ACS Infectious Diseases最新文献

The urgent need for rapidly acting compounds in the development of antimalarial drugs underscores the significance of such compounds in overcoming resistance issues and improving patient adherence to antimalarial treatments. The present study introduces a high-throughput screening (HTS) approach using 1536-well plates, employing Plasmodium falciparum lactate dehydrogenase (PfLDH) combined with nitroreductase (NTR) and fluorescent probes to evaluate inhibition of the growth of the asexual blood stage of malaria parasites. This method was adapted to efficiently assess the speed of action profiling (SAP) in a 384-well plate format, streamlining the traditionally time-consuming screening process. By successfully screening numerous compounds, this approach identified fast-killing hits early in the screening process, addressing challenges associated with artemisinin-based combination therapies. The high-throughput SAP method is expected to be of value in continuously monitoring fast-killing properties during structure-activity relationship studies, expediting the identification and development of novel, rapidly acting antimalarial drugs within phenotypic drug discovery campaigns.

In recent years, naturally occurring darobactins have emerged as a promising compound class to combat infections caused by critical Gram-negative pathogens. In this study, we describe the in vivo evaluation of derivative D22, a non-natural biosynthetic darobactin analogue with significantly improved antibacterial activity. We found D22 to be active in vivo against key critical Gram-negative human pathogens, as demonstrated in murine models of Pseudomonas aeruginosa thigh infection, Escherichia coli peritonitis/sepsis, and urinary tract infection (UTI). Furthermore, we observed the restored survival of Acinetobacter baumannii-infected embryos in a zebrafish infection model. These in vivo proof-of-concept (PoC) in diverse models of infection against highly relevant pathogens, including drug-resistant isolates, highlight the versatility of darobactins in the treatment of bacterial infections and show superiority of D22 over the natural darobactin A. Together with a favorable safety profile, these findings pave the way for further optimization of the darobactin scaffold toward the development of a novel antibiotic.

Antimicrobial resistance (AMR) poses a serious threat to global health. The rapid emergence of resistance contrasts with the slow pace of antimicrobial development, emphasizing the urgent need for innovative drug discovery approaches. This study addresses a critical bottleneck in early drug development by introducing integral solvent-induced protein precipitation (iSPP) to rapidly assess the target-engagement of lead compounds in extracts of pathogenic microorganisms under close-to-physiological conditions. iSPP measures the change in protein stability against solvent-induced precipitation in the presence of ligands. The iSPP method for bacteria builds upon established SPP procedures and features optimized denaturation gradients and minimized sample input amounts. The effectiveness of the iSPP workflow was initially demonstrated through a multidrug target-engagement study. Using quantitative mass spectrometry (LC-MS/MS), we successfully identified known drug targets of seven different antibiotics in cell extracts of four AMR-related pathogens: the three Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and the Gram-positive bacterium Staphylococcus aureus. The iSPP method was ultimately applied to demonstrate target-engagement of compounds derived from target-based drug discovery. We employed five small molecules targeting three enzymes in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway─a promising focus for anti-infective drug development. The study showcases iSPP adaptability and efficiency in identifying anti-infective drug targets, advancing early-stage drug discovery against AMR.

Shiga-toxin-producing Escherichia coli (STEC) are important human pathogens causing diarrhea, hemorrhagic colitis, and severe hemolytic uremic syndrome. Timely detection of the multifaceted STEC is of high importance but is challenging and labor-intensive. An easy-to-perform rapid test would be a tremendous advance. Here, the major STEC virulence factor Shiga toxins (Stx), RNA-N-glycosidases targeting the sarcin ricin loop (SRL) of 28S rRNA, was used for detection. We designed synthetic FRET-based ssDNA SRL substrates, which conferred a fluorescence signal after cleavage by Stx. Optimal results using bacterial culture supernatants or single colonies were achieved for substrate StxSense 4 following 30 to 60 min incubation. Stx1 and Stx2 subtypes, diverse STEC serotypes, and Shigella were detected. Within a proof-of-principle study, a total of 94 clinical strains were tested, comprising 65 STEC, 11 Shigella strains, and 18 strains of other enteropathogenic bacteria without Stx. In conclusion, the assay offers rapid and facile STEC detection based on a real-time readout for Stx activity. Therefore, it may improve STEC risk evaluation, therapy decisions, outbreak, and source detection and simplify research for antimicrobials.

The proteasome is essential for eukaryotic cell proteostasis, and inhibitors of the 20S proteasome are progressing preclinically and clinically as antiparasitics. We screenedTrypanosoma brucei, the causative agent of human and animal African trypanosomiasis, in vitro with a set of 27 carmaphycin B analogs, irreversible epoxyketone inhibitors that were originally developed to inhibit thePlasmodium falciparum20S (Pf20S). The structure-activity relationship was distinct from that of the human c20S antitarget by the acceptance of d-amino acids at the P3 position of the peptidyl backbone to yield compounds with greatly decreased toxicity to human cells. For the three most selective compounds, binding to the Tb20S β5 catalytic subunit was confirmed by competition with a fluorescent activity-based probe. For one compound, J-80, with its P3 d-configuration, the differential binding to the parasite's β5 subunit was supported by both covalent and noncovalent docking analysis. Further, J-80 was equipotent against both Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense in vitro. In a mouse model of Stage 1 T. brucei infection, a single intraperitoneal (i.p.) dose of 40 mg/kg J-80 halted the growth of the parasite, and when given at 50 mg/kg i.p. twice daily for 5 days, parasitemia was decreased to below the detectable limit, with parasite recrudescence 48 h after the last dose. The in vivo proof of principle demonstrated by a potent, selective, and irreversible inhibitor of Tb20S reveals an alternative path to the development of kinetoplastid proteasome inhibitors that differs from the current focus on allosteric reversible inhibitors.

Well-tolerated and novel antimalarials that can combat multiple stages of the parasite life cycle are desirable but challenging to discover and develop. Herein, we report results for natural product-inspired novel tambjamine antimalarials. We show that they are potent against liver, asexual erythrocytic, and sexual erythrocytic parasite life cycle stages. Notably, our lead candidate 1 (KAR425) displays excellent oral efficacy with complete clearance of parasites within 72 h of treatment in the humanized Plasmodium falciparum (NOD-scid) mouse model at 50 mg/kg × 4 days. Profiling of compound 1 demonstrated a fast in vitro killing profile. In addition, several other tambjamine analogues cured erythrocytic Plasmodium yoelii infections after oral doses of 30 and 50 mg/kg × 4 days in a murine model while exhibiting good safety and metabolic profiles. This study presents the first account of multiple-stage antiplasmodial activities with rapid killing profile in the tambjamine family.

Enterovirus D68 (EV-D68) has had several outbreaks worldwide, yet no FDA-approved antiviral is available for treating this viral infection. EV-D68 infection typically leads to respiratory illnesses and, in severe cases, can cause neurological complications and even death, particularly in children. This study identified a small molecule, A-967079, as an EV-D68 antiviral through phenotypical screening. A-967079 has shown potent and broad-spectrum antiviral activity with a high selectivity index against multiple strains of EV-D68. Pharmacological characterization of the mechanism of action involving time-of-addition, resistance selection, and differential scanning fluorimetry assays suggests that viral 2C protein is the drug target. Overall, A-967079 represents a promising candidate for further development as an EV-D68 antiviral.

The Zika virus (ZIKV) has garnered significant public attention, particularly following the outbreak in Brazil, due to its potential to cause severe damage to the central nervous system and its ability to cross the placental barrier, resulting in microcephaly in infants. Despite the urgency, there remains a lack of targeted therapies or vaccines for the prevention or treatment of ZIKV infection and its related diseases. Fangchinoline (FAN), an alkaloid derived from traditional Chinese medicinal herbs, has a range of biological activities. In this study, we employed both in vitro and in vivo infection models to demonstrate the efficacy of FAN in inhibiting ZIKV. Our findings indicate that FAN effectively suppresses the replication of ZIKV viral RNA and protein, thereby validating its anti-ZIKV capabilities in living organisms. Further analysis through dosing time assays and infectious inhibition assays revealed that FAN exerts its antiviral effects by impeding the early stages of infection, specifically by inhibiting the internalization of ZIKV. These results underscore the potential of FAN as a candidate for anti-ZIKV drug development and offer novel insights into drug design strategies that target the virus's internalization process.

The coronavirus pandemic outbreak of 2019 highlighted the critical importance of preparedness for current and future public health threats (https://www.mmv.org/mmv-open/global-health-priority-box/about-global-health-priority-box). While the main attention for the past few years has been on COVID-19 research, this focus has reduced global resources on research in other areas, including malaria and neglected tropical diseases (NTDs). Such a shift in focus puts at risk the hard-earned progress in global health achieved over the past two decades (https://www.who.int/news-room/spotlight/10-global-health-issues-to-track-in-2021). To address the urgent need for new drugs to combat drug-resistant malaria, emerging zoonotic diseases, and vector control, Medicines for Malaria Venture (MMV) and Innovative Vector Control Consortium (IVCC) assembled a collection of 240 compounds and, in August 2022, launched the Global Health Priority Box (GHPB). This collection of compounds has confirmed activity against emerging pathogens or vectors and is available free of charge. This valuable tool enables researchers worldwide to build on each other's work and save precious time and resources by providing a starting point for the further development of treatments and insecticides. Furthermore, this open access box aligns with two of the many priorities outlined by the World Health Organization (WHO) (https://www.who.int/news-room/spotlight/10-global-health-issues-to-track-in-2021).

In early 2024, the National Academies of Sciences, Engineering, and Medicine (NASEM) released a roadmap for the future of research into mapping ribonucleic acid (RNA) modifications, which underscored the importance of better defining these diverse chemical changes to the RNA macromolecule. As nearly all mature RNA molecules harbor some form of modification, we must understand RNA modifications to fully appreciate the functionality of RNA. The NASEM report calls for massive mobilization of resources and investment akin to the transformative Human Genome Project of the early 1990s. Like the Human Genome Project, a concerted effort in improving our ability to assess every single modification on every single RNA molecule in an organism will change the way we approach biological questions, accelerate technological advance, and improve our understanding of the molecular world. Consequently, we are also at the start of a revolution in defining the impact of RNA modifications in the context of host-microbe and even microbe-microbe interactions. In this perspective, we briefly introduce RNA modifications to the infection biologist, highlight key aspects of the NASEM report and exciting examples of RNA modifications contributing to host and pathogen biology, and finally postulate where infectious disease research may benefit from this exciting new endeavor in globally mapping RNA modifications.