Bindzus, Marc

ABSTRACT Background and Objective Previously, the novel small molecule ISFP10 has been shown to inhibit fungal phosphoglucomutase (PGM) activity in Aspergillus fumigatus and Pneumocystis spp. With 50-fold selectivity over the human PGM molecule due to the presence of a unique yet conserved cysteine residue present in a number pathogenic fungal PGMs, use of this compound may provide a novel broad-spectrum approach to treating fungal infections. Accordingly, we sought to determine the tolerability in test animals receiving this compound, as well as the potential antifungal activity of ISFP10 on cultures of the common fungal pathogens Candida albicans and Candida glabrata . Methods C57BL6 mice received once daily intraperitoneal (IP) injections of 100 mL of vehicle control (DMSO) or ISFP10 at a concentration of 10 mg/kg. Body weights were recorded daily for 7 days of treatment. On the final day, mice were weighed and euthanized. Postmortem blood collection was conducted via cardiac puncture and distributed to EDTA and lithium heparin tubes for complete blood count (CBC) and comprehensive blood chemistry panels, respectively. Liver, kidney, and lung tissue were also harvested and placed in 10% formalin for H&E staining and blinded histopathologic scoring. Lung samples were further analyzed for proinflammatory cytokines using enzyme-linked immunosorbent assays (ELISA) and quantitative PCR (qPCR). Furthermore, ISPF10 was tested for antifungal activity via 8-hour growth curve analysis in a concentration-dependent fashion against Candida albicans and Candida glabrata . Results There was no significant difference in the daily or final body weights of the mice receiving 10 mg/kg of ISFP10 compared to those of the vehicle control group. Extracellular matrix (ECM) transcripts for IL-6 and TNFα were statistically similar via qPCR. ELISA results of proinflammatory cytokines for IL-6 was not significant whereas TNFα levels in lung tissue from the ISFP10 treatment group were significantly reduced, indicating a potential anti-inflammatory effect of ISFP10 at this dosage. Overall, blood chemistry and CBC analysis revealed no overall significant differences between the two groups, except for increased neutrophil counts and decreased potassium levels in samples collected from ISFP10 treated animals compared to the vehicle control group. These laboratory abnormalities were not of clinical significance to the test animals. Blinded histopathological examination revealed no abnormalities or evidence of critical organ toxicity from all groups. Inhibition of C. albicans and C. glabrata culture growth by ISFP10 was concentration-dependent in YPD liquid media containing the ISFP10 compared to vehicle control. Conclusions Our preliminary testing of ISFP10 revealed no inherent safety or toxicology concerns within the observed parameters. These data further support significant culture suppressive activity against C. albicans and C. glabrata . Taken together, these observations of ISFP10 further indicate that targeting PGM might be a novel and viable therapeutic strategy for serious fungal infections. Key points An inhibitor specific to fungal PGM enzymes, termed ISFP10, was generally well tolerated when administered via intraperitoneal (IP) injection in mice. ISFP10 displays antifungal activity in a concentration-dependent manner against C. albicans and C. glabrata .

ABSTRACT Pneumocystis jirovecii pneumonia (PJP) remains a significant cause of morbidity and mortality during AIDS. In AIDS, the absence of CD4 immunity results in exuberant and often fatal PJP. In addition, organism clearance requires a balanced macrophage response since excessive inflammation promotes lung injury and respiratory failure. Corticosteroids given in addition to antibiotics significantly improve outcomes during PJP. However, concerns exist that corticosteroids further suppress immunity and increase co-infections. New strategies to promote killing and clearance of Pneumocystis while balancing lung inflammation are required. Prior studies have shown that innate immunity to Pneumocystis is mediated by C-type lectin receptors (CLRs) on macrophages and involves downstream CARD9 activation. CARD9 can be targeted by a novel specific small molecule inhibitor (BRD5529) that significantly reduces inflammatory signaling by macrophages. CARD9 serves as the central intracellular molecule through which Dectin-1, Dectin-2, Mincle, and other CLRs signal. Dectin-1 CLR is activated through its own intracytoplasmic domain, whereas other innate CLRs (e.g., Dectin-2 and Mincle) require interactions with a common Fc-gamma receptor (FcγR) accessory chain to mediate responses. We now observe that mice double deficient in both Dectin-1 and Fcer1g (which lack the FcγR gamma chain) exhibit markedly reduced organism clearance compared with Card9 −/− infected animals. These mice also possess deficiencies in immunoglobulin (Ig) Fc receptors directly mediating antibody responses, further implicating altered humoral responses in Pneumocystis killing. We further demonstrate in the Pneumocystis pneumonia (PCP) mouse model that BRD5529 administration successfully suppresses inflammatory cytokines. Our data support that innate immune responses through the CLR-CARD9 axis and humoral response act together to mediate effective responses resulting in optimal organism killing and generation of host inflammatory responses. Furthermore, host lung inflammation during PCP may be successfully reduced with a novel CARD9 small molecule inhibitor. IMPORTANCE Pneumocystis pneumonia (PCP) causes severe respiratory impairment in hosts with suppressed immunity, particularly those with CD4 deficiencies, such as HIV. In addition to lymphocytic immunity, both innate and humoral immunities also participate in host defense against Pneumocystis . In the current studies, we defined the relative roles of CLR receptor-mediated inflammation, as well as FcgR-related inflammation and clearance of Pneumocystis organisms. Our studies reveal important roles for CLR activities for inducing lung inflammation, which can be ameliorated with a novel small molecule inhibitor of the CARD9 adaptor protein that is necessary for CLR signaling. In contrast, FcgR has a dominant role in organism clearance, underscoring an integral role of humoral responses for the elimination of this infection.