Timothy M. Willson’s research while affiliated with University of North Carolina at Chapel Hill and other places

Text One in eight women will be diagnosed with breast cancer in their lifetimes. Estrogen receptor alpha (ER) drives breast cancer pathology and is expressed in approximately seventy percent of tumors. Hormone therapies are used to treat and prevent the metastasis of ER+ breast cancers and these patients have favorable 5-year survivals. However, most breast cancer patient deaths are ER+. While the next generation of antiestrogens have shown promise in the advanced setting, more work needs to be done to fully address these mortalities. ER is a ligand-dependent master transcriptional regulator, whereby ligand-specific ER conformational ensembles redirect multiple programs to produce oncogenic or therapeutic endpoints. To better understand ER structure-activity relationships, we have developed new antiestrogenic small molecules based on a thienopyrimidine scaffold. Comprehensive structural, ER activity, and cancer endpoint profiling shows that these new molecules adopt a unique ligand binding pose, which perturbs new structural elements within the orthosteric hormone binding pocket. These molecules downregulate ER target genes and halt the proliferation of ER+ breast cancer cells. Date of Presentation October 17, 2024

Alphaviruses are mosquito-borne RNA viruses that pose a significant public health threat, with no FDA-approved antiviral therapeutics available. The non-structural protein 2 helicase (nsP2hel) is an enzyme involved in unwinding dsRNA essential for alphavirus replication. This study reports the discovery and optimization of first-in-class oxaspiropiperidine inhibitors targeting nsP2hel. Structure-activity relationship (SAR) studies identified potent cyclic sulfonamide analogs with nanomolar antiviral activity against chikungunya virus (CHIKV). Biochemical analyses of nsP2hel ATPase and RNA unwindase activities showed these compounds act by a non-competitive mode suggesting that they are allosteric inhibitors. Viral resistance mutations mapped to nsP2hel and a fluorine-labeled analog exhibited direct binding to the protein by ¹⁹ F NMR. The lead inhibitor, 2o , demonstrated broad-spectrum antialphaviral activity, reducing titers of CHIKV, Mayaro virus (MAYV), and Venezuelan equine encephalitis virus (VEEV). These findings support nsP2hel as a viable target for development of broad-spectrum direct-acting antialphaviral drugs. Graphical Abstract

Candida albicans is the most common cause of life-threatening fungal infection in the developed world but remains a therapeutic challenge. Protein kinases have been rewarding drug targets across diverse indications but remain untapped for antifungal development. Previously, screening kinase inhibitors against C. albicans revealed a 2,3-aryl-pyrazolopyridine, GW461484A (GW), which targets casein kinase 1 (CK1) family member Yck2. Here, we report optimization of GW via two complementary approaches, synthesis of bioisosteres possessing an imidazo[1,2-a]pyridine core, and R-group substitution of GW’s pyrazolo[1,5-a]pyridine core. Characterization of compounds reveals two 6-cyano derivatives with improved pharmacological properties that retain whole-cell bioactivity and selectivity for fungal Yck2 compared to human CK1α. Efficacy studies in mice indicate both analogs possess single-agent activity against C. albicans resistant to first-line echinocandin antifungals and potentiate non-curative echinocandin treatment. Results validate Yck2 as an antifungal target and encourage further development of inhibitors acting by this previously unexploited mode of action.

Chikungunya is a mosquito-borne viral disease that causes fever and severe joint pain for which there is no direct acting drug treatments. Vinyl sulfone SGC-NSP2PRO-1 (3) was identified as a potent inhibitor of the nsP2 cysteine protease (nsP2pro) that reduced viral titer against infectious isolates of Chikungunya and other alphaviruses. The covalent warhead in 3 captured the active site C478 and inactivated nsP2pro with a kinact/Ki ratio of 5950 M–1 s–1. The vinyl sulfone 3 was inactive across a panel of 23 other cysteine proteases and demonstrated remarkable proteome-wide selectivity by two chemoproteomic methods. A negative control analog SGC-NSP2PRO-1N (4) retained the isoxazole core and covalent warhead but demonstrated > 100-fold decrease in enzyme inhibition. Both 3 and 4 were stable across a wide range of pH in solution and upon prolonged storage as solids. Vinyl sulfone 3 and its negative control 4 will find utility as high-quality chemical probes to study the role of the nsP2pro in cellular studies of alphaviral replication and virulence.

Helicases are essential motor enzymes that couple nucleoside-triphosphate hydrolysis with DNA or RNA strand unwinding. Helicases are integral to replication, transcription, splicing, and translation of the genome, play crucial roles in the proliferation of cancer cells and propagation of viral pathogens, and are implicated in neurodegenerative diseases. Despite their therapeutic potential, drug discovery efforts targeting helicases face significant challenges due to their dynamic enzymatic cycles, the transient nature of their conformational states, and the conservation of their active sites. Analysis of cocrystal structures of inhibitor–helicase complexes revealed four distinct mechanisms of inhibition: allosteric, ATP-competitive, RNA-competitive, and interfacial inhibitors. While these static X-ray structures reveal potential binding pockets that may support the development of selective drugs, the application of advanced techniques such as cryo-EM, single-molecule analysis, and computational modeling will be essential for understanding helicase dynamics and designing effective inhibitors.

RA-0002034 ( 1 ) is a potent covalent inhibitor targeting the alphavirus nsP2 cysteine protease. The species-dependent pharmacokinetics and metabolism of 1 were investigated to evaluate its therapeutic potential. Pharmacokinetic profiling revealed rapid clearance in mice, predominantly mediated by glutathione S -transferase (GST)-catalyzed conjugation. This metabolic liability contrasted with slower clearance observed in human hepatocytes and preclinical species such as rats, dogs, and monkeys. Cross-species studies confirmed the dominance of GST-driven metabolism in mice, whereas oxidative pathways were more pronounced in dogs. Despite rapid systemic clearance, 1 achieved antiviral efficacy in mice, reducing CHIKV viral loads in multiple tissues. Initial estimations of human hepatic clearance and half-life extrapolated from animal data indicate that b.i.d. dosing of 1 will be possible to maintain concentrations sufficient for antiviral activity in humans. These cross-species pharmacokinetic and metabolism studies support the continued evaluation of 1 as a promising anti-alphaviral therapeutic. Graphical Abstract

Candida albicans is a growing health concern as the leading causal agent of systemic candidiasis, a life-threatening fungal infection with a mortality rate of ∼40% despite best available therapy. Yck2, a fungal casein kinase 1 (CK1) family member, is the cellular target of inhibitors YK-I-02 (YK) and MN-I-157 (MN). Here, multiplexed inhibitor beads paired with mass spectrometry (MIB/MS) employing ATP-competitive kinase inhibitors were used to define the selectivity of these Yck2 inhibitors across the global C. albicans proteome. The MIB matrix captured 89% of the known and predicted C. albicans protein kinases present in cell lysate. In MIB/MS competition assays, YK and MN demonstrated exquisite selectivity across the C. albicans fungal kinome with target engagement of only three CK1 homologs (Yck2, Yck22, and Hrr25) and a homolog of human p38α (Hog1). Additional chemoproteomics using a custom MN-kinobead identified only one additional C. albicans protein, confirming its remarkable fungal proteome-wide selectivity. To identify new Yck2 inhibitors with selectivity over Hog1, thirteen human CK1 kinase inhibitors were profiled for fungal kinase-binding activity using MIB/MS competition assays and in-cell NanoBRET target engagement assays. A new chemotype of family-selective Yck2 inhibitors with antifungal activity was identified. Together, these findings expand the application of MIB/MS proteomic profiling for non-human kinomes and demonstrate its utility in the discovery and development of selective inhibitors of fungal kinases with potential antimicrobial activity. GRAPHICAL ABSTRACT

Non-structural protein 2 (nsP2), which plays an essential role in replication of CHIKV, contains a protease, helicase, and methyltransferase-like domain. We executed a simple a screen using malachite green to detect compounds that decreased ATP hydrolysis and tested a library of diverse compounds to find inhibitors of CHIKV nsP2 helicase.

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