Structure-Guided Discovery of ( S) -3 - Structure-Guided Discovery - PowerPoint PPT Presentation

OH B O ABX NH 2 Structure-Guided Discovery of ( S) -3 - Structure-Guided Discovery of ( S) -3 - ( am inom ethyl) benzo[ c] [ 1 ,2 ] oxaborol- ( am inom ethyl) benzo[ c] [ 1 ,2 ] oxaborol- 1 ( 3 H) -ol hydrochloride ( ABX) : A First in 1 ( 3 H) -ol hydrochloride ( ABX) : A First in Class Gram -negative Antibacterial Class Gram -negative Antibacterial Vincent Hernandez Senior Scientist, Medicinal Chemistry Anacor Pharmaceuticals, Inc. Palo Alto, CA, USA vhernandez@anacor.com

Talk Summary Talk Summary OH B O ABX NH 2 � Anacor Pharmaceuticals has a rich pipeline of boron-containing therapeutics in the clinic � Boron is underexploited in medicinal chemistry and has tremendous potential in drug discovery � ABX inhibits bacterial Leucyl tRNA synthetase and represents a new class of Gram-negative antibacterial agents � This novel mechanism of action means ABX is not affected by existing modes of bacterial resistance � ABX is efficacious in vivo against E. coli and Pseudomonas in mouse models of infection � AN3365 has advanced to Phase I clinical development for the treatment of Gram-negative bacterial infections 2

Anacor Has a Large Pipeline of Novel Anacor Has a Large Pipeline of Novel Boron-containing Candidates Boron-containing Candidates Research Research Preclinical Preclinical Phase 1 Phase 1 Phase 2 Phase 2 Phase 3 Phase 3 Topical Anti-Fungals AN2 6 9 0 Onychomycosis AN2 7 1 8 Skin & Onychomycosis Fungal Infections Topical Anti-I nflam m atories AN2 7 2 8 Psoriasis Atopic Dermatitis AN2 8 9 8 Psoriasis Atopic Dermatitis System ic Anti-Biotics AN3365 Antibacterial 2 Antibacterial 3 Antibacterial 4 3

Boron is Commonly Found in Our Environment Boron is Commonly Found in Our Environment � In nature, boron is present as boric acid � Boric acid is the main ingredient of Goop – Children’s brightly colored toy, that they squeeze through their fingers � Boric acid is used as a preservative in eye wash and vaginal creams � Boric acid has an LD 50 similar to regular table salt (~ 3000 mg/ kg) � Boron is an essential plant nutrient � We consume up to 4 mg of boron a day, primarily from fruits, vegetables and nuts � At Anacor, we found background levels of 200 ng/ mL of boron in mouse plasma 4

Boron has a Unique Bonding Orbital Boron has a Unique Bonding Orbital Configuration: An Empty P-Orbital Configuration: An Empty P-Orbital Trigonal Planar Tetrahedral � Boron has an empty p -orbital & can form a dative bond under specific conditions � The dative bond forms a tetrahedral structure � Exploitation of p- orbital expands drug design possibilities 5

History and Overview of Organo-boron Drug History and Overview of Organo-boron Drug Discovery Efforts Discovery Efforts � Design of boronic acid protease inhibitors initiated in 1990s � Multiple disease targets have been pursued – Thrombin – Factor Xa – Bacterial β -lactamases – HCV protease O O H H – DPP4 N N B N O H H – Arginase O N Velcade � Only Velcade has reached FDA approval � Lack of success despite substantial efforts attributed to poor drug-like properties of boronic acids Baker et al. (2009) Future Medicinal Chemistry, 1(7), 1275-1288 6

AN2690 was Found to be a Broad Spectrum AN2690 was Found to be a Broad Spectrum Antifungal Agent Antifungal Agent Minimum Inhibitory Concentration Against Fungal Pathogens (µg/mL) AN2690 T. rubrum T. mentagrophytes C. albicans C. neoformans A. fumigatus OH B 1 1 0.5 0.25 0.25 O F � AN2690 has shown good efficacy, safety and tolerability in Phase I and II clinical trials � AN2690 is scheduled to begin Phase III 7

Antifungal Validated the LeuRS Editing Site Antifungal Validated the LeuRS Editing Site as a Novel Drug Target as a Novel Drug Target LeuRS has two active sites OH • Editing site B • Synthetic site O F AN2690 All mutations mapped to the editing domain Genetic study in Saccharomyces cerevisiae identified the cytoplasmic LeuRS gene ( CDC60 ) Rock et al. (2007) Science 316: 1759-1761 8

The Target: Leucyl-tRNA Synthetase (LeuRS) The Target: Leucyl-tRNA Synthetase (LeuRS) � Aminoacyl-tRNA synthetase - Structurally related to isoleucyl-tRNA synthetase and valyl- tRNA synthetase � Leucyl-tRNA synthetase attaches leucine to the 3’ end of tRNA Leu � Essential enzyme in protein synthesis � Enzyme has two active sites - Aminoacylation active site - Editing active site (proofreading) � Editing activity ensures fidelity of protein synthesis - Editing mutants are supersensitive to leucine analogues, like norvaline 9

X-ray Structure Revealed A tRNA Leu Adduct in the X-ray Structure Revealed A tRNA Leu Adduct in the Editing Site of Leucyl tRNA Synthetase Editing Site of Leucyl tRNA Synthetase AN2690-A76 Adduct In Editing Site NH 2 Leucine N in N Synthesis Site tRNA N O N O P O O O - O O B O F LeuRS tRNA Leu Rock et al. ( 2 0 0 7 ) Science 3 1 6 :1 7 5 9 -1 7 6 1 10

AN2690 Traps tRNA Leu In The Editing Site Thus AN2690 Traps tRNA Leu In The Editing Site Thus Inhibiting Aminoacylation And Editing Inhibiting Aminoacylation And Editing O xa bo role t R NA t rapping in editing Under normal conditions tRNA is domain free to move through domains Editing Editing X X Exit Exit Aminoacylation Aminoacylation AN2690 “OBORT” 11

X-ray Structure of AN2690 in LeuRS Revealed a X-ray Structure of AN2690 in LeuRS Revealed a Key Binding Site was not Utilized Key Binding Site was not Utilized Tyr-327 Tyr-327 Leu-329 Leu-329 Ile-337 Asp-347 Asp-347 Ile-337 Val-340 Tyr-332 Val-340 Thr-252 Thr-248 Thr-248 Thr-252 Met-338 Met-338 AN2690-AMP Norvaline post-transfer substrate analogue Lincecum et al. (2003) Molecular Cell 1 1 : 951-963 Rock et al. (2007) Science 316: 1759-1761 12

3-Aminomethyl Substitution was Added to 3-Aminomethyl Substitution was Added to Gain These Key H-bonds Gain These Key H-bonds Nva2 aa AN2 6 9 0 Nva2 aa ABX 13

Synthesis of ABX Synthesis of ABX ABX 14

Aminomethyl Group of ABX Makes Three Aminomethyl Group of ABX Makes Three Hydrogen Bonds with LeuRS Hydrogen Bonds with LeuRS Leu-327 Glu-329 Asp-342 Asp-345 Tyr-330 Val-338 NH 2 Met-336 Thr-248 N N Thr-252 N O N HO P O O O - Ser-227 O O Thr-247 O B H 2 N 15

Addition of Aminomethyl Group Provided Greatly Improved Inhibition For the S-Isomer I C 5 0 * ( µM) MI C ( µg/ m L) E. coli K1 2 tolC P. aeruginosa P. aeruginosa ATCC 2 7 8 5 3 E. coli K1 2 E. coli Com pound OH B 27.5 22.3 16 16 > 64 O OH B O 1.0 2.8 2 2 1 NH 2 ABX OH B O 48.0 > 100 16 32 16 NH 2 * IC 50 determined after 20 minutes pre-incubation with enzyme and tRNA 16

Enzyme Kinetics Shows Slow Tight Binding Enzyme Kinetics Shows Slow Tight Binding Inhibition and Slow Off-Rate Inhibition and Slow Off-Rate I C 5 0 I C 5 0 Enzym e Com pound ( 2 0 m ins) ( 6 0 m ins) Recovery ( t½ , hr) OH B 27.5 µM 26.3 µM 0.2 O OH B O 1.0 µM 0.4 µM 5.5 NH 2 ABX 17

Gram-negative MIC 90 Panel Demonstrates Gram-negative MIC 90 Panel Demonstrates Broad-spectrum Activity Broad-spectrum Activity Piperacillin/ Amoxycillin/ Levofloxacin Gentamycin Ceftazidime tazobactam clavulanate Tigeccyline Imipenem Cefepime Ampicillin ABX No. of Strain strains 1 16 1 8 2 4 16 32 >64 >64 P. aeruginosa (WT) 50 1 >16 32 >32 >16 >16 >32 >128 >64 >64 P. aeruginosa (MbL-) 25 1 >16 >64 >32 >16 >16 >32 >128 >64 >64 P. aeruginosa (MbL+) 26 >128 1 0.25 8 4 2 16 8 32 32 A. baumannii (WT) 25 >128 8 64 >32 >16 >16 >32 >128 >64 >64 Acinetobacter spp. (MDR) 26 1 1 >64 >32 4 >16 >32 >128 >64 >64 S. maltophilia (WT) 50 4 4 16 32 8 >16 16 32 >64 >64 B. cepacia 50 1 0.25 0.12 ≤ 1 >16 2 ≤ 1 8 32 >64 E. coli (WT) 27 2 0.25 0.25 >32 >16 >16 >32 128 64 >64 E. coli (ESBL) 25 1 0.5 0.25 ≤ 1 ≤ 0.5 1 ≤ 1 16 8 >64 Klebsiella spp. (WT) 25 1 2 1 >32 16 >16 >32 >128 64 >64 Klebsiella spp. (ESBL) 15 2 1 >64 >32 >16 16 >32 >128 >64 >64 Klebsiella spp. (KPC) 10 1 0.5 1 ≤ 1 ≤ 0.5 ≤ 0.5 2 8 >64 >64 Enterobacter spp. (WT) 25 1 4 0.5 8 >16 >16 >32 >128 >64 >64 Enterobacter spp. (AmpC) 26 1 0.5 1 ≤ 1 1 >16 2 16 >64 >64 Citrobacter spp. (WT) 36 0.5 0.5 1 2 16 2 >32 128 >64 >64 Citrobacter spp. (AmpC) 16 128 4 2 ≤ 1 2 2 ≤ 1 0.5 8 >64 P. mirabilis (WT) 42 >128 4 2 >32 >16 >16 >32 4 64 >64 P. mirabilis (ESBL) 11 >128 2 2 ≤ 1 ≤ 0.5 1 ≤ 1 0.5 16 >64 P. vulgaris (WT) 20 2 2 4 ≤ 1 4 2 4 2 >64 >64 M. morganii (WT) 17 16 2 2 ≤ 1 16 4 ≤ 1 4 >64 >64 Indole positive Proteae 14 0.5 1 1 ≤ 1 1 1 2 32 >64 >64 S. marcenscens (WT) 38 0.5 2 1 4 4 >16 >32 64 >64 >64 S. marcenscens (AmpC) 16 18