Summary Tumor necrosis factor α (TNFα) has both positive and negative roles in human disease. In certain cancers, TNFα is infused locally to promote tumor regression, but dose-limiting inflammatory effects limit broader utility. In autoimmune disease, anti-TNFα antibodies control inflammation in most patients, but these benefits are offset during chronic treatment. TAK1 acts as a key mediator between survival and cell death in TNFα-mediated signaling. Here, we describe Takinib, a potent and selective TAK1 inhibitor that induces apoptosis following TNFα stimulation in cell models of rheumatoid arthritis and metastatic breast cancer. We demonstrate that Takinib is an inhibitor of autophosphorylated TAK1 that binds within the ATP binding pocket, yet is non-competitive, and inhibits by slowing down the rate-limiting step of TAK1 activation. Overall, Takinib is an attractive starting point for the development of inhibitors that sensitize cells to TNFα-induced cell death, with general implications for cancer and autoimmune disease treatment.

Malaria remains a global health burden partly due to parasite resistance to first-line therapeutics. The molecular chaperone heat shock protein 90 (Hsp90) has emerged as an essential protein for blood-stage parasites, but details about its function during malaria's elusive liver stage are unclear. We used target-based screens to identify compounds that bind to and human Hsp90, which revealed insights into chemotypes with species-selective binding. Using cell-based malaria assays, we demonstrate that all identified Hsp90-binding compounds are liver- and blood-stage inhibitors. Additionally, the Hsp90 inhibitor SNX-0723 in combination with the phosphatidylinositol 3-kinase inhibitor PIK-75 synergistically reduces the liver-stage parasite load. Time course inhibition studies with the Hsp90 inhibitors and expression analysis support a role for Hsp90 in late-liver-stage parasite development. Our results suggest that Hsp90 is essential to liver- and blood-stage parasite infections and highlight an attractive route for development of species-selective Hsp90 inhibitors that may act synergistically in combination therapies to prevent and treat malaria.

Highlights d PfPK9-binding compounds were discovered d PfPK9-binding compounds inhibit K63-linked ubiquitination in Plasmodium d Takinib and PfPK9-selective HS220 inhibit liver-stage Plasmodium d Takinib and PfPK9-selective HS220 increase liver-stage parasite size

An in silico screen of 350,000 commercially available compounds was conducted with an unbiased approach to identify potential malaria inhibitors that bind to the Plasmodium falciparum protein kinase 5 (PfPK5) ATP-binding site. PfPK5 is a cyclin-dependent kinase-like protein with high sequence similarity to human cyclin-dependent kinase 2 (HsCDK2), but its precise role in cell cycle regulation remains unclear. After two-dimensional fingerprinting of the top scoring compounds, 182 candidates were prioritized for biochemical testing based on their structural diversity. Evaluation of these compounds demonstrated that 135 bound to PfPK5 to a similar degree or better than known PfPK5 inhibitors, confirming that the library was enriched with PfPK5-binding compounds. A previously reported triazolodiamine HsCDK2 inhibitor and the screening hit 4-methylumbelliferone were each selected for an analog study. The results of this study highlight the difficult balance between optimization of PfPK5 affinity and binding selectivity for PfPK5 over its closest human homolog HsCDK2. Our approach enabled the discovery of several new PfPK5-binding compounds from a modest screening campaign and revealed the first scaffold to have improved PfPK5/HsCDK2 selectivity. These steps are critical for the development of PfPK5-targeting probes for functional studies and antimalarials with reduced risks of host toxicity.

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