Opportunistic fungal infections caused by are a significant source of mortality in immunocompromised patients. They are challenging to treat because of a limited number of antifungal drugs, and novel and more effective anticryptococcal therapies are needed. Ciclopirox olamine, a -hydroxypyridone, has been in use as an approved therapeutic agent for the treatment of topical fungal infections for more than two decades. It is a fungicide, with broad activity across multiple fungal species. We synthesized 10 -hydroxypyridone derivatives to develop an initial structure-activity understanding relative to efficacy as a starting point for the development of systemic antifungals. We screened the derivatives for antifungal activity against and and counter-screened for specificity in and two species. Eight of the ten show inhibition at 1-3 μM concentration (0.17-0.42 μg per mL) in both species and in , but poor activity in the species. In , the -hydroxypyridones are fungicides, are not antagonistic with either fluconazole or amphotericin B, and are synergistic with multiple inhibitors of the mitochondrial electron transport chain. They appear to function primarily by chelating iron within the active site of iron-dependent enzymes. This preliminary structure-activity relationship points to the need for a lipophilic functional group at position six of the -hydroxypyridone ring and identifies positions four and six as sites where further substitution may be tolerated. These molecules provide a clear starting point for future optimization for efficacy and target identification.

Checkpoint blockade immunotherapy relies on the empowerment of the immune system to fight cancer. Why some patients fail to achieve durable clinical responses is not well understood, but unique individual factors such as diet, obesity, and related metabolic syndrome could play a role. The link between obesity and patient outcomes remains controversial and has been mired by conflicting reports and limited mechanistic insight. We addressed this in a C57BL/6 mouse model of diet-induced obesity using a Western diet high in both fats and sugars. Obese mice bearing B16 melanoma or MC38 carcinoma tumors had impaired immune responses to immunotherapy and a reduced capacity to control tumor progression. Unexpectedly, these compromised therapeutic outcomes were independent of body mass and, instead, were directly attributed to dietary fructose. Melanoma tumors in mice on the high-fructose diet were resistant to immunotherapy and showed increased expression of the cytoprotective enzyme heme oxygenase-1 (HO-1). This increase in HO-1 protein was recapitulated in human A375 melanoma cells exposed to fructose in culture. Induced expression of HO-1 shielded tumor cells from immune-mediated killing and was critical for resistance to checkpoint blockade immunotherapy, which could be overcome using a small-molecule inhibitor of HO-1. This study reveals dietary fructose as a driver of tumor immune evasion, identifying HO-1 expression as a mechanism of resistance and a promising molecular target for combination cancer immunotherapy..

Antitumor immunity is impaired in obese mice. Mechanistic insight into this observation remains sparse and whether it is recapitulated in patients with cancer is unclear because clinical studies have produced conflicting and controversial findings. We addressed this by analyzing data from patients with a diverse array of cancer types. We found that survival after immunotherapy was not accurately predicted by body mass index or serum leptin concentrations. However, oxidized low-density lipoprotein (ox-LDL) in serum was identified as a suppressor of T-cell function and a driver of tumor cytoprotection mediated by heme oxygenase-1 (HO-1). Analysis of a human melanoma gene expression database showed a clear association between higher (HO-1) expression and reduced progression-free survival. Our experiments using mouse models of both melanoma and breast cancer revealed HO-1 as a mechanism of resistance to anti-PD1 immunotherapy but also exposed HO-1 as a vulnerability that could be exploited therapeutically using a small-molecule inhibitor. In conclusion, our clinical data have implicated serum ox-LDL as a mediator of therapeutic resistance in patients with cancer, operating as a double-edged sword that both suppressed T-cell immunity and simultaneously induced HO-1-mediated tumor cell protection. Our studies also highlight the therapeutic potential of targeting HO-1 during immunotherapy, encouraging further translational development of this combination approach..

The Hepatitis B Virus (HBV) ribonuclease H (RNaseH) is a promising but unexploited drug target. Here, we synthesized and analyzed a library of 57 amide-containing α-hydroxytropolones (αHTs) as potential leads for HBV drug development. Fifty percent effective concentrations ranged from 0.31 to 54 μM, with selectivity indexes in cell culture of up to 80. Activity against the HBV RNaseH was confirmed in semi-quantitative enzymatic assays with recombinant HBV RNaseH. The compounds were overall poorly active against human ribonuclease H1, with 50% inhibitory concentrations of 5.1 to >1,000 μM. The αHTs had modest activity against growth of the fungal pathogen Cryptococcus neoformans, but had very limited activity against growth of the Gram – bacterium Escherichia coli and the Gram + bacterium Staphylococcus aureus, indicating substantial selectivity for HBV. A molecular model of the HBV RNaseH templated against the Ty3 RNaseH was generated. Docking the compounds to the RNaseH revealed the anticipated binding pose with the divalent cation coordinating motif on the compounds chelating the two Mn ions modeled into the active site. These studies reveal that that amide αHTs can be strong, specific HBV inhibitors that merit further assessment toward becoming anti-HBV drugs.

can cause fatal meningoencephalitis in patients with AIDS or other immunocompromising conditions. Current antifungals are suboptimal to treat this disease; therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall and is required for survival in the mammalian host and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds, we developed a robust and novel cell-based flow cytometry screening method to identify small-molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in We used flow cytometry-based counterscreens and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the G-protein-coupled receptor Gpr4 and, via the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis and could lead to potential novel therapeutics against is a fungal pathogen that kills ∼200,000 people every year. The cell wall is an essential organelle that protects fungi from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of the cryptococcal cell wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in Two of these compounds were confirmed using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan biosynthesis in and validates that this screening approach could be used to find potential antifungal agents.