Biology subjects

Donlin, M. J.

Publications and source records attributed to Donlin, M. J..

3 recordsLinked to original sources

bioRxiv2026

Mitochondrial cardiolipin sequestration of caspofungin underlies Cryptococcus neoformans inherent resistance and may contribute to cardiotoxicity

Cryptococcus exhibits inherent resistance to the echinocandin, caspofungin, which inhibits the synthesis of (1,3)-{beta}-D-glucan, a key component of the polysaccharide cell wall. The essential FKS1 gene encodes the catalytic subunit of (1,3)-{beta}-D-glucan synthase and caspofungin effectively inhibits its activity in vitro, yet the drug remains ineffective against Cryptococcus, suggesting mechanisms beyond target insensitivity. The underlying mechanisms of caspofungin resistance remain unknown, although altered regulation of cell-wall remodeling genes, plasma membrane modifications, drug efflux pathways, and melanin biosynthesis have been suggested. Using boron dipyrromethene (BD-) and fluorescein (F-) labelled caspofungin, we demonstrate that caspofungin enters the cryptococcal cell and primarily accumulates in the mitochondrial inner membrane rather than the plasma membrane. We further establish that this mitochondrial accumulation is driven by a specific interaction between caspofungin and cardiolipin, a phospholipid found in mitochondrial membranes. We demonstrate that this unforeseen localization indicates that mitochondrial sequestration diminishes the effective drug concentrations at the intended target. Notably, the interaction between caspofungin and cardiolipin also takes place in human cells, establishing a mechanistic connection to caspofungin-related cardiotoxicity. Our findings reveal a previously unrecognized mechanism of echinocandin resistance in Cryptococcus and emphasize cardiolipin as an important factor in caspofungin effectiveness.

microbiology↗

bioRxiv2022

Metal Coordinating Inhibitors of Rift Valley Fever virus Replication

Rift Valley Fever Virus (RVFV) is a veterinary and human pathogen and is an agent of bioterrorism concern. Currently, RVFV treatment is limited to supportive care, so new drugs to control RVFV infection are urgently needed. RVFV is a member of the Bunyavirales order, and replication of these viruses depends on the viral endonuclease activity of the viral L protein. Screening for RVFV replication inhibitors among compounds with divalent cation-coordinating motifs similar to known viral nuclease inhibitors identified 31 novel RVFV inhibitors with selective indexes from 5 - 402 and 50% effective concentrations of 0.54 - 56 {micro}M in Vero cells, primarily -Hydroxytropolones and N-Hydroxypyridinediones. Inhibitor activity and selective index was validated in the human cell line A549. To evaluate specificity, select compounds were tested against another Bunyavirus, La Crosse Virus (LACV). Conservation of the enzymatic activity such as the cap-snatching mechanism among the Bunyavirales implies that the -Hydroxytropolone and N-Hydroxypyridinedione chemotypes hold potential for development into treatments for related pathogens, including Hantaan Virus, Severe fever with thrombocytopenia syndrome virus, Crimean-Congo Hemorrhagic Fever Virus, and LACV. Keywords: Rift Valley Fever Virus 1, La Crosse virus 2, Cap-snatching endonuclease 3, Replication inhibitors 4, -Hydroxytropolones 5, N-Hydroxypyridinediones 6.

microbiology↗

bioRxiv2019

The aminoalkylindole, BML-190, negatively regulates chitosan synthesis via the cAMP/PKA1 pathway in Cryptococcus neoformans

Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immune-compromising 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, 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.\n\nTo 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 C. neoformans. We used flow cytometry-based counter and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits.\n\nOne 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 C. neoformans G-protein coupled receptor, Gpr4, and via the cAMP/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 C. neoformans.\n\nImportanceCryptococcus neoformans is a fungal pathogen that kills [~]200,000 people every year. The cell wall is an essential organelle that protects fungus from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of cryptococcal cells 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 C. neoformans. Two of these compounds were validated 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 in C. neoformans, and validates that this screening approach could be used to find potential antifungal agents.

microbiology↗