Design, Synthesis, and Characterization of Novel Tetrahydropyran-Based Bacterial Topoisomerase Inhibitors with Potent Anti-Gram-Positive Activity

, 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. Gram-negative pathogens have become an increased focus for antibiotic development with the continued erosion of the efficacy of current therapies (1). Current options to treat Gramnegative infections are becoming alarmingly limited due to the organisms' abilities to evade existing antibiotic classes by employing a broad array of resistance mechanisms (2). Multidrug-resistant (MDR) Gram-negative bacteria represent important nosocomial pathogens and are responsible for a significant proportion of infections in patients in hospital and intensive care unit (ICU) settings (3). It is clear that additional agents effective against Gram-negative organisms, in particular Pseudomonas aeruginosa, are needed (4).The bacterial topoisomerases have proven to be very effective targets for the fluoroquinolone class of antibiotics (5, 6). Bacterial type II topoisomerases are enzymes that mediate transient double-strand DNA breaks and participate in DNA replication and decatenation reactions (7). DNA gyrase can introduce negative supercoils and controls the level of supercoiling in the bacterial chromosomal DNA (8, 9). Topoisomerase IV is most efficient in decatenating activity, and participates in daughter chromosome separation (10, 11). DNA gyrase is a heterotetramer composed of two copies of each of two protein subunits, GyrA and GyrB (12). Topoisomerase IV is similarly a tetramer of two homodimeric subunits, designated ParC and ParE (13). The fluoroquinolone antibiotics inhibit DNA replication by forming complexes of the drug with DNA bound to the topoisomerase enzyme. This complex acts as a poison for DNA replication, blocking the progression of the replication fork and subsequently inducing the formation of double-strand breaks in the chromosome (14). Despite clinical success, the utility of the fluoroquinolones has eroded over time with use, due primarily to point mutations in the two target enzymes, bacterial gyrase and topoisomerase IV, as well as drug efflux pump mechanisms (15).In this report, we describe the properties of a novel bacterial type II topoisomerase inhibitor (NBTI), NBTI 5463. Mechanistic studies with NBTIs have revealed that members of this class are similar to the fluoroquinolones in tha...

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NBTI 5463 Is a Novel Bacterial Type II Topoisomerase Inhibitor with Activity against Gram-Negative Bacteria and In Vivo Efficacy

Dougherty

Nayar

Newman

et al. 2014

“…In one study, for example, NBTI analogs displayed equipotent target affinity for Streptococcus pneumoniae gyrase and TopoIV but an asymmetrical profile toward the Staphylococcus aureus enzymes, with greater affinity for S. aureus gyrase (40). Another group concluded that an asymmetrical target profile in S. aureus led to unacceptably high resistance frequencies (3 ϫ 10 Ϫ6 at 4ϫ MIC) (41). This group directed compound optimization toward a balanced target profile in S. aureus and demonstrated that an analog with equipotent target affinities had a reduced spontaneous resistance mutation frequency (5 ϫ 10 Ϫ8 at 4ϫ MIC).…”

Section: Discussionmentioning

confidence: 99%

Target-Based Resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a Novel Bacterial Type II Topoisomerase Inhibitor

Nayar

Dougherty

Reck

et al. 2015

), 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.

“…Similarly to fluoroquinolones, they bind near the catalytic center of DNA gyrase and topoisomerase IV but are structurally and mechanistically distinct from fluoroquinolones. Due to this differential action, they possess enhanced potency against Grampositive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) as well as penicillin-and fluoroquinolone-resistant Streptococcus pneumoniae (1,(3)(4)(5).The goal of the present experiments was to assess the in vivo pharmacokinetics (PK)/pharmacodynamics (PD) characteristics of two topoisomerase inhibitors under development in the mouse thigh infection model against two target bacterial species, S. aureus and S. pneumoniae.(Part of this work was presented at the 55th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, September 2015 [21,22]. )…”

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confidence: 99%

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confidence: 99%

In Vivo Pharmacodynamic Target Investigation of Two Bacterial Topoisomerase Inhibitors, ACT-387042 and ACT-292706, in the Neutropenic Murine Thigh Model against Streptococcus pneumoniae and Staphylococcus aureus

Lepak

Seiler

Surivet

et al. 2016

bACT-387042 and ACT-292706 are two novel bacterial topoisomerase inhibitors with broad-spectrum activity against Gram-positive and -negative bacteria, including methicillin-resistant Staphylococcus aureus and penicillin-and fluoroquinolone-resistant Streptococcus pneumoniae. We used the neutropenic murine thigh infection model to characterize the pharmacokinetics (PK)/ pharmacodynamics (PD) of these investigational compounds against a group of 10 S. aureus and S. pneumoniae isolates with phenotypic resistance to beta-lactams and fluoroquinolones. The in vitro activities of the two compounds were very similar (MIC range, 0.03 to 0.125 mg/liter). Plasma pharmacokinetics were determined for each compound by using four escalating doses administered by the subcutaneous route. In treatment studies, mice had 10 7.4 to 10 8 CFU/thigh at the start of therapy with ACT-387042 and 10 6.7 to 10 8.3 CFU/thigh at the start of therapy with ACT-292706. A dose-response relationship was observed with all isolates over the dose range. Maximal kill approached 3 to 4 log 10 CFU/thigh compared to the burden at the start of therapy for the highest doses examined. There was a strong relationship between the PK/PD index AUC/MIC ratio (area under the concentration-time curve over 24 h in the steady state divided by the MIC) and therapeutic efficacy in the model (R 2 , 0.63 to 0.82). The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-387042 against S. aureus and S. pneumoniae were 43 and 10, respectively. The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-292706 against S. aureus and S. pneumoniae were 69 and 25, respectively. The stasis PD targets were significantly lower for S. pneumoniae (P < 0.05) for both compounds. The 1-log-kill AUC/MIC ratio targets were ϳ2-to 4-fold higher than stasis targets. Methicillin, penicillin, or ciprofloxacin resistance did not alter the magnitude of the AUC/MIC ratio required for efficacy. These results should be helpful in the design of clinical trials for topoisomerase inhibitors. ACT-387042 and ACT-292706 are two novel bacterial topoisomerase inhibitors (NBTIs) with related structures (compounds 32d and 32a, respectively, in reference 1). These compounds belong to a class of antibiotics discovered in the late 1990s (2). Similarly to fluoroquinolones, they bind near the catalytic center of DNA gyrase and topoisomerase IV but are structurally and mechanistically distinct from fluoroquinolones. Due to this differential action, they possess enhanced potency against Grampositive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) as well as penicillin-and fluoroquinolone-resistant Streptococcus pneumoniae (1,(3)(4)(5).The goal of the present experiments was to assess the in vivo pharmacokinetics (PK)/pharmacodynamics (PD) characteristics of two topoisomerase inhibitors under development in the mouse thigh infection model against two target bacterial species, S. aureus and S. pneumoniae.(Part of this work was presented at the 55th Interscience Conferen...