AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target.Keywords: acetyl CoA synthetase; antifungal; C. albicans; chemical genetics

Cryptococcus neoformans is one of the most important human fungal pathogens; however, no new therapies have been developed in over 50 years. Fungicidal activity is crucially important for an effective anticryptococal agent and, therefore, we screened 361,675 molecules against C. neoformans using an adenylate kinase release assay that specifically detects fungicidal activity. A set of secondary assays narrowed the set of hits to molecules that interfere with fungal cell wall integrity and identified three benzothioureas with low in vitro mammalian toxicity and good in vitro anticryptococcal (minimum inhibitory concentration = 4 μg/mL). This scaffold inhibits signaling through the cell wall integrity MAP kinase cascade. Structure–activity studies indicate that the thiocarbonyl moiety is crucial for activity. Genetic and biochemical data suggest that benzothioureas inhibit signaling upstream of the kinase cascade. Thus, the benzothioureas appear to be a promising new scaffold for further exploration in the search for new anticryptococcal agents.Keywords: antifungal; cell wall integrity pathway; Cryptococcus neoformans; high-throughput screening; mitogen-activated protein kinase; yeast cell wall

Although protein kinases have recently emerged as important drug targets, the anti-infective potential of protein kinase inhibitors has not been developed extensively. We identified the mammalian PDK1 inhibitor KP-372-1 as a potent antifungal molecule with activity against yeast and fungal biofilms using a screening strategy for protein kinase inhibitors that block the cell wall stress response in yeast. Genetic and biochemical studies indicate that KP-372-1 inhibits fungal PDK1 orthologs (Pkh kinases) as part of its mode of action and support a role for Pkh kinases in eisosome assembly. Two other structurally distinct molecules that inhibit PDK1, OSU-03012 and UCN-01, also have antifungal activity. Taken together, these data indicate that fungal PDK1 orthologs are promising targets for new antifungal drug development.

•Review of the current state of anti-cryptococcal therapy.•Proposal for ideal anti-cryptococcal therapy.•Review of recent progress toward new therapies.Cryptococcosis is one of the most important fungal infections of humans. It primarily, but not exclusively, afflicts people with compromised immune function. Cryptococcosis is most commonly caused by Cryptococcus neoformans var. grubii with C. neoformans var. neoformans and C. gatti also contributing to the disease. Cryptococcosis is primarily manifested as meningoencephalitis although pneumonia occurs frequently as well. Globally, the burden of disease is highest among those living with HIV/AIDS and is one of the most common causes of death in this patient population. Cryptococcal meningitisis almost invariably fatal if untreated. The current gold standard therapy is amphotericin B combined with 5-flucytosine. Unfortunately, this therapy has significant toxicity and is not widely available in resource-limited regions. Fluconazole, which is associated with poorer outcomes, is frequently as an alternative. Here, I present the characteristics of an ideal anti-cryptococcal agent and review recent progress toward identifying both novel and repurposed drugs as potential new therapies.