Screening of ultra-low-molecular weight ligands (MiniFrags) successfully identified viable chemical starting points for a variety of drug targets. Here we report the electrophilic analogues of MiniFrags that allow the mapping of potential binding sites for covalent inhibitors by biochemical screening and mass spectrometry. Small electrophilic heterocycles and their N-quaternized analogues were first characterized in the glutathione assay to analyze their electrophilic reactivity. Next, the library was used for systematic mapping of potential covalent binding sites available in human histone deacetylase 8 (HDAC8). The covalent labeling of HDAC8 cysteines has been proven by tandem mass spectrometry measurements, and the observations were explained by mutating HDAC8 cysteines. As a result, screening of electrophilic MiniFrags identified three potential binding sites suitable for the development of allosteric covalent HDAC8 inhibitors. One of the hit fragments was merged with a known HDAC8 inhibitor fragment using different linkers, and the linker length was optimized to result in a lead-like covalent inhibitor.

The STAT family of transcription factors are important signaling hubs, with several of them, particularly STAT3, being emerging oncotargets already investigated in clinical trials. The modular structure of STAT3 nominates several of its protein domains as possible drug targets, but their exploitation with potential small-molecule inhibitors has been unevenly distributed so far, with past efforts highly favoring the conserved SH2 domain. Here, we have targeted a sparsely studied binding site at the junction of the coiled-coil and DNA-binding domains and discovered several new lead-like covalent inhibitors by virtual screening. The most favorable hit compound has been explored via structure-guided hit expansion and optimized into a low micromolar inhibitor. This compound can serve as a chemical biology tool against this site in future exploratory studies or form the basis of a more advanced stage of lead optimization.

Metabolites resulting from the bacterial fermentation of dietary fibers, such as short-chain fatty acids, especially butyrate, play important roles in maintaining gut health and regulating various biological effects in the skin. However, butyrate is underutilized due to its unpleasant odor. To circumvent this organoleptic unfavorable property, phenylalanine butyramide (PBA), a butyrate precursor, has been synthesized and is currently available on the market. We evaluated the inhibition of mushroom tyrosinase by butyrate and PBA through in vitro assays, finding IC₅₀ values of 34.7 mM and 120.3 mM, respectively. Docking calculations using a homology model of human tyrosinase identified a putative binding mode of PBA into the catalytic site. The anti-aging and anti-spot efficacy of topical PBA was evaluated in a randomized, double-blind, parallel-arm, placebo-controlled clinical trial involving 43 women affected by photo-damage. The results of this study showed that PBA significantly improved skin conditions compared to the placebo and was well tolerated. Specifically, PBA demonstrated strong skin depigmenting activity on both UV and brown spots (UV: −12.7% and −9.9%, Bs: −20.8% and −17.7% after 15 and 30 days, respectively, p < 0.001). Moreover, PBA brightened and lightened the skin (ITA°: +12% and 13% after 15 and 30 days, respectively, p < 0.001). Finally, PBA significantly improved skin elasticity (Ua/Uf: +12.4% and +32.3% after 15 and 30 days, respectively, p < 0.001) and firmness (Uf: −3.2% and −14.9% after 15 and 30 days, respectively, p < 0.01).

Covalent mechanism of action is a powerful way to modulate challenging drug targets. Here, we present a streamlined workflow that combines covalent fragment screening (electrophile first approach) and ligand-first strategy to identify covalent inhibitors targeting lysine residues in the allosteric latch site of SHP2 phosphatase. Supported by complementary computational and experimental analyses, this strategy enabled us to identify the first potent cell active allosteric covalent inhibitors acting at this site. Demonstrating the covalent tractability of the latch site opens further avenues for future optimization and therapeutic exploration of high-value allosteric SHP2 inhibitors.