Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries
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Bibliographic record
Abstract
Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and mu-calpain rapidly (k(2)/K(i) = 131,000 and 16,500 m(-1) s(-1), respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
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 it