Identification of fungal natural products with potent inhibition in Toxoplasma gondii.

In an effort to identify novel compounds with potent inhibition against Toxoplasma gondii, a phenotypic screen was performed utilizing a library of 683 pure compounds derived primarily from terrestrial and marine fungi. An initial screen with a fixed concentration of 5 µM yielded 91 hits with inhibition comparable to an equal concentration of artemisinin. These compounds were then triaged based on known biological and chemical concerns and liabilities. From these, 49 prioritized compounds were tested in a dose response format with T. gondii and human foreskin fibroblasts (HFFs) for cytotoxicity. Ten compounds were identified with an IC50 less than 150 nM and a selectivity index (SI) greater than 100. An additional eight compounds demonstrated submicromolar IC50 and SI values equal to or greater than 35. While the majority of these scaffolds have been previously implicated against apicomplexan parasites, their activities in T. gondii were largely unknown. Herein, we report the T. gondii activity of these compounds with chemotypes including xanthoquinodins, peptaibols, heptelidic acid analogs, and fumagillin analogs, with multiple compounds demonstrating exceptional potency in T. gondii and limited toxicity to HFFs at the highest concentrations tested. IMPORTANCE: Current therapeutics for treating toxoplasmosis remain insufficient, demonstrating high cytotoxicity, poor bioavailability, limited efficacy, and drug resistance. Additional research is needed to develop novel compounds with high efficacy and low cytotoxicity. The success of artemisinin and other natural products in treating malaria highlights the potential of natural products as anti-protozoan therapeutics. However, the exploration of natural products in T. gondii drug discovery has been less comprehensive, leaving untapped potential. By leveraging the resources available for the malaria drug discovery campaign, we conducted a phenotypic screen utilizing a set of natural products previously screened against Plasmodium falciparum. Our study revealed 18 compounds with high potency and low cytotoxicity in T. gondii, including four novel scaffolds with no previously reported activity in T. gondii. These new scaffolds may serve as starting points for the development of toxoplasmosis therapeutics but could also serve as tool compounds for target identification studies using chemogenomic approach.

Antiplasmodial peptaibols act through membrane directed mechanisms.

Our previous study identified 52 antiplasmodial peptaibols isolated from fungi. To understand their antiplasmodial mechanism of action, we conducted phenotypic assays, assessed the in vitro evolution of resistance, and performed a transcriptome analysis of the most potent peptaibol, HZ NPDG-I. HZ NPDG-I and 2 additional peptaibols were compared for their killing action and stage dependency, each showing a loss of digestive vacuole (DV) content via ultrastructural analysis. HZ NPDG-I demonstrated a stepwise increase in DV pH, impaired DV membrane permeability, and the ability to form ion channels upon reconstitution in planar membranes. This compound showed no signs of cross resistance to targets of current clinical candidates, and 3 independent lines evolved to resist HZ NPDG-I acquired nonsynonymous changes in the P. falciparum multidrug resistance transporter, pfmdr1. Conditional knockdown of PfMDR1 showed varying effects to other peptaibol analogs, suggesting differing sensitivity.

Appraisal of Fungus-Derived Xanthoquinodins as Broad-Spectrum Anti-Infectives Targeting Phylogenetically Diverse Human Pathogens.

Xanthoquinodins make up a distinctive class of xanthone-anthraquinone heterodimers reported as secondary metabolites from several fungal species. Through a collaborative multi-institutional screening program, a fungal extract prepared from a Trichocladium sp. was identified that exhibited strong inhibitory effects against several human pathogens (Mycoplasma genitalium, Plasmodium falciparum, Cryptosporidium parvum, and Trichomonas vaginalis). This report focuses on one of the unique samples that exhibited a desirable combination of biological effects: namely, it inhibited all four test pathogens and demonstrated low levels of toxicity toward HepG2 (human liver) cells. Fractionation and purification of the bioactive components and their congeners led to the identification of six new compounds [xanthoquinodins NPDG A1-A5 (1-5) and B1 (6)] as well as several previously reported natural products (7-14). The chemical structures of 1-14 were determined based on interpretation of their 1D and 2D NMR, HRESIMS, and electronic circular dichroism (ECD) data. Biological testing of the purified metabolites revealed that they possessed widely varying levels of inhibitory activity against a panel of human pathogens. Xanthoquinodins A1 (7) and A2 (8) exhibited the most promising broad-spectrum inhibitory effects against M. genitalium (EC50 values: 0.13 and 0.12 µM, respectively), C. parvum (EC50 values: 5.2 and 3.5 µM, respectively), T. vaginalis (EC50 values: 3.9 and 6.8 µM, respectively), and P. falciparum (EC50 values: 0.29 and 0.50 µM, respectively) with no cytotoxicity detected at the highest concentration tested (HepG2 EC50 > 25 µM).

Human Polo-like Kinase Inhibitors as Antiplasmodials.

Protein kinases have proven to be a very productive class of therapeutic targets, and over 90 inhibitors are currently in clinical use primarily for the treatment of cancer. Repurposing these inhibitors as antimalarials could provide an accelerated path to drug development. In this study, we identified BI-2536, a known potent human polo-like kinase 1 inhibitor, with low nanomolar antiplasmodial activity. Screening of additional PLK1 inhibitors revealed further antiplasmodial candidates despite the lack of an obvious orthologue of PLKs in Plasmodium. A subset of these inhibitors was profiled for their in vitro killing profile, and commonalities between the killing rate and inhibition of nuclear replication were noted. A kinase panel screen identified PfNEK3 as a shared target of these PLK1 inhibitors; however, phosphoproteome analysis confirmed distinct signaling pathways were disrupted by two structurally distinct inhibitors, suggesting PfNEK3 may not be the sole target. Genomic analysis of BI-2536-resistant parasites revealed mutations in genes associated with the starvation-induced stress response, suggesting BI-2536 may also inhibit an aminoacyl-tRNA synthetase.

Mpox Vaccine Interest Survey Prioritization and Data Flow: Maricopa County, Arizona, July-August 2022.

With increasing mpox cases in Maricopa County, Arizona, the county's health department launched a survey on July 11, 2022, to gather eligibility and contact data and provide clinic information to those interested in JYNNEOS as postexposure prophylaxis (PEP) or expanded postexposure prophylaxis(PEP++). Survey data were matched to case and vaccination data. Overall, 343 of the 513 respondents (66.9%) who reported close contact with an mpox case patient received PEP and 1712 of the 3379 respondents (50.7%) who were unsure of their contact status received PEP++. This outreach intervention connected potential close contacts unknown to MCDPH with PEP or PEP++. (Am J Public Health. 2023;113(5):504-508. https://doi.org/10.2105/AJPH.2023.307224).

Cyclic Tetrapeptide HDAC Inhibitors with Improved Plasmodium falciparum Selectivity and Killing Profile.

Cyclic tetrapeptide histone deacetylase inhibitors represent a promising class of antiplasmodial agents that epigenetically disrupt a wide range of cellular processes in Plasmodium falciparum. Unfortunately, certain limitations, including reversible killing effects and host cell toxicity, prevented these inhibitors from further development and clinical use as antimalarials. In this study, we present a series of cyclic tetrapeptide analogues derived primarily from the fungus Wardomyces dimerus that inhibit P. falciparum with low nanomolar potency and high selectivity. This cyclic tetrapeptide scaffold was diversified further via semisynthesis, leading to the identification of several key structural changes that positively impacted the selectivity, potency, and in vitro killing profiles of these compounds. We confirmed their effectiveness as HDAC inhibitors through the inhibition of PfHDAC1 catalytic activity, in silico modeling, and the hyperacetylation of histone H4. Additional analysis revealed the in vitro inhibition of the most active epoxide-containing analogue was plasmodistatic, exhibiting reversible inhibitory effects upon compound withdrawal after 24 or 48 h. In contrast, one of the new diacetyloxy semisynthetic analogues, CTP-NPDG 19, displayed a rapid and irreversible action against the parasite following compound exposure for 24 h.

Antiplasmodial Compounds from Deep-Water Marine Invertebrates.

Novel drug leads for malaria therapy are urgently needed because of the widespread emergence of resistance to all available drugs. Screening of the Harbor Branch enriched fraction library against the Plasmodium falciparum chloroquine-resistant strain (Dd2) followed by bioassay-guided fractionation led to the identification of two potent antiplasmodials; a novel diterpene designated as bebrycin A (1) and the known C21 degraded terpene nitenin (2). A SYBR Green I assay was used to establish a Dd2 EC50 of 1.08 ± 0.21 and 0.29 ± 0.02 µM for bebrycin A and nitenin, respectively. Further analysis was then performed to assess the stage specificity of the inhibitors antiplasmodial effects on the Dd2 intraerythrocytic life cycle. Exposure to bebrycin A was found to block parasite maturation at the schizont stage if added any time prior to late schizogony at 42 hours post invasion, (HPI). In contrast, early life cycle exposure to nitenin (prior to 18 HPI) was identified as crucial to parasite inhibition, suggesting nitenin may target the maturation of the parasite during the transition from ring to early trophozoite (6-18 HPI), a novel property among known antimalarials.

Leveraging Peptaibol Biosynthetic Promiscuity for Next-Generation Antiplasmodial Therapeutics.

Malaria remains a worldwide threat, afflicting over 200 million people each year. The emergence of drug resistance against existing therapeutics threatens to destabilize global efforts aimed at controlling Plasmodium spp. parasites, which is expected to leave vast portions of humanity unprotected against the disease. To address this need, systematic testing of a fungal natural product extract library assembled through the University of Oklahoma Citizen Science Soil Collection Program has generated an initial set of bioactive extracts that exhibit potent antiplasmodial activity (EC50 < 0.30 µg/mL) and low levels of toxicity against human cells (less than 50% reduction in HepG2 growth at 25 µg/mL). Analysis of the two top-performing extracts from Trichoderma sp. and Hypocrea sp. isolates revealed both contained chemically diverse assemblages of putative peptaibol-like compounds that were responsible for their antiplasmodial actions. Purification and structure determination efforts yielded 30 new peptaibols and lipopeptaibols (1-14 and 28-43), along with 22 known metabolites (15-27 and 44-52). While several compounds displayed promising activity profiles, one of the new metabolites, harzianin NPDG I (14), stood out from the others due to its noteworthy potency (EC50 = 0.10 µM against multi-drug-resistant P. falciparum line Dd2) and absence of gross toxicity toward HepG2 at the highest concentrations tested (HepG2 EC50 > 25 µM, selectivity index > 250). The unique chemodiversity afforded by these fungal isolates serves to unlock new opportunities for translating peptaibols into a bioactive scaffold worthy of further development.