Potent and Selective Tyk2 Inhibitors Block Th1- and Th17- Mediated Immune Responses and Reduce Disease Progression in Rodent Models of Delayed-Type Hypersensitivity and Psoriasis

Wenyan Miao1, Craig E. Masse1, Jeremy Greenwood2, Rosana Kapeller1, William F. Westlin1

1Nimbus Therapeutics, Inc., Cambridge, MA, USA; 2Schrödinger, Inc., New York, NY, USA

BACKGROUND/PURPOSE

SUMMARY

METHODS

RESULTS

1. Human Genetic Studies Implicate Tyk2 in RA and SLE Susceptibility

2. Broad Potential of Tyk2 Inhibition Across Autoimmune Indications of High Unmet Medical Need

3. Tyk2: Key Mediator of Th17 and Th1 Pathogenesis

4. Free Energy Perturbation (FEP) Used to Develop Quantitative Selectivity Model to Drive SAR

6. Potency, Selectivity, and Drug-Like Properties of NDI-031301

5. NDI-031301 is a Potent and Selective Tyk2 Inhibitor

7. NDI-031301 Reduced Inflammation in the mBSA-induced Delayed Type Hypersensitivity Model

8. NDI-031301 Reduced Inflammation in the IL-23-induced Mouse Epidermal Hyperplasia Model

9. NDI-031301 Reduced Disease in Imiquimod-induced Psoriasis Model

CONCLUSION

P=10-25 in >30,000 case-control

samples

P=10-18 in ~15,000

Ass

ocia

tion

(-log

P) 2

2

41 Type 1 diabetes2 Type 1 diabetic ketoacidosis3 Insulin pump user

4 Psoriasis vulgaris 5 Rheumatod arthritis& related disorders6 Rheumatod_arthritis

5

6

4321

OR<

1O

R>1

0

GAA 0.008

CGA 0.038

GGC 0.086

GGA 0.871 2 3 F

0.4 0.6 0.8 1 1.2 0.4 0.6 0.8 1 1.20.2OROR

ref

GGAA 0.15

GAAA 0.006

CGAA 0.036

GGAC 0.711 2 3 F

GGCA 0.083

4ref

case-control samples

Infectious and ParasiticNeoplasmsEndocrine, nutritional and metabolic; immunity disordersBlood and blood-forming organsMental disordersNervous system and sense organs

PheWAS phenotypes (ICD9 Classification)Circulatory systemRespiratory systemDigestive systemGenitourinary systemSkin and subcutaneous tissueMusculoskeletal system and connective tissue

600

JAK

2 Se

lect

ivity

(Fol

d O

ver K

i)~560x

3503002500 50 100 150 2000

500

400

300

200

100

Number of Compounds Synthesized

Use of FEP for Tyk2 Program

Additional ProprietaryCo-crystal Structures

New sub-seriesthat takesadvantage ofadditionalinteractions withnon-conservedresidues

Psor

iasis

Sco

re

10

8

6

4

2

00 1 2 3 4 9 10

Day

Sple

en W

eigh

t (m

g)

200

150

100

50

0

VehicleNDI-031301 30 mg/kgNDI-031301 100 mg/kg

NaïveDexamethasone

5 6 7 8

**** ** **

**

*************

* ** ** ** ** **

** p < 0.01* p < 0.05

Vehicle

NDI-031301 30 mg/kgNDI-031301 100 mg/kg

NaïveDexamethasone

** p < 0.01

** **

**

0.0

0.2

0.4

0.6

0.8

Hours After mBSA Challenge

Vehicle

Paw

Sw

ellin

g (m

m)

-1 23

Dexamethasone 3 mg/kgNDI-031301 10 mg/kgNDI-031301 30 mg/kgNDI-031301 100 mg/kg

**** p = 1x10-8

*** p = 2x10-6

** p = 2x10-4

* p = 1x10-3****

***

***

Vehicle

0.0

0.1

0.2

0.3

0.4

Num

ber o

f Cel

ls (M

illio

n)

CD4+IFNγ-IL17A+

NDI-031301 100 mg/kg

CD4+IFNγ+IL17A- CD4+CD25+FOXP3+

0

1000

2000

4000

6000

INFγ

(pg/

mL)

VehicleDexamethasone 1 mg/kgNDI-031301 100 mg/kg

3000

5000

0

mBSA (μg/mL

1.2 3.7 11 33 100

* p < 0.05

*

* p < 0.05

*

*

*

* p < 0.01

*

*

0.0

0.1

0.2

0.3

0.4

Ear S

wel

ling

(mm

)

Vehicle Dex 3 mg/kg

NDI-03130110 mg/kg

NDI-03130130 mg/kg

NDI-031301100 mg/kg

0.0

0.1

0.2

0.3

0.4

Ear S

wel

ling

(mm

)

Vehicle Dex 3 mg/kg

NDI-03130110 mg/kg

NDI-03130130 mg/kg

NDI-031301100 mg/kg

*

NDI-031301

Biochemical Tyk2 Kinase Assay Tyk2 Ki (nM) 0.53

JAK Family Kinase Biochemical Selectivity (Fold over Ki)

Fold Selectivity over JAK2 85x

Fold Selectivity over JAK1 107x

Fold Selectivity over JAK3 15x

Plasma Protein Binding Human PPB (%bound) 75

Human in vitro metabolism Clint(mL/min/kg)

Microsomes 3.0Hepatocytes 9.1

Mouse PK

IV 3 mg/kg Clobs (mL/min/kg) 39

PO 30 mg/kg

T1/2 (h) 2.9Cmax (μM ) 13

F (%) 100%AUC (μM*hr) 38

0

600

800

1000

1200

Foot

pad

Swel

ling

(μm

)

SalinemBSA

Tyk2+/+ Tyk2+/- Tyk2-/-

400

200

***

*

*

#

#

Ear S

wel

ling

(x10

-2m

m)

25

20

15

10

5

00 1 2 3 4

Day

**

***

***

*** ***

###

######

BSA, Tyk2+/+BSA, Tyk2-/-IL-23, Tyk2+/+IL-23, Tyk2-/-

Ear T

hick

ness

(μm

)

1000

800

600

400

00 1 2 3 7

Day

***

***

*** ### ###

4 5 6

******

***

###

###******

********

######

Control, Tyk2+/+Control, Tyk2-/-IMQ, Tyk2+/+IMQ, Tyk2-/-

Ki (nM)0.1 to 0.5

5 to 10

10 to 150

NNDDII--003311330011

97

56

460

% A

ctiv

ity

125

0

100

75

50

25

-2510-4 10-3 10-2 10-1 100 101 102

NDI-031301Tofacitinib

Compound (μM)

% A

ctiv

ity

125

0

100

75

50

25

-2510-4 10-3 10-2 10-1 100 101 102

NDI-031301Tofacitinib

Compound (μM)

% A

ctiv

ity

125

0

100

75

50

25

-2510-6 10-4 10-2 100 102

NDI-031301Tofacitinib

Compound (μM)

IC50 (nM)

PBMC_IL12/pSTAT4

NK92_IL12/IFNγ

hWB_IL12/IFNγ

Tyk2 JAK3

NDI-031301

0.25x

17x

15x

IL-12

GMCSF

IL-2

Fold Selec.vs.

Tofacitinib

LOF

LOF

Crohn’s Disease

Systemic Lupus

Erythematosus

Mutliple Sclerois

PBC

Type 1 Diabetes

Systemic Sclerosis

Psoriasis OR of 1.4

OR of 1.12

OR of 1.2

OR of 1.32

OR of 1.91

OR of 0.86

OR of 0.59

A genome-wide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1

Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease susceptibility loci

Association of NCF2, IKZF1, IRF8, IFIH1, and TYK2 with systemic lupus erythematosus

Replication analysis identifies TYK2 as a multiple sclerosis susceptibility factor [increased risk with more active Tyk2 variant]

Dense fine-mapping study identfies new susceptibility loci for primary biliary cirrhosis

The imprinted DLK1-MEG3 gene region on chromosome 14q32.2 alters susceptibility to type 1 diabetes

Influence of TYK2 in systemic sclerosis susceptibility: a new locus in the IL-12 pathway

IL-12

p35 subunit

p40 subunit

IL-23

INF /

p40 subunit

p19 subunit

Tyk2

Jak2

Jak2

Tyk2 Jak1

Tyk2

STAT 1-5

P

via Th17 Th1

25 First St #404, Cambridge, MA 02141, USA • www.nimbustx.com • Tel: 857.999.2009 • info@nimbustx.com

Tyk2 is a member of the JAK family kinases and a key mediator of IL-12, IL-23, and type I interferon signaling. These cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as lupus, psoriasis and inflammatory bowel diseases. Supported by compelling data from human genetic association studies, Tyk2 inhibition is an attractive therapeutic strategy for these diseases.

One of the challenges of developing selective Tyk2 inhibitors is the high sequence homology of the active site among the members of the JAK family kinases. We utilized cutting edge proprietary structure-based drug design tools to identify highly potent and selective inhibitors of Tyk2. These inhibitors were character-ized for their potency and selectivity in the enzyme and cell-based assays, and in mouse models of delayed type hypersensitivity and psoriasis.

We have identified Tyk2 inhibitors with up to 720-, 540-, and 210-fold selectivity against JAK1, JAK2, and JAK3 respectively, with potent cellular activity and ex-cellent cellular selectivity against other JAK family kinases in human peripheral blood mononuclear cells. NDI-031301 is a Tyk2 inhibitor with a Ki of 0.5 nM that is 107-, 85-, and 15-fold selective against JAK1, JAK2, and JAK3 respectively. It blocks IL-12 induced phospho-STAT4 and GM-CSF induced phospho-STAT5 in human PBMCs with IC50 of 0.1 mM and 2.6 mM, respectively. NDI-031301 has ex-cellent selectivity against a panel of 364 kinases, showing less than 70% inhibi-tion at 300 nM against all but 16 of the kinases tested. It also showed less than 50% inhibition up to 30 mM against human CYP enzymes and hERG channel. In addition, NDI-031301 has an attractive PK profile with good oral bioavailability in rodents and dogs. Studies with humans carrying inactive forms of Tyk2 and mice deficient in Tyk2 revealed a role in Th1 and Th17 polarization. We investigated the in vivo activity and mechanism of action of Nimbus Tyk2 inhibitors in a meth-ylated-BSA induced mouse delayed type hypersensitivity model. At 100 mg/kg dose, orally administered NDI-031301 reduced paw swelling and paw weight, as well as Th1 (IFNγ) and Th17 (IL-17A and IL-22) cytokines in the inflamed paws by more than 50%. It also dramatically reduced Th1 cells in the draining lymph nodes and suppressed over 85% of in vitro antigen-induced IFNγ response in the drain-ing lymph node cells. In an IL-23-induced mouse psoriasis model, NDI-031301 dose-dependently reduced skin inflammation with up to 76% inhibition of ear swelling at 100 mg/kg, highlighting the role of Tyk2 inhibition in Th17 pathogen-esis. Finally, NDI-031301 was highly efficacious in an imiquimod-induced mouse psoriasis model, showing dose-dependent reduction of psoriasis score, spleen weight, and improved skin histology. 30 mg/kg of NDI-031301 treatment blocked disease progression and 100 mg/kg treatment reversed the disease.

• Tyk2 is a sought after target for treatment of autoimmune diseases• Given the high degree of structural homology amongst the JAK kinase family

members, designing potent and selective inhibitors has remained a challenge• Using a physics-based computational approach, Nimbus has uncovered previ-

ously unexploited drivers of potency and JAK family selectivity• Potent inhibition of IL-12-induced STAT4 phosphorylation and cytokine pro-

duction was observed in human PBMC and whole blood• In vivo proof of mechanism and efficacy was demonstrated in mouse models

of delayed type hypersensitivity and psoriasis• Nimbus compounds have excellent drug-like properties, are well tolerated,

and are candidates for further development for inflammatory diseases with excessive IL-12, IL-23 and/or type I interferon signaling

Utilizing unique and innovative structure-based drug design technologies, we rapidly designed highly potent and selective Tyk2 inhibitors for use as potential therapeutics in inflammatory disorders involving Th1, Th17, and type I interferon pathogenesis.

All kinase assays were performed in the radiometric for-mat with peptide substrates. PK study was conducted in C57BL/6 mice.

Kinome selectivity is based on radiometric peptide kinase assays. For cell-based assays, human PBMC were pre-incubated with the compound for one hour and stimulated with IL-12 for 30 minutes, or GM-CSF or IL-2 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 IC50 calculation. NK92 assay was performed by incubating the compound with the IL-2 starved NK92 cells for one hour following by IL-12 stimulation for 24 hrs. Human and mouse whole blood assay was performed by incubating the compound with blood for one hour followed by IL-12 stimulation for 24 hours on anti-CD3 antibody coated plate. At the end of the incubation, IFNγ level in the supernatant was quantified by ELISA.

C57BL/6 mice were immunized with methylated BSA/CFA emulsion at lower back on day 0. Mice were challenged on day 5 by injecting mBSA/PBS solution into one hind paw and PBS into the other hind paw. Vehicle (20% HP-b-CD 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. Draining lymph node cells were harvested and T cell subsets were phenotyped by flow cytometry. The in vitro mBSA recall response was measured by incubating the draining lymph node cells with mBSA for 72 hours and supernatant collected for IFNγ measurement. p values are student t-test vs. the vehicle.

Ear swelling was induced by injecting recombinant murine IL-23 into the ears of C57BL/6 mice once daily for 4 days. Vehicle (20% HP-b-CD in saline) or NDI-031301 were dosed twice daily orally and Dexamethasone was dosed once daily IP. Ear thickness was measured prior to the first IL-23 injection and one day post the last IL-23 injection, and ear swelling was the difference of the two measurements. Ears were harvested and homogenized one hour post the last compound dose. IL-17A in the tissue homogenate was measured by Luminex kit. p values are student t-test vs. the vehicle. Ear skin was harvested for histology.

Psoriasis was induced by topical application of Imiquimod cream once daily on the shaved back skin of Balb/c mice for 11 days. Vehicle (20% HP-b-CD in saline) or NDI-031301 were dosed twice daily and Dexamethasone was dosed once daily orally. Skin inflammation was scored daily. Spleens were collected one hour post the last compound dose and weighed. p values are student t-test vs. the vehicle. Back skin was harvested for histology.

• 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

• Dexamethasone was dosed IP QD for 5 days

• Paw swelling is the difference between the mBSA injected vs. the saline injected paw from the same mouse

• p values are student t-test vs. the vehicle

Ishizaki M, et al. (2011) J. Immunol.,187: 181

Ishizaki M, et al. (2011) J. Immunol.,187: 181

Ishizaki M, et al. (2014) Int. Immunol., 26: 257

PBMC: peripheral blood mononuclear cells; hWB: human whole blood; mWB: mouse whole blood

Diogo D, et al. (2015) PLoS ONE 10: e0122271

Kinome Selectivity Cell-Based PotencyMultiple Alleles Protect from RA Multiple Alleles Protect from SLE

No Obvious Adverse Events in ~30K Patients

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

Human PBMC Cellular SelectivityIL-12 Induced pSTAT4 (Tyk2/JAK2) GM-CSF Induced pSTAT5 (JAK2/JAK2) IL-2 Induced pSTAT5 (JAK1/JAK3)

Paw Swelling Draining Lymph Node T Cell Subsets

In vitro mBSA Recall IFNγ Response

Ear Thickness IL-17A in the Ears

Ear Histology

PBS Injected IL-23 Injected + Vehicle IL-23 Injected + ND-031301

Psoriasis Score Spleen Weight

HistologyNaïve Vehicle HP-b-CDPBS Dexamethasone

NDI-031301 30 mg/kg

NDI-031301 100 mg/kg