Saturation mutagenesis of α-synuclein reveals monomer fold that modulates aggregation.

α-Synuclein (aSyn) aggregation underlies neurodegenerative synucleinopathies. aSyn seeds are proposed to replicate and propagate neuronal pathology like prions. Seeding of aSyn can be recapitulated in cellular systems of aSyn aggregation; however, the mechanism of aSyn seeding and its regulation are not well understood. We developed an mEos-based aSyn seeding assay and performed saturation mutagenesis to identify with single-residue resolution positive and negative regulators of aSyn aggregation. We not only found the core regions that govern aSyn aggregation but also identified mutants outside of the core that enhance aggregation. We identified local structure within the N terminus of aSyn that hinders the fibrillization propensity of its aggregation-prone core. Based on the screen, we designed a minimal aSyn fragment that shows a ~4-fold enhancement in seeding activity and enabled discrimination of synucleinopathies. Our study expands the basic knowledge of aSyn aggregation and advances the design of cellular systems of aSyn aggregation to diagnose synucleinopathies based on protein conformation.

Identification of a Class of WNK Isoform-Specific Inhibitors Through High-Throughput Screening.

Introduction: WNK [with no lysine (K)] kinases are serine/threonine kinases associated with familial hyperkalemic hypertension (FHHt). WNKs are therapeutic targets for blood pressure regulation, stroke and several cancers including triple negative breast cancer and glioblastoma. Here, we searched for and characterized novel WNK kinase inhibitors. Methods: We used a ~210,000-compound library in a high-throughput screen, re-acquisition and assay, commercial specificity screens and crystallography to identify WNK-isoform-selective inhibitors. Results: We identified five classes of compounds that inhibit the kinase activity of WNK1: quinoline compounds, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-derived compounds. The compounds are strongly pan-WNK selective, inhibiting all four WNK isoforms. A class of quinoline compounds was identified that further shows selectivity among the WNK isoforms, being more potent toward WNK3 than WNK1. The crystal structure of the quinoline-derived SW120619 bound to the kinase domain of WNK3 reveals active site binding, and comparison to the WNK1 structure reveals the potential origin of isoform specificity. Discussion: The newly discovered classes of compounds may be starting points for generating pharmacological tools and potential drugs treating hypertension and cancer.

Synthesis and Structural Characterization of Novel Trihalo-sulfone Inhibitors of WNK1.

With No lysine (K) [WNK] kinases are structurally unique serine/threonine protein kinases that have therapeutic potential for blood pressure regulation and cancer. A novel class of trihalo-sulfone compounds was identified by high-throughput screening. Trihalo-sulfone 1 emerged as an effective inhibitor of WNK1 with an IC50 value of 1.6 µM. Herein, we define chemical features necessary for inhibition of WNK1 using chemical synthesis and X-ray crystallography. Analogues that probed the role of specific functional groups to the inhibitory activity were synthesized. X-ray structures of trihalo-sulfone 1 and a second trihalo-sulfone 23 bound to WNK1 revealed active site binding to two of the three previously defined canonical inhibitor binding pockets as well as a novel binding site for the trihalo-sulfone moiety. The elucidation of these novel interaction sites may allow for the strategic design of even more selective and potent WNK inhibitors.