ACS Infectious Diseases

The pathogen Mycobacterium abscessus (Mab) can cause severe and difficult to treat chronic lung infections. Despite the rising incidence and clinical concern of Mab infections, treatment options are limited and often ineffective. Treatment is complicated by Mabs ability to persist in a non-replicative, drug-resistant state. Several {beta}-lactam antibiotics are potently bactericidal against Mab but are underutilized because their molecular mechanisms of action against Mab are incompletely understood. In the current study, we used {beta}-lactam-derived activity-based probes and chemoproteomics to report the first comprehensive list of enzymes in Mab targeted by {beta}-lactams. We compared {beta}-lactam targets across two Mab subspecies in actively replicating and non-replicative cultures, using a new carbon starvation model of persistence. We identified 17 targets that were active in every condition tested, seven of which were previously unknown to bind {beta}-lactams. Lastly, we characterized the {beta}-lactamase activity and {beta}-lactam inhibition profiles of nine Mab enzymes, demonstrating that imipenem inhibits these targets more effectively than cefoxitin. These findings provide clarity on the mechanisms of action of clinically relevant {beta}-lactams in Mab, a crucial step toward fully realizing their potential for treating infections caused by this opportunistic pathogen.

Artemisinin resistant Plasmodium falciparum (Pf) is spreading despite combination chemotherapy (ACT). Here we report the design of artezomibs, single-molecule hybrids of an artemisinin and a Pf-selective proteasome inhibitor. Artezomibs exert a novel mode of action inside the malaria parasites. The artemisinin component covalently modifies parasite proteins, which become substrates of the Pf proteasome. The proteasomal degradation products that bear the proteasome inhibitor component of the hybrid then inhibit Pf proteasomes, including those with mutations that reduce binding affinity of the proteasome inhibitor component on its own. We demonstrated that artezomibs circumvent both artemisinin resistance conferred by Kelch13 polymorphism and resistance to the proteasome inhibitor associated with mutations in Pf proteasomes. This mode of action may enable the use of a single molecule with one pharmacokinetic profile to prevent the emergence of resistance.

Chlamydia trachomatis, an obligate intracellular bacterium with limited metabolic capabilities, possesses the futalosine pathway for menaquinone biosynthesis. Futalosine pathway enzymes have promise as narrow spectrum targets, but the activity and essentiality of chlamydial menaquinone biosynthesis have yet to be established. In this work, menaquinone-7 (MK-7) was identified as a C. trachomatis-produced quinone through LC-MS/MS. An immunofluorescence-based assay revealed that treatment of C. trachomatis-infected HeLa cells with futalosine pathway inhibitor docosahexaenoic acid (DHA) reduced inclusion number, inclusion size, and infectious progeny. Supplementation with MK-7 nanoparticles rescued the effect of DHA on inclusion number, indicating that the futalosine pathway is a target of DHA in this system. These results open the door for menaquinone biosynthesis inhibitors to be pursued in antichlamydial development.

Malaria treatments are compromised by drug resistance, creating an urgent need to discover new drugs. We used a phenotypic high-throughput screening (HTS) platform to identify new antimalarials, uncovering three related pyrrole-, indole-, and indoline-based series with a shared -azacyclic acetamide core. These compounds showed fast-killing activity on asexual blood-stage Plasmodium falciparum parasites, were not cytotoxic, and disrupted parasite intracellular pH and Na+ regulation similarly to cipargamin (KAE609), a clinically advanced inhibitor of the P. falciparum Na+ pump (PfATP4). PfATP4 is localized to the parasite plasma membrane and is essential for maintaining a low cytosolic Na+ concentration. Resistance selections on P. falciparum parasites with two -azacyclic acetamide analogs identified mutations in PfATP4, and cross-resistance was observed across the -azacyclic acetamides and KAE609, confirming PfATP4 as the target. PfATP4 is a well-established antimalarial target, and identification of additional PfATP4 inhibitors provides alternative avenues to disrupt its function.

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.

{beta}-Lactams are the most widely used antibiotics for the treatment of bacterial infections because of their proven track record of safety and efficacy. However, susceptibility to {beta}-lactam antibiotics is continually eroded by resistance mechanisms. Emerging multidrug-resistant (MDR) Neisseria gonorrhoeae strains possessing altered penA alleles (encoding PBP2) pose a global health emergency as they threaten the utility of ceftriaxone, the last remaining outpatient antibiotic. Here we disclose a novel benzoxaborinine-based penicillin-binding protein inhibitor series (boro-PBPi) that is envisioned to address penA-mediated resistance while offering protection against evolution and expansion of {beta}-lactamases. Optimization of boro-PBPi led to the identification of compound 21 (VNRX-14079) that exhibits potent antibacterial activity against MDR N. gonorrhoeae achieved by high affinity binding to the PBP2 target. Boro-PBPi/PBP2 complex structures confirmed covalent interaction of the boron atom with Ser310 and the importance of the {beta}3-{beta}4 loop for improved affinity. 21 elicits bactericidal activity, a low frequency of resistance, a good safety profile, suitable pharmacokinetic properties, and in vivo efficacy in a murine infection model against ceftriaxone-resistant N. gonorrhoeae. 21 is a promising anti-gonorrhea agent poised for further advancement.

The high burden of malaria and growing resistance to frontline antimalarials demand new drugs with reduced propensities for generating resistance. An attractive approach to identifying chemical hits as starting points for antimalarial drug discovery involves the repositioning and chemical optimization of compounds used in other disease areas that are active against the human malaria parasite Plasmodium falciparum. Here we show that the human checkpoint kinase 1 (Chk1) inhibitor CHIR-124 is active in vitro against both drug-sensitive and drug-resistant asexual blood stage parasites and competitively binds to several Plasmodium kinases, including P. falciparum aurora-related kinase-1 (PfArk1). The compound also shows moderate activity against both the liver and gametocyte forms of the parasite. Further target investigation for CHIR-124 via conditional knockdown experiments confirmed that PfArk1 is implicated in its parasiticidal activity. Notably, CHIR-124 also inhibits {beta}-hematin (synthetic hemozoin) formation and causes a dose-dependent increase in free heme that correlates with inhibition of parasite growth. These findings suggest that polypharmacology is involved in the activity of CHIR-124 against P. falciparum via the dual inhibition of Plasmodium PfArk1 and hemozoin formation, both essential for parasite proliferation. This is further supported by in vitro drug combination experiments, morphological studies and resistance generation attempts. This study demonstrates the feasibility of designing dual Plasmodium kinase/hemozoin formation inhibitors active against resistant strains with decreased resistance risks in the fight against malaria.

We report a systematic, cellular phenotype-based antimalarial screening of the MMV Pathogen Box collection, which facilitated the identification of specific blockers of late stage intraerythrocytic Plasmodium falciparum maturation. First, from standard growth inhibition asays, we discovered 62 additional antimalarials (EC50 [≤] 10M) over previously known antimalarial candidates from Pathogen Box. A total of 90 potent molecules (EC50 [≤] 1M) were selected for evaluating their stage-specific effects during the intra-erythrocytic development of P. falciparum. None of these molecules had significant effect on ring-trophozoite transition, 10 molecules inhibited trophozoite-schizont transition, and 21 molecules inhibited schizont-ring transition at 1M. These compounds were further validated in secondary assays by flow cytometry and microscopic imaging of treated cells to prioritize 12 molecules as potent and selective blockers of schizont-ring transition. Seven of these were found to strongly inhibit calcium ionophore induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via similar mechanism in the two parasites, which can be further exploited for target identification studies. Two of these molecules, with previously unknown mechanism of action, MMV020670 and MMV026356, were found to induce fragmentation of DNA in developing merozoites. Further mechanistic studies would facilitate therapeutic exploitation of these molecules as broadly active inhibitors targeting development and egress of apicomplexan parasites relevant to human health.

Mycobacterium tuberculosis and pathogenic nontuberculous mycobacteria pose a growing challenge to human health, and new antibiotics that target new pathways with novel mechanisms of action are urgently needed. Acylaminooxadiazole derivatives have previously been shown to inhibit the trans-translation ribosome rescue pathway and kill M. tuberculosis. Here, we show that modifications to the acylaminooxadiazole scaffold can improve potency and tune mycobacterial species specificity, resulting in molecules that kill M. avium, M. abscessus, and M. tuberculosis clinical isolates. Free iron was previously shown to antagonize antibacterial activity and decrease the inhibition of trans-translation by acylaminooxadiazoles, but we found that biologically relevant iron sources such as hemin and transferrin do not affect activity. Mutants depleted for tmRNA and mutants defective in siderophore-mediated iron utilization are both hypersusceptible to acylaminooxadiazole-based trans-translation inhibitors, indicating a dual mechanism of action involving both direct inhibition of trans-translation and metal starvation. These findings establish acylaminooxadiazoles as dual-mechanism antimycobacterial agents that couple inhibition of trans-translation with disruption of iron homeostasis.

Ecological interactions between bacteria and nematodes in many environments provide a basis for the prediction that diverse bacteria produce anti-nematode compounds. The discovery of microbial secondary metabolites with broad-spectrum nematostatic or nematicidal properties can be hastened by drug screening approaches that include several nematode species and phenotypes. We cultured a collection of 22 soil-derived bacterial isolates that carry in their genomes putative pathways for production of unknown secondary metabolites. Isolates were cultured in various media to enhance natural product diversity and yield, and we evaluated culture filtrates for activity against two evolutionarily distinct nematode species: Clade V free-living nematode Caenorhabditis elegans and Clade III mammalian parasitic nematodes in the genus Brugia. Partitioned extracts from Pseudomonas sp. strain TE4607 stunted C. elegans development and caused motility defects in both blood-circulating larval and adult stages of Brugia. The primary active compound was identified as labradorin 1, an indole with known antibacterial and anticancer properties that had not been previously described as affecting nematodes. Notably, filtrates of Pseudomonas sp. TE4607 cultures attracted free-living nematodes in sensory assays, adding to evidence that certain Pseudomonas species modulate the behavior of free-living nematodes. These findings underscore the need to further explore the link between nematode sensory responses and whole-organism effects of microbial metabolites, with potential applications in anthelmintic discovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/700618v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1909e29org.highwire.dtl.DTLVardef@17f9badorg.highwire.dtl.DTLVardef@c59d79org.highwire.dtl.DTLVardef@1e5abbf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Salmonellosis, including typhoid and paratyphoid fever, causes significant diarrheal disease worldwide underscoring the urgent need for effective treatments in the face of rising antibiotic resistance. Host-targeted therapies, such as AR-12, offer a promising solution to combat drug resistance. AR-12 has demonstrated broad-spectrum antimicrobial activity against various bacterial pathogens, including Salmonella enterica serovar Typhimurium, S. Typhi, Francisella tularensis, and F. novicida, as well as protozoan parasites and fungal pathogens. Our research has developed AR-12 analogs for pre-clinical development, focusing on enhancing potency and decreasing cytotoxicity. This involved systematic optimization of various points of diversity and the pyrazole core, resulting in significant improvements in combating resistant intracellular S. Typhimurium. Primary screening of 81 AR-12 analogs assessed changes in intracellular S. Typhimurium burden (IC50), host cell viability (LC50), and direct effects on planktonic S. Typhimurium (MIC50), calculating selectivity (LC50/IC50) to determine host-directed potency versus cytotoxicity. Of the full library, only seven affected planktonic Salmonella growth below 20 {micro}M, suggesting host-directed activity in most of the compounds. Further, 38 analogs were found to be both more potent and less cytotoxic than parent compound AR-12, while only three were less potent and more cytotoxic. Twelve analogs were chosen for secondary screening in MDR S. Typhimurium. Compounds 372, 373, and 378 demonstrated remarkable selectivity, with values exceeding 1500 for both susceptible and MDR S. Typhimurium, compared to AR-12s selectivity of around 20. This approximately 100-fold improvement, coupled with improved potency against intracellular Salmonella, suggests these analogs have significantly greater host-directed activity than direct antibacterial effects. Proteomic analysis for the two most potent compounds, 341 and 370 revealed enrichment of vesicle-mediated transport proteins, specifically with respect to retrograde transport at the trans-Golgi-network and intra-Golgi traffic. These results suggest that the analogs reduce intracellular S. Typhimurium replication by disrupting its exploitation of the host cells vesicle-mediated transport system.

Tuberculosis (TB) is a leading cause of death among infectious diseases worldwide due to latent TB infection, which is the critical step for the successful pathogenic cycle. In this stage, Mycobacterium tuberculosis resides inside the host in a dormant and antibiotic-tolerant state. Latent TB infection can lead to a multisystemic diseases because M. tuberculosis invades virtually all organs, including ocular tissues. Ocular tuberculosis (OTB) occurs when the dormant bacilli within ocular tissues reactivate, originally seeded by hematogenous spread from pulmonary TB. Timely and accurate diagnosis as well as efficient chemotherapies are crucial in preventing poor visual outcomes of OTB patients. Histological evidence suggests that retinal pigment epithelium (RPE) cells play a central role in immune privilege and in the protection from the antibiotic effects, making them an anatomical niche for invading M. tuberculosis. RPE cells exhibit high tolerance to environmental redox stresses, allowing phagocytosed M. tuberculosis bacilli to maintain viability in a dormant state. However, the microbiological and metabolic mechanisms determining the interaction between the RPE intracellular environment and phagocytosed M. tuberculosis are largely unknown. Here, liquid chromatography mass spectrometry (LC-MS) metabolomics was used to illuminate the metabolic state within RPE cells reprogrammed to harbor dormant M. tuberculosis bacilli and enhance the antibiotic tolerance. The results have led to propose a novel therapeutic option to synthetically kill the dormant M. tuberculosis inside the RPE cells by modulating the phenotypic state of M. tuberculosis, thus laying the foundation for a new, innovative regimen for treating OTB. ImportanceUnderstanding the metabolic environment within the retinal pigment epithelium (RPE) cells altered by infection with M. tuberculosis and mycobacterial dormancy is crucial to identify new therapeutic methods to cure OTB. The present study showed that RPE cellular metabolism is altered to foster intracellular M. tuberculosis to enter into the dormant and drug tolerant state, thereby blunting the efficacy of anti-TB chemotherapy. RPE cells serve as an anatomical niche as the cells protect invading bacilli from antibiotic treatment. LC-MS metabolomics of RPE cells after co-treatment with H2O2 and M. tuberculosis infection showed that intracellular environment within RPE cells is enriched with greater level of oxidative stress. The antibiotic tolerance of intracellular M. tuberculosis within RPE cells can be restored by a metabolic manipulation strategy such as co-treatment of antibiotic with the most downstream glycolysis metabolite, phosphoenolpyruvate.

The Resistance-Nodulation-Division (RND) efflux pump superfamily is pervasive among Gram-negative pathogens and contributes extensively to clinical antibiotic resistance. The opportunistic pathogen Pseudomonas aeruginosa contains 12 RND-type efflux systems, with four contributing to resistance including MexXY-OprM which is uniquely able to export aminoglycosides. At the site of initial substrate recognition, small molecule probes of the inner membrane transporter (e.g., MexY) have potential as important functional tools to understand substrate selectivity and a foundation for developing adjuvant efflux pump inhibitors (EPIs). Here, we optimized the scaffold of berberine, a known but weak MexY EPI, using an in-silico high-throughput screen to identify di-berberine conjugates with enhanced synergistic action with aminoglycosides. Further, docking and molecular dynamics simulations of di-berberine conjugates reveal unique contact residues and thus sensitivities of MexY from distinct P. aeruginosa strains. This work thereby reveals di-berberine conjugates to be useful probes of MexY transporter function and potential leads for EPI development.

Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate the indispensable role of lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, in the survival of Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We show that genetic and functional ablation of RibH in M. tb cannot be compensated by exogenous supply of Riboflavin, or co-factors Flavin Adenine Dinucleotide (FAD) or Flavin Mononucleotide (FMN). Capitalizing on the essentiality of RibH, we employ a high-throughput molecular docking approach to screen [~]600,000 compounds and identify inhibitors of RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 inhibitors that exhibit potent antimycobacterial activity. These compounds effectively also eradicate intracellular M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these 3 compounds synergistically enhance the bactericidal effect of first-line anti-TB drugs, Isoniazid and Rifampicin. Corroborating with the in silico predicted high docking scores along with favorable ADME and toxicity profiles, all 3 compounds demonstrate exceptional binding affinity towards purified lumazine synthase enzyme in vitro, and display an acceptable safety profile in mammalian cells, with a high selective index. By providing mechanistic evidence for the essentiality of RibH in M. tb survival, and discovering potent RibH inhibitors with outstanding antimycobacterial activity, our study contributes to the development of superior TB treatment strategies and advances the global fight against this devastating disease.

Cryptosporidium species, mostly C. parvum and C. hominis in humans, are intestinal apicomplexan parasites that cause life-threatening diarrhea in young children and people with cell-mediated immune defects, such as due to AIDS. There is only one approved treatment for cryptosporidiosis, but it is ineffective for immunocompromised people and only modestly effective for children. In this study, screening 278 compounds from the Merck KGaA, Darmstadt, Germany collection and accelerated follow-up work enabled by prior investigation of the compounds resulted in identification of a series of pyrazolopyrimidine human phosphodiesterase (PDE)-V inhibitors with potent anticryptosporidial activity and efficacy following oral administration in C. parvum-infected immunocompromised mice. The novel PDE inhibitor leads (compounds PDEi2 and PDEi5) affect parasite egress from infected host cells. They have comparable activity against C. parvum and C. hominis, rapidly eliminate C. parvum in tissue culture, and have minimal off-target effects in a panel of safety screening assays. In comparison, the potent human PDE-V inhibitors sildenafil and the 4-aminoquinoline compound 7a have no useful activity against C. parvum. Based on homology modeling and in silico compound docking, PDEi5 interacts directly with an active-site metal ion and docks well to two C. parvum PDEs. In contrast, larger amino acid side groups (Val900/Tyr11128 and His884/Asn1112) in both C. parvum PDEs replace alanine in human PDE-V and block sildenafil binding, explaining its lack of efficacy. These results identify a promising new drug target and lead series for anticryptosporidial drug development and validates a route to target-based optimization.

Fungal cyclotetrapeptide nectriatide and its synthetic linear derivatives (nectriatide-based compounds, NCTs), having no antifungal activity, potentiated antifungal activity of amphotericin B (AmB) against Candida albicans. The mechanism of action was investigated using fluorescein-conjugated and biotin-tagged probes. Microscopy revealed fluorescent-probe localization at the C. albicans cell membrane. The biotinyl probe binding assay towards membrane lipids showed the highest affinity for ergosterol but no affinity for cholesterol. Ergosterol binding was confirmed by a liposome disruption assay. LC-MS quantification of AmB in C. albicans revealed that NCTs increased AmB binding to C. albicans. These results indicate that NCTs bind to ergosterol on the fungal plasma membrane, subsequently localizing AmB, explaining the observed potentiation of AmB fungicidal activity.

The shikimate pathway, a metabolic pathway absent in humans, is responsible for the production of chorismate, a branch point metabolite. In the malaria parasite, chorismate is postulated to be a direct precursor in the synthesis of p-aminobenzoic acid (folate biosynthesis), p-hydroxybenzoic acid (ubiquinone biosynthesis), menaquinone, and aromatic amino acids. While the potential value of the shikimate pathway as a drug target is debatable, the metabolic dependency of chorismate in P. falciparum remains unclear. Current evidence suggests that the main role of chorismate is folate biosynthesis despite ubiquinone biosynthesis being active and essential in the malaria parasite. Our goal in the present work was to expand our knowledge of the ubiquinone head group biosynthesis and its potential metabolic dependency on chorismate in P. falciparum. These data led us to further characterize the mechanism of action of MMV688345, a compound from the open-access \"Pathogen Box\" collection from Medicine for Malaria Venture. We systematically assessed the development of both asexual and sexual stages of P. falciparum in a defined medium in the absence of an exogenous supply of chorismate end-products and present biochemical evidence suggesting that the benzoquinone ring of ubiquinones in this parasite may be synthesized through a yet unidentified route.

Rapid emergence of drug resistance in Mycobacterium tuberculosis (Mtb) is one of the most significant healthcare challenges of our time. The cause of drug resistance is multifactorial, with the long course anti-tubercular therapy required to treat tuberculosis (TB) constituting a major contributing factor. Introduction of pyrazinamide (PZA) resulted in shortening of TB treatment from twelve to six months and consequently played a critical role in curbing drug resistance that developed over long course therapy. Nevertheless, because PZA is a prodrug activated by a nonessential amidase, PncA, resistance to PZA develops and frequently results in treatment failure. Here, we leveraged a whole cell drug screening approach to identify anti-tuberculars with unconventional mechanisms of action or activation that could be further developed into compounds effective at killing Mtb resistant to PZA. We discovered an amide containing prodrug, DG160, that was activated by the amidase, Rv2888c (AmiC). This amidase was capable of metabolizing a variety of amide containing compounds including a novel pyrazinoic acid-isoquinolin-1-amine prodrug, JSF-4302, which we developed as a potential PncA-independent replacement for PZA. As predicted, AmiC activation of JSF-4302 led to the generation of POA in Mtb including in a PZA resistant clinical isolate, thereby successfully delivering the active component of PZA while bypassing the need for activation by PncA. This work provides a framework for a new approach to drug development and prodrug activation in Mtb. SIGNIFICANCEPyrazinamide (PZA) is a vital component of Mycobacterium tuberculosis (Mtb) treatment since its inclusion shortened tuberculosis therapy by six months. However, PZA is a prodrug and resistance develops at a high frequency due to mutations in its activator PncA. Here, we present the discovery of amide-containing anti-tubercular prodrugs that are activated intracellularly by the Mtb amidase, AmiC. Taking advantage of this finding, we successfully designed and synthesized pyrazinoic acid (POA) prodrugs that were activated by AmiC and found that these compounds delivered intracellular POA to PZA- resistant Mtb isolates that contained a nonfunctional PncA. This new approach to prodrug development provides a method for delivering conjugated drugs into Mtb with the potential to overcome clinical drug resistance.

Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and participate in regulating DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi (TcBDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350 ranked compounds were further analyzed in a dose-response assay. From this analysis, 7 compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significative trypanocidal activity, all showed bona fide interaction with TcBDF2 with dissociation constants between 1 and 3 M validating these assays to search for bromodomain inhibitors.

Toxoplasma gondii is a protozoan parasite capable of infecting most warm-blooded animals, including humans, and can cause severe disease in immunocompromised individuals and the developing fetus. Current treatments for toxoplasmosis are effective only against the acute stage of infection and have limited or no activity against the latent bradyzoite stage found within tissue cysts. The mitochondrion of T. gondii is a validated drug target, and the clinically used drug atovaquone acts by inhibiting the mitochondrial electron transport chain (ETC) at the coenzyme Q:cytochrome c oxidoreductase (bc1 complex). In this study, we evaluate two legacy 4(1H)-quinolones: ICI 56,780 and WR 243246, previously shown to inhibit the Plasmodium falciparum bc1 complex, for their efficacy against T. gondii. Both compounds inhibit tachyzoite growth with low-nanomolar EC values and disrupt parasite mitochondrial function by blocking cytochrome c reduction and collapsing the mitochondrial membrane potential. Notably, ICI 56,780 protects mice from lethal infection with type I RH tachyzoites. Importantly, ICI 56,780 also exhibits potent activity against chronic-stage parasites, reducing cyst size and bradyzoite viability in vitro and showing low-nanomolar EC values against in vivo-derived bradyzoites. In mice chronically infected with T. gondii, treatment with ICI 56,780 significantly decreases brain cyst burden. Although these 4(1H)-quinolones display some pharmacokinetic limitations, our findings highlight their potential as promising chemotypes active against both acute and chronic stages of T. gondii and provide a framework for future medicinal chemistry efforts to improve drug-like properties while preserving or enhancing anti-bradyzoite activity.