Biology subjects

Kottom, T.

Publications and source records attributed to Kottom, T..

5 recordsLinked to original sources

bioRxiv2026

Pneumocystis phosphoglucomutase links glucose-phosphate metabolism to β-1,3-glucan synthesis and cell-wall integrity

The fungal cell wall is a dynamic, carbohydrate-rich structure whose composition is closely coordinated with cellular metabolism. Phosphoglucomutase (PGM) interconverts glucose-1-phosphate and glucose-6-phosphate, linking carbon metabolism with UDP-glucose-dependent cell-wall biosynthesis. We previously identified functional Pgm2 homologs from Pneumocystis jirovecii and Pneumocystis murina. Here, using a Saccharomyces cerevisiae pgm2{Delta} complementation model, we examined the relationship between Pgm2 activity, {beta}-1,3-glucan, and cell-wall integrity. Pgm2 deficiency reduced {beta}-1,3-glucan content and altered zymolyase sensitivity without major changes in cell-wall thickness. Bulk RNA sequencing revealed that the pgm2{Delta} mutant exhibited coordinated metabolic and cell-wall transcriptional responses, including increased SLT2/MPK1, RLM1, FKS2/GSC2, GFA1, CHS1, CRH1, SED1, and CWP1 expression and decreased FKS1, GAS1, GAS3, and CHS2 expression. Expression of either Pjpgm2 or Pmpgm2 broadly shifted these responses toward wild-type levels. Selected transcriptional changes were further evaluated by qRT-PCR, which supported the major stress and cell-wall responses identified by RNA sequencing. FKS2/GSC2 showed the same directional pattern, although differences were not statistically significant by qRT-PCR. Despite this compensatory response, Pgm2-deficient cells were markedly more susceptible to sublethal caspofungin, whereas either Pneumocystis Pgm2 homolog restored tolerance toward wild-type levels. These findings link Pgm2-dependent glucose-phosphate metabolism with {beta}-1,3-glucan levels and cell-wall function. Given the importance of {beta}-1,3-glucan to the Pneumocystis cyst wall and its limited cell-wall biosynthetic pathways, these findings further support Pgm2 as an important component of Pneumocystis cell-wall biology and a potential antifungal target.

microbiology↗

bioRxiv2025

The Maximum-Tolerated Dose and Pharmacokinetics of ISFP10 a Novel Inhibitor of Fungal Phosphoglucomutase (PGM)

BackgroundThe newly identified small molecule ISFP10 has demonstrated the ability to inhibit fungal phosphoglucomutases (PGM), leading to decreased fungal growth and survival. Exhibiting 50 times greater selectivity for fungal PGM compared to the human homolog, ISFP10 shows promise as a broad-spectrum antifungal and a candidate for preliminary testing in rodent models of fungal infections, including Pneumocystis pneumonia (PCP). In preparation for targeted and relevant administration of the drug in fungal mouse models of infection, the current study describes the maximum-tolerated-dose (MTD) and pharmacokinetics (PK) of ISFP10 in mice. MethodsFor the MTD study, 24 C57BL/6 mice were randomly distributed into 6 groups (2M/2F per group): Placebo (vehicle; 10% DMSO with 90% of 0.5% methylcellulose in 0.9% NaCl) at various doses (12.5, 25, 50, 100, 200 mg/kg), were administered twice daily by intraperitoneally (IP) for 7 consecutive days. For the PK study, 108 mice were administered either vehicle or ISFP10 in single-dose intraperitoneal (IP) injections twice daily for 7 consecutive days at concentrations of 0, 12.5, 25, 50, and 100 mg/kg. Pharmacokinetics were assessed in plasma, and epithelial lining fluid (ELF). Analysis of this test compound was performed using LC-MS/MS for PK evaluation on blood samples drawn from mice at multiple time points. ResultsIP administered, ISFP10 did not produce significant changes in body weight, food consumption or adverse events in the MTD up to 100 mg/kg dosing. The plasma PK study demonstrated Tmax values ranging from 2 to 8 h. Non-compartmental analysis (NCA) yielded elimination half-life values, t1/2, of 4.39 and 5.31 h for the 12.5 and 50 mg/kg dose groups. Concentration dependent peak-blood concentration (Cmax) at the dosing and length tested ranged from 3.450-5.140 ug/ml. ConclusionsIn conclusion, ISFP10, administered intraperitoneally to mice twice daily at doses up to 100 mg/kg, exhibited no inherent safety concerns based on the analyzed parameters. Pharmacokinetic analysis revealed slow absorption and distribution kinetics, with plasma Tmax values ranging from 2 to 8 hours and elimination half-lives of approximately 4-5 hours at lower doses. These data support broader in vivo testing of the inhibitor as potential novel antifungal in fungal diseases including PCP.

pharmacology and toxicology↗

bioRxiv2025

Preclinical and Toxicology Assessment of ISFP10, an Inhibitor of Fungal Phosphoglucomutase (PGM)

Background and ObjectivePreviously, 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. MethodsC57BL6 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. ResultsThere 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 TNF were statistically similar via qPCR. ELISA results of proinflammatory cytokines for IL-6 was not significant whereas TNF 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. ConclusionsOur 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 pointsAn 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.

pharmacology and toxicology↗

bioRxiv2022

Blood Chemistry Analysis in an Active Pneumocystis Pneumonia (PCP) Model Treated with Selective CARD9 Inhibitor BRD5529

BackgroundPneumocystis pneumonia (PCP) in AIDS and other immunosuppressive states that result from absence of CD4 lymphocytic immunity, continues to be a significant cause of morbidity and mortality. We and others have shown the importance of CARD9 in PCP and other fungal infections, respectively. BRD5529 has been shown to be an effective in vitro and in vivo (18 hour) inhibitor of Pneumocystis {beta}-glucans induced proinflammatory response. These recent results, along with recent general safety and toxicology assessments suggests the application of BRD5529 in an active PCP mouse model of infection to assess initial blood toxicology parameters. MethodsTo assess preliminary blood toxicology, mice were injected intraperitoneally (IP) daily either with vehicle or BRD5529 at 1.0 mg/kg for one week starting at the 6th week of the PCP mouse model. After one week, mice were sacrificed, and blood collection postmortem was performed for blood chemistry analysis. ResultsAnalysis of blood chemistry showed a significant reduction in blood urea nitrogen (BUN) in the BRD5529 IP treated PCP mice cohort compared to the vehicle control group. All other blood chemistry parameters were not significantly different between the two groups. ConclusionsBRD5529 in this preliminary PCP treatment model displayed only significant changes in BUN levels in the BRD5529 treatment group versus the vehicle control group. All other blood chemistry parameters were statistically similar between the two groups.

immunology↗

bioRxiv2021

Preclinical and Toxicology Studies of BRD5529, a Selective Inhibitor of CARD9

BackgroundExuberant inflammation during Pneumocystis pneumonia leads to lung injury. CARD9 is a central mediator of inflammatory signaling mediated by C-type lectin receptors. CARD9 inhibitor BRD5529 has been shown to be an effective in vitro inhibitor of Pneumocystis {beta}-glucan-induced proinflammatory signaling and downstream TNF-alpha production, suggesting its viability as a candidate for preliminary drug testing as an anti- inflammatory agent in the rodent Pneumocystis pneumonia model (PCP). MethodsTo assess for potential toxicity, mice were injected intraperitoneally (IP) daily either with vehicle or BRD5529 at 0.1 mg/kg or 1.0 mg/kg for two weeks. Mouse weights were taken daily. At day 14, mice were euthanized, weighed, and analyzed by flexiVent for lung stiffness. Lungs, liver, and kidney were then harvested for H&E staining and pathology scoring. Lung samples were further analyzed for proinflammatory cytokines via ELISA and extracellular matrix generation via quantitative PCR (q-PCR). Blood collection postmortem was performed for blood chemistry analysis. ResultsBRD5529 at both doses of IP administration resulted in no significant changes in daily or final weight gain. Analysis of lung stiffness by flexiVent showed no significant differences between the control or treated groups. Furthermore, ELISA results for proinflammatory IL-1 Beta, IL-6, and TNF-alpha showed no major differences in the respective groups. qPCR analysis of extracellular matrix transcripts collagen type I, alpha 1 (Col1a1) and fibronectin (Fn) were statically similar as well in the treated and control groups. Examination and pathology scoring of H&E slides from lung, liver, and kidney from the each of the mice in all groups and subsequent pathology scoring showed no significant change. Blood chemistry analysis revealed similar, non-significant patterns. ConclusionsBRD5529 in our initial general safety and toxicology assessments displayed no inherent safety concerns in the analyzed parameters. These data support broader in vivo testing of the inhibitor as a timed adjunct therapy to the deleterious proinflammatory host immune response often associated with anti-Pneumocystis therapy.

pharmacology and toxicology↗