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Mass Spectrometry Methods and Mathematical PK/PD Model for Decision Tree-Guided Covalent Drug Development

ABSTRACT: Covalent drug discovery efforts are growing rapidly but have major unaddressed limitations. These include high false positive rates during hit-to-lead identification; the inherent uncoupling of covalent drug concentration and effect [i.e., uncoupling of pharmacokinetics (PK) and pharmacodynamics (PD)]; and a lack of bioanalytical and modeling methods for determining PK and PD parameters. We present a covalent drug discovery workflow that addresses these limitations. Our bioanalytical methods are based upon a mass spectrometry (MS) assay that can measure the percentage of drug-target protein conjugation (% target engagement) in biological matrices. Further we develop an intact protein PK/PD model (iPK/PD) that outputs PK parameters (absorption and distribution) as well as PD parameters (mechanism of action, protein metabolic half-lives, dose, regimen, effect) based on time-dependent target engagement data. Notably, the iPK/PD model is applicable to any measurement (e.g., bottom-up MS and other drug binding studies) that yields % of target engaged. A Decision Tree is presented to guide researchers through the covalent drug development process. Our bioanalytical methods and the Decision Tree are applied to two approved drugs (ibrutinib and sotorasib); the most common plasma off-target, human serum albumin; three protein targets (KRAS, BTK, SOD1), and to a promising SOD1-targeting ALS drug candidates.

INSTRUMENT(S): 6220 Time-of-Flight LC/MS

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Blood Cell, Blood

DISEASE(S): Amyotrophic Lateral Sclerosis Type 1

SUBMITTER: Md Amin Hossain

LAB HEAD: Jeffrey N.

PROVIDER: PXD046903 | Pride | 2025-01-23

REPOSITORIES: pride

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Project description:Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, two cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, “S-XL6,” was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24 °C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A’s in vivo half-life; and that S-XL6 crosses the blood-brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS.

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Mass Spectrometry Methods and Mathematical PK/PD Model for Decision Tree-Guided Covalent Drug Development

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