Bacterial efflux pump modulators prevent bacterial growth in macrophages and under broth conditions that mimic the host environment.

New approaches for combating microbial infections are needed. One strategy for disrupting pathogenesis involves developing compounds that interfere with bacterial virulence. A critical molecular determinant of virulence for Gram-negative bacteria are efflux pumps of the resistance-nodulation-division family, which includes AcrAB-TolC. We previously identified small molecules that bind AcrB, inhibit AcrAB-TolC, and do not appear to damage membranes. These efflux pump modulators (EPMs) were discovered in an in-cell screening platform called SAFIRE (Screen for Anti-infectives using Fluorescence microscopy of IntracellulaR Enterobacteriaceae). SAFIRE identifies compounds that disrupt the growth of a Gram-negative human pathogen, Salmonella enterica serotype Typhimurium (S. Typhimurium), in macrophages. We used medicinal chemistry to iteratively design ~200 EPM35 analogs and test them for activity in SAFIRE, generating compounds with nanomolar potency. Analogs were demonstrated to bind AcrB in a substrate binding pocket by cryo-electron microscopy. Despite having amphipathic structures, the EPM analogs do not disrupt membrane voltage, as monitored by FtsZ localization to the cell septum. The EPM analogs had little effect on bacterial growth in standard Mueller Hinton Broth. However, under broth conditions that mimic the micro-environment of the macrophage phagosome, acrAB is required for growth, the EPM analogs are bacteriostatic, and the EPM analogs increase the potency of antibiotics. These data suggest that under macrophage-like conditions, the EPM analogs prevent the export of a toxic bacterial metabolite(s) through AcrAB-TolC. Thus, compounds that bind AcrB could disrupt infection by specifically interfering with the export of bacterial toxic metabolites, host defense factors, and/or antibiotics.IMPORTANCEBacterial efflux pumps are critical for resistance to antibiotics and for virulence. We previously identified small molecules that inhibit efflux pumps (efflux pump modulators, EPMs) and prevent pathogen replication in host cells. Here, we used medicinal chemistry to increase the activity of the EPMs against pathogens in cells into the nanomolar range. We show by cryo-electron microscopy that these EPMs bind an efflux pump subunit. In broth culture, the EPMs increase the potency (activity), but not the efficacy (maximum effect), of antibiotics. We also found that bacterial exposure to the EPMs appear to enable the accumulation of a toxic metabolite that would otherwise be exported by efflux pumps. Thus, inhibitors of bacterial efflux pumps could interfere with infection not only by potentiating antibiotics, but also by allowing toxic waste products to accumulate within bacteria, providing an explanation for why efflux pumps are needed for virulence in the absence of antibiotics.

Bacterial Efflux Pump Modulators Prevent Bacterial Growth in Macrophages and Under Broth Conditions that Mimic the Host Environment.

New approaches for combatting microbial infections are needed. One strategy for disrupting pathogenesis involves developing compounds that interfere with bacterial virulence. A critical molecular determinant of virulence for Gram-negative bacteria are efflux pumps of the resistance-nodulation-division (RND) family, which includes AcrAB-TolC. We previously identified small molecules that bind AcrB, inhibit AcrAB-TolC, and do not appear to damage membranes. These efflux pump modulators (EPMs) were discovered in an in-cell screening platform called SAFIRE (Screen for Anti-infectives using Fluorescence microscopy of IntracellulaR Enterobacteriaceae). SAFIRE identifies compounds that disrupt the growth of a Gram-negative human pathogen, Salmonella enterica serotype Typhimurium (S. Typhimurium) in macrophages. We used medicinal chemistry to iteratively design ~200 EPM35 analogs and test them for activity in SAFIRE, generating compounds with nanomolar potency. Analogs were demonstrated to bind AcrB in a substrate binding pocket by cryo-electron microscopy (cryo-EM). Despite having amphipathic structures, the EPM analogs do not disrupt membrane voltage, as monitored by FtsZ localization to the cell septum. The EPM analogs had little effect on bacterial growth in standard Mueller Hinton Broth. However, under broth conditions that mimic the micro-environment of the macrophage phagosome, acrAB is required for growth, the EPM analogs are bacteriostatic, and increase the potency of antibiotics. These data suggest that under macrophage-like conditions the EPM analogs prevent the export of a toxic bacterial metabolite(s) through AcrAB-TolC. Thus, compounds that bind AcrB could disrupt infection by specifically interfering with the export of bacterial toxic metabolites, host defense factors, and/or antibiotics.

Identification of a 1,2,4-Oxadiazole with Potent and Specific Activity against Clostridioides difficile, the Causative Bacterium of C. difficile Infection, an Urgent Public Health Threat.

The discovery of compound 57, a new, totally synthetic 1,2,4-oxadiazole antibacterial agent, is described. This oxadiazole displays highly selective, bactericidal killing of Clostridioides (Clostridium) difficile, the bacterium that causes C. difficile infection (CDI) in both hospital and community settings. The narrow spectrum of activity exhibited by 57 should avoid any disruption of commensal anaerobic bacteria in the gut microbiome, minimizing chances for recurrent CDI.

The Synthesis of Functionalized 3-Aryl- and 3-Heteroaryloxazolidin-2-ones and Tetrahydro-3-aryl-1,3-oxazin-2-ones via the Iodocyclocarbamation Reaction: Access to Privileged Chemical Structures and Scope and Limitations of the Method.

3-Aryl- and 3-heteroaryloxazolidin-2-ones, by virtue of the diverse pharmacologic activities exhibited by them after subtle changes to their appended substituents, are becoming increasingly important and should be considered privileged chemical structures. The iodocyclocarbamation reaction has been extensively used to make many 3-alkyl-5-(halomethyl)oxazolidin-2-ones, but the corresponding aromatic congeners have been relatively underexplored. We suggest that racemic 3-aryl- and 3-heteroaryl-5-(iodomethyl)oxazolidin-2-ones, readily prepared by the iodocyclocarbamation reaction of N-allylated N-aryl or N-heteroaryl carbamates, may be useful intermediates for the rapid preparation of potential lead compounds with biological activity. We exemplify this point by using this approach to prepare racemic linezolid, an antibacterial agent. Herein, we report the results of our systematic investigation into the scope and limitations of this process and have identified some distinguishing characteristics within the aryl/heteroaryl series. We also describe the first preparation of 3-aryloxazolidin-2-ones bearing new functionalized C-5 substituents derived from conjugated 1,3-dienyl and cumulated 1,2-dienyl carbamate precursors. Finally, we describe the utility of the iodocyclocarbamation reaction for making six-membered tetrahydro-3-aryl-1,3-oxazin-2-ones.

The oxazolidinones: past, present, and future.

The success of linezolid stimulated significant efforts to discover new agents in the oxazolidinone class. Over a dozen oxazolidinones have reached the clinic, but many were discontinued due to lack of differentiated potency, inadequate pharmacokinetics, and safety risks that included myelosuppression. Four oxazolidinones are currently undergoing clinical evaluation. The Trius Therapeutics compound tedizolid phosphate (formerly known as torezolid phosphate, TR-701, DA-7218), the most advanced, is in phase 3 clinical trials for acute bacterial skin and skin structure infections. Rib-X completed two phase 2 studies for radezolid (Rx-01_667, RX-1741) in uncomplicated skin and skin structure infections and community-acquired pneumonia. Pfizer and AstraZeneca have each identified antitubercular compounds that have completed phase 1 studies: sutezolid (PNU-100480, PF-02341272) and AZD5847 (AZD2563), respectively. The oxazolidinones share a relatively low frequency of resistance largely due to the requirement of mutations in 23S ribosomal RNA genes. However, maintaining potency against strains carrying the mobile cfr gene poses a challenge for the oxazolidinone class, as well as other 50S ribosome inhibitors that target the peptidyl transferase center.

5-(2-Pyrimidinyl)-imidazo[1,2-a]pyridines are antibacterial agents targeting the ATPase domains of DNA gyrase and topoisomerase IV.

Dual inhibitors of bacterial gyrB and parE based on a 5-(2-pyrimidinyl)-imidazo[1,2-a]pyridine template exhibited MICs (microg/mL) of 0.06-64 (Sau), 0.25-64 (MRSA), 0.06-64 (Spy), 0.06-64 (Spn), and 0.03-64 (FQR Spn). Selected examples were efficacious in mouse sepsis and lung infection models at <50mg/kg (PO dosing).

Synthesis of (-)-PNU-286607 by asymmetric cyclization of alkylidene barbiturates.

PNU-286607 is the first member of a promising, novel class of antibacterial agents that act by inhibiting bacterial DNA gyrase, a target of clinical significance. Importantly, PNU-286607 displays little cross-resistance with marketed antibacterial agents and is active against methicillin-resistant staphylococcus aureus (MRSA) and fluoroquinoline-resistant bacterial strains. Despite the apparent stereochemical complexity of this unique spirocyclic barbituric acid compound, the racemic core is accessible by a two-step route employing a relatively obscure rearrangement of vinyl anilines, known in the literature as the "tert-amino effect." After a full investigation of the stereochemical course of the racemic reaction, starting with the meso cis-dimethylmorpholine, a practical asymmetric variant of this process was developed.

Discovery and characterization of QPT-1, the progenitor of a new class of bacterial topoisomerase inhibitors.

QPT-1 was discovered in a compound library by high-throughput screening and triage for substances with whole-cell antibacterial activity. This totally synthetic compound is an unusual barbituric acid derivative whose activity resides in the (-)-enantiomer. QPT-1 had activity against a broad spectrum of pathogenic, antibiotic-resistant bacteria, was nontoxic to eukaryotic cells, and showed oral efficacy in a murine infection model, all before any medicinal chemistry optimization. Biochemical and genetic characterization showed that the QPT-1 targets the beta subunit of bacterial type II topoisomerases via a mechanism of inhibition distinct from the mechanisms of fluoroquinolones and novobiocin. Given these attributes, this compound represents a promising new class of antibacterial agents. The success of this reverse genomics effort demonstrates the utility of exploring strategies that are alternatives to target-based screens in antibacterial drug discovery.

Antibacterial oxazolidinones possessing a novel C-5 side chain. (5R)-trans-3-[3-Fluoro-4- (1-oxotetrahydrothiopyran-4-yl)phenyl]-2- oxooxazolidine-5-carboxylic acid amide (PF-00422602), a new lead compound.

Oxazolidinones possessing a C-5 carboxamide functionality (reverse amides) represent a new series of compounds that block bacterial protein synthesis. These reverse amides also exhibited less potency against monoamine oxidase (MAO) enzymes and thus possess less potential for the side effects associated with MAO inhibition. The title compound (14) showed reduced in vivo myelotoxicity compared to linezolid in a 14-day safety study in rats, potent in vivo efficacy in murine systemic infection models, and excellent pharmacokinetic properties.

Novel oxazolidinone-quinolone hybrid antimicrobials.

Antimicrobial compounds incorporating oxazolidinone and quinolone pharmacophore substructures have been synthesized and evaluated. Representative analogues 2, 5, and 6 display an improved potency versus linezolid against gram-positive and fastidious gram-negative pathogens. The compounds are also active against linezolid- and ciprofloxacin-resistant Staphylococcus aureus and Enterococcus faecium strains. The MOA for these new antimicrobials is consistent with a combination of protein synthesis and gyrase A/topoisomerase IV inhibition, with a structure-dependent degree of the contribution from each inhibitory mechanism.

The effect of remote chirality on the antibacterial activity of indolinyl, tetrahydroquinolyl and dihydrobenzoxazinyl oxazolidinones.

The oxazolidinones are promising agents for the treatment of infections caused by gram-positive bacteria, including multidrug-resistant strains. In ongoing studies we have discovered that a strategically placed chiral center of appropriate absolute configuration improves the antibacterial activity of indolinyl oxazolidinone analogues (gram-positive MIC's<0.5 microg/mL for the most potent congeners). The design, synthesis, antibacterial activity and pharmacokinetic profile of a selected series of alpha-methylated indoline derivatives and a related set of tetrahydroquinolyl and dihydrobenzoxazinyl analogues are discussed.

Oxazolidinone structure-activity relationships leading to linezolid.

The development of bacterial resistance to currently available antibacterial agents is a growing global health problem. Of particular concern are infections caused by multidrug-resistant Gram-positive pathogens which are responsible for significant morbidity and mortality in both the hospital and community settings. A number of solutions to the problem of bacterial resistance are possible. The most common approach is to continue modifying existing classes of antibacterial agents to provide new analogues with improved attributes. Other successful strategies are to combine existing antibacterial agents with other drugs as well as the development of improved diagnostic procedures that may lead to rapid identification of the causative pathogen and permit the use of antibacterial agents with a narrow spectrum of activity. Finally, and most importantly, the discovery of novel classes of antibacterial agents employing new mechanisms of action has considerable promise. Such agents would exhibit a lack of cross-resistance with existing antimicrobial drugs. This review describes the work leading to the discovery of linezolid, the first clinically useful oxazolidinone antibacterial agent.

Identification of phenylisoxazolines as novel and viable antibacterial agents active against Gram-positive pathogens.

A new and promising group of antibacterial agents, collectively known as the oxazolidinones and exemplified by linezolid (PNU-100766, marketed as Zyvox), have recently emerged as important new therapeutic agents for the treatment of infections caused by Gram-positive bacteria. Because of their significance, extensive synthetic investigations into the structure-activity relationships of the oxazolidinones have been conducted at Pharmacia. One facet of this research effort has focused on the identification of bioisosteric replacements for the usual oxazolidinone A-ring. In this paper we describe studies leading to the identification of antibacterial agents incorporating a novel isoxazoline A-ring surrogate. In a gratifying result, the initial isoxazoline analogue prepared was found to exhibit in vitro antibacterial activity approaching that of the corresponding oxazolidinone progenitor. The synthesis and antibacterial activity profile of a preliminary series of isoxazoline analogues incorporating either a C-C or N-C linkage between their B- and C-rings will be presented. Many of the analogues exhibited interesting levels of antibacterial activity. The piperazine derivative 54 displayed especially promising in vitro activity and in vivo efficacy comparable to the activity and efficacy of linezolid.