Abstract Invasive candidiasis is a serious fungal infection caused by Candida species that can enter the bloodstream and affect major organs, particularly in immunocompromised patients. Development of resistance to standard antifungal therapies, especially with C. auris, has become a major public health issue. While antimicrobial peptides such as the human cathelicidin, LL-37, could represent a possible therapeutic strategy, they are often inactivated when delivered systemically. One alternative therapeutic strategy is antimicrobial peptoids. We studied in vitro activity of three peptoid compounds, oligomers of N-substituted glycines, which mimic the activity of LL-37. Our aim was to establish these peptoid compounds as a potential treatment of invasive candidiasis and compare them to LL-37′s antifungal activity. In vitro, LL-37 did not exhibit antifungal activity (MIC > 64) against all C. auris strains and three species of Candida. In contrast, three peptoids were tested against ten C. auris strains and six other Candida species and showed good activity. MICs measured as low as 4 μg/mL for TM5 against C. auris strain 381. Mechanistic studies using TEM and fluorescent confocal microscopy of peptoids against C. auris clinical isolates showed membrane disruption and consolidation of organelles. Peptoids TM4 and TM5 showed strong synergistic effects with fluconazole and caspofungin, and amphotericin B. Serial daily passage of TM4 and TM5 at sub-MIC concentrations showed no resistance developing for up to 3 weeks. Fungicidal kinetics revealed complete killing of fungal colonies after around 1 h. A systemic candidiasis mouse model treated subcutaneously with TM5 3 h after C. auris infection resulted in clearing of infection, indicated by colony forming units (cfu) per gram of kidney, at 24 h with the 5 mg/kg dose. This demonstrates that antimicrobial peptoids are active systemically and have potential to be an antifungal treatment for candidemia, particularly as an alternative therapy for resistant strains.
Abstract “Pathogen-hopping” is an approach in infectious disease research with potential to expedite drug discovery. This is done by testing agents which are active against one or more pathogens in a new pathogen, exploiting the fact that orthologues of protein targets essential for survival exist across different pathogens. In some cases, these compounds possess selectivity versus the human orthologue, further aiding the process. To identify new lead compounds for our schistosomiasis drug discovery program, we adopted this approach by first mining the literature to find antimalarial compounds which had not been evaluated against Schistosoma. Next, a filtering process was applied to remove undesirable or potentially toxic compounds. The triaged set of compounds was then screened against Schistosoma mansoni schistosomula and adult worms. Two chemotypes displayed schistosomula and adult worm paralysis activity. Hit assessment was completed for one of these multistage actives to assess its potential for development as an antischistosomal treatment.
Mosquito-borne diseases, particularly malaria, remain a major global health challenge. Among different mosquitoes, Anopheles are solely responsible for malaria. Insecticide-based interventions such as insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are central to mosquito control. However, the widespread emergence of insecticide resistance, especially to pyrethroids, the most commonly used insecticides for mosquito worldwide, threatens their effectiveness. While genetic and metabolic mechanisms of resistance are well characterized, behavioral and sequestration-based mechanisms, particularly those involving chemosensory proteins (CSPs), are less understood. CSPs and odorant-binding proteins (OBPs) are integral components of the mosquito olfactory system, mediating host-seeking and other behavioral processes by transporting odorant molecules to olfactory receptors. Recent evidence suggests that CSPs may also bind insecticides like pyrethroids, contributing to resistance through sequestration. In this study, we investigated the structural and functional roles of CSPs in Anopheles mosquitoes. We determined three high-resolution X-ray crystal structures (apo-form) of CSPs (<2 Å) from Anopheles culicifacies and Anopheles gambiae, representing the first crystal structures of CSPs from any Anopheles species reported to date. Using structural, biophysical and computational approaches, we assessed CSP-ligand recognition of various insecticides, specifically pyrethroids and identified key residues potentially involved in pyrethroid interaction through site-directed mutagenesis. The moderate micromolar binding affinities of deltamethrin and permethrin, compared to other insecticides, support the role of CSPs in pyrethroid sequestration. Our findings offer mechanistic insights into pyrethroid sequestration and provide a foundation for developing novel vector control strategies that target CSP-insecticide interactions to combat pyrethroid resistance.
Abstract The kinetoplastid neglected tropical diseases, Chagas disease, African trypanosomiasis, and leishmaniasis, are vector-borne diseases that remain a persistent global health challenge. Progress has been made in combating human African trypanosomiasis, but efforts to contain other kinetoplastid diseases have been slower. The current drugs used for treatment have limitations, such as drug resistance, toxicity, high cost, and limited access. To overcome these challenges, alternative therapeutic strategies are being explored, including drug combinations, drug repurposing, and nanotechnology, which have shown promise in other diseases, like cancer. Combination therapies, both conventional and nanobased, have the potential to enhance antiparasitic efficacy, reduce side effects, and prevent resistance development. This review focuses on the efficacy and safety of drug combinations for kinetoplastid neglected tropical diseases, highlighting current conventional combination therapies and ongoing research on both conventional and nanobased combination therapies in various stages of development. The review aims to provide novel insights and promote future research for the development of innovative, safe, and effective drug combination therapies. This will help in accelerating progress toward the elimination of Kinetoplastid neglected tropical diseases, in line with Sustainable Development Goal 3.3 to ensure healthy lives and promote well-being for all, at every stage of life and to end the epidemics of neglected tropical diseases by 2030.
Abstract The development of antibacterials with unique mechanisms of action is key to addressing the threat posed by antibiotic resistance. Among the wide array of natural products that target bacterial cell wall biosynthesis, bacitracin A is the preeminent example of an antibiotic that functions by selectively targeting and sequestering the key bacterial phospholipid undecaprenyl pyrophosphate (C55PP). Historically, the bacitracins have been isolated from fermentations of Bacillus species. We recently discovered a series of structurally distinct bacitracin-like peptide antibiotics produced by members of the Paenibacillus genus, termed the paenitracins. Here, we report the total synthesis of paenitracin B (1), enabling definitive stereochemical validation of the previously proposed structure and delivering quantities of material suitable for more extensive antibacterial testing. The synthetic route developed was also applied to the preparation of paenitracin analogues, with some showing significantly enhanced antibacterial activity, particularly against VanA-type vancomycin-resistant Enterococcus faecium clinical isolates.
Abstract The continued spread of drug-resistant Mycobacterium tuberculosis (Mtb) necessitates the development of new chemotypes with novel modes of action. Here, we report 5-carboxamide-substituted barbituric acid as a promising scaffold for developing new drugs against Mtb. Fenoxacrim, the initial hit compound identified through phenotypic screening, demonstrated potent activity against Mtb. It is active against drug-susceptible and multidrug-resistant clinical Mtb isolates as well as against intracellular Mtb in infected THP-1 derived macrophages. Fenoxacrim demonstrates high selectivity (>73-fold) for the pathogen over several tested mammalian cell lines. Other than Mtb, fenoxacrim exhibits strong bactericidal activity against several Gram-positive pathogens, including methicillin-resistantStaphylococcus aureus (MRSA), with in vitro potency comparable to, or exceeding several clinically used antibiotics. Medicinal chemistry optimization efforts yielded new fenoxacrim analogues with improved Mtb specificity, enhanced potency, and reduced cytotoxicity. Mode-of-action studies suggest that fenoxacrim inhibits undecaprenyl pyrophosphate synthase (UPPS) and dissipates the bacterial membrane potential. Together, these findings highlight the potential of 5-carboxamidebarbituric acid derivatives as a foundation for developing new antibacterial agents with novel modes of action for the treatment of tuberculosis and other drug-resistant bacterial infections.
Adenovirus infections are a leading cause of respiratory and gastrointestinal diseases, representing a significant global health challenge. Rapid and accurate detection of adenovirus types is essential for timely diagnosis and effective management. Traditional diagnostic methods, such as PCR, are often time-consuming and require complex laboratory infrastructure, limiting their application in resource-limited settings. In this study, we present a CRISPR/Cas12a-based assay integrated with a hydrogel microarray for the simultaneous detection of six common adenovirus types (1, 2, 3, 4, 7, and 14). After amplification of adenoviral DNA using recombinase polymerase amplification (RPA), the amplified DNA enters the hydrogel, where it activates the Cas12a-crRNA complex trapped within the gel. This activation leads to the cleavage of an ssDNA reporter, generating a fluorescent signal. The use of a hydrogel microarray enables efficient and multiplexed detection of adenovirus types in a single assay. The method demonstrated high sensitivity, with detection limits ranging from 10 to 50 copies/μL across the six adenovirus types. It also showed excellent specificity, with no cross-reactivity observed with other respiratory viruses. Clinical validation with 30 human adenovirus samples revealed 100% specificity and high concordance with qPCR results. This CRISPR/Cas12a-based hydrogel microarray platform offers a rapid, cost-effective, and highly specific diagnostic tool with significant potential for clinical and public health applications.
Abstract Plasmepsin V is an essential aspartyl protease that cleaves the Plasmodium export element in proteins targeted for export from Plasmodium parasites into the host red blood cell. Substrate-mimicking peptidomimetics have been developed that potently inhibit plasmepsin V but show modest parasite activity. To advance these inhibitors, we optimized their potency by introducing a 2-chloro aryl group at P2 and an N-terminal sulfonamide that yielded WM960, which showed markedly improved plasmepsin V inhibition while retaining high selectivity over plasmepsin IX and X and human aspartyl proteases. WM960 also demonstrated enhanced activity against asexual blood-stage parasites and effectively suppressed protein export. However, WM960 displayed low metabolic stability and limited systemic exposure in mice, highlighting the need to improve pharmacokinetic properties in future development of peptidomimetic plasmepsin V targeted antimalarials.
Abstract Farnesol is a quorum-sensing sesquiterpene produced by Candida albicans that inhibits filamentation, a morphogenetic transition underlying fungal invasion and virulence. Notably, several farnesyl-derived sesquiterpenes bearing functional groups other than the original alcohol of farnesol have also been reported to suppress filamentation, indicating that this activity is not unique to farnesol itself but may instead reflect shared properties of the farnesyl scaffold. This observation prompted us to probe whether differences in the intracellular distribution of farnesyl-derived molecules might accompany differences in their morphogenesis-suppressing activity. To address this question, we synthesized two fluorescent farnesyl-derived probes, F-1 and F-2, together with dodecyl-chain analogues as controls. Both probes preserve the native farnesyl isoprenoid scaffold but differ in fluorophore identity, with F-1 labeled with a tetramethylrhodamine (TAMRA) dye and F-2 labeled with a 7-diethylaminocoumarin dye. Counterintuitively, in live C. albicans cells, F-1, despite bearing the more sterically demanding TAMRA dye, inhibited filamentation and displayed broad cytosolic distribution with enrichment along the tubular mitochondrial network. Inhibition by F-1 was reversed by exogenous dbcAMP, consistent with a mechanism like that of farnesol involving the Ras1-cAMP signaling pathway. In contrast, F-2 showed no antifilamentation activity and localized predominantly to cytosolic membranous compartments with irregular morphology. Notably, F-1 retained mitochondrial enrichment and filamentation-inhibitory activity in drug-resistant strains, and its intracellular accumulation at biologically active concentrations was not substantially diminished by enhanced efflux. Together, these findings demonstrate that chemical modification of farnesyl-derived sesquiterpenes, including incorporation of substituents with substantial molecular weight and steric bulk, can preserve morphogenesis-inhibitory activity. These results support a model in which the pleiotropic morphogenesis-inhibitory effects of farnesyl-derived compounds involve not only plasma membrane-associated signaling but also access to intracellular compartments, including mitochondria.
Trimethoprim-sulfamethoxazole (SXT) remains central to melioidosis eradication therapy, yet the genetic basis of resistance evolution in Burkholderia pseudomallei is not fully defined. Using Burkholderia thailandensis as a biosafe surrogate, we subjected populations to stepwise in vitro evolution under increasing SXT concentrations. Whole-genome sequencing revealed rapid population diversification followed by selective sweeps leading to fixation of fitter resistant variants. Early adaptation involved mutations in regulators of RND efflux systems and folate-associated genes, consistent with multifactorial resistance. Despite this heterogeneity, all evolutionary trajectories converged on a single nonsynonymous substitution, I99L, in dihydrofolate reductase (DHFR/FolA), which rose to fixation at high SXT concentrations. This substitution mirrors changes reported in SXT-resistant B. pseudomallei isolates from chronic infections. Allelic reconstruction demonstrated that Bt-DHFR(I99L) is sufficient to confer a 4- to 16-fold increase in trimethoprim MIC. Biochemical and structural analyses showed preserved catalytic activity but reduced trimethoprim binding, consistent with an allosteric resistance mechanism. Together, these findings establish DHFR as a dominant evolutionary target under SXT pressure and support B. thailandensis as a robust model for dissecting resistance evolution in B. pseudomallei.
Nipah virus (NiV), a World Health Organization priority pathogen, is a lethal enveloped RNA virus within the Paramyxoviridae family. NiV attachment G and fusion F glycoproteins execute viral-cell or cell-cell membrane fusion during viral entry and syncytia formation, respectively. Despite past studies, several aspects of the molecular-level orchestration underlying this process, such as how F, G, and their interactions regulate fusion, remain poorly understood, obscured by challenges in decoupling fusion determinants in complex cellular environments. Here, we report a reconstitution-based approach for mimicking the NiV-host interface, employing virus-like particles with NiV M, F, and/or G proteins and plasma membrane vesicles derived from NiV-permissive host cells. We found this minimal system can accurately recapitulate NiV-host cell fusion, and used this platform for biophysical dissection of NiV fusion using wild-type or mutant F and G proteins affecting fusion at distinct intermediate steps. We show that F and G affect hemifusion and pore formation stages of NiV fusion in distinct ways, revealing a tightly regulated fusion landscape. Our approach also delivers one of the first direct measurements of temperature thresholds for NiV fusion, providing novel insights into energetic requirements underlying viral fusogenicity. These findings demonstrate the power of this approach in dissecting aspects of NiV fusion not accessible via cell-based assays. Thus, this study serves as a blueprint for comprehensive fusion analysis of other viruses with multicomponent fusion machinery and host protein receptors, and could be repurposed into therapeutic screening platforms targeting their fusion mechanisms, necessary for developing novel antivirals.
Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to South America caused by thermodimorphic fungi of the genus Paracoccidioides, particularly P. brasiliensis. Calcineurin inhibition by cyclosporine A (CsA) perturbs fungal thermodimorphism and growth. Here, we asked whether pre-exposure of P. brasiliensis yeasts to CsA modulates host-pathogen interactions during infection. Mice infected with CsA-pre-exposed fungi exhibited significantly reduced levels of pro- and anti-inflammatory cytokines, along with decreased recruitment and activation of innate and adaptive immune cells at both time points. Although tissue inflammation and lesion areas were diminished, fungal burdens in lungs and liver were similar between groups. Importantly, yeasts recovered from lungs 72 h after infection with CsA-pretreated fungi displayed reduced in vitro colony growth, suggesting that prior CsA exposure induces a persistent slow-growth physiological state. This altered phenotype may impair effective immune recognition and inflammatory activation. As host immunosuppression was excluded in the experimental design, fungus-intrinsic phenotypes consistent with transient calcineurin pathway inhibition: altered growth/morphogenesis and putatively modified PAMP exposure that together blunt early immune activation. We interpret late-phase differences as downstream of these early pathway-dependent shifts rather than as heritable reprogramming. Overall, pre-exposure to CsA modulates P. brasiliensis-host interaction, dissociating tissue injury from fungal burden, highlights the role of calcineurin in fungal adaptation, and demonstrates how environmental exposure to bioactive compounds can influence fungal virulence and disease outcomes.
Staphylococcus aureus is a leading cause of antibiotic-resistant infections worldwide. Therefore, there is a constant need to develop new growth inhibitors. Pentamidine analogs (PAs) composed of thiourea-linked amidines were evaluated for activity against methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria. S. aureus, MRSA, Staphylococcus epidermidis, Streptococcus pyogenes, Mycobacterium, and Bacillus were highly susceptible with minimal inhibitory concentration (MIC) values ranging from 1.56 to 6.25 μM, whereas Enterococcus species and Clostridioides difficile were not as susceptible with MICs of 50 to ≥100 μM. For S. aureus, MRSA and VISA strains, PAs showed ∼4-fold lower MICs when compared to pentamidine (PNT). The concentration of PAs needed to lyse 50% of red blood cells (HC50) was ∼16-32-fold higher than their corresponding MICs. For PNT, a lower HC50/MIC range of 1.25-5 was observed. Approximately 60% of MRSA was cleared from infected Caenorhabditis elegans by the PAs. PAs also provided 100% protection in a septicemia model of infected mice. Morphological changes induced by these compounds indicate disruption of cell membrane integrity, where >80% of treated bacterial cells took up propidium iodide in a permeation assay. Importantly, there was a lack of resistance observed with the PAs, compared to the fairly rapid and stable resistance occurrence for drugs commonly used to treat S. aureus infection. These results reflect the potential of such compounds as potential new tools to optimize and combat the menace of drug-resistant S. aureus.
Since Fleming's discovery of penicillin, antibiotic resistance has remained an inevitable biological reality; however, our guidelines and diagnostic practices are not keeping pace with the rising medical need. This viewpoint uses a real family case of recurrent Group C streptococcal pharyngitis to illustrate these gaps in an outpatient setting. The situation is obviously more dire with life threatening infections. Addressing the fragile antibiotic pipeline requires updated, accessible guidelines, routine culturing upon relapse, and public education to support fair pricing and sustained investment in novel antibiotics and companion diagnostics.
To decentralize tuberculosis surveillance in resource-limited and tropical regions, a rapid, robust, and cold-chain-independent near-point-of-care biosensor is urgently required to overcome the instability of conventional liquid-formulation diagnostics. Herein, we developed a ready-to-use lyophilized reaction mix Loop-Mediated Isothermal Amplification (LAMP) matrix integrated with a Lateral Flow Biosensor (LFB) for the instrument-free visual detection of Mycobacterium tuberculosis (M. tuberculosis). The freeze-dried reagents, stabilized with an optimized trehalose-BSA matrix, were hermetically packaged and systematically evaluated for long-term thermal stability across four regulated profiles (-20 °C, 4 °C, 25 °C, and 37 °C) over a 12-month period. Diagnostic viability was further validated in a double-blinded study using 191 clinical sputum specimens against gold-standard culture and line probe assays. The stabilized platform demonstrated an analytical sensitivity down to 102 CFU/mL and maintained full functional activity for up to 12 months at -20 °C and 4 weeks at 4 °C, with robust short-term resilience under tropical stress (25 °C) for up to 1 week. However, the formulation was unable to withstand prolonged exposure to harsh environmental conditions at 37 °C, resulting in a progressive decline in amplification performance following phase-transition instability. Crucially, clinical validation yielded an outstanding diagnostic sensitivity of 98.92% and a positive predictive value (PPV) of 100.00%. These findings validate the lyophilized LAMP-LFB platform as a highly reliable, sustainable, and distributable molecular diagnostic tool capable of overcoming the cold-chain bottleneck in field-based tuberculosis detection. Although the developed platform shows strong potential for near-point-of-care deployment, further optimization of the lyophilization formulation and process will be necessary to enable prolonged storage and transportation under ambient-temperature conditions.
Influenza, an acute respiratory infectious disease caused by influenza viruses, poses a serious public health threat with high infectivity and virulence. Existing antivirals suffer from numerous limitations, such as the frequent emergence of drug resistance and inconvenient administration, highlighting the urgent need for the development of next-generation anti-influenza agents. As a core component of the RNA-dependent RNA polymerase (RdRp) complex, the PB2 subunit mediates cap binding in the cap-snatching process, a prerequisite for viral mRNA transcription. Owing to its indispensable biological roles, high sequence conservation, and distinct structural differences from host proteins, PB2 serves as an attractive therapeutic target for antiviral drug development. Recently, the approval of onradivir, the first-in-class PB2 inhibitor, has not only validated the scientific rationale and feasibility of drug discovery targeting the PB2 subunit but also underscored the considerable clinical potential of this novel class of agents. In this review, we systematically summarize the research advances in PB2 inhibitors and discuss the challenges and prospects for their broader clinical application, with the aim of providing new insights into the development of novel anti-influenza drugs.
Abstract Pseudomonas aeruginosa (P.a) is a common opportunistic pathogen causing serious infection and complications in diverse populations, especially under immunodeficient conditions. Previous studies indicate that mitophagy activation is related to multiple conditions and processes in the human body, including infectious diseases. It has been recently implicated that the PARL-PGAM5-PINK1 axis activates mitophagy, but its regulatory role in pneumonia is unclear. We hypothesize that mitophagy receptor PHB2 orchestrates an autophagic process through PARL and PGAM5 to limit P.a. infection and inflammation. By knocking down PHB2, we explored the regulatory role of mitophagy in P.a infection and evaluated the underlying mechanism of the PARL-PGAM5-PINK1 signals both in vitro and in P.a pneumonia mice. Our study also reveals that the critical factors, such as PARL and PINK1, may have potential for therapeutic targeting during Pseudomonas infection.
Abstract Antisense oligomers show promise for the treatment of infectious diseases but face challenges as therapeutic agents due to limited uptake in bacteria. Although cell-penetrating peptides can serve as effective delivery vehicles, they may exhibit nonspecific effects and can contribute to resistance development. Aminoglycoside-peptide nucleic acid (PNA) conjugates represent a complementary delivery strategy that leverages the intrinsic cationic and membrane-interacting properties of aminoglycosides to enhance cellular uptake. In this work, we synthesized tobramycin-PNA (TOB-PNA) conjugates targeting the translation initiation region of the acyl carrier protein gene (acpP) in Enterobacterales. Conjugation of TOB to PNA enhanced target-binding stability, improved the inhibition of translation in vitro, and enhanced activity against Gram-negative bacteria. TOB-PNA activity was further enhanced in combination with the outer membrane-permeabilizing antibiotic polymyxin B (PMB), leading to improved antibacterial activity against clinically relevant Gram-negative pathogens, including drug-resistant isolates. Resistance studies demonstrated that the TOB-PNA conjugate, both alone and in combination with PMB, maintains antibacterial activity over prolonged exposure, suggesting a higher barrier to resistance evolution than conventional TOB treatment. Mammalian toxicity studies in HEK293T cells showed IC50 > 100 μM. Morphological analyses indicated that TOB-PNA treatment induces cellular stress and division abnormalities consistent with disruption of essential metabolic pathways, while PMB increased membrane permeability. In addition, TOB-PNA behaved synergistically with other compounds targeting distinct cellular processes, including fatty acid biosynthesis (triclosan), folate metabolism (trimethoprim), and cell morphology (A22).
