0.002 |
| Meta-epidemiology (broad) | 0.002 | 0.002 |
| Bibliometrics | 0.002 | 0.002 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.002 | 0.002 |
| Open science | 0.004 | 0.002 |
| Research integrity | 0.002 | 0.005 |
| Insufficient payload (model declined to judge) | 0.067 | 0.038 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".