ACS Infectious Diseases最新文献

The type II NADH-dehydrogenase enzyme in Mycobacterium tuberculosis plays a critical role in the efficient functioning of the oxidative phosphorylation pathway. It acts as the entry point for electrons in the electron transport chain, which is essential for fulfilling the energy requirements of both replicating and nonreplicating mycobacterial species. Due to the absence of the type II NADH-dehydrogenase enzyme in mammalian mitochondria, targeting the type II NADH-dehydrogenase enzyme for antitubercular drug discovery could be a vigilant approach. Utilizing type II NADH-dehydrogenase inhibitors in antitubercular therapy led to bactericidal response, even in monotherapy. However, the absence of the cryo-EM structure of Mycobacterium tuberculosis type II NADH-dehydrogenase has constrained drug discovery efforts to rely on high-throughput screening methods, limiting the use of structure-based drug discovery. Here, we have delineated the literature-reported Mycobacterium tuberculosis type II NADH-dehydrogenase inhibitors and the rationale behind selecting this specific enzyme for antitubercular drug discovery, along with shedding light on the architecture of the enzyme structure and functionality. The gap in the current research and future research direction for TB treatment have been addressed.

Schistosomiasis is the infection caused by Schistosoma mansoni and constitutes a worldwide public health problem. The parasitological recommended method and serological methods can be used for the detection of eggs and antibodies, respectively. However, both have limitations, especially in low endemicity areas. Thus, new approaches for the diagnosis of schistosomiasis are essential. In this study, a six-amino acid peptide and derived sequences from SmATPDase1 were synthesized for the evaluation of immunogenicity. SmATPDase1 is included in a protein group in S. mansoni tegument; therefore, its peptides could be potential candidates for diagnostic antigens. In the hypothetical SmATPDase1 three-dimensional structure, peptides are located in a region exposed and accessible to antibody binding. In addition, peptide amino acid sequences are conserved in the most relevant Schistosoma species and have low identity with human NTPDases isoforms. Swiss mice immunization resulted in significant anti-peptide polyclonal antibodies production, which recognized a 63 kDa protein in tegument and adult worm preparations. By immunofluorescence microscopy, polyclonal antibodies also identified this enzyme in cercariae. Sera of infected animals presented high seropositivity in ELISA-peptides, with an area under curve (AUC) greater than 0.96 for all peptides. In mice with low parasite burden, we observed a seropositivity AUC > 0.9. Reactivity in the prepatent period exhibited AUC values greater than 0.94 for all peptides. Anti-P1425 monoclonal antibodies were successfully produced, and mAbs recognized the integral protein in ELISA and Western blots. The data indicate that peptides from SmATPDase1 are potential biomarkers for schistosomiasis, and anti-peptide antibodies are interesting tools for the detection of the infection.

Half the world's population is at risk of developing a malaria infection, which is caused by parasites of the genus Plasmodium. Currently, resistance has been identified to all clinically available antimalarials, highlighting an urgent need to develop novel compounds and better understand common mechanisms of resistance. We previously identified a novel tetrahydro-β-carboline compound, PRC1590, which potently kills the malaria parasite. To better understand its mechanism of action, we selected for and characterized resistance to PRC1590 in Plasmodium falciparum. Through in vitro selection of resistance to PRC1590, we have identified that a single-nucleotide polymorphism on the parasite's multidrug resistance protein 1 (PfMDR1 G293V) mediates resistance to PRC1590. This mutation results in stereospecific resistance and sensitizes parasites to other antimalarials, such as mefloquine, quinine, and MMV019017. Intraerythrocytic asexual stage specificity assays have revealed that PRC1590 is most potent during the trophozoite stage when the parasite forms a single digestive vacuole (DV) and actively digests hemoglobin. Moreover, fluorescence microscopy revealed that PRC1590 disrupts the function of the DV, indicating a potential molecular target associated with this organelle. Our findings mark a significant step in understanding the mechanism of resistance and the mode of action of this emerging class of antimalarials. In addition, our results suggest a potential link between resistance mediated by PfMDR1 and PRC1590's molecular target. This research underscores the pressing need for future research aimed at investigating the intricate relationship between a compound's chemical scaffold, molecular target, and resistance mutations associated with PfMDR1.

Antimicrobial drug resistance is a significant global health challenge, causing hundreds of thousands of deaths annually and severely impacting healthcare systems worldwide. Several reported antimicrobial compounds have a guanidine motif, as the positive charge on guanidine promotes cell lysis. Therefore, pyrrole- and indole-based allylidene hydrazine carboximidamide derivatives with guanidine motifs are proposed as antimicrobial agents that mimic cationic antimicrobial peptides (CAMPs). A total of 72 derivatives having pyrrol-2-yl-phenyl allylidene hydrazine carboximidamide and indol-3-yl-phenyl allylidene hydrazine carboximidamide scaffolds were assessed for their inhibitory potential against a panel of Gram-positive and Gram-negative bacteria. Analogs 1j, 1k, 1s, 2j, 2q, 4a, 4c, 4h, 5b, 6a, and 6d exhibited potent broad-spectrum antimicrobial activity better than the standard antibiotics. Also, these compounds showed no cytotoxicity up to 3-fold of the minimum inhibitory concentration, and structure-activity relationship was established. Further, the most active compound, 6a, showed a strong biofilm disruption, acted on the bacterial membrane, and lysed it. The further development of these compounds as novel antimicrobial agents is warranted.

New antimalarial combination therapies with novel modes of action are required to counter the emergence and spread of Plasmodium drug resistance against existing therapeutics. Here, we present a study to evaluate the preventive activity of a combination of clinical antimalarial drug candidates, cabamiquine and ganaplacide, that have multistage activity against the liver and blood stages of Plasmodium infection. Cabamiquine (DDD107498, M5717) inhibits parasite protein synthesis, and ganaplacide (KAF156) inhibits protein trafficking, blocks the establishment of new permeation pathways, and causes endoplasmic reticulum expansion. The pharmacodynamic parameters of a combination of the two compounds were assessed employing a pharmacometrics approach in conjunction with in vitro-in silico checkerboard analysis. The in vitro study was performed on a previously established 3D infection platform based on human hepatic cell lines that sustain infection by rodent P. berghei parasites. The in vivo efficacy of this drug combination was assessed against the liver stage of the P. berghei. Our results show that the combination of both drugs at the tested concentrations does not interfere with the drugs respective mode of action or affect hepatocyte cell viability. The drug combination was fully effective in preventing the appearance of blood stage parasites when a systemic plasma C_av0-24/EC₅₀ ratio >2 for ganaplacide and >5 for cabamiquine was achieved. These findings demonstrate that chemoprevention using a combination of cabamiquine and ganaplacide has the potential to target the asymptomatic liver stage of Plasmodium infection and prevent the development of parasitemia.

New World alphaviruses, including Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV), are mosquito-transmitted viruses that cause disease in humans. These viruses are endemic to the western hemisphere, and disease in humans may lead to encephalitis and long-term neurological sequelae. There are currently no FDA-approved vaccines or antiviral therapeutics available for the prevention or treatment of diseases caused by these viruses. The alphavirus nonstructural protein 2 (nsP2) functions as a protease, which is critical for the establishment of a productive viral infection by enabling accurate processing of the nsP123 polyprotein. Owing to the essential role played by nsP2 in the alphavirus infectious process, it is also a valuable therapeutic target. In this article, we report the synthesis and evaluation of novel small molecule inhibitors that target the alphavirus nsP2 protease via a covalent mode of action. The two lead compounds demonstrated robust inhibition of viral replication in vitro. These inhibitors interfered with the processing of the nsP123 polyprotein as determined using VEEV TC-83 as a model pathogen and are active against EEEV and WEEV. The compounds were found to be nontoxic in two different mouse strains and demonstrated antiviral activity in a VEEV TC-83 lethal challenge mouse model. Cumulatively, the outcomes of this study provide a compelling rationale for the preclinical development of nsP2 protease inhibitors as direct-acting antiviral therapeutics against alphaviruses.

The pathway of bacterial cysteine biosynthesis is gaining traction for the development of antibiotic adjuvants. Bacterial cysteine biosynthesis is generally facilitated by two enzymes possessing O-acetyl-l-serine sulfhydrylases (OASS), CysK and CysM. In Staphylococcus aureus, there exists a single OASS homologue, SaCysK. Knockout of SaCysK was found to increase sensitivity to oxidative stress, making it a relevant target for inhibitor development. SaCysK also forms two functional complexes via interaction with the preceding enzyme in the pathway serine acetyltransferase (CysE) or the transcriptional regulator of cysteine metabolism (CymR). These interactions occur through insertion of a C-terminal peptide of CysE or CymR into the active site of SaCysK, inhibiting OASS activity, and therefore represent an excellent starting point for developing SaCysK inhibitors. Here, we detail the characterization of CysE and CymR-derived C-terminal peptides as inhibitors of SaCysK. Using a combination of X-ray crystallography, surface plasmon resonance, and enzyme inhibition assays, it was determined that the CymR-derived decapeptide forms extensive interactions with SaCysK and acts as a potent inhibitor (K_D = 25 nM; IC₅₀ = 180 nM), making it a promising lead for the development of SaCysK inhibitors. To understand the determinants of this high-affinity interaction, the structure-activity relationships of 16 rationally designed peptides were also investigated. This identified that the C-terminal pentapeptide of CymR facilitates the high-affinity interaction with SaCysK and that subtle structural modification of the pentapeptide is possible without impacting potency. Ultimately, this work identified CymR pentapeptides as a promising scaffold for the development of antibiotic adjuvants targeting SaCysK.

Developing new classes of drugs that are active against infections caused by Mycobacterium tuberculosis is a priority for treating and managing this deadly disease. Here, we describe screening a small library of 20 DNA gyrase inhibitors and identifying new lead compounds. Three structurally diverse analogues were identified with minimal inhibitory concentrations of 0.125 μg/mL against both drug-susceptible and drug-resistant strains of M. tuberculosis. These lead compounds also demonstrated antitubercular activity in ex vivo studies using infected THP-1 macrophages with minimal cytotoxicity in THP-1, HeLa, and HepG2 cells (IC₅₀ ≥ 128 μg/mL). The molecular target of the lead compounds was validated through biochemical studies of select analogues with purified M. tuberculosis gyrase and the generation of resistant mutants. The lead compounds were assessed in combination with bedaquiline and pretomanid to determine the clinical potential, and the select lead (158) demonstrated in vivo efficacy in an acute model of TB infection in mice, reducing the lung bacterial burden by approximately 3 log₁₀ versus untreated control mice. The advancement of DNA gyrase inhibitors expands the field of innovative therapies for tuberculosis and may offer an alternative to fluoroquinolones in future therapeutic regimens.

Gastrointestinal nematodes (GINs) are among the most common parasites of humans, livestock, and companion animals. GIN parasites infect 1-2 billion people worldwide, significantly impacting hundreds of millions of children, pregnant women, and adult workers, thereby perpetuating poverty. Two benzimidazoles with suboptimal efficacy are currently used to treat GINs in humans as part of mass drug administrations, with many instances of lower-than-expected or poor efficacy and possible resistance. Thus, new anthelmintics are urgently needed. However, screening methods for new anthelmintics using human GINs typically have low throughput. Here, using our novel screening pipeline that starts with human hookworms, we screened 30,238 unique small molecules from a wide range of compound libraries, including ones with generic diversity, repurposed drugs, natural derivatives, known mechanisms of action, as well as multiple target-focused libraries (e.g., targeting kinases, GPCRs, and neuronal proteins). We identified 55 compounds with broad-spectrum activity against adult stages of two evolutionary divergent GINs, hookworms (Ancylostoma ceylanicum) and whipworms (Trichuris muris). Based on known databases, the targets of these 55 compounds were predicted in nematode parasites. One novel scaffold from the diversity set library, F0317-0202, showed good activity (high motility inhibition) against both GINs. To better understand this novel scaffold's structure-activity relationships (SAR), we screened 28 analogs and created SAR models highlighting chemical and functional groups required for broad-spectrum activity. These studies validate our new and efficient screening pipeline at the level of tens of thousands of compounds and provide an important set of new GIN-active compounds for developing novel and broadly active anthelmintics.

Arthropod-borne members of the genus Orthoflavivirus cause significant human disease. Four serotypes of dengue virus are endemic globally, and approximately 50 percent of the world's population lives in a dengue-affected area. Complications from immunoenhancement occurring after a secondary infection with a different dengue serotype make vaccine development challenging. Antiviral therapies that target features conserved in all four serotypes would, therefore, be beneficial. Computational studies identified multiple potential G-quadruplex sites that are conserved in the RNA genome sequences of members of the genus Orthoflavivirus. Biophysical studies confirmed that the NS5-B quadruplex sequences obtained from viruses of each dengue serotype can form quadruplexes in vitro, and binding data showed that known quadruplex binders stabilized NS5-B quadruplexes for all four dengue serotypes.