Characterization of the Second-Generation Covalent Fragment Library (CovLib Gen2): Thiol Reactivity Profiling and...

Covalent Fragment-Based Drug Discovery (FBDD) has emerged as a powerful strategy for unlocking challenging pharmacological targets and engaging shallow or "cryptic" binding pockets. In this study, we present the design and characterization of the Second Generation Covalent Fragment Library (CovLib Gen2), an expanded collection of 81 structurally diverse electrophiles tailored for Covalent Fragment-Based Drug Discovery (FBDD) using an electrophile-first approach. The library spans five distinct warhead classes, including epoxides, vinyl sulfones, acrylamides, α-cyanoacrylamides, and a core set of SɴAr-reactive heteroarenes.

We comprehensively profiled the library for physicochemical properties and intrinsic thiol reactivity using high-throughput 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and high-performance liquid chromatography (HPLC)-based glutathione (GSH) reactivity assays. To demonstrate the library's utility, we performed differential scanning fluorimetry (DSF) screening against the oncogenic, thermally unstable p53-Y220C mutant and subsequent specificity testing with two control mutants.

The library exhibited a broad dynamic range of reactivities with a clear correlation between the assay methods. Additionally, we identified 12 fragments with desirable mild reactivity profiles (t_1/2GSH = 1-10 h). The DSF screen yielded 15 hits, primarily SɴAr-reactive heteroarenes and vinyl sulfones. Notably, the fragment SN054 emerged as the most potent stabilizer, inducing a maximal thermal shift of 4.5 °C. Specificity was confirmed using a cysteine-light variant (T-p53C-Y220C-CL), where SN054 retained significant stabilizing activity.

Our findings validate CovLib Gen2 as a versatile tool for ligand discovery, including electrophilic fragments covering a broad range of reactivity, and provide tractable starting points for the pharmacological rescue of p53-Y220C.