Victor Nizet, MD Professor & Vice Chair for Basic Research, Department of Pediatrics Chief, Division of Host-Microbe Systems & Therapeutics Professor, Skaggs School of Pharmacy & Pharmaceutical RadySciences Children’s Hospital University of California, San Diego San Diego

UCSD Town & Gown Luncheon Series | October 10, 2017

“Resistance will “Antibiotic-resistance “A National Action “A post-antibiotic era result in $100 trillion is a fundamental Plan to preserve the would mean the end in lost economic threat to global health utility of antibiotics of modern medicine productivity” and safety” must be enacted” as we know it ”

The Human Microbiome is Diverse and Dynamic

Human vs. Bacterial Pathogen

J. Sullivan “Cells Alive”

Seeking alternatives to classical antibiotics, especially very broad-spectrum agents, that kill bacteria or block their growth

* Drugs to block specific pathogen immune resistance factors Sensitize pathogens to clearance by normal host innate defenses More targeted therapy, avoid “collateral damage” to microbiome

* Modulation of innate immunity to treat bacterial Can we pharmacologically boost function?

* Explore “repurposing” existing drugs for the above properties

* These approaches can work in concert with classical antibiotics

> 40 million users in USA in 2015

with C. Glass Lab

Chow et al. Cell Host Microbe (2010) Chow et al. Cell Host Microbe (2010) Chow et al. Cell Host Microbe (2010) Tamoxifen Induces NETs By Increasing Intracellular Ceramide Levels

Corriden et al. Nat Commun 2015 Tamoxifen Boosts Host Defense Against Staphylococcal in vivo

Corriden et al. Nat Commun 2015 Proc Soc Exp Biol Med, 48: 330-333 (1941)

30.0% Beef infusion 1.75% Casein hydrosylate 0.15% Starch 1.70% Agar pH to neutral at 25oC

Later – cation-adjusted (for Pseudomonas) Calcium 20-25 mg/L Magnesium 10-12.5 mg/L A SINGLE TEST, MIC/MBC TESTING IN BACTERIOLOGIC MEDIA (i.e. CA-MHB), EFFECTIVELY DELIMITS PHARMACOTHERAPY OF HUMAN BACTERIAL INFECTIONS

ANTIBIOTIC DISCOVERY AND DEVELOPMENT

WHICH DRUGS CHOSEN FOR HOSPITAL FORMULARY

WHICH INFORMATION IS PROVIDED TO DOCTORS WHEN THE PATHOGEN IS CULTURED FROM THE PATIENT Before a patient has even seen the doctor ...

... their infection is already being treated by dozens of antibiotics with R. Gallo Lab Reintroduction of β-Lactam Antibiotics in Refractory M.R.S.A. Bacteremia – With Surprising Results

Day 1 Day 12 Day 17 Day 21 Day 22 Day 23-76

VAN MIC 1 Persistent (+) blood VAN MIC 4.0 Bacteremia Cure DAP MIC 0.5 cultures for MRSA DAP MIC 2.0 Resolved (isolate D592) (isolate Vancomycin Daptomycin Daptomycin D712) dosed for 6 mg/kg 8 mg/kg + serum trough Gentamicin Daptomycin 15-20 mg/L 10 mg/kg + Nafcillin 2 g IV q 4hr

George Sakoulas, MD

Sakoulas et al. J Mol Med 2014 Nafcillin Increases Binding to MRSA by Rhodamine-Labeled Cathelicidin LL-37

MRSA + LL-37 MRSA + LL-37 + Naf 10

Sakoulas et al. J Mol Med 2014 Sublethal Nafcillin Sensitizes MRSA/VISA Strains to Whole Blood, Neutrophil & Keratinocyte Killing

Whole Blood Killing Neutrophil Killing Human Keratinocyte (HaCat) Survival at 2h No Abx No Abx 60 150 Sanger 252 MRSA NAF 2 NAF 2 200

100 150 40 ** P < 0.02 P < 0.05 *** 100 * ** 20 *** 50 50

Relative survival % 0 vs. cell free control % survival (1 h) (1 survival % 0 0 No NAF NAF No NAF NAF D592 D712 Sanger 252 min) (90 survival % D592 D712 Abx 5 20 Abx 5 20 VISA VISA MRSA VISA VISA MOI = 1.0 MOI = 0.1

Sakoulas et al. J Mol Med 2014 Sublethal Nafcillin (Monotherapy) Influences MRSA Lesion Development in Mouse Skin Infection Models

Antibiotic pretreatment of Sanger 252 MRSA Representative gross appearance of skin followed by mouse subcutaneous challenge lesions at 48 h time point 15 No abx

) NAF 2 VAN 10 n.s.

n.s. 5 ** ** None NAF None VAN NAF VAN *P < 0.05 * n.s. Lesion (mm size 0 1 2 3 4 5 6 7 Time after infection (d)

Mouse s.c. challenge with Sanger 252 Mouse s.c. challenge with Sanger 252 MRSA +/- antibiotic treatment MRSA +/- antibiotic treatment 6

) 30 2 )

No abx 7 NAF

20 4 P = 0.0185

* 10 *P = 0.026 2

Lesion size (mm size Lesion 0 (cfu/g (cfu/g tissue x 10 1 2 3 4 cfu MRSA Surviving 0 Time after infection (d) No Abx Nafcillin Sakoulas et al. J Mol Med 2014

Dramatic Differences in Azithromycin Activity vs. Multidrug-Resistant Gram-Negative Rods in Tissue Culture Media vs. Bacteriologic Media

Leo Lin (UCSD MSTP) Lin et al. eBiomedicine 2015 Azithromycin is Cidal for MDR Gram-Negative Rods at low Concentrations in RPMI + 5% LB

MDR K. pneumoniae MDR A. baumannii

10 10 8 8 6 6 *** *** 4 4

Log CFU/ml 2 Log CFU/ml Log 2 < limit < limit 0 0 0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24 Time (h) Time (h)

Ca-MHB No abx Ca-MHB No abx RPMI (5% LB) AZM 1 RPMI (5%LB) AZM 0.5

Lin et al. eBiomedicine 2015 Synergy Between Azithromycin and LL-37 in Killing MDR Gram-Negative Rods

Lin et al. eBiomedicine 2015 Azithromycin Synergy with LL-37: Increased Cell Wall Permeability and Azithromycin Entry

MDR Acinetobacter baumannii

DAPI SYTOX Green Untreated AZM 0.5 LL-37 (5 μM) AZM + LL-37 6 *** 40 T T 5 *** 30 4 *** 3 20 DAPI *** 2 10 1 Relative intensity Relative intensity 0 0 AZM 0.5 LL-37 (5 μM)

Toroid nuclei NBD

SYTOX Green SYTOX 1 μΜ g 20 *** 8 *** f 15 6 Untreated NBD-AZM 5 LL-37 (5 μM) NBD-AZM + LL-37 10 4

% of total 5 2

0 Relative intensity 0 NBD NBD-AZM 5 1 μΜ LL-37 (5 μM)

Lin et al. eBiomedicine 2015 Azithromycin Monotherapy Reduces CFU, Lung and Mortality in Mouse Model of A. baumannii Pneumonia

MDR A. baumannii MDR A. baumannii MDR A. baumannii 8 lung infection 3 lung infection 5 lung infection *

7 *** *** 5 *** Start PMN 4 PBS 6 ** 2 AZM 100 5 Mac 3 4 2 * 3 1 2

Log CFU (24h) Log CFU 1 BAL CellsBAL x 10 1 ng/ml in BAL 0 0 0 PBS AZM 50 AZM 100 PBS AZM 100 IL-1β IL-6 MIP-2

MDR A. baumannii MDR A. baumannii MDR A. baumannii lung infection 100 Mac PMN 80 AZM 100 q 24h x 2

PBS Mac

AZM 100 60 – *** – A. baumannii 40 PBS control A. baumannii PMN survival % 20

BAL FluidBAL 100μm 100μm 0 BAL FluidBAL 0 1 2 3 4 5

Lin et al. eBiomedicine 2015

Munguia & Nizet Trends Pharm Sci 2017 Concept: The Collaborative to Halt Antibiotic-Resistant Microbes

@ Mining novel sources of natural product chemical diversity (the ocean!)

Fundamental microbiology of antibiotic resistance mechanisms, alternative model systems, active genetics Innovative medicinal chemistry approaches to make smarter, safer, targeted antibiotics

High-throughput screens, chemical genomics, extend to parasitic & emerging viral diseases Treat infection while preserving the microbiome, modulate microbiome to increase host resistance

Systems biology, big data “–omics” approaches, engineering solutions to diagnosis/treatment Treat infection as a host-pathogen interaction, block virulence and boost immune clearance

Analytics of patient data, personalized medicine, novel clinical lab testing, phage therapeutics

Robert Schooley Monika Kumaraswamy Clinical Therapeutics Antibiotic Mechanisms Antibiotic Stewardship Phage Therapy Clinical Therapeputics Infection Control New NIH/NIAID U01 (Palsson/Nizet/Pogliano/Sakoulas/Dorrestein/Knight/Feist) “Systems Biology Approach to Redefine Susceptibility Testing and Treatment of MDR Pathogens in the Context of Host Immunity”

OVERARCHING GOAL

Establish UC San Diego and its La Jolla Mesa Partners as a global leader in innovative, paradigm-shifting, research-driven solutions to combat the Antibiotic Resistance Crisis, which will inevitably become a public health and national security emergency for the coming generation. Selected to move forward - new Health Sciences Strategic, with potential for Institute of Engineering in Medicine to partner Fundraising strategy to be developed – meetings this week w/

Ruben Flores (Office of Innovation & Commercialization) Working with CARB-X (NIH/DOD/Wellcome Trust) Accelerator – 3 projects already funded with UCSD investigators International Partners lined up (CCCID-China, Amrita U-India, Utrecht, Queensland, Pasteur, Karolinska) Launch website and social media presence by Winter Quarter ’18 Philanthropy key (meeting w/ Emily Bass) FORMER LAB http://nizetlab.ucsd.edu

Yung-Chi Chang (National Taiwan U.) Ismael Secundino (UNAM-Cuernava) COLLABORATORS Ajit Varki, Richard Gallo, Mark Walker, Randy Johnson, Nina van Sorge (Utrecht Univ.) Suzan Rooijakkers (Utrecht Univ.) Partho Ghosh, Michael Karin, Bipin Nair, Geetha Kumar, Jeff Perry, Pieter Dorrestein. Ethan Bier, Joe Pogliano, Maren von Köckritz-Blickwede (U. Hanover) Cheryl Okumura (Occidental) George Liu (Cedars-Sinai) Kelly Doran (San Diego State) Carole Peyssonnaux (Instiute Cochin) Annelies Zinkernagel (U. Zurich) Laura Crotty Alexander (UCSD) Amanda Lewis (Wash. U. St. Louis) Shauna McGillivray (Texas Christian U.) David Gonzalez (UC San Diego)