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Infectious Diseases Drug Discovery: An AstraZeneca Perspective

Infectious Diseases Drug Discovery: An AstraZeneca Perspective. Tomas Lundqvist GSC LG-DECS AstraZeneca R&D Mölndal Stewart L. Fisher Infection Discovery AstraZeneca R&D Boston. AstraZeneca R&D Boston. History AZ’s newest research facility Construction initiated August 1998 (Astra)

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Infectious Diseases Drug Discovery: An AstraZeneca Perspective

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  1. Infectious Diseases Drug Discovery:An AstraZeneca Perspective Tomas Lundqvist GSC LG-DECS AstraZeneca R&D Mölndal Stewart L. Fisher Infection Discovery AstraZeneca R&D Boston

  2. AstraZeneca R&D Boston History • AZ’s newest research facility • Construction initiated August 1998 (Astra) • Building completed March 2000 (AstraZeneca) • Three Research Areas • Infection Discovery (Global Center) • Oncology • Discovery Informatics • Building expansion completed 2003 • Increased resourcing for Oncology • Approximately 450 employees • Expansion underway: • $100 mil investment in capital (buildings) • Increased resource for Infection Research

  3. Medical Need Business Opportunity Social Responsibility Why Focus on Infectious Disease?

  4. Medical need • 41% of global disease burden is due to infection (WHO, 2002) • Outside EU & US the disease burden from infection is greater than the total of all other therapy areas combined Causes of Death Percentage of all deaths worldwide Ref. WHO Data

  5. A Major Issue for All

  6. The Golden Age & Today • The pipeline for new antibacterials is drying up • Resistance to antibacterials continues to rise • There is a clear & present danger of import to both individual patients and the public health The Golden Age of Antibiotic Discovery was very brief, mid 1930s- early 1960s penicillin, cephalosporin, streptomycin, erythromycin, tetracycline, vancomycin

  7. Target Based Approaches • 1990’s: Dominant lead generation approach • “Genomic era” • Combinatorial/parallel chemistry = large compound libraries • Automated screening technologies provided economy of scale • Structural approaches most amenable to bacterial targets • Soluble • High yield overproduction/purification • 2000-present • Approach seen as “not delivering the pipeline” • Many reasons for “failure” • Poor compound libraries (not as clean as envisioned) • Difficult to choose the “druggable” targets • Enzyme inhibition ≠ antimicrobial activity (efflux) • Sufficient patience in the industry?

  8. Cell Based Approaches • 1990’s: Diminished activity due to target-based approaches • Hit followup appeared “messy” relative to target based • Identification of novel antibiotics increasingly difficult • Major efforts in combinatorial biosynthesis • Genetic manipulation of natural product producers • 2000-present – renewed interest • Less faith in target based approaches (e.g. lessons from GSK FabI) • Improvements in genomic technologies allows facile hit followup • Regulated gene libraries • Target identification via resistance gene mapping • Automated screening technologies affords novel approaches • Approach amenable to pathways and difficult targets

  9. “Look Back” Programs • Revisiting past discoveries, finding new value • Ramoplanin, Tiacumicin B – value of C. difficile in 1980s? • Daptomycin – value of MRSA in 1980’s • Advances in chemistry make intractable scaffolds amenable • ADEPs • Anisomycin • Moiramide

  10. Target-Based Approaches: Pipeline Target Identification HitIdentification Lead Identification Lead Optimisation Preclinical/ Clinical Peptide Deformylase GyrB/ParE MurA MurB MurC MurD MurE MurF MurG MurA-F pathway MurG MraY-PBPII pathway DdlB FtsZ FtsZ/ZipA LpxC RNA Polymerase (RNAP) DNA Polymerase (DNAP) DnaB Phe-tRNAS Trp-tRNAS Met-tRNAS GyrB PanK H. pylori MurI FabI/K Phe-tRNAS Ile-tRNAS GyrB many (100’s) see genomic patents FabDFGAI pathway FabI AcpS FtsZ Mur Pathway

  11. First Step: Define the Problem Target Product Profile Target Identification HitIdentification Lead Identification Lead Optimisation Preclinical/ Clinical • Definition of a Target Product Profile • Define the disease & unmet medical need • Set the requirements for the drug • Find targets that fit the requirements

  12. Causative agent for stomach ulcers • Implicated in gastric cancer • Current therapy effective (~ 90%) if properly completed • Poor patient compliance due to complicated regimen and side • effects • Resistance • Metronidazole 20 - 60%, Clarithromycin 10 -15% Proton pump inhibitor (O) + two antibiotics: Clarithromycin (C), Amoxicillin (A), Metronidazole (M) Need for New Therapeutic Strategies Therapy for Helicobacter pylori Infections

  13. Target Product Profile (H. pylori TPP) Deliver a candidate drug with this profile: • Monotherapy • Oral dose, once a day (Patient Compliance) • High Selectivity • Minimize gut flora disturbance (Patient compliance) • Novel target • No pre-existing resistance (General Utility) • No threat to current antibiotic regimens (Cross-Resistance) • No target based toxicity issues (Patient Safety)

  14. Target Identification • Target Identification • Genomics-based selection • Validation of essentiality in relevant organisms • Cloning and expression of target proteins • Production of target proteins Phases of Target-Based Approach: Target Identification

  15. Glutamate Racemase (MurI) UDP-GlcNAc Fosfomycin Attributes • Novel target for drug discovery • Essential target • Pathway is specific to bacteria • Clinically validated Cons • Cytoplasmic target (Drug penetration?) • Bacterial kingdom conservation (Selectivity?) UDP-MurNAc MurC UDP-MurNAc-(L) Ala MurI L-Glu D-Glu MurD UDP-MurNAc-(L) Ala-(D) Glu B-lactam classes glycopeptides peptidoglycan

  16. Bacillus subtilis Bacillus anthracis Staphylococcus haemolyticus Staphylococcus aureus Bacillus sphaericus Streptococcus pneumoniae Gram +ve Streptococcus pyogenes Enterococcus faecalis Lactobacillus brevis Pediococcus pentosaceus Lactobacillus fermentum Mycobacterium leprae Mycobacterium tuberculosis Haemophilus influenzae Escherichia coli Shewanella putrefaciens Vibrio cholerae Gram -ve Treponema pallidum Borrelia burgdorferi Deinococcus radiodurans Pseudomonas aeruginosa Porphyromonas gingivalis Campylobacter jejuni Helicobacter pylori H. pylori Aquifex aeolicus Genomic-based Hypotheses for Selectivity • Low sequence identity observed across bacterial species • Lowest sequence identity of all mur pathway genes • H. pylori MurI in a distinct phylogenic clade • Facile protein expression and production • Gram-scale quantities achieved in high purity (>99% pure)

  17. Phases of Target-Based Approach: Hit Identification Target Identification HitIdentification • Hit Identification • Biophysical and biochemical characterization of targets • Development of primary assay and secondary assays for evaluation of hits • Kinetic mechanism studies for enzyme targets • Screening (e.g. HTS, virtual) and chem-informatic analysis • Limited SAR generation

  18. Results from Biochemical and Biophysical Characterization: • Active protein is a dimer • No cofactors required for activity • Kinetic analysis of enzyme reaction indicates an unusual profile • Assays required for forward and reverse reaction H. pylori MurI: an Enigma • Novel Enzyme Crystal Structure Solved – 1998 • Crystal Structure Features • Dimeric enzyme • Active sites occluded from solvent • Selective binding of D-Glu

  19. Enzyme Mechanism and Assays L-Glutamate D-Glutamate Cys 70 Cys 181 -S SH S- HS SH HS 70 181 70 70 181 181 Carbanion intermediate Coupled Assay with L-Glutamate dehydrogenase Measure NADH Preferred HTS Assay Coupled Assay with MurD Measure Pi or ADP Resource intensive, Expensive

  20. Kinetic Analysis of Native H. pylori MurI D-Glu L-Glu L-Glu D-Glu D-Glu KM = 63 mM kcat = 12 min-1 KIS = 5.8 mM L-Glu KM = 700 mM kcat = 88 min-1 kcat/KM = 185 mM-1 min-1 kcat/KM = 126 mM-1 min-1

  21. E.L-glu Energy E+L-glu E+D-glu Energy E.D-glu E+L-glu E+D-glu Reaction Coordinate L-Glutamate D-Glutamate E.L-glu E.D-glu Reaction Coordinate -S SH S- HS SH HS 70 181 70 70 181 181 H. pylori MurI Glutamate Racemases: Biochemistry

  22. No obvious avenues HTS Assay? Poor Inhibition Profile Novel Assay Format Implications of Unique Biochemical Profile • Screening unlikely to identify substrate-competitive inhibitors • Enzyme:Substrate complex = dominant population • Free Enzyme levels = very low • Active site is not drug-friendly • Highly charged • Small • Accessibility • Options: • Structural / Rational Design • HTS – non-competitive or uncompetitive inhibitors? • Suicide substrate / mechanism-based inhibitors HTS of corporate collection using novel assay

  23. blank 0.2mM S 0.5mM S 2.0mM S Suicide Substrate HTS Assay • HTS Assay • All reagents commercially available • Linear time course (irreversible) • Excellent Assay Window • Amenable to 384-well HTS format x4000 x1 Screened corporate collection for inhibitors (~150,000 cpds)

  24. Pyrimidinediones:Features of the Hit Cluster • Hit Attributes: • in vitro inhibition confirmed in multiple, orthogonal assay formats • Whole cell activity in H. pylori • Confirmed mode of action in whole cells • Amenable to MPS routes • Drug-Like Scaffold Compound A IC50 = 1.4 mM MIC = 8 mg/mL

  25. Lead Identification HitIdentification Target Identification • Lead Identification • Biochemical mode of inhibition understood • Facile synthetic strategies in-place (combichem, MPS) • Whole-cell activity • Confirmed target-mediated mode of action in cells • Early drug metabolism/pharmacokinetics (DMPK) studies • Hit Identification • Biophysical and biochemical characterization of targets • Development of primary HTS assay and secondary assays for evaluation of hits • Kinetic mechanism studies for enzyme targets • HTS Screening and chem-informatic analysis • Limited SAR generation • Target Identification • Genomics-based selection • Validation of essentiality in relevant organisms • Cloning and expression of target proteins • Production of target proteins Phases of Target-Based Approaches: Lead Identification

  26. Mechanism of Inhibition? ≠ Substrate Inhibitor

  27. Protein NMR – Foundational Work glutamate free 1.8 mM D-Glutamate • Double (15N, 2H) & Triple-labeled (15N, 13C, 2H) protein prepared in high yield • D-Glutamate titration produced a highly resolved spectrum • All backbone resonances assigned; homodimer ~ 60kD NMR indicates multiple conformations at room temperature D-Glutamate stabilizes protein – consistent with kinetic profile

  28. Protein NMR Demonstrates Substrate Dependence • Titration of compound reveals specific shifts only when substrate present • Spectrum remains unresolved when compound titration with apo protein • Assignment of resonances allows binding site mapping Black = D-Glu + MurI Red = D-Glu + MurI + Inh Compound binding requires substrate Binding site distal from active site

  29. Inhibitor:Enzyme Co-Crystal Structure: The “Where” • Cryptic binding site identified ~7.5Å from active site • Consistent with NMR binding studies - C-Terminal helix movement • Catalytic residues unchanged relative to apo structure. • Supported biochemically: • Isothermal Titration Calorimetry • Intrinsic Protein Fluoresence Quenching • Uncompetitive inhibition KI = Kd

  30. Cryptic Binding Site – Detailed View MurI + D-Glutamate MurI + D-Glutamate + Inhibitor Unexpected allosteric inhibition mechanism – impact of HTS

  31. Increasing [Inh] Rate (RFU/min) [D-Glu] (μM) ΔRFU [Inhibitor] μM KI = IC50 Biochemical Confirmation of Inhibition Mode • Binding mode confirmed in multiple formats: • Intrinsic Protein Fluorescence Quenching • Isothermal Titration Calorimetry • Kinetic Mechanism Consistent with Uncompetitive Inhibition

  32. Inhibitor Hinge Mode of Inhibition: The “How” • Catalytic activity dependent on hinge movement • Compounds bind at domain interface – lock hinge movement

  33. Bacterial Growth Inhibition Mode of Action Confirmation PeptidoglycanBiosynthesis Pentapeptide UDP-MurNAc UDP-MurNAc-(L) Ala MurC UDP-MurNAc-(L) Ala MurI MurD D-Glu L-Glu A254nm UDP-MurNAc-(L) Ala-(D) Glu MurE + Inhibitor UDP-MurNAc-(L) Ala-(D) Glu-mDap MurF UDP-MurNAc-(L) Ala-(D) Glu-mDap-(D) Ala-(D) Ala * Growth inhibition through MurI inhibition

  34. Lead Identification Lead Optimization HitIdentification Target Identification • Lead Identification • Facile synthetic strategies in-place (combichem, MPS) • Biochemical mode of inhibition understood • Whole-cell activity • Confirmed target-mediated mode of action in cells • Early drug metabolism/pharmacokinetics (DMPK) studies • Lead Optimization • Focus on analogs of central scaffold(s) • Activity in animal disease-state model • Assess potential for resistance • in vivo DMPK studies for human dosing estimation • in vitro toxicological studies • Scale up synthesis; process chemistry • Hit Identification • Biophysical and biochemical characterization of targets • Development of primary HTS assay and secondary assays for evaluation of hits • Kinetic mechanism studies for enzyme targets • HTS Screening and chem-informatic analysis • Limited SAR generation • Target Identification • Genomics-based selection • Validation of essentiality in relevant organisms • Cloning and expression of target proteins • Production of target proteins Phases of Target-Based Approaches: Lead Optimization

  35. Trojan Horse or Goldmine? Can we improve potency? What is the potential for resistance? Can we achieve the desired selectivity margin?

  36. Potency Enhancements • Established parallel synthesis approaches to rapidly diversify all 4 positions • Short synthesis, clean reactions • Amenable to MPS and readily diversified • Compounds easily purified by preparative HPLC • Guided by co-crystal structure Site partially open to solvent but has potential for specific H-bond interactions (Glu, Ser, H2O) R4 Exposed to solvent R1 Deep large hydrophobic pocket R3 R2 Site mainly surrounded by hydrophobic groups with a polar terminus (His, Lys)

  37. IC50 = 67 nM IC50 = 503 nM Cl Glu150 IC50 = 6 nM • Combination of best R3 and R4 resulted in • 250-fold improvement in potency from Hit SAR - Highlights IC50 = 2200 nM IC50 = 103 nM Potent inhibitors used to assess resistance

  38. Novel Pocket Concerns: Resistance Rates Resistance Potential (single step selection): • Acceptable (very low) resistance rates observed • Despite the low resistance rate, mutations in murI were identified at low [Inhibitor] [Inhibitor] ≈ 2 x MIC

  39. A35T A75T A75V E151K C162Y I178T G180S L186F L206P Q248R Biochemical Analysis of Resistance Mutants - Mapping onto crystal structure did not yield an obvious answer: Not in the substrate binding pocket Not in the inhibitor binding pocket (L186F) - Two were chosen for biochemical characterization: A75T (most prevalent) E151K (most dramatic)

  40. A75T H. pylori MurI Kinetic Profile D-Glu L-Glu L-Glu D-Glu D-Glu KM = 275 mM (63 mM) kcat = 4 min-1 (12 min-1) KIS = 660 mM(5.8 mM) L-Glu KM = 7400mM (700 mM) kcat = 106 min-1 (88 min-1) kcat/KM = 14.5 mM-1 min-1 kcat/KM = 14.3 mM-1 min-1 Inhibition elevation: (IC50A75T/IC50wt) ~9 fold MIC elevation: ~4 – 8 fold

  41. E151K H. pylori MurI Kinetic Profile D-Glu L-Glu L-Glu D-Glu D-Glu KM = 280 mM (63 mM) kcat = 5 min-1 (12 min-1) (5.8 mM) L-Glu KM = 7300mM (700 mM) kcat = 136 min-1 (88 min-1) kcat/KM = 18 mM-1 min-1 kcat/KM = 18 mM-1 min-1 Inhibition elevation: (IC50E151K/IC50wt) ~15 fold MIC elevation: ~8 - 16 fold

  42. E151K Energy Decreased Stability Resistance impact A75T E WT ES Reaction Coordinate Destabilization of ES Complex

  43. MurI Resistance Mechanism D-Glu (MurI*•D-Glu) MurI* MurI Substrate inhibited L-Glu D-Glu (MurI*•L-Glu) (MurI•D-Glu) Resistance mutants disfavor [ES]/[FS] species: - Higher Km - Reduced/Eliminated Substrate Inhibition Reduced [ES] = less inhibition! But… increased potency can overcome effect

  44. High D-Glu (5mM) Low D-Glu (50mM) 26 nM 23 nM 31 nM 170 nM Direct Binding Measurements with Inhibitors 10000 8000 6000 ΔRFU 4000 2000 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 [Inhibitor] uM Dissociation Constant (Kd) MurI Enzyme Native A75T Mutant

  45. Bacterial Selectivity Requirement What about the selectivity profile?

  46. Organism IC50 (nM) MIC (mg/mL) H. pylori 9.2 0.5 E. coli >400000 >64 H. influenzae >64 M. catarrhalis >64P. aeruginosa >64S. aureus >400000 >64 S. pneumoniae >64 S. pyogenes >64 E. faecalis >400000 C. albicans >64 Selectivity Profile • Excellent selectivity profile observed in series: • in vitro (IC50) > 50,000-fold • Whole cell > 128-fold • Basis for selectivity understood – variations in inhibitor binding pocket • Binding pocket sequence divergence • Limited flexibility to form pocket across species

  47. Trojan Horse or Goldmine? Can we improve potency? YES! What is the potential for resistance? Low Can we achieve the desired selectivity margin? YES! So, where’s the drug?

  48. Target Inhibitor  Drug • biochemical properties • bona fide enzyme inhibition • potency, spectrum • physical properties • molecular size • lipophilicity • solubility • in-vivo properties • plasma protein binding • absorption • metabolism • excretion • pharmacokinetics • safety • microbiological properties • potency, spectrum • bona fide inhibition of bacterial growth (MOA) • resistance frequency • population MICs (MIC90)

  49. Pharmacokinetic Profiles in Mouse in vivo Drug Levels in Mouse Plasma 10 iv 5 mg/kg 8 po 40 mg/kg Cl = 14 µl/min/kg t½ = 0.7 hr F = 76 % 6 Concentration (mg/ml) 4 2 MIC 0 0 1 2 3 4 5 6 Time (h) • Improved PK in dogs • Total drug levels above MIC for extended period of time

  50. Requirements for Efficacy: Free Fraction in vivo Drug Levels in Mouse Plasma 10 po 40 mg/kg, free 8 po 40 mg/kg, total Cl = 14 µl/min/kg t½ = 0.7 hr F = 76 % fu < 3 % 6 Concentration (mg/ml) 4 2 MIC 0 0 1 2 3 4 5 6 Time (h) • Free drug levels in plasma below MIC • Difficult to achieve balance between protein binding and potency

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