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The polyprenyl lipid undecaprenyl phosphate (C55P) is the universal carrier lipid for the biosynthesis of bacterial cell wall polymers. C55P is synthesized in its pyrophosphate form by undecaprenyl pyrophosphate synthase (UppS), an essential cis-prenyltransferase that is an attractive target for antibiotic development. We previously identified a compound (MAC-0547630) that showed promise as a novel class of inhibitor and an ability to potentiate β-lactam antibiotics. Here, we provide a structural model for MAC-0547630's inhibition of UppS and a structural rationale for its enhanced effect on UppS from Bacillus subtilis versus Staphylococcus aureus. We also describe the synthesis of a MAC-0547630 derivative (JPD447), show that it too can potentiate β-lactam antibiotics, and provide a structural rationale for its improved potentiation. Finally, we present an improved structural model of clomiphene's inhibition of UppS. Taken together, our data provide a foundation for structure-guided drug design of more potent UppS inhibitors in the future.

The growing challenge of microbial resistance emphasizes the importance of new antibiotics or reviving strategies for the use of old ones. Macrolide antibiotics are potent bacterial protein synthesis inhibitors with a formidable capacity to treat life-threatening bacterial infections; however, acquired and intrinsic resistance limits their clinical application. In the work presented here, we reveal that bicarbonate is a potent enhancer of the activity of macrolide antibiotics that overcomes both acquired and intrinsic resistance mechanisms. With a focus on azithromycin, a highly prescribed macrolide antibiotic, and using clinically relevant pathogens, we show that physiological concentrations of bicarbonate overcome drug resistance by increasing the intracellular concentration of azithromycin. We demonstrate the potential of bicarbonate as a formulation additive for topical use of azithromycin in treating a murine wound infection caused by

The spread of antimicrobial resistance continues to be a priority health concern worldwide, necessitating exploration of alternative therapies. Cannabis sativa has long been known to contain antibacterial cannabinoids, but their potential to address antibiotic resistance has only been superficially investigated. Here, we show that cannabinoids exhibit antibacterial activity against MRSA, inhibit its ability to form biofilms and eradicate pre-formed biofilms and stationary phase cells persistent to antibiotics. We show that the mechanism of action of cannabigerol is through targeting the cytoplasmic membrane of Gram-positive bacteria and demonstrate in vivo efficacy of cannabigerol in a murine systemic infection model caused by MRSA. We also show that cannabinoids are effective against Gram-negative organisms whose outer membrane is permeabilized, where cannabigerol acts on the inner membrane. Finally, we demonstrate that cannabinoids work in combination with polymyxin B against multi-drug resistant Gram-negative pathogens, revealing the broad-spectrum therapeutic potential for cannabinoids.

Therapeutics targeting Gram-negative bacteria have the challenge of overcoming a formidable outer membrane (OM) barrier. Here, we characterize the action of SPR741, a novel polymyxin B (PMB) analogue shown to potentiate several large-scaffold antibiotics in Gram-negative pathogens. Probing the surface topology of Escherichia coli using atomic force microscopy revealed substantial OM disorder at concentrations of SPR741 that lead to antibiotic potentiation. Conversely, very little cytoplasmic membrane depolarization was observed at these same concentrations, indicating that SPR741 acts predominately on the OM. Truncating the lipopolysaccharide (LPS) core with genetic perturbations uniquely sensitized E. coli to SPR741, suggesting that LPS core residues keep SPR741 at the OM, where it can potentiate a codrug, rather than permit its entry to the cytoplasmic membrane. Further, a promoter activity assay revealed that SPR741 challenge induced the expression of RcsAB, a stress sensor for OM perturbation. Together, these results indicate that SPR741 interacts predominately with the OM, in contrast to the dual action of PMB and colistin at both the outer and cytoplasmic membranes.

Discovering new Gram-negative antibiotics has been a challenge for decades. This has been largely attributed to a limited understanding of the molecular descriptors governing Gram-negative permeation and efflux evasion. Herein, we address the contribution of efflux using a novel approach that applies multivariate analysis, machine learning, and structure-based clustering to some 4,500 molecules (actives) from a small-molecule screen in efflux-compromised Escherichia coli. We employed principal-component analysis and trained two decision tree-based machine learning models to investigate descriptors contributing to the antibacterial activity and efflux susceptibility of these actives. This approach revealed that the Gram-negative activity of hydrophobic and planar small molecules with low molecular stability is limited to efflux-compromised E. coli. Furthermore, molecules with reduced branching and compactness showed increased susceptibility to efflux. Given these distinct properties that govern efflux, we developed the first efflux susceptibility machine learning model, called Susceptibility to Efflux Random Forest (SERF), as a tool to analyze the molecular descriptors of small molecules and predict those that could be susceptible to efflux pumps in silico. Here, SERF demonstrated high accuracy in identifying such molecules. Furthermore, we clustered all 4,500 actives based on their core structures and identified distinct clusters highlighting side-chain moieties that cause marked changes in efflux susceptibility. In all, our work reveals a role for physicochemical and structural parameters in governing efflux, presents a machine learning tool for rapid in silico analysis of efflux susceptibility, and provides a proof of principle for the potential of exploiting side-chain modification to design novel antimicrobials evading efflux pumps.

Staphylococcus aureus is a leading cause of bacterial infections world-wide. Staphylococcal infections are preferentially treated with β-lactam antibiotics, however, methicillin-resistant S. aureus (MRSA) strains have acquired resistance to this superior class of antibiotics. We have developed a growth-based, high-throughput screening approach that directly identifies cell wall synthesis inhibitors capable of reversing β-lactam resistance in MRSA. The screen is based on the finding that S. aureus mutants lacking the ClpX chaperone grow very poorly at 30°C unless specific steps in teichoic acid synthesis or penicillin binding protein (PBP) activity are inhibited. This property allowed us to exploit the S. aureus clpX mutant as a unique screening tool to rapidly identify biologically active compounds that target cell wall synthesis. We tested a library of ∼50,000 small chemical compounds and searched for compounds that inhibited growth of the wild type while stimulating growth of the clpX mutant. Fifty-eight compounds met these screening criteria, and preliminary tests of 10 compounds identified seven compounds that reverse β-lactam resistance of MRSA as expected for inhibitors of teichoic acid synthesis. The hit compounds are therefore promising candidates for further development as novel combination agents to restore β-lactam efficacy against MRSA.

The antibacterial properties of sodium bicarbonate have been known for years, yet the molecular understanding of its mechanism of action is still lacking. Utilizing chemical-chemical combinations, we first explored the effect of bicarbonate on the activity of conventional antibiotics to infer on the mechanism. Remarkably, the activity of 8 classes of antibiotics differed in the presence of this ubiquitous buffer. These interactions and a study of mechanism of action revealed that, at physiological concentrations, bicarbonate is a selective dissipater of the pH gradient of the proton motive force across the cytoplasmic membrane of both Gram-negative and Gram-positive bacteria. Further, while components that make up innate immunity have been extensively studied, a link to bicarbonate, the dominant buffer in the extracellular fluid, has never been made. Here, we also explored the effects of bicarbonate on components of innate immunity. Although the immune response and the buffering system have distinct functions in the body, we posit there is interplay between these, as the antimicrobial properties of several components of innate immunity were enhanced by a physiological concentration of bicarbonate. Our findings implicate bicarbonate as an overlooked potentiator of host immunity in the defense against pathogens. Overall, the unique mechanism of action of bicarbonate has far-reaching and predictable effects on the activity of innate immune components and antibiotics. We conclude that bicarbonate has remarkable power as an antibiotic adjuvant and suggest that there is great potential to exploit this activity in the discovery and development of new antibacterial drugs by leveraging testing paradigms that better reflect the physiological concentration of bicarbonate.

The dramatic rise in microbial drug resistance in recent years has led to ongoing searches for novel drugs to add to the armory against infectious disease. Nevertheless, a paucity of new antibacterial drugs in discovery and development pipelines using traditional approaches has prompted a variety of unconventional and disruptive strategies for antibacterial drug discovery. Herein, we review recent nontraditional approaches that have been piloted for early drug discovery efforts. These unique methodologies open new avenues for finding the next generation of antimicrobials.

Objectives To assess the ability of meropenem to potentiate aminoglycoside (AG) activity against laboratory and AG-resistant cystic fibrosis (CF) isolates of Pseudomonas aeruginosa and to elucidate its mechanism of action. Methods AG resistance gene deletions were engineered into P. aeruginosa laboratory and CF isolates using standard gene replacement technology. Susceptibility to AGs ± meropenem (at ½ MIC) was assessed using a serial 2-fold dilution assay. mexXY expression and MexXY-OprM efflux activity were quantified using quantitative PCR and an ethidium bromide accumulation assay, respectively. Results A screen for agents that rendered WT P. aeruginosa susceptible to a sub-MIC concentration of the AG paromomycin identified the carbapenem meropenem, which potentiated several additional AGs. Meropenem potentiation of AG activity was largely lost in a mutant lacking the MexXY-OprM multidrug efflux system, an indication that it was targeting this efflux system in enhancing P. aeruginosa susceptibility to AGs. Meropenem failed to block AG induction of mexXY expression or MexXY-OprM efflux activity, suggesting that it may be interfering with some MexXY-dependent process linked to AG susceptibility. Meropenem potentiated AG activity versus AG-resistant CF isolates, enhancing susceptibility to at least one AG in all isolates and susceptibility to all tested AGs in 50% of the isolates. Notably, meropenem potentiation of AG activity was linked to MexXY in some but not all CF isolates in which this was examined. Conclusions Meropenem potentiates AG activity against laboratory and CF strains of P. aeruginosa, both dependent on and independent of MexXY, highlighting the complexity of AG resistance in this organism.

SummaryPathways of bacterial energy metabolism, such as the proton motive force (PMF), have largely remained unexplored as drug targets, owing to toxicity concerns. Here, we elaborate on a methodical and systematic approach for targeting the PMF using chemical combinations. We began with a high-throughput screen to identify molecules that selectively dissipate either component of the PMF, ΔΨ or ΔpH, in Staphylococcus aureus. We uncovered six perturbants of PMF, three that countered ΔΨ and three that selectively dissipated ΔpH. Combinations of dissipators of ΔΨ with dissipators of ΔpH were highly synergistic against methicillin-resistant S. aureus. Cytotoxicity analyses on mammalian cells revealed that the dose-sparing effect of the observed synergies could significantly reduce toxicity. The discovery and combination of modulators of ΔΨ and ΔpH may represent a promising strategy for combating microbial pathogens.

The increasing use of polymyxins1 in addition to the dissemination of plasmid-borne colistin resistance threatens to cause a serious breach in our last line of defence against multidrug-resistant Gram-negative pathogens, and heralds the emergence of truly pan-resistant infections. Colistin resistance often arises through covalent modification of lipid A with cationic residues such as phosphoethanolamine-as is mediated by Mcr-1 (ref. 2)-which reduce the affinity of polymyxins for lipopolysaccharide3. Thus, new strategies are needed to address the rapidly diminishing number of treatment options for Gram-negative infections4. The difficulty in eradicating Gram-negative bacteria is largely due to their highly impermeable outer membrane, which serves as a barrier to many otherwise effective antibiotics5. Here, we describe an unconventional screening platform designed to enrich for non-lethal, outer-membrane-active compounds with potential as adjuvants for conventional antibiotics. This approach identified the antiprotozoal drug pentamidine6 as an effective perturbant of the Gram-negative outer membrane through its interaction with lipopolysaccharide. Pentamidine displayed synergy with antibiotics typically restricted to Gram-positive bacteria, yielding effective drug combinations with activity against a wide range of Gram-negative pathogens in vitro, and against systemic Acinetobacter baumannii infections in mice. Notably, the adjuvant activity of pentamidine persisted in polymyxin-resistant bacteria in vitro and in vivo. Overall, pentamidine and its structural analogues represent unexploited molecules for the treatment of Gram-negative infections, particularly those having acquired polymyxin resistance determinants.

Rising drug resistance is limiting treatment options for infections by methicillin-resistant Staphylococcus aureus (MRSA). Herein we provide new evidence that wall teichoic acid (WTA) biogenesis is a remarkable antibacterial target with the capacity to destabilize the cooperative action of penicillin-binding proteins (PBPs) that underlie β- lactam resistance in MRSA. Deletion of gene tarO, encoding the first step of WTA synthesis, resulted in the restoration of sensitivity of MRSA to a unique profile of β-lactam antibiotics with a known selectivity for penicillin binding protein 2 (PBP2). Of these, cefuroxime was used as a probe to screen for previously approved drugs with a cryptic capacity to potentiate its activity against MRSA. Ticlopidine, the antiplatelet drug Ticlid, strongly potentiated cefuroxime, and this synergy was abolished in strains lacking tarO. The combination was also effective in a Galleria mellonella model of infection. Using both genetic and biochemical strategies, we determined the molecular target of ticlopidine as the N- acetylglucosamine-1-phosphate transferase encoded in gene tarO and provide evidence that WTA biogenesis represents an Achilles heel supporting the cooperative function of PBP2 and PBP4 in creating highly cross-linked muropeptides in the peptidoglycan of S. aureus. This approach represents a new paradigm to tackle MRSA infection.

A screen for agents that potentiated the activity of paromomycin (PAR), a 4,5-linked aminoglycoside (AG), against wild-type Pseudomonas aeruginosa identified the RNA polymerase inhibitor rifampin (RIF). RIF potentiated additional 4,5-linked AGs, such as neomycin and ribostamycin, but not the clinically important 4,6-linked AGs amikacin and gentamicin. Potentiation was absent in a mutant lacking the AmgRS envelope stress response two-component system (TCS), which protects the organism from AG-generated membrane-damaging aberrant polypeptides and, thus, promotes AG resistance, an indication that RIF was acting via this TCS in potentiating 4,5-linked AG activity. Potentiation was also absent in a RIF-resistant RNA polymerase mutant, consistent with its potentiation of AG activity being dependent on RNA polymerase perturbation. PAR-inducible expression of the AmgRS-dependent genes htpX and yccA was reduced by RIF, suggesting that AG activation of this TCS was compromised by this agent. Still, RIF did not compromise the membrane-protective activity of AmgRS, an indication that it impacted some other function of this TCS. RIF potentiated the activities of 4,5-linked AGs against several AG-resistant clinical isolates, in two cases also potentiating the activity of the 4,6-linked AGs. These cases were, in one instance, explained by an observed AmgRS-dependent expression of the MexXY multidrug efflux system, which accommodates a range of AGs, with RIF targeting of AmgRS undermining mexXY expression and its promotion of resistance to 4,5- and 4,6-linked AGs. Given this link between AmgRS, MexXY expression, and pan-AG resistance in P. aeruginosa , RIF might be a useful adjuvant in the AG treatment of P. aeruginosa infections.

Combinations of antibiotics are commonly used in medicine to broaden antimicrobial spectrum and generate synergistic effects. Alternatively, combination of nonantibiotic drugs with antibiotics offers an opportunity to sample a previously untapped expanse of bioactive chemical space. We screened a collection of drugs to identify compounds that augment the activity of the antibiotic minocycline. Unexpected synergistic drug combinations exhibited in vitro and in vivo activity against bacterial pathogens, including multidrug-resistant isolates.

The thienopyridine antiplatelet agent, ticlopidine and its analog, clopidogrel, have been shown to potentiate the action of β-lactam antibiotics, reversing the methicillin-resistance phenotype of methicillin-resistant Staphylococcus aureus (MRSA), in vitro. Interestingly, these thienopyridines inhibit the action of TarO, the first enzyme in the synthesis of wall teichoic acid, an important cell wall polymer in Gram-positive bacteria. In the human body, both ticlopidine and clopidogrel undergo a rapid P450-dependent oxidation into their respective antiplatelet-active metabolites, resulting in very low plasma concentrations of intact drug. Herein, a series of analogs of ticlopidine and clopidogrel that would avoid oxidative metabolism were designed, prepared and evaluated as inhibitors of TarO. Specifically, we replaced the P450-labile thiophene ring of ticlopidine and clopidogrel to a more stable phenyl group to generate 2-(2-chlorobenzyl)-1,2,3,4-tetrahydro-isoquinoline) (6) and (2-chloro-phenyl)-(3,4-dihydro-1H-isoquinolin-2-yl)-acetic acid methyl ester (22), respectively. The latter molecules displayed inhibitory activity against TarO and formed the basis of a library of analogs. Most synthesized compounds exhibited comparable efficacy to ticlopidine and clopidogrel. So far, it was introduction of a trifluoromethyl group to compound 6, to generate 2-(2-trifluoromethyl-benzyl)-1,2,3,4-tetrahydro-isoquinoline (13) that exhibited enhanced activity against TarO. Compound 13 represents a novel stable inhibitor of TarO with synergistic impact on β-lactam antibiotics against MRSA and low potential for P-450 metabolism.

SummaryWhile cell-based screens have considerable power in identifying new chemical probes of biological systems and leads for new drugs, a major challenge to the utility of such compounds is in connecting phenotype with a cellular target. Here, we present a systematic study to elucidate the mechanism of action of uncharacterized inhibitors of the growth of Escherichia coli through careful analyses of interactions with compounds of known biological activity. We studied growth inhibition with a collection of 200 antibacterial compounds when systematically combined with a panel of 14 known antibiotics of diverse mechanism and chemical class. Our work revealed a high frequency of synergistic chemical-chemical interactions where the interaction profiles were unique to the various compound pairs. Thus, the work revealed that chemical-chemical interaction data provides a fingerprint of biological activity and testable hypotheses regarding the mechanism of action of the novel bioactive molecules. In the study reported here, we determined the mode of action of an inhibitor of folate biosynthesis and a DNA gyrase inhibitor. Moreover, we identified eight membrane-active compounds, found to be promiscuously synergistic with known bioactives.