β‑Glucocerebrosidase Modulators Promote Dimerization of β‑Glucocerebrosidase and Reveal an Allosteric Binding Site

β-Glucocerebrosidase (GCase) mutations cause Gaucher’s disease and are a high risk factor in Parkinson’s disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, <b>JZ-4109</b>, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (<b>JZ-5029</b>) for <i>in vitro</i> mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher’s disease and Parkinson’s disease

Pharmacokinetic Benefits of 3,4-Dimethoxy Substitution of a Phenyl Ring and Design of Isosteres Yielding Orally Available Cathepsin K Inhibitors

Rational structure-based design has yielded highly potent inhibitors of cathepsin K (Cat K) with excellent physical properties, selectivity profiles, and pharmacokinetics. Compounds with a 3,4-(CH₃O)₂Ph motif, such as <b>31</b>, were found to have excellent metabolic stability and absorption profiles. Through metabolite identification studies, a reactive metabolite risk was identified with this motif. Subsequent structure-based design of isoteres culminated in the discovery of an optimized and balanced inhibitor (indazole, <b>38</b>)