3,000 Proprietary Co-crystal Structures 90 0x >120x 6MM Selective Tyk2 Lead Series Compound Ideas Milestone Achieved Compounds Synthesized Selectivity over JAK2 Tyk2 Potency 2.5 nM 0 50 100 150 % Activity JAK2 Tyk2 Cellular Selectivity 31,979 400 >500x 0.3 nM 6 10 0 Months 12,400 260 >250x 0.5 nM 14 In vivo PoM GMCSF/ pSTAT5 IL-12/ pSTAT4 0 60 80 100 % Activity 20 40 N D I - 0 3 1 2 3 2 N D I - 0 3 1 3 0 1 B i o c h e m i c a l T y k 2 K i n a s e A s s a y T y k 2 K i ( n M ) 0.14 0.53 J A K F a m i l y K i n a s e B i o c h e m i c a l S e l e c t i v i t y ( F o l d o v e r K i ) F o l d S e l e c t i v i t y o v e r J A K 2 210x 85x F o l d S e l e c t i v i t y o v e r J A K 1 93x 107x F o l d S e l e c t i v i t y o v e r J A K 3 24x 15x P l a s m a P r o t e i n B i n d i n g H u m a n P P B ( % b o u n d ) 83 74 H u m a n i n v i t r o m e t a b o l i s m C l i n t ( m L / m i n / k g ) M i c r o s o m e s 5 3 H e p a t o c y t e s 2 9 M o u s e P K I V 3 m g / k g C l o b s ( m L / m i n / k g ) 35 39 P O 3 0 m g / k g T 1 / 2 ( h ) 1.6 4.8 C m a x ( μ M ) 15 13 F ( % ) 85 100 A U C ( μ M * h r ) 29 38 P h y s i c a l P r o p e r t i e s M W ( D a ) ; s o l u b i l i t y ( μ M ) 400-450; 10 400-450; 302 Vehicle - IL-12 Vehicle + IL-12 10 mg/kg NDI-031232 + IL-12 30 mg/kg Tofacitinib + IL-12 0 50 100 150 JAK2 pSTAT5 pSTAT4 Tyk2/JAK2 GMCSF IL-12 K i (nM) 0.1 to 0.5 5 to 10 10 to 150 Tyk2 JAK3 • IL-12 induced pSTAT4 IC 50 = 17 nM • GMCSF induced pSTAT5 IC 50 ~ 1.2 μM • Selectivity Tyk2/JAK2 = 70x 0 25 50 75 100 125 Activity (%) NDI-031232 (μM) 100 0.001 0.01 0.1 1 10 IL-12/pSTAT4 GMCSF/pSTAT5 IFNγ (pg/ml) pY pY IL-12/32 JAK2 Tyk2 Tyk2 JAK3 K i (nM) 0.1 to 0.5 5 to 10 10 to 150 • IL-12 induced pSTAT4 IC 50 = 74 nM • GMCSF induced pSTAT5 IC 50 ~ 4.5 μM • Selectivity Tyk2/JAK2 = 61x 0 25 50 75 100 Activity (%) NDI-031301 (μM) 100 0.0001 0.01 0.1 1 10 IL-12/pSTAT4 GMCSF/pSTAT5 0.001 IC 50 = 0.4 μM -1000 0 200 300 400 IFNγ (pg/ml) NDI-031301 (μM) 100 0.001 0.01 0.1 1 10 100 IL-12 p35 subunit p40 subunit IL-23 INF / p40 subunit p19 subunit Tyk2 Jak2 Jak2 Tyk2 Jak1 Tyk2 STAT 1-5 P via Th 17 Th 1 600 JAK2 Selectivity (Fold Over K i ) ~560x 350 300 250 0 50 100 150 200 0 500 400 300 200 100 Number of Compounds Synthesized Use of FEP for Tyk2 Program Additional Proprietary Co-crystal Structures New sub-series that takes advantage of additional interactions with non-conserved residues Free Energy Perturbation (FEP) Calculates Differences in Binding Energy Between Two Structures via Intermediary States P=10 -25 in >30,000 case-control samples P=10 -18 in ~15,000 Association (-logP) 2 2 4 1 Type 1 diabetes 2 Type 1 diabetic ketoacidosis 3 Insulin pump user 4 Psoriasis vulgaris 5 Rheumatod arthritis & related disorders 6 Rheumatod_arthritis 5 6 4 3 2 1 OR<1 OR>1 0 GAA 0.008 CGA 0.038 GGC 0.086 GGA 0.87 123 F 0.4 0.6 0.8 1 1.2 0.4 0.6 0.8 1 1.2 0.2 OR OR ref GGAA 0.15 GAAA 0.006 CGAA 0.036 GGAC 0.71 123 F GGCA 0.083 4 ref case-control samples 1200 1000 800 600 400 200 0 Footpad Swelling (μm) Tyk2 +/+ Tyk2 +/- Tyk2 -/- *** * * # ### Saline mBSA Infectious and Parasitic Neoplasms Endocrine, nutritional and metabolic; immunity disorders Blood and blood-forming organs Mental disorders Nervous system and sense organs Circulatory system Respiratory system Digestive system Genitourinary system Skin and subcutaneous tissue Musculoskeletal system and connective tissue PheWAS phenotypes (ICD9 Classification) 0.0 0.2 0.4 0.6 0.8 Hours After mBSA Challenge Vehicle Paw Swelling (mm) -1 23 Dexamethasone 3 mg/kg ND-031301 10 mg/kg ND-031301 30 mg/kg ND-031301 100 mg/kg **** p = 1x10 -8 *** p = 2x10 -6 ** p = 2x10 -4 * p = 1x10 -3 **** * ** *** Identification of Highly Potent and Selective Tyk2 Inhibitors for the Treatment of Autoimmune Diseases Through Structure-based Drug Design Craig E. Masse 1 , Wenyan Miao 1 , Jeremy Greenwood 2 , Mee Shelley 2 , Joshua Kennedy-Smith 2 , Rosana Kapeller 1 Nimbus Therapeutics, Inc., Cambridge, MA, USA 1 ; Schrödinger, Inc., New York, NY, USA 2 25 First St #404, Cambridge, MA 02141, USA • www.nimbustx.com • Tel: 857.999.2009 • [email protected] ABSTRACT CONCLUSIONS • Tyk2 is a sought after target for the treatment of autoimmune dis- eases with compelling human genetic data from GWAS/PheWAS studies • Given the high degree of structural homology amongst the JAK ki- nase family members, designing potent and selective inhibitors has remained a considerable challenge • Using a physics-based computational approach, Nimbus has uncov- ered previously unexploited drivers of potency and JAK family se- lectivity • Potent inhibition of IL-12-induced STAT4 phosphorylation and cyto- kine production was observed in human PBMC and whole blood and the Nimbus compounds maintain high levels of functional selectivity over JAK2 • In vivo proof of mechanism was demonstrated in a mouse delayed type hypersensitivity model • Nimbus compounds have good drug-like properties and are candi- dates for further development for inflammatory diseases The JAK family kinase Tyk2 is essential for IL-12 and IL-23 signaling, which are associated with Th1 and Th17 cell differentiation and activa- tion. The Th1 and Th17 pathways have been implicated in the patho- genesis of psoriasis and inflammatory bowel diseases (IBD) thereby making Tyk2 a highly attractive target for the treatment of these disor- ders. However, given the high degree of sequence homology between the JAK family kinases, designing potent and selective Tyk2 inhibitors remains a challenge. Using an innovative structure-based approach, we have designed, synthesized and characterized small molecule in- hibitors optimized for JAK family selectivity using computational free energy perturbation (FEP) methods and medicinal chemistry SAR. We have identified selective Tyk2 inhibitors with pM activity against Tyk2 (K i =140-520 pM) and >100 fold selectivity over JAK2 and JAK1 with more moderate selectivity over JAK3. These analogs are orally bio- available (85%F) with suitable drug-like properties. NDI-031232 was determined to be highly selective across 359 kinases, and is a potent inhibitor of IL-12 induced pSTAT4 in hPBMCs (IC 50 =17 nM) and IL-12 induced IFNg in human whole blood (IC 50 =520 nM). NDI-031232 also demonstrated robust efficacy in blocking IL-12-mediated IFNg pro- duction in an ex vivo mouse model. Therefore, selective inhibitors of Tyk2 retain the anti-inflammatory activity while reducing potential for dose-limiting side effects observed with non-selective JAK inhibitors. All kinase assays were performed with radiolabeled peptides. PK study was conducted in C57BL/6 mice. The ve- hicle for NDI-031232 was 20% DMSO/60% PEG400 in saline, and the vehicle for NDI-031302 was 20% HPbCD in saline. 1. Tyk2: Key Mediator of Th17 and Th1 Pathogenesis 2. Free Energy Perturbation (FEP) Used to Develop Quantitative Selectivity Model to Drive SAR 3. Highly Selective Lead Series Identified in ~10 Months 4. Potency, Selectivity, and Drug-Like Properties of Tyk2 Lead Candidates 5. NDI-031232 Is A Potent and Selective Tyk2 Inhibitor Kinome selectivity is based on radio-peptide kinase assays. For cell-based assays, human PBMC were pre-in- cubated with NDI-031232 for one hour and stimulated with 1.67 ng/ml of IL-12 for 30 minutes or 10 ng/ml of GMCSF for 10 minutes. Cell lysates were prepared and phospho and total STAT protein was measured by MSD. The ratio of pSTAT/total STAT was used for IC 50 calculation. For the ex vivo study, C57BL/6 mice were dosed orally with vehicle (20% DMSO/60% PEG400 in saline), NDI-031232, or Tofacitinib. Whole blood was collected 30 minutes post dose and stimulated with 2 ng/ml IL-12 on anti-CD3 antibody coated plate for 24 hours. At the end of the incubation, IFNg level in the supernatant was quantified by MSD. 6. NDI-031301 Is a Potent and Selective Tyk2 Inhibitor 7. NDI-031301 Reduces Inflammation in the mBSA-induced Delayed Type Hypersensitivity Model Kinome selectivity is based on radio-peptide kinase assays. Cell selectivity assays were performed with human PBMC as above. Human whole blood assay was performed by incubating NDI-031301 with human whole blood for one hour followed by 0.5 ng/ml of IL-12 stimulation for 24 hours on anti-CD3 antibody coated plate. At the end of the incubation, IFNg level in the supernatant was quantified by ELISA. C57BL/6 mice were immunized with 0.11 mg of methylated BSA/CFA emulsion at lower back on day 0. Mice were challenged on day 5 by injecting 0.1 mg mBSA/PBS solution into one hind paw and PBS into the other hind paw. Vehicle (20% HPbCD in saline) or NDI-031301 were dosed twice daily orally and Dexamethasone was dosed once daily IP on day 0 through day 5. Paw thickness was measured one day post the challenge (day 6) and paw swelling was calculated by subtracting the thickness of the PBS paw from the mBSA paw of the same mouse. Mice received a final dose on day 6 and plasma was collected one hour post the dose and analyzed for NDI-031301 by LC/M/MS. p values are student t-test vs. the vehicle. Similarity of Tofacitinib Co-Crystal Structure with JAK1, 2, 3 and Tyk2 FEP-enabled Selectivity Breakthrough (Tyk2 vs JAK2) Psoriasis and Crohn’s Pathology Lupus Pathology Multiple Alleles Protect from RA Multiple Alleles Protect from SLE No Obvious Adverse Events in ~30K Patients Diogo D, et al. (2015) PLoS ONE 10: e0122271 • Active sites virtually identical • Gatekeeper residue (methionine) conserved across JAK family See details in Wang, L., et al. (2015) J. Am. Chem. Soc.,137: 2695 Kinome Selectivity Cell-Based Assay Cellular Selectivity Blocks ex vivo IL-12-induced INFa in the Whole Blood of NDI-031232 Dosed Mice Kinome Selectivity Cellular Selectivity Human Whole Blood Activity mBSA-induced DTH Model Ishizaki M, et al. (2011) J. Immunol.,187: 181