Discovery and Optimization of DNA Gyrase and Topoisomerase IV Inhibitors with Potent Activity against Fluoroquinolone-Resistant Gram-Positive Bacteria.

Herein, we describe the discovery and optimization of a novel series that inhibits bacterial DNA gyrase and topoisomerase IV via binding to, and stabilization of, DNA cleavage complexes. Optimization of this series led to the identification of compound 25, which has potent activity against Gram-positive bacteria, a favorable in vitro safety profile, and excellent in vivo pharmacokinetic properties. Compound 25 was found to be efficacious against fluoroquinolone-sensitive Staphylococcus aureus infection in a mouse thigh model at lower doses than moxifloxacin. An X-ray crystal structure of the ternary complex formed by topoisomerase IV from Klebsiella pneumoniae, compound 25, and cleaved DNA indicates that this compound does not engage in a water-metal ion bridge interaction and forms no direct contacts with residues in the quinolone resistance determining region (QRDR). This suggests a structural basis for the reduced impact of QRDR mutations on antibacterial activity of 25 compared to fluoroquinolones.

Topoisomerase Inhibitors Addressing Fluoroquinolone Resistance in Gram-Negative Bacteria.

Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region.

Size Matters and How You Measure It: A Gram-Negative Antibacterial Example Exceeding Typical Molecular Weight Limits.

Monobactam antibiotic 1 is active against Gram-negative bacteria even though it has a higher molecular weight (MW) than the limit of 600 Da typically applied in designing such compounds. On the basis of 2D NMR data, the compound is able to adopt a compact conformation. The dimensions, projection area, and dipole moment derived from this conformation are compatible with porin permeation, as are locations of polar groups upon superimposition to the crystal structure of ampicillin bound to E. coli OmpF porin. Minimum inhibitory concentration (MIC) shifts in a porin knock-out strain are also consistent with 1 predominately permeating through porins. In conclusion, we describe a carefully characterized case of a molecule outside default design parameters where MW does not adequately represent the 3D shape more directly related to permeability. Leveraging 3D design criteria would open up additional chemical space currently underutilized due to limitations perceived in 2D.

Identification of Mutations in the mrdA Gene Encoding PBP2 That Reduce Carbapenem and Diazabicyclooctane Susceptibility of Escherichia coli Clinical Isolates with Mutations in ftsI (PBP3) and Which Carry blaNDM-1.

Penicillin-binding proteins (PBPs) are essential for bacterial cell wall biosynthesis, and several are clinically validated antibacterial targets of ß-lactam antibiotics. We identified mutations in the mrdA gene encoding the PBP2 protein in two Escherichia coliblaNDM-1 clinical isolates that reduce susceptibility to carbapenems and to the intrinsic antibacterial activity of a diazabicyclooctane (DBO) PBP2 and ß-lactamase inhibitor. These mutations coexisted with previously described mutations in ftsI (encoding PBP3) that reduce susceptibility to monobactams, penicillins, and cephalosporins. Clinical exposure to ß-lactams is driving the emergence of multifactorial resistance that may impact the therapeutic usefulness of existing antibacterials and novel compounds that target PBPs.IMPORTANCE Emerging antibacterial resistance is a consequence of the continued use of our current antibacterial therapies, and it is limiting their utility, especially for infections caused by multidrug-resistant isolates. ß-Lactams have enjoyed extensive clinical success, but their broad usage is linked to perhaps the most extensive and progressive example of resistance development for any antibacterial scaffold. In Gram-negative pathogens, this largely involves constant evolution of new ß-lactamases able to degrade successive generations of this scaffold. In addition, more recently, alterations in the targets of these compounds, penicillin-binding proteins (PBPs), are being described in clinical isolates, which often also have multiple ß-lactamases. This study underscores the multifactorial nature of ß-lactam resistance by uncovering alterations of PBP2 that reduce susceptibility to carbapenems in E. coli clinical isolates that also have alterations of PBP3 and express the NDM-1 ß-lactamase. The changes in PBP2 also reduced susceptibility to the intrinsic antibacterial activity of some diazabicyclooctane (DBO) compounds that can target PBP2. This may have implications for the development and use of the members of this relatively newer scaffold that are inhibitors of PBP2 in addition to their inhibition of serine-ß-lactamases.

A Unified Framework for the Incorporation of Bioorthogonal Compound Exposure Probes within Biological Compartments.

Compartmentalization is a crucial facet of many biological systems, and key aspects of cellular processes rely on spatial segregation within the cell. While many drug targets reside in specific intracellular compartments, the tools available for assessing compound exposure are generally limited to whole-cell measurements. To address this gap, we recently developed a bioorthogonal chemistry-based method to assess compartment-specific compound exposure and demonstrated its use in Gram-negative bacteria. To expand the applicability of this approach, we report here novel bioorthogonal probe modalities which enable diverse probe incorporation strategies. The probes we developed utilize a cleavable thiocarbamate linker to connect localizing elements such as metabolic substrates to a cyclooctyne moiety which enables the detection of azide-containing molecules. Adducts between the probe and azide-bearing compounds can be recovered and affinity purified after exposure experiments, thus facilitating the mass-spectrometry based analysis used to assess compound exposure. The bioorthogonal system reported here thus provides a valuable new tool for interrogating compartment-specific compound exposure in a variety of biological contexts while retaining a simple and unified sample preparation and analysis workflow.

IID572: A New Potentially Best-In-Class ß-Lactamase Inhibitor.

Resistance in Gram-negative bacteria to ß-lactam drugs is mediated primarily by the expression of ß-lactamases, and co-dosing of ß-lactams with a ß-lactamase inhibitor (BLI) is a clinically proven strategy to address resistance. New ß-lactamases that are not impacted by existing BLIs are spreading and creating the need for development of novel broader spectrum BLIs. IID572 is a novel broad spectrum BLI of the diazabicyclooctane (DBO) class that is able to restore the antibacterial activity of piperacillin against piperacillin/tazobactam-resistant clinical isolates. IID572 is differentiated from other DBOs by its broad inhibition of ß-lactamases and the lack of intrinsic antibacterial activity.

Mode of Action of the Monobactam LYS228 and Mechanisms Decreasing In Vitro Susceptibility in Escherichia coli and Klebsiella pneumoniae.

The monobactam scaffold is attractive for the development of new agents to treat infections caused by drug-resistant Gram-negative bacteria because it is stable to metallo-ß-lactamases (MBLs). However, the clinically used monobactam aztreonam lacks stability to serine ß-lactamases (SBLs) that are often coexpressed with MBLs. LYS228 is stable to MBLs and most SBLs. LYS228 bound purified Escherichia coli penicillin binding protein 3 (PBP3) similarly to aztreonam (derived acylation rate/equilibrium dissociation constant [k2/Kd ] of 367,504 s-1 M-1 and 409,229 s-1 M-1, respectively) according to stopped-flow fluorimetry. A gel-based assay showed that LYS228 bound mainly to E. coli PBP3, with weaker binding to PBP1a and PBP1b. Exposing E. coli cells to LYS228 caused filamentation consistent with impaired cell division. No single-step mutants were selected from 12 Enterobacteriaceae strains expressing different classes of ß-lactamases at 8× the MIC of LYS228 (frequency, <2.5 × 10-9). At 4× the MIC, mutants were selected from 2 of 12 strains at frequencies of 1.8 × 10-7 and 4.2 × 10-9 LYS228 MICs were ≤2 µg/ml against all mutants. These frequencies compared favorably to those for meropenem and tigecycline. Mutations decreasing LYS228 susceptibility occurred in ramR and cpxA (Klebsiella pneumoniae) and baeS (E. coli and K. pneumoniae). Susceptibility of E. coli ATCC 25922 to LYS228 decreased 256-fold (MIC, 0.125 to 32 µg/ml) after 20 serial passages. Mutants accumulated mutations in ftsI (encoding the target, PBP3), baeR, acrD, envZ, sucB, and rfaI These results support the continued development of LYS228, which is currently undergoing phase II clinical trials for complicated intraabdominal infection and complicated urinary tract infection (registered at ClinicalTrials.gov under identifiers NCT03377426 and NCT03354754).

Impact of Inducible blaDHA-1 on Susceptibility of Klebsiella pneumoniae Clinical Isolates to LYS228 and Identification of Chromosomal mpl and ampD Mutations Mediating Upregulation of Plasmid-Borne blaDHA-1 Expression.

Twenty-three Klebsiella pneumoniae (blaDHA-1) clinical isolates exhibited a range of susceptibilities to LYS228, with MICs of ≥8 µg/ml for 9 of these. Mutants with decreased susceptibility to LYS228 and upregulated expression of blaDHA-1 were selected from representative isolates. These had mutations in the chromosomal peptidoglycan recycling gene mpl or ampD Preexisting mpl mutations were also found in some of the clinical isolates examined, and these had strongly upregulated expression of blaDHA-1.

In Vitro Activity of LYS228, a Novel Monobactam Antibiotic, against Multidrug-Resistant Enterobacteriaceae.

LYS228 is a novel monobactam with potent activity against Enterobacteriaceae LYS228 is stable to metallo-ß-lactamases (MBLs) and serine carbapenemases, including Klebsiella pneumoniae carbapenemases (KPCs), resulting in potency against the majority of extended-spectrum ß-lactamase (ESBL)-producing and carbapenem-resistant Enterobacteriaceae strains tested. Overall, LYS228 demonstrated potent activity against 271 Enterobacteriaceae strains, including multidrug-resistant isolates. Based on MIC90 values, LYS228 (MIC90, 1 µg/ml) was ≥32-fold more active against those strains than were aztreonam, ceftazidime, ceftazidime-avibactam, cefepime, and meropenem. The tigecycline MIC90 was 4 µg/ml against the strains tested. Against Enterobacteriaceae isolates expressing ESBLs (n = 37) or displaying carbapenem resistance (n = 77), LYS228 had MIC90 values of 1 and 4 µg/ml, respectively. LYS228 exhibited potent bactericidal activity, as indicated by low minimal bactericidal concentration (MBC) to MIC ratios (MBC/MIC ratios of ≤4) against 97.4% of the Enterobacteriaceae strains tested (264/271 strains). In time-kill studies, LYS228 consistently achieved reductions in CFU per milliliter of 3 log10 units (≥99.9% killing) at concentrations ≥4× MIC for Escherichia coli and K. pneumoniae reference strains, as well as isolates encoding TEM-1, SHV-1, CTX-M-14, CTX-M-15, KPC-2, KPC-3, and NDM-1 ß-lactamases.

Molecular Probes for the Determination of Subcellular Compound Exposure Profiles in Gram-Negative Bacteria.

The Gram-negative cell envelope presents a formidable barrier to xenobiotics, and achieving sufficient compound exposure inside the cell is a key challenge for the discovery of new antibiotics. To provide insight on the molecular determinants governing compound exposure in Gram-negative bacteria, we developed a methodology leveraging a cyclooctyne-based bioorthogonal probe to assess compartment-specific compound exposure. This probe can be selectively localized to the periplasmic or cytoplasmic compartments of Gram-negative bacteria. Once localized, the probe is used to test azide-containing compounds for exposure within each compartment by quantifying the formation of click-reaction products by mass spectrometry. We demonstrate this approach is an accurate and sensitive method of determining compartment-specific compound exposure profiles. We then apply this technology to study the compartment-specific exposure profiles of a small panel of azide-bearing compounds with known permeability characteristics in Gram-negative bacteria, demonstrating the utility of the system and the insight it is able to provide regarding compound exposure within intact bacteria.

Optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae - Identification of LYS228.

Metallo-ß-lactamases (MBLs), such as New Delhi metallo-ß-lactamase (NDM-1) have spread world-wide and present a serious threat. Expression of MBLs confers resistance in Gram-negative bacteria to all classes of ß-lactam antibiotics, with the exception of monobactams, which are intrinsically stable to MBLs. However, existing first generation monobactam drugs like aztreonam have limited clinical utility against MBL-expressing strains because they are impacted by serine ß-lactamases (SBLs), which are often co-expressed in clinical isolates. Here, we optimized novel monobactams for stability against SBLs, which led to the identification of LYS228 (compound 31). LYS228 is potent in the presence of all classes of ß-lactamases and shows potent activity against carbapenem-resistant isolates of Enterobacteriaceae (CRE).

Synthesis of ciprofloxacin dimers for evaluation of bacterial permeability in atypical chemical space.

We describe the synthesis and evaluation of a library of variably-linked ciprofloxacin dimers. These structures unify and expand on the use of fluoroquinolones as probes throughout the antibiotic literature. A dimeric analog (19) showed enhanced inhibition of its intracellular target (DNA gyrase), and translation to antibacterial activity in whole cells was demonstrated. Overall, cell permeation was governed by physicochemical properties and bacterial type. A principal component analysis demonstrated that the dimers occupy a unique and privileged region of chemical space most similar to the macrolide class of antibiotics.

Genetic and chemical validation identifies Mycobacterium tuberculosis topoisomerase I as an attractive anti-tubercular target.

DNA topoisomerases perform the essential function of maintaining DNA topology in prokaryotes. DNA gyrase, an essential enzyme that introduces negative supercoils, is a clinically validated target. However, topoisomerase I (Topo I), an enzyme responsible for DNA relaxation has received less attention as an antibacterial target, probably due to the ambiguity over its essentiality in many organisms. The Mycobacterium tuberculosis genome harbors a single topA gene with no obvious redundancy in its function suggesting an essential role. The topA gene could be inactivated only in the presence of a complementing copy of the gene in M. tuberculosis. Furthermore, down-regulation of topA in a genetically engineered strain of M. tuberculosis resulted in loss of bacterial viability which correlated with a concomitant depletion of intracellular Topo I levels. The topA knockdown strain of M. tuberculosis failed to establish infection in a murine model of TB and was cleared from lungs in two months post infection. Phenotypic screening of a Topo I overexpression strain led to the identification of an inhibitor, thereby providing chemical validation of this target. Thus, our work confirms the attractiveness of Topo I as an anti-mycobacterial target.

Target-based resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a novel bacterial type II topoisomerase inhibitor.

In a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657-2664, 2014), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.

Overexpression of Pseudomonas aeruginosa LpxC with its inhibitors in an acrB-deficient Escherichia coli strain.

In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.

Optimization of physicochemical properties and safety profile of novel bacterial topoisomerase type II inhibitors (NBTIs) with activity against Pseudomonas aeruginosa.

Type II bacterial topoisomerases are well validated targets for antimicrobial chemotherapy. Novel bacterial type II topoisomerase inhibitors (NBTIs) of these targets are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. We now disclose the optimization of a class of NBTIs towards Gram-negative pathogens, especially against drug-resistant Pseudomonas aeruginosa. Physicochemical properties (pKa and logD) were optimized for activity against P. aeruginosa and for reduced inhibition of the hERG channel. The optimized analogs 9g and 9i displayed potent antibacterial activity against P. aeruginosa, and a significantly improved hERG profile over previously reported analogs. Compound 9g showed an improved QT profile in in vivo models and lower clearance in rat over earlier compounds. The compounds show promise for the development of new antimicrobial agents against drug-resistant Pseudomonas aeruginosa.

NBTI 5463 is a novel bacterial type II topoisomerase inhibitor with activity against gram-negative bacteria and in vivo efficacy.

The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial type II topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O'Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections.

Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II with reduced pK(a): antibacterial agents with an improved safety profile.

Novel non-fluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. N-Linked amino piperidines, such as 7a, generally show potent antibacterial activity, including against quinolone-resistant isolates, but suffer from hERG inhibition (IC(50) = 44 µM for 7a) and QT prolongation in vivo. We now disclose the finding that new analogues of 7a with reduced pK(a) due to substitution with an electron-withdrawing substituent in the piperidine moiety, such as R,S-7c, retained the Gram-positive activity of 7a but showed significantly less hERG inhibition (IC(50) = 233 µM for R,S-7c). This compound exhibited moderate clearance in dog, promising efficacy against a MRSA strain in a mouse infection model, and an improved in vivo QT profile as measured in a guinea pig in vivo model. As a result of its promising activity, R,S-7c was advanced into phase I clinical studies.

Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II: broad-spectrum antibacterial agents with reduced hERG activity.

Novel non-fluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. Aminopiperidines that have a bicyclic aromatic moiety linked through a carbon to an ethyl bridge, such as 1, generally show potent broad-spectrum antibacterial activity, including quinolone-resistant isolates, but suffer from potent hERG inhibition (IC(50)= 3 µM for 1). We now disclose the finding that new analogues of 1 with an N-linked cyclic amide moiety attached to the ethyl bridge, such as 24m, retain the broad-spectrum antibacterial activity of 1 but show significantly less hERG inhibition (IC(50)= 31 µM for 24m) and higher free fraction than 1. One optimized analogue, compound 24l, showed moderate clearance in the dog and promising efficacy against Staphylococcus aureus in a mouse thigh infection model.

Exploring Left-Hand-Side substitutions in the benzoxazinone series of 4-amino-piperidine bacterial type IIa topoisomerase inhibitors.

An SAR survey at the C-6 benzoxazinone position of a novel scaffold which inhibits bacterial type IIa topoisomerase demonstrates that a range of small electron donating groups (EDG) and electron withdrawing groups (EWG) are tolerated for antibacterial activity. Cyano was identified as a preferred substituent that affords good antibacterial potency while minimizing hERG cardiac channel activity.