Pathway to Alter Bacterial Membranes: Structure and Inhibition of Lipid A Enzymes Pei Zhou, Ph.D.; Department of Biochemistry Duke University Medical Center SSS STAR STI CTG 2017 Programmatic Meeting on Antimicrobial Resistance in Neisseria gonorrhoeae April 13, 2017; NIH

The Escherichia coli Cell Envelope Lipid A

Solution Structure of LpxC with TU-514, A Substrate Analog Inhibitor Insert I TU-514 Domain I Insert II Catalytic Zn2+ Domain II Coggins et al. Nat Struct Biol 2003; 10, 645-651. Coggins et al. Biochemistry 2005; 44, 1114-1126.

Features of LpxC-substrate Interaction Hydrophobic passage: I186, I189, L200, T203, V205, Y212 Hydrophobic patch: F155, F180, F182 Basic patch: H253, K227, R137, R250 Coggins et al. Biochemistry 2005; 44, 1114-1126.

CHIR-090 Exploits Conserved Features of LpxC for Inhibition Barb, Jiang, Raetz and Zhou. PNAS 2007; 104, 18433-18438.

Structural Basis of CHIR-090 Resistance Barb, Jiang, Raetz and Zhou. PNAS 2007; 104, 18433-18438.

Improving the Spectrum of Inhibition Lee et al, Chem Biol. 2011; 18: 38-47.

Inhibitor Design from A Cryptic Ligand Envelope Lee et al., Nature Communications 2016;7:10638.

Inhibitor Design from A Cryptic Ligand Envelope Lee et al., Nature Communications 2016;7:10638.

LPC-058 is a Potent Antibiotic Lee et al., Nature Communications 2016;7:10638.

LpxC Inhibitor In A Murine Sepsis Model Courtesy of Prof. Vance Fowler, Duke

LpxC as a Target for N. gonorrhoeae?

LpxC-targeting Antibiotics against N. gonorrhoeae

lpxC is an Essential Gene in Neisseria gonorrhoeae pUC18us-NgLpxC-E(kanr) Knock-out construct pUC18us-NgLpxC-B(kanr) Transformation control construct Construct Transformation Results pUC18us-NgLpxC-E(kanr) - pUC18us-NgLpxC-B(kanr) +++

LpxC is the primary target of LPC-067 in N. gonorrhoeae E. coli LpxC is potently inhibited by diacetylene LpxC inhibitors R. leguminosarum LpxC is poorly inhibited by LpxC inhibitors

Species-specific Conformation Hints the Tail Group as Key Determinant ~10 Å E. coli P. aeruginosa A. aeolicus Lee CJ et al, Chemistry and Biology 2011; 18: 38-47.

Lead Optimization against N. gonorrhoeae Plasma Protein Binding 94.2  0.5 % T1/2~1.4 hrs (rat)

Strains of Neisseria gonorrhoeae Characteristics FA19 Antibiotic-susceptible strain 35/02 Ceftriaxone intermediate-resistant strain H041 Ceftriaxone-resistant strain (MIC=2-4 mg/ml) F89 Ceftriaxone-resistant strain (MIC=2 mg/ml)

LpxC Inhibitors Overcome Antibiotic Resistance MIC (mg/mL) FA19 35/02 F89 H041 LPC-169 0.015 0.03 LPC-174 0.025 0.1 LPC-201 0.06 LPC-211 0.002 0.005 0.008 Cefixime 0.0015 0.3 4 8 Ceftriaxone 0.001 0.12 ~1-2 2

Efficacy of LPC-215 against Ceftriaxone-Resistant H041 in the Mouse Model MICH041: 0.008 mg/mL

Pharmacokinetics of LPC-215: An Oral LpxC Inhibitor   Plasma Concentration of LPC-211 (ng/mL) LPC-215 Route 1 hr 4 hr 8 hr 40 mg/kg IV 318 10 Oral 290 40 6

Oral Efficacy of LPC-215 against Ceftriaxone-resistant H041 Infection   n % Clearance p-value (vs HBC control) (vs GEN) Lpc-215 18 94% < 0.0001 0.4 GEN 40% HBC 16 6% H041 (200 mg/kg, oral, 1X daily) Log-rank (Mantel-Cox): p < 0.0001

Resistance Mechanisms to LpxC Inhibition Clements et al. Antimicrob Agents Chemother. 2002; 46: 1793-9. Zeng D et al, J Biol Chem. 2013; 288: 5475-86.

Resistance to LpxC Inhibition By Rebalancing Homeostasis FabZ FabZ LpxC LpxC Zeng D et al, J Biol Chem. 2013; 288: 5475-86.

Resistance Mechanisms to LpxC Inhibition Clements et al. Antimicrob Agents Chemother. 2002; 46: 1793-9. Zeng D et al, J Biol Chem. 2013; 288: 5475-86.

Constitutive Enzymes of Lipid A Biosynthesis LpxG LpxI Constitutive Enzymes of Lipid A Biosynthesis (Raetz Pathway)

Chemical Biology of Lipid A Enzymes LpxG LpxI Nature Microbiology 2016; 1: 16154. Nature 2014; 505: 422. PNAS 2012; 109: 12956. Biochemistry 2013; 52: 2280. J Biol Chem 2014; 289: 24059.

Prof. Christian R. H. Raetz (1946-2011)

Acknowledgements - The Lipid A Team Prof. Rob Nicholas (UNC) Dr. Shauna Swanson Kate Newns Prof. Eric Toone (Duke) Dr. Xiaofei Liang Dr. Xin Chen Dr. Ramesh GopalaSwamy Dr. Frank Navas III Prof. Ann Jerse (USUHS) Dr. Kristie Connolly Zhou Laboratory (Duke) Dr. Chul-Jin Lee, Dr. Brian Coggins, Dr. Adam Barb, Dr. Ling Jiang, Dr. Amanda McClerren Dr. Daina Zeng, Dr. Jinshi Zhao, Javaria Najeeb NIAID AI094475; Special Thanks to Dr. Thomas Hiltke @ NIH