ACS Infectious Diseases

Conditionally essential pathways - such as the biotin biosynthesis - represent promising targets for new antibiotics. However, the chemical interrogation of the biotin pathway with an orally effective lead remains elusive, and the preclinical development of biotin inhibitors for mycobacterial infections in vivo is challenging due to the unusually high concentration of biotin in standard mouse models. Structure-guided optimization was applied to develop the first oral lead targeting aminotransferase BioA, a key enzyme in bacterial biotin biosynthesis, resulting in C48, a picomolar inhibitor displaying sub-micromolar MICs against Mycobacterium tuberculosis (Mtb). Mechanism of action was confirmed by biochemical, structural, and genetic studies. C48 demonstrated favorable pharmacokinetics and excellent oral bioavailability resulting in over 39,000-fold improved exposure. We next developed an easy-to-operate, low-biotin mouse model that recapitulates human biotin physiology. C48 significantly reduced Mtb burden in this low-biotin mouse model, providing the first in vivo proof-of-concept for targeting biotin biosynthesis in Mtb.

The virulence of Toxoplasma gondii and other apicomplexan parasites relies on a unique form of cellular motility driven by MyoA, an unconventional class XIV myosin motor protein. To identify new chemical probes for investigating the molecular mechanisms of parasite motility, we screened over 50,000 small molecules for inhibitors of T. gondii MyoA (TgMyoA). The top hit from the screen, UCB-9721, is almost 40-fold more potent as an inhibitor of TgMyoA actin-activated ATPase activity than the previously described TgMyoA inhibitor, KNX-002, and 45-fold more potent at inhibiting parasite motility, with no detectable toxicity towards mammalian cells. UCB-9721 also inhibited the motility and/or growth of the related apicomplexan parasites Plasmodium falciparum, Cryptosporidium parvum, and Babesia duncani, suggesting that this compound will be a useful new chemical probe for studying motility and MyoA function in apicomplexan parasites more broadly. While UCB-9721 and KNX-002 were identified independently, they share a similar chemical scaffold. To determine why UCB-9721 is so much more potent than KNX-002 and to inform future development of this inhibitor class, we undertook comparative molecular docking analyses, targeted TgMyoA mutagenesis, and a directed structure-activity relationship analysis. The results identified the sulfonamide group of UCB-9721 and its hydrogen bond interactions with R249, E275 and a stabilized water network within the TgMyoA binding pocket as key to the compounds increased potency. Further development of UCB-9721, informed by the results presented here, may transform this promising new chemical class into actionable drug development leads against this important group of human and animal pathogens.

Accurate quantitation of therapeutic bacteriophages (phages) remains a challenge for clinical development. Plaque-based enumeration is the current standard but is laborious, host-dependent, and variable, particularly when distinguishing individual phages in cocktails. Targeted mass spectrometry of virion structural proteins offers an orthogonal, structure-based approach amenable to reproducible and scalable phage quantitation. Here, we describe a targeted proteomic liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for host-independent quantitation of the Pseudomonas aeruginosa podovirus LUZ19. Proteomic characterization was performed on an LTQ Orbitrap XL to assess sequence coverage and select surrogate peptide candidates based on specificity and sensitivity. High-resolution peptide mapping identified multiple structural proteins of LUZ19 and provided 55% sequence coverage for the major head protein (YP_001671977.1). Fifteen peptides were detected and evaluated, from which the tryptic peptide EVAELDGQELAR was selected based on abundance, stability, and chromatographic performance. Quantitative analysis was conducted on a QTRAP 7500+ using optimized multiple reaction monitoring transitions for targeted peptide detection. Back-calculated concentrations met accuracy criteria across a validated range of 0.008 to 80 pg/mL, with bias spanning -8.2 to 8.2%, intra-day precision ranging from 0.5 to 9.8%, and inter-day precision ranging from 6.3 to 9.7%. Peptide concentrations from digested lysate samples were related to phage concentrations determined by double layer agar assay, yielding an estimated three copies of the major head protein per virion. ImportanceBacteriophages are the most abundant biological entities on the planet and represent a promising therapeutic class for combating drug-resistant bacterial infections. Realizing the clinical potential of bacteriophage therapy requires analytical methods capable of meeting the standards of modern drug development. Targeted mass spectrometry offers unmatched specificity and resolution for precise quantitation of individual bacteriophages within complex biological samples, a capability that conventional enumeration methods cannot match. Only one prior study has applied mass spectrometry to bacteriophage quantitation, using a well-characterized model bacteriophage at a single concentration without calibration or a validated analytical range. Using Pseudomonas aeruginosa podovirus LUZ19, we present the first targeted mass spectrometry-based bacteriophage quantitation assay developed and validated following FDA bioanalytical guidance. This work establishes a rigorous analytical foundation that moves bacteriophage therapy closer to the standards required for informed dose selection, candidate evaluation, and clinical development.

Klebsiella pneumoniae (Kp) is a common antibiotic-resistant pathogen that colonizes the gastrointestinal tract and can disseminate to peripheral sites, causing a range of infections including bacteremia, urinary tract infections, and pneumonia. Intestinal colonization with Kp is a risk factor for subsequent infection, as the colonizing strain frequently corresponds to the infecting isolate. Accordingly, targeting Kp prior to dissemination at the site of colonization through decolonization strategies offers a promising approach to mitigate infection risk. In this study, we evaluated the repurposing of existing drugs with previously uncharacterized antibacterial activity as candidates for Kp decolonization. To this end, we screened an antiviral compound library for their activity against Kp. We identified and validated six compounds with previously uncharacterized activity against Kp. Then, we screened a library of clinical Kp strains against a subset of these compounds and found that their activity was strain-specific to degrees that differed based on the compound. Finally, we tested the activity of these compounds in conditions relevant to the human gut. We determined the activity of these candidates was dependent on biological context. Collectively, these findings support further investigation of antiviral drugs as potential gut decolonization therapies for Kp.

New antitubercular agents acting through previously unexploited mechanisms are urgently needed. Using a drug-repurposing platform, we identified TI-374, a hydroxamic acid containing compound that inhibits Mycobacterium tuberculosis (Mtb) with sub-micromolar potency. Systems analysis, resistance mapping, supplementation assays, and biochemical studies showed that TI-374 inhibits two PLP-dependent aminotransferases, AlaA and HisC1. However, its activity is driven primarily by irreversible inhibition of AlaA, whereas HisC1 inhibition is only partially reversible, revealing differential reversibility between the two targets. Optimization yielded TI-801, a low-nanomolar AlaA inhibitor. Both compounds remained active against intracellular Mtb in a macrophage infection model, where alanine supplementation did not rescue growth, indicating that host-derived alanine is unlikely to bypass AlaA inhibition. Genetic deletion of alaA attenuated Mtb survival in a murine infection model. Together, these findings support AlaA as a host-relevant metabolic vulnerability in Mtb and TI-801 as a mechanistic chemical probe for its validation as an antitubercular target.

Resistance to {beta}-lactam antibiotics, including carbapenems, mediated by metallo-{beta}-lactamases (MBLs), including the New Delhi metallo-{beta}-lactamase (NDM) MBL subfamily, is increasing. No MBL inhibitors are currently approved for clinical use with most reported MBL inhibitors are metal ion chelators, acting either at the Zn(II) ion active site and/or in solution. The hexokinase inhibitor 3-bromopyruvate (3-BP) is reported to inhibit NDM-1. We found that 3-BP selectively restored the antimicrobial activity of meropenem against carbapenem resistant Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii strains, obtained from clinical and environmental isolates from Tanzania and Malawi, containing genes that encode NDM-1 or NDM-5, but not against strains containing genes encoding for serine {beta}-lactamases. Mass spectrometry studies with NDM-1 and NDM-5 support a mechanism involving covalent reaction of 3-BP with an active site cysteine residue. The results will promote work on the development of covalently reacting MBL inhibitors, a strategy that has been successful for inhibition of the nucleophilic serine {beta}-lactamases.

Antimicrobial resistant infections present a growing threat to public health and were associated with or directly caused 6 million deaths globally in 2021. This huge death toll highlights the need for novel strategies to address AMR infections. Interfering with the regulation of resistance mechanisms could provide an alternative approach to treat drug-resistant infections. PhoQ, a sensor histidine kinase ubiquitous amongst gram-negative bacteria, regulates several virulence factors, as well as resistance to outer membrane-targeting antibiotics, making it an attractive target for adjuvant therapy development. However, the identification of potent small molecule inhibitors is limited by the assays available for in vitro assessment of binding and activity inhibition in PhoQ. Thus, we sought to investigate the use of a fluorescence-based assay to evaluate enzymatic activity, as well as a thermal shift assay to assess inhibitor-protein binding in PhoQ. Together, these newly implemented protocols are valuable contributors to the toolbox of methods available for the development of PhoQ-targeted inhibitors to block this major contributor to antimicrobial resistance.

Malaria remains a major global health challenge, with emerging partial resistance to first-line therapies in Africa threatening current control efforts. Drug combinations are essential to improve treatment efficacy and restrain resistance development. However, in vitro assays that quantify parasite viability after drug exposure and characterize pharmacodynamic drug interactions are labor- and resource-intensive, with standard approaches such as the parasite reduction ratio assay limiting systematic, high-resolution evaluation of drug combinations. We present the MUltidimensional Luminescence Test for integration of interactions (MULT-i2), an in vitro assay that enables scalable, high-resolution assessment of parasite viability across multidimensional drug concentration spaces. For dual drug combinations, the MULT-i2 assay characterizes interaction surfaces while requiring [~]50-fold fewer resources and more than two-fold less time than conventional methods, enabling exploration of broader combination scenarios. The assay combines a highly sensitive chemiluminescence readout with inducible reporter expression in Plasmodium falciparum, supporting potential extension to multidimensional combination testing. Using the general pharmacodynamic interaction (GPDI) model, the MULT-i2 assay quantified interaction potency and directionality, confirming and refining the known synergy between atovaquone and proguanil, and revealing detailed interaction patterns for additional drug combinations. Overall, this approach provides an efficient framework for testing and characterizing pharmacodynamic drug interactions and supports the rational development of antimalarial combination therapies.

The prokaryotic ubiquitin-like protein (Pup) conjugation system (PPS), which is essential for Mycobacterium tuberculosis (Mtb) virulence but absent in humans, presents an attractive drug target. Here, we report the discovery of ARQ-501, a quinone-based, covalent, substrate-competitive inhibitor of the Pup ligase PafA. ARQ-501 exhibited potent anti-mycobacterial activity against Mtb under host-mimicking stress conditions and within macrophages. We further identified the catalase-peroxidase KatG, essential for activation of the frontline prodrug isoniazid (INH), as a pupylation substrate. ARQ-501 inhibits KatG pupylation, causing its accumulation and creating a selective synergy with INH. This quantity over quality mechanism successfully rescued INH activation by the clinically prevalent KatG S315T mutant in enzymatic assays and enhanced INH efficacy against clinical S315T isolates to variable degrees. This work identifies a novel class of PafA inhibitors and a previously unrecognized role of pupylation in regulating KatG, offering a potential therapeutic avenue to combat drug-resistant tuberculosis.

The persistence of P. aeruginosa infections is largely driven by the secretion of several factors during invasion, including the redox-active phenazine pyocyanin (PYO), which promotes biofilm formation and oxidative stress. Biofilms contribute to chronic infections and antibiotic resistance, limiting the efficacy of conventional therapies. We found that a synthetic compound, I-152, a conjugate of N-acetyl-L-cysteine (NAC) and S-acetylcysteamine (also known as S-acetyl-{beta}-mercaptoethylamine; SMEA), effectively restored colistin susceptibility against P. aeruginosa by altering biofilm nanomechanical properties. These perturbations in matrix integrity were associated with I-152s ability to hinder the phenazine redox cycle, shifting PYO to a reduced state and promoting chemical interactions (S-conjugates). The compound decreased PYO accumulation in bacterial cultures and PYO-generated reactive oxygen species (ROS) in macrophage cells. Together with PYO, LPS is another driver of ROS-dependent inflammatory signaling in the host, which leads to an uncontrolled cytokine response and organ damage, especially in patients with cystic fibrosis. I-152 treatment downregulated the expression of LPS-induced inflammatory cytokines, i.e., IL-6 and TNF-, in bone marrow-derived macrophages (BMDM) isolated from transgenic CFTR-/- and CFTR+/+ mice. Consistently, I-152 partially counteracted the inflammatory response in the P. aeruginosa LPS-induced acute lung injury murine model. Taken together, these results support I-152 as an adjunctive treatment for P. aeruginosa respiratory infections through a dual mechanism: combating antimicrobial resistance in biofilms and dampening host inflammation in the respiratory system.

F. tularensis is a highly infectious Gram-negative bacterial pathogen that causes tularemia, a re-emerging zoonosis of public health concern. Here we identify respiratory complex I as a selective vulnerability in Francisella and define the mechanism of action of a pyrazole compound, tolfenpyrad, with species-specific antibacterial activity. Using F. novicida as a surrogate model, we demonstrated that tolfenpyrad selectively inhibits growth with no measurable effect on E. coli or P. aeruginosa. Tolfenpyrad rapidly suppressed oxygen consumption, depleted ATP, collapsed proton motive force, and induced reactive oxygen species, indicating disruption of bacterial metabolism. Biochemical assays demonstrated selective inhibition of NADH-dependent respiration and membrane-associated NADH oxidation, whereas succinate-driven respiration was unaffected. Moreover, the alternative NADH dehydrogenase (ndh) was not required for tolfenpyrad activity. Structural docking identified a potential tolfenpyrad-binding pocket within the membrane subunit NuoM. These findings reveal species-specific inhibition of Francisella complex I and establish respiratory metabolism as a promising antimicrobial target in these bacteria.

In vitro methods to characterize drug combinations typically involve phenotypic screenings using checkerboard assays (CBA) or, more recently, DiaMOND. Such approaches rely on the Fractional Inhibitory Concentration Index (FICI), a fixed-time measurement of growth inhibition that, nonetheless, necessitates secondary validation by time-kill assays (TKA). Longitudinal time-kinetics of bacterial killing are considered the gold standard in vitro proxy for antimicrobial activity, but they required increased assay complexity, particularly against the slow growing Mycobacterium tuberculosis. Here, we developed a new methodology named OPTIKA (Optimized Time Kill Assays) that enhances the capacity of traditional TKA by over 1000-fold. This allows for easy and dynamic examination of n-way drug interactions by simultaneously monitoring bactericidal and sterilizing capacities in a longitudinal manner. We then replicated previous DiaMOND studies and performed comparisons using CBA and OPTIKA methodologies. We demonstrate that selection of the efficacy parameters (either routed on bacteriostatic, bactericidal or sterilizing properties) affects the interpretation of in vitro drug interactions and, consequently, its potential translational value. The increased assay throughput provided by OPTIKA offers a novel framework for developing tuberculosis treatment regimens. TeaserOPTIKA is a new methodology that increases time-kill assay performance against Mycobacterium tuberculosis by over 1,000-fold

Periodontitis is a chronic inflammatory disease driven by complex subgingival multispecies biofilms, in which Porphyromonas gingivalis plays a central role in coordinating microbial interactions. Together with host-associated factors, these microbial communities create dual constraints that limit antimicrobial efficacy. In this study, we engineered a library of recombinant lysins by fusing membrane-destabilizing peptides to the periodontal pathogen-derived lysin LysPd078 and identified four optimized variants (PlyPd06, PlyPd19, PlyPd27, and PlyPd44) with enhanced salt tolerance, environmental stability, and bactericidal activity. In a clinically derived polymicrobial oral biofilm, PlyPd44 at only 25 g/mL eradicated 96.6% of P. gingivalis. In a humanized oral microbiota mouse model of periodontitis, selected PlyPds significantly reduced inflammation and inhibited alveolar bone loss compared with LysPd078 and minocycline. Collectively, these results establish membrane-destabilizing peptide-enabled lysins as a promising platform for developing microenvironment-adapted precision antimicrobials for periodontitis.

Mycobacterium tuberculosis possesses a flexible metabolic system helping it to survive inside the host. The type II NADH dehydrogenase, composed of Ndh and NdhA, essential for bacilli, is a promising drug target. Based on ATP depletion values, two quinoline scaffolds were shortlisted after screening of a library of drug like molecules. Structurally, both 64-9C and 64-9D carry ester moieties at the 5- and 8-positions of the quinoline core, respectively. Ease to re-synthesise 64-9D resulted in synthesis of a focused library of compounds, with MIC values of 0.25-4 g/mL, consistent with ATP depletion. These compounds exhibited bactericidal activity against non-replicating mycobacteria, and showed potent efficacy against multidrug-resistant isolates. Altered, intracellular NADH/NAD+ ratio and reduced respiration was indicative of oxidative phosphorylation inhibition. Inhibition of the purified recombinant NDH protein uncompetitively, SNPs in gene encoding NDH-2 for selected one step mutants and, molecular modelling of 4FQN and 2FQN validated NDH-2 as a target for these compounds. The derivative 2FQN exhibited dose-dependent bactericidal efficacy in mice, underscoring the potential of the series as a promising anti-tuberculosis candidates.

Antibiotic resistance in Mycobacterium tuberculosis is a pressing global health challenge demanding new therapeutic strategies. The bacterial respiratory chain comprises promising antibacterial targets, with dual inhibition of the terminal oxidases cytochrome bcc:aa3 and cytochrome bd (cyt bd) showing bactericidal activity. While bcc:aa3 inhibitors such as Q203 have advanced clinically, cyt bd remains underexplored due to difficulties in assigning activity of the purified enzyme and structurally resolving the quinol substrate binding site. Here, we report a rapid in vitro screening platform for cyt bd inhibitors by engineering a minimal respiratory system that couples the activity of cyt bd to that of a type 2 NADH dehydrogenase. This coupled assay enables spectroscopic monitoring of NADH oxidation as a proxy for cyt bd activity, allowing rapid screening of over 10,000 compounds. Screening identified WSL017, a fragment with low micromolar potency against both M. tuberculosis and E. coli cyt bd. Kinetic and structural analyses revealed competitive inhibition at the quinol-binding site, providing the first structural insights into cyt bd inhibition by a non-quinone scaffold. WSL017 displayed growth inhibition of M. tuberculosis H37ra, corroborating oxidase inhibition as a promising therapeutic strategy. This work establishes a pipeline for cyt bd inhibitor discovery and highlights new opportunities for structure-guided drug development against cytochrome bd oxidases.

Gonorrhea is the second most common bacterial sexually transmitted infection and affects about 80 million people worldwide annually. The causative agent, Neisseria gonorrhoeae, has become resistant to almost every antibiotic used for its treatment. There is no licensed vaccine against gonorrhea. Therefore, there is an urgent need to develop novel prevention and treatment strategies to curb the spread of gonorrhea. The gonococcus has evolved several mechanisms to evade complement, a key arm of immune defenses against this pathogen, including binding of the human complement inhibitors Factor H (FH) and C4b-binding protein (C4BP). We previously showed that chimeric molecules fusing the gonococcal binding domains of FH and C4BP to IgG Fc and IgM Fc, respectively, mediate complement-dependent killing of gonococci in vitro and attenuate gonococcal colonization of mouse vaginas when administered topically. Here, we fused C4BP domains 1 and 2, which contain the gonococcal binding region, to IgG Fc bearing the IgM tail-piece to facilitate Fc hexamerization. This molecule, called C4BP-Hexa IgG Fc, showed [~]650-fold greater complement-dependent bactericidal activity on a molar basis than monomeric C4BP-IgG1 Fc. C4BP-Hexa IgG Fc enhanced association with and uptake by human neutrophils in a complement-independent manner. Despite off-target complement activation in solution, C4BP-Hexa IgG Fc reduced both the duration and the bacterial burden of gonococcal vaginal colonization in human FH and C4BP transgenic mice when administered intravaginally daily. In conclusion, we show proof-of-concept of the efficacy of a hexameric C4BP IgG Fc fusion molecule against N. gonorrhoeae, which could aid in the fight against this multidrug-resistant pathogen.

Scrub typhus is a potentially fatal infectious disease caused by the obligate intracellular bacterium Orientia tsutsugamushi. While antibiotic treatment is generally effective, it requires extended treatment, and drug resistance and treatment failures have emerged. O. tsutsugamushi encodes a deubiquitylating enzyme, OtDUB, which interferes with host ubiquitin-dependent pathways. OtDUB cleaves ubiquitin from various substrates, but whether this activity can be selectively targeted by small molecules is unknown. Here we have screened a chemically diverse small-molecule library using a fluorescence-based deubiquitylation assay to identify potential inhibitors of OtDUB. Two compounds, gentisic acid and amiloride hydrochloride, inhibited OtDUB activity at low dosage, with little effect on the related Wolbachia CidB or yeast Ulp1 enzymes. Computational docking predicted the compounds engage regions near the OtDUB catalytic pocket, suggesting a competitive mode of inhibition; this was supported by enzyme kinetic analyses. Neither compound caused detectable cytotoxicity in mammalian cells. Amiloride hydrochloride treatment reduced both total cellular deubiquitylating activity and the O. tsutsugamushi bacterial load in infected cells. While the identified compounds are not optimized inhibitors, they establish that bacterial pathogen-encoded deubiquitylating enzymes can be targeted by small molecules. Overall, our results provide a framework for using selective inhibitors as tools to study DUB function in genetically intractable intracellular bacteria and as potential treatments for scrub typhus.

Insecticide-based vector control remains the cornerstone of malaria prevention, averting approximately 1.2 billion cases between 2000 and 2025. These interventions primarily reduce transmission by killing mosquitoes; however, widespread reliance on a limited number of compounds has driven the emergence of insecticide resistance. This has prompted the development of new insecticides with novel modes of action. Notably, the pyrrole insecticide chlorfenapyr has been shown to affect both the mosquito vector and the malaria parasite, suggesting that compounds with dual activity could provide an additional strategy to suppress transmission. Here, we present a medium-throughput discovery pipeline that integrates in vitro Plasmodium sporozoite motility assays with machine-learning-based analysis, alongside in vivo exposure of infected Anopheles mosquitoes and quantification of parasite development. Screening 32 insecticidal chemistries identified five compounds that significantly impaired sporozoite motility, including three avermectin endectocides, the mitochondrial complex III inhibitor hydramethylnon, and tralopyril, the active form of chlorfenapyr. Several compounds transiently increased motility, indicating that parasite physiology is frequently influenced by insecticide exposure. In vivo exposure to abamectin reduced parasite numbers in both the haemolymph and salivary glands and impaired productive motility. Importantly, this inhibition was confirmed in Plasmodium falciparum-infected mosquitoes, where exposure significantly reduced salivary gland invasion. These findings reveal that parasite-directed activity among insecticides may be more common than previously appreciated and demonstrate a scalable approach to identify compounds capable of simultaneously killing mosquitoes and suppressing parasite transmission. Significance StatementVector control relies heavily on insecticides that kill mosquitoes, yet rising resistance threatens their effectiveness. Here we show that several insecticides also affect the malaria parasite itself. Using a scalable screening pipeline combining machine learning-assisted sporozoite motility analysis with mosquito infection assays, we found that 15% of tested insecticides significantly impaired parasite motility, including compounds with distinct modes of action. Among these hits, the avermectin abamectin reduced parasite dissemination in mosquitoes and limited salivary gland invasion in both Plasmodium berghei and the human malaria parasite P. falciparum. These findings reveal that parasite-directed activity among insecticides may be more widespread than expected and highlight the potential to develop vector control tools that simultaneously kill mosquitoes and block parasite transmission.

Maintaining the efficacy of {beta}-lactam antibiotics against Staphylococcus aureus is a clinical priority given the prevalence of methicillin-resistant S. aureus (MRSA). We previously showed that the pyrimidine analogues 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUrd) synergize with {beta}-lactams. Here, we extended this by evaluating additional nucleotide metabolism-targeting agents. Gemcitabine (Gem) and mitomycin C (Mito), like 5-FU and 5-FUrd, exhibited intrinsic anti-MRSA activity and potentiated {beta}-lactams, whereas the purine analogue 6-thioguanine (6-TG) showed distinct, often antagonistic effects. Transcriptomic analysis revealed that pyrimidine-targeting agents repress lysine and glutamate biosynthesis, while 6-TG induced these pathways, implicating amino acid metabolism in {beta}-lactam potentiation. Consistent with this, pyrimidine analogues also suppressed GlmS expression, potentially limiting UDP-GlcNAc production required for cell wall synthesis, and synergized with fosfomycin. Fluorescence microscopy confirmed that the potentiation of oxacillin activity by pyrimidine-targeting agents, but not 6-TG, was accompanied by impaired peptidoglycan synthesis. Additionally, glutathione-mediated attenuation of killing implicated reactive oxygen species in the bactericidal activity of cloxacillin combinations. Finally, these agents displayed strong anti-biofilm activity, further enhanced in combination with daptomycin and rifampicin. Together, these findings highlight the potential of pyrimidine analogues to potentiate cell wall-targeting antibiotics and identify an important role for modulation of cell wall precursor pathways in this anti-MRSA activity. ImportanceDrug interactions can complicate the treatment of antimicrobial resistant infections in patients undergoing treatment for cancer highlighting the importance of understanding the effects of anti-cancer drugs on pathogens like MRSA. Here, we investigated several drugs that target nucleotide metabolism and are used to treat cancer, fungal, and viral infections, both alone and in combination with commonly used penicillin-type antibiotics. We found that pyrimidine analogue drugs enhanced the activity of these antibiotics against MRSA, whereas the purine analogue 6-thioguanine reduced antibiotic effectiveness. These drugs altered the bacterial cell wall and other metabolic pathways linked to antibiotic susceptibility. Our findings reveal the potential to repurpose certain anticancer drugs to improve treatment of MRSA infections, while also cautioning that some drug combinations may interfere with antibiotic therapy.

Klebsiella pneumoniae is an opportunistic bacterial pathogen associated with high morbidity and mortality, exacerbated by the rapid emergence of resistance to last-resort antibiotics, such as carbapenems. Adaptation to nutrient limitation, particularly fluctuations in metal availability, is critical for bacterial survival and virulence, yet the regulatory mechanisms coordinating these responses remain incompletely understood. Protein phosphorylation represents a key post-translational modification governing bacterial physiology and offers a promising avenue for identifying novel antimicrobial targets. Here, we applied mass spectrometry-based phosphoproteomics to define nutrient-responsive signaling networks in K. pneumoniae under varying iron and zinc conditions. This analysis identified iron-dependent phosphorylation of TolQ, a conserved inner membrane component of the Tol-Pal system that maintains cell envelope integrity. Structural modeling predicted that phosphorylation modulates TolQ-TolR conformation, suggesting a mechanism by which iron availability regulates Tol-Pal function. Functional characterization demonstrated that deletion of tolQ results in reduced bacterial viability, increased susceptibility to host immune clearance, and heightened sensitivity to antibiotic treatment. To further explore the therapeutic potential of this pathway, we integrated high-throughput compound screening with computational modeling and identified small molecules that phenocopy {Delta}tolQ. Collectively, these findings reveal a previously unrecognized link between iron availability and phosphoregulation of the Tol-Pal system and establish TolQ as a critical mediator of bacterial survival. This work highlights phosphoproteomics as a powerful strategy to uncover regulatory vulnerabilities and identify targets for antimicrobial development in drug-resistant pathogens.