Against Penicillin-Binding Proteins of Gram-Negative Bacteria E

ASM microbe 2018, June 10th 2018 Contact: [email protected] Session 412-AAR08 Tel. 819-821-8000, ext. 61202 SUN-556 E. Lacasse et al. In Vitro Activity of Tebipenem (SPR859) Against Penicillin-Binding Proteins of Gram-Negative Bacteria E. Lacasse*1, E. Brouillette1, T. R. Parr Jr2, A. Rubio2, F. Malouin1 1Université de Sherbrooke, QC, Canada; 2Spero Therapeutics, Inc., Cambridge, MA, US

ABSTRACT RESULTS CONCLUSIONS

Background: Carbapenems are potent antibacterial agents with broad spectrum activity. SPR859 is the microbiologically SPR859 0 0.0008 0.004 0.02 0.2 2 20 200 µg/mL active form of tebipenem-pivoxil prodrug SPR994, an orally available carbapenem with activity against extended spectrum β- | | | | | | | | • Potency of SPR859 and MEM against E. coli was correlated with their high affinity lactamase (ESBL) producing Enterobacteriaceae. In this study, SPR859 was characterized in vitro by assessing its potency CTRL -1a/b for PBP2: SPR859 MIC 0.015-0.03 µg/mL, IC50 0.022 ± 0.007 µg/mL; MEM MIC 0.03 against putative target penicillin-binding proteins (PBPs) and other assays. (a) PBP 1a/b- ns ns µg/mL, IC50 0.015 ± 0.003 µg/mL. Methods: PBPs were labeled using fluorescent Bocillin FL (BoFL). The binding affinity of β-lactams (SPR859, meropenem 2- -2 -3 [MEM], ceftazidime [CAZ], mecillinam [MEC]) for PBPs was measured by adding increasing concentrations of the test drugs 3- • MEC showed binding to PBP2 only (MIC 0.12-0.25 µg/mL, IC 0.04 µg/mL) and CAZ to membrane preparations of Escherichia coli K12 and Klebsiella pneumoniae ATCC 13883. The concentration of the drugs 50 4- -4 had the highest affinity for PBP3 (MIC 0.25 µg/mL, IC50 0.11 ± 0.02 µg/mL). SPR859 needed to block 50% of the binding of BoFL to each PBP (IC50) was estimated by quantification of fluorescence after gel electrophoresis. MICs were also determined using a broth microdilution technique according to CLSI guideline M7-A10 and and MEM also had good binding to PBP3: IC50 0.56 ± 0.28 µg/mL and 0.45 ± 0.42 -5/6 changes in bacterial cell morphology in presence of 1×MIC were recorded by microscopic examination after a 4 h exposure 5/6- µg/mL, respectively. to the antibiotics. | | | | | | | | Results: Potency of SPR859 and MEM against E. coli was correlated with their high affinity for PBP2: SPR859 MIC 0.015- Meropenem 0 0.0008 0.004 0.02 0.2 2 20 200 µg/mL • Binding to PBPs 1a/b was equivalent for CAZ, SPR859 and MEM (IC50 range 2.5 to | | | | | | | | 0.03 µg/mL, IC50 0.022 ± 0.007 µg/mL; MEM MIC 0.03 µg/mL, IC50 0.015 ± 0.003 µg/mL. MEC showed binding to PBP2 only 3.5 µg/mL) but CAZ had no significant binding to PBPs 4 and 5/6 (IC >20 µg/mL) CTRL 50 (MIC 0.12-0.25 µg/mL, IC50 0.04 µg/mL) and CAZ had the highest affinity for PBP3 (MIC 0.25 µg/mL, IC50 0.11 ± 0.02 (b) PBP 1a/b- -1a/b compared to SPR859 and MEM (IC range 0.32 to 2.1 µg/mL). ns ns 50 µg/mL). SPR859 and MEM also had good binding to PBP3: IC50 0.56 ± 0.28 µg/mL and 0.45 ± 0.42 µg/mL, respectively. Binding to PBPs 1a/b was equivalent for CAZ, SPR859 and MEM (IC range 2.5 to 3.5 µg/mL) but CAZ had no significant 50 2- -2 • K. pneumoniae PBP2 and PBP3 were also the primary targets of SPR859, with IC binding to PBPs 4 and 5/6 (IC50 >20 µg/mL) compared to SPR859 and MEM (IC50 range 0.32 to 2.1 µg/mL). K. pneumoniae 3- -3 50 PBP2 and PBP3 were also the primary targets of SPR859, with IC50 values similar to that determined for these PBPs in E. values similar to that determined for these PBPs in E. coli. coli. As expected for β-lactams having high affinity for PBP2, exposure to SPR859, MEM and MEC caused rounding of cells, 4- -4 whereas preferential binding to PBP3 by CAZ provoked cell filamentation. • As expected for β-lactams having high affinity for PBP2, exposure to SPR859, MEM Conclusion: SPR859 was found to be a potent inhibitor of multiple PBPs, but primarily a PBP2 inhibitor, similar to other 5/6- -5/6 and MEC caused rounding of cells, whereas preferential binding to PBP3 by CAZ compounds in this class. These data support further clinical development of SPR994 to become the first oral carbapenem for provoked cell filamentation. treatment of serious Gram-negative infections. Fig 1. PBP binding competition assay between BoFL and SPR859 (a) and meropenem (b) using E. coli K12 cell membranes. The fluorescent BoFL signal decreases with increasing antibiotic concentrations. The calculated • SPR859 was found to be a potent inhibitor of multiple PBPs, but primarily a PBP2 inhibitor, similar to other compounds in this class. These data support further IC50 are reported in Table 1 below. The control (CTRL) are the unlabeled membranes (no BoFL and antibiotic), INTRODUCTION showing non-specific (ns) autofluorescent bands that are not PBPs. clinical development of SPR994 to become the first oral carbapenem for treatment of serious Gram-negative infections.

Carbapenems are potent antibacterial agents with broad spectrum S activity [1]. SPR859 is the microbiologically active form of the N Table 1. Binding of test antibiotics to PBPs of Gram-negative bacteria tebipenem-pivoxil prodrug SPR994, an orally available carbapenem OH CH N with activity against extended spectrum β-lactamase (ESBL) 3 b H H Binding affinity (IC50 in µg/mL) for the specified PBPs REFERENCES producing Enterobacteriaceae. S MIC Antibiotica PBP1a/b PBP2 PBP3 PBP4 PBP5/6 In this study, SPR859 was characterized in vitro by assessing its N (µg/mL) [1] El-Gamal, M.I. et al. Recent updates of carbapenem antibiotics. Eur. J. Med. Chem. 131, O potency against putative target penicillin-binding proteins (PBPs). 185–195 (2017). OH E. coli SPR859 0.015-0.03 3.32 ± 2.55 0.022 ± 0.007 0.56 ± 0.28 0.65 ± 0.25 2.07 ± 1.02 O [2] Zhao, G., T.I. Meier, S.D. Kahl, K.R. Gee and L.C. Blaszczak. Bocillin FL, a sensitive MEM 0.03 2.59 ± 1.66 0.015 ± 0.003 0.45 ± 0.42 0.32 ± 0.18 2.10 ± 1.21 and commercially available reagent for detection of penicillin-binding proteins. Antimicrob. METHODS MEC 0.12-0.25 >20 0.040 ± 0.009 >20 >20 >20 Agents. Chemother. 43, 1124-1128 (1999). CAZ 0.25 2.56 ± 1.90 >20 0.11 ± 0.02 >20 >20 PBPs were labeled using fluorescent Bocillin FL (BoFL) [2]. The binding affinity of β-lactams (SPR859, meropenem [MEM], ceftazidime [CAZ], mecillinam [MEC]) for PBPs was measured by 1a: 3.58 ± 1.53 adding increasing concentrations of the test drugs to membrane preparations of Escherichia coli K. pneumoniae SPR859 0.12 1b: 18.2 ± 1.10 0.012 ± 0.009 0.92 ± 0.31 --- 3.31 ± 0.95 K12 and Klebsiella pneumoniae ATCC 13883. The concentration of the drugs needed to block 50% ACKNOWLEDGMENTS of the binding of BoFL to each PBP (IC50) was estimated by quantification of fluorescence after gel a MEC: mecillinam, CAZ: ceftazidime, MEM: meropenem. electrophoresis using the Quantity One 1-D analysis software. b the “” sign indicates that the IC50 was greater than the highest antibiotic concentration tested. This study was supported by a research contract between Université de Sherbrooke and Spero Therapeutics Inc.

This work was also supported by a team grant from the Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT) to F. Malouin. no drug Ceftazidime (CAZ) Meropenem (MEM) SPR859 E. Lacasse received a studentship from FRQNT.

MICs were also determined using a broth microdilution technique according to CLSI guideline M7- Fig 2. Microscopy of E. coli K12 incubated with 1×MIC for 4h. CAZ mainly targets the PBP3 (Table 1), leading to A10 and changes in bacterial cell morphology in presence of 1×MIC were recorded by microscopic cell filamentation. MEM and SPR859 target PBP2, causing the rounding of the cells. examination after a 4 h exposure to the antibiotics.