NABATIVI – Novel Approaches to Bacterial Target Identification, Validation and Inhibition

1. NABATIVI – Novel Approaches to Bacterial Target Identification, Validation and Inhibition Alessandra Bragonzi, PhD Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Disease San Raffaele Scientific Institute

2. OUTLINE • The problem • Anti-microbial drug resistance • Pharmaceutics' response • EU’s response • How we built the NABATIVI project • International collaboration • Multidisciplinary approach • Scientific excellence • Major achievement

3. OUTLINE • The problem • Anti-microbial drug resistance • Pharmaceutics' response • EU’s response • How we built the NABATIVI project • International collaboration • Multidisciplinary approach • Scientific excellence • Major achievement

4. The world-wide threat of resistant pathogens

5. What is boosting antimicrobial resistance? superbugs Insufficient surveillance, prevention and control Insufficient research and development activities Insufficient funding Insufficient coordination of EU efforts Absence of new drugs 5

6. Why is antimicrobial resistance a global concern? • kills • hampers the control of infectious diseases • threatens a return to the pre-antibiotic era • increases the costs of health care • jeopardizes health-care gains to society

7. Facts on antimicrobial resistance • 4 million patients every year • 25 000 deaths • economic losses € 1.5 billion

8. Who is responsible? Enterococcusfaecium Staphylococcusaereus Klebsiellapneumoniae Acinetobacterbaumanni Pseudomonas aeruginosa Enterobacter species

9. OUTLINE • The problem • Anti-microbial drug resistance • Pharmaceutics' response • EU’s response • How we built the NABATIVI project • International collaboration • Multidisciplinary approach • Scientific excellence • Major achievement

10. Pharmaceutics’ response to antimicrobial-resistance • The industry is cutting research in antimicrobial discovery and development • Only few big pharmaceutical companies are still involved in antibiotic discovery • Antibiotic R&D is a lengthy, costly, and risky process due to: - length of time (10-15 years) from the discovery phase to market - huge cost of bringing a new drug to market ($800 million to 1.7 billion) - low reimbursement due to the small market - low success rate Payne et al.Nature Reviews Drug Discovery6, 2007

11. Antibacterial pipeline, Big Pharma New systemic antibacterial agents approved by the US Food and Drug Administration per 5-year period, through 2012. Boucher H W et al. Clin Infect Dis. 2013;56:1685-1694

12. How do we fill the gap ?

13. OUTLINE • The problem • Anti-microbial drug resistance • Pharmaceutics' response • EU’s response • How we built the NABATIVI project • International collaboration • Multidisciplinary approach • Scientific excellence • Major achievement

14. EU’s response –6th and 7th Framework Programme • Basic Science • Prudent use of antibiotics • New antimicrobials • Point of care diagnostic tests • Vaccines

15. EU’s response –Translational research for health (unit F3) • 2.3.1 – Anti-microbial drug resistance • Management of Gram negative multi-drug resistant infections. 2.3. TRANSLATIONAL RESEARCH IN MAJOR INFECTIOUS DISEASES: TO CONFRONT MAJOR THREATS TO PUBLIC HEALTH 2.3.1. Anti-microbial drug resistance including fungal pathogens 2.3.1-1: Novel targets for drugs against Gram negative bacteria. The objective is to identify and validate novel drug targets in order to select lead compounds, which may be derived from natural sources or from synthetic compounds, for future development of a new class of anti-infective drugs against Gram-negative bacteria. Significant industrial involvement, particularly by SMEs, is foreseen in this topic. Funding scheme: Collaborative projects (Small or medium-scale focused research projects with maximum EC contribution of € 6,000,000/project). SME ≥30%

16. OUTLINE • The problem • Anti-microbial drug resistance • Pharmaceutics' response • EU’s response • How we built the NABATIVI project • International collaboration • Multidisciplinary approach • Scientific excellence • Major achievement

17. How we built the NABATIVI project • International Collaboration Academic SMEs Alessandra Bragonzi, San Raffaele Scientific Institute, Milano Natalia Nekohotieva, KDevExploratory, Stockholm Miguel Cámara, University of Nottingham Gerd Döring, Eberhard Karls Univerität Tübingen Do Quoc-Tuan, Greenpharma S.A., Orléans, John A. Robinson and Leo Eberl, University of Zürich Giovanni Bertoni, Università degli Studi di Milano Daniel Obrecht, Polyphor, Allschwil Peter E. Nielsen, University of Copenhagen

18. How we built the NABATIVI project • Multidisciplinary approach Molecular microbiology Clinical research Genomics Biochemistry Pre-clinical research Drugs Structural biology High-throughput technology Bioinformatics Cell biology

19. NABATIVI approaches in response to Call FP7-HEALTH-2007-B • Discovery Phase Approach A: “from target to lead compounds” • Target identification/validation is done at the beginning • Discovery Phase Approach B: “from drugs to targets” • Target identification runs in parallel to the discovery phase Discovery phase A Target identification Library selection/design Screening cascade Hit identification Lead optimization Hit-to-lead B Pre-clinical and clinical phases Pre-clinical development Phase I Phase II Phase III NDA Launch 19

20. Pseudomonas aeruginosa selected from ESKAPE pathogens • 5 million cases in Europe, USA and Japan every year • Responsible of ≈ 30% of world-wide hospital-acquired infections • High risk of disease in people with cystic fibrosis, in immunocompromised, burn patients, patients with cancer, and with HIV • Multi-drug resistant strains

21. Scientific approach –Genome - based approach for target identification PAO1 6.3 Mb 5,570 ORFs 6,7% function experimentally demonstrated (Class 1) Stover et al. Nature. 2000 Aug 31;406(6799):959-64.

22. NABATIVI scientific approach –Step 1: genome-wide screening for targets identification Transposon libraries Antisense libraries Screenings Università degli Studi di Milano University of Nottingham Results

23. NABATIVI scientific approach –Step 2: pathogenicity in different model system University of Nottingham San Raffaele Scientific Institute Università degli Studi di Milano Genome-wide screening 57.360 University of Zürich 404 43 Eberhard Karls Univerität Tübingen San Raffaele Scientific Institute 27 Eberhard Karls Univerität Tübingen San Raffaele Scientific Institute 5 23

24. Genomic Target database (GTD) NABATIVI scientific approach –Step 3: target inhibition by high-throughput technology 24

25. Genomic Target database (GTD) Purified target-based HTS: • Plant extracts (800) • Pure natural compounds (480) • Synthetic compounds (45000) • Database AMBINTER 18 M compounds Determination of the druggability of selected targets N H B Greenpharma S.A., Orléans, N O O N H KDevExploratory, Stockholm B N O Greenpharma S.A., Orléans, PNA O Antisense targeting of selected targets by peptide nucleic acids University of Copenhagen NABATIVI scientific approach –Step 3: target inhibition by high-throughput technology 25

26. NABATIVI approaches in response to Call FP7-HEALTH-2007-B • Discovery Phase Approach B: “from drugs to targets” • Target identification runs in parallel to the discovery phase • Most antibiotics have been identified by this approach Synthesis of a library of Protegrin I analogues by PEM technology Initial hits with broad spectrum antimicrobial activity and lack of hemolytic activity Selective anti-Pseudomonas compounds with novel SAR POL7080 POL7001 MIC determination Target identification Library selection/design Screening cascade Hit identification Lead optimization Hit-to-lead LptD, a β-barrel outer membrane transporter is the target of POL7001 and POL7080 Pre-clinical development Phase I Phase II Phase III NDA Launch Efficacy studies in animal models of sepsis and respiratory infections POL7080: preparations for a first Phase IIa clinical trial under NABATIVI 26

27. NABATIVI scientific approach –Step 4: target identification running in parallel to the discovery phase LptD Srinivas N, et al. Science. 2010 Bragonzi A. Sci. Transl. Med. 2010 Polyphor Ltd. University of Zurich • LptD is an essential gene in P. aeruginosa and the target for novel drug POL7001 and POL7080 27

28. NABATIVI scientific approach –Step 4: translational research up to clinical trial Polyphor Ltd. San Raffaele Scientific Institute     POL7080 POL7001   Discovery phase Pre-IND Clinical phase • Novel drug POL7001 is active against MDR P. aeruginosa strains • In pre-clinical studies including models of airway infections and septicemia, POL7001 showed higher efficacy when compared to clinically approved antibiotics 28

29. POL7080 indications for P. aeruginosa infections Indications for Pseudomonas infections

30. NABATIVI - Major achievements • A genomic target database of P. aeruginosa is assembled • Novel hits and PNAs are identified and are currently under validation • LptD as novel target for drug with novel mechanism of action is identified • POL7080 successfully completed Phase I and will start Phase II at the end of 2013 Discovery phase A Target identification Library selection/design Screening cascade Hit identification Lead optimization Hit-to-lead Pre-clinical and clinical phases B Pre-clinical development Phase I Phase II Phase III NDA Launch

31. NABATIVI – novel drug

32. NABATIVI – hit the target !

33. Critical success factors for NABATIVI project • Integrated multidisciplinary research at European level was successful in novel drug discovery; • Academia and a small innovative company worked together productively to fill the gap left by the migration of big pharmaceuticals away from antibacterial development; • Combined scientific approaches, including exploitation of post-genomic information, were successful in identifying novel drugs; • Optimal translation of the results into the clinic through appropriate pre-clinical models Molecular microbiology Clinical research Genomics Pre-clinical research Biochemistry Drugs Structural biology High-throughput technology Cell biology Bioinformatics