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1249. Genetic Evidence That Gepotidacin Shows Well-balanced Dual Targeting against DNA Gyrase And Topoisomerase IV in Neisseria gonorrhoeae

Abstract Background Gepotidacin (GEP) is a novel triazaacenaphthylene bacterial type II topoisomerase inhibitor targeting both bacterial DNA gyrase and topoisomerase IV by a different mechanism from fluoroquinolone antibiotics. Although in vitro frequency of resistance to GEP in Neisseria gonorrhoeae (NG) is low, during a phase 2 trial, clinical resistance to gepotidacin in NG emerged in a subset of fluoroquinolone-resistant NG isolates that contained a pre-existing ParC D86N mutation by introduction of a new GyrA A92T mutation. The objective of this study was to evaluate the role of GyrA A92T & Parc D86N mutations in resistance to GEP. Methods We utilized the high frequency of natural transformation to introduce GyrA A92T and ParC D86N mutations, individually and in combination, into NG isolates either with GyrA S91F D95G mutations or with wild type (WT) GyrA by selection on ciprofloxacin (CIP) or GEP to generate isogenic strains for susceptibility evaluation. Results Results are summarized in enclosed table. Overall, GyrA A92T and ParC D86N mutations alone did not confer a significant (>4-fold) increase in GEP MIC; whereas together they gave >16-fold increases in GEP MIC. Importantly, quinolone target mutations (GyrA S91F D95G and ParC D86N) together showed no significant effect on the GEP MIC; while they gave >1000-fold increase in CIP MIC. As expected, GyrA A92T and ParC D86N mutations alone or together in WT GyrA background had no significant effect on CIP susceptibility. Susceptibility of isogenic NG strains to gepotidacin and ciprofloxacin Conclusion Our results indicated that unlike fluoroquinolones that primarily target DNA gyrase in NG, there is no obvious primary target for GEP, supporting well-balanced dual targeting of DNA gyrase and topoisomerase IV by GEP in NG. Though, the pre-existing ParC D86N mutation is a potential risk marker for clinical resistance development, as this mutation compromises dual targeting of GEP, our studies provide mechanistic insight for appropriate clinical dose selection to potentially suppress further resistance development in this subset of clinical isolates. Disclosures Pan Chan, PhD, GlaxoSmithKline (Employee, Shareholder) Karen Ingraham, MS, GlaxoSmithKline (Employee, Shareholder) Sharon Min, MS, GlaxoSmithKline (Employee, Shareholder) Nicole Scangarella-Oman, MS, GlaxoSmithKline plc. (Employee, Shareholder) Steve Rittenhouse, PhD, GlaxoSmithKline (Employee, Shareholder) Jianzhong Huang, PhD, GlaxoSmithKline (Employee, Shareholder)

Conclusion: Preliminary analysis of these 41 cases indicate that oral ibrexafungerp provides a favorable therapeutic response in the majority of patients with difficult to treat Candida spp. infections, including those caused by non-albicans Candida species.
Disclosures. Barbara D. Alexander, MD, MHS, SCYNEXIS, Inc. (Employee, Scientific Research Study Investigator, Research Grant or Support) Oliver Cornely, Prof., Poster Abstracts • OFID 2020:7 (Suppl 1) • S643 Collegeville, Pennsylvania; 2 GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania Session: P-58. Novel Agents Background. Gepotidacin (GEP) is a novel triazaacenaphthylene bacterial type II topoisomerase inhibitor targeting both bacterial DNA gyrase and topoisomerase IV by a different mechanism from fluoroquinolone antibiotics. Although in vitro frequency of resistance to GEP in Neisseria gonorrhoeae (NG) is low, during a phase 2 trial, clinical resistance to gepotidacin in NG emerged in a subset of fluoroquinolone-resistant NG isolates that contained a pre-existing ParC D86N mutation by introduction of a new GyrA A92T mutation. The objective of this study was to evaluate the role of GyrA A92T & Parc D86N mutations in resistance to GEP.
Methods. We utilized the high frequency of natural transformation to introduce GyrA A92T and ParC D86N mutations, individually and in combination, into NG isolates either with GyrA S91F D95G mutations or with wild type (WT) GyrA by selection on ciprofloxacin (CIP) or GEP to generate isogenic strains for susceptibility evaluation.
Results. Results are summarized in enclosed table. Overall, GyrA A92T and ParC D86N mutations alone did not confer a significant (>4-fold) increase in GEP MIC; whereas together they gave >16-fold increases in GEP MIC. Importantly, quinolone target mutations (GyrA S91F D95G and ParC D86N) together showed no significant effect on the GEP MIC; while they gave >1000-fold increase in CIP MIC. As expected, GyrA A92T and ParC D86N mutations alone or together in WT GyrA background had no significant effect on CIP susceptibility.
Susceptibility of isogenic NG strains to gepotidacin and ciprofloxacin

Conclusion.
Our results indicated that unlike fluoroquinolones that primarily target DNA gyrase in NG, there is no obvious primary target for GEP, supporting well-balanced dual targeting of DNA gyrase and topoisomerase IV by GEP in NG. Though, the pre-existing ParC D86N mutation is a potential risk marker for clinical resistance development, as this mutation compromises dual targeting of GEP, our studies provide mechanistic insight for appropriate clinical dose selection to potentially suppress further resistance development in this subset of clinical isolates.
Disclosures. Pan Chan, PhD, GlaxoSmithKline (Employee, Shareholder) Karen Ingraham, MS, GlaxoSmithKline (Employee, Shareholder) Sharon Min, MS, GlaxoSmithKline (Employee, Shareholder) Nicole Scangarella-Oman, MS, GlaxoSmithKline plc. (Employee, Shareholder) Steve Rittenhouse, PhD, GlaxoSmithKline (Employee, Shareholder) Jianzhong Huang, PhD, GlaxoSmithKline (Employee, Shareholder) Background. Catalytic mechanisms of serine β-lactamases (SBL; classes A, C and D) and metallo-β-lactamases (MBLs) have directed divergent strategies towards inhibitor design. SBL inhibitors act as high affinity substrates that -as in BATSIs-form a reversible, dative covalent bond with the conserved active site Ser. MBL inhibitors bind the active-site Zn 2+ ions and displace the nucleophilic OH -. Herein, we explore the efficacy of a series of BATSI compounds with a free-thiol group at inhibiting both SBL and MBL.

Novel Boronic Acid Transition State Analogs (BATSI) with in vitro inhibitory activity against class A, B and C β-lactamases
Methods. Exploratory compounds were synthesized using stereoselective homologation of (+) pinandiol boronates to introduce the amino group on the boron-bearing carbon atom, which was subsequently acylated with mercaptopropanoic acid. Representative SBL (KPC-2, ADC-7, PDC-3 and OXA-23) and MBL (IMP-1, NDM-1 and VIM-2) were purified and used for the kinetic characterization of the BATSIs. In vitro activity was evaluated by a modified time-kill curve assay, using SBL and MBLproducing strains.
Results. Kinetic assays revealed that IC 50 values ranged from 1.3 µM to >100 µM for this series. The best compound, s08033, demonstrated inhibitory activity against KPC-2, VIM-2, ADC-7 and PDC-3, with IC50 in the low μM range. Reduction of at least 1.5 log 10 -fold of viable cell counts upon exposure to sub-lethal concentrations of antibiotics (AB) + s08033, compared to the cells exposed to AB alone, demonstrated the microbiological activity of this novel compound against SBL-and MBL-producing E. coli (Table 1). Table 1 Conclusion. Addition of a free-thiol group to the BATSI scaffold increases the range of these compounds resulting in a broad-spectrum inhibitor toward clinically important carbapenemases and cephalosporinases.

Prevention of Pneumocystis Pneumonia by Ibrexafungerp in a Murine Prophylaxis Model
Katyna Borroto-Esoda, PhD 1 ; Nkechi Azie, MD 1 ; Alan Ashbaugh, PhD 2 ; Melanie Cushion, PhD 2 ; David A. Angulo, MD 1 ; 1 SCYNEXIS, Inc., Jersey City, NJ; 2 University of Cincinnati, Cincinnati, Ohio Session: P-58. Novel Agents Background. Pneumocystis pneumonia (PCP) is an opportunistic fungal infection that affects immunocompromised patients. Ibrexafungerp (IBX) is an oral and intravenous antifungal from a novel class of glucan synthase inhibitors, triterpenoids, and has shown activity against Candida, Aspergillus, and PCP in a murine therapy model. We evaluated the ability of IBX to prevent PCP in a prophylaxis model of murine PCP.
Methods. Experiment 1: Balb/c mice (10 mice/group) were infected by intranasal inoculation with Pneumocystis murina, immune-suppressed with dexamethasone in acidified drinking water and treated with 30-, 15-and 7.5 mg/kg, IBX/BID. Control groups treatment included TMP-SMX (50/250 mg/kg QD) and vehicle. After 6 weeks, mice were sacrificed, and prevention was determined by organism burdens (asci and total nuclei). Experiment 2: Balb/c mice were immune-suppressed and infected as in Exp. 1. Treatment groups included: 1) 30 mg/kg BID x 6wk; 2) 30 mg/kg/BID x 6wk followed by cessation of treatment with IBX but with immune-suppression for 3 additional weeks; 3) 15 mg/kg BID 1 week prior and 6 wks after infection and immune suppression; 4) 15mg/kg BID for 8 wks; 5) 15 mg/kg BID for 6 wks then IBX was discontinued but with immune suppression; 6) untreated, vehicle control.
Results. Experiment 1: No P. murina nuclei or asci were observed after 6 weeks of treatment at a dose of 30 mg/kg/BID in the prophylaxis mouse model of PCP, similar to positive control, TMP/SMX. Some nuclei and asci were observed in the lower dose IBX groups. Experiment 2: To investigate whether any P. murina remained after different regimens of prophylaxis, treatment of IBX was withdrawn at both doses for an additional 3 wks of immune suppression to provoke the growth of any remaining fungi. Group 1 showed reduction in total nuclei and asci to undetectable. Group 2 did not result in any recrudescence of infection. Group 3 and 4 showed similar reduction in organism burden. Group 5 was similar to untreated control.
Conclusion. These results demonstrate that 30 mg/kg BID IBX prevented PCP in a murine model. We suggest that IBX could be a viable option for preventing PCP in immunocompromised patients.