Divya Chaudhary1, Brad Geddes1, Shaughnessy Robinson2, Craig E. Masse1, Matthew D. Wessel2, Identi cation of Highly Potent and Selective Interleukin-1 Receptor-Associated K. Shawn Watts2, Jeremy R. Greenwood2, Mee Shelley2, Mark L. Brewer2, Carolyn M. McQuaw2, Geraldine Harriman1, Leah L. Frye2, Ronald T. Wester1, Rosana Kapeller1, and Donna Romero1 Kinase 4 Inhibitors for the Treatment of Rheumatic Diseases 1Nimbus Discovery, Inc., Cambridge, MA, 2Schrödinger, Inc., New York, NY

ABSTRACT ND-2110 & ND-2158 Inhibit TNFα Production in Human PBMCs ND-2110 & ND-2158 Inhibit TNFα ND-2110 Inhibits LPS-Induced TNFα IRAK4 Inhibitors are Ef cacious in MSU Air Pouch Model of Human Gout Background/Purpose: Interleukin-1 receptor-associated kinase 4 (IRAK4) is a key mediator of the innate immune Production in Human Whole Blood Production in Rats PO Dosing response orchestrated by interleukin-1 receptor (IL-1R), interleukin-18 receptor (IL-18R), IL-33 receptor (IL-33R), 6000 8.0x1006 AB1200 LPS Stimulation CpG Stimulation R848 Stimulation ED50= 7.5 mg/kg and Toll-like receptors (TLRs). IRAK4 activation is mediated by MYD88, a common signaling adaptor protein down- 1200 2000 stream of these receptors. Mutations leading to inactivation or activation of MYD88 have been reported in patients 1000 1000 6.0x1006 with immune defi ciencies and cancer, respectively. In addition, IRAK4-defi cient humans are protected from chronic 1500 Figure 8: Balb/c mice were used to create a sterile air pouch, 4000 **** * and mice were dosed for 6 days as indicated. Following last infl ammatory diseases. Thus, IRAK4 is an attractive therapeutic target for the treatment of autoimmune diseases 800 800 ****

pg/mL **** dose mice were anesthetized and the air pouch was injected such as lupus. Historically, identifi cation of potent and selective IRAK4 inhibitors has been challenging due to struc- 4.0x1006 pg/mL pg/mL 600 pg/mL with MSU suspension. Four hours later the air pouch exudate tural features in the catalytic binding site that block access to the hydrophobic back pocket. We have developed 600 1000 ****

Total WBC **** TNF 2000 was collected and an aliquot is analyzed for cell counts. **** p

new structure-activity relationship (SAR) insights, including the identifi cation of unstable (high-energy) hydration TNF TNF 400 TNF 400 < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05 2.0x1006 **** sites, which guide the design of potent and selective small molecule ligands. ND-1659 ND-1659 500 200 ND-2110 200 ND-2110 ND-2110 Methods: Using this innovative structure-based approach, we designed, synthesized and tested small molecule in- ND-2158 ND-2158 ND-2158 0 0 0 0 0 hibitors based on hits originating from a virtual screen. These novel compounds were profi led for IRAK4 kinase in- 0.001 0.01 0.1 110 100 LPS 0.001 0.01 0.1 110 100 0.001 0.01 0.1 110 100 Naive hibition, selectivity, and drug-like properties. Furthermore, selected compounds were tested in THP1 cells, human Vehicle 10 mpk 30 mpk Concentration M 100 mpk Colchine Concentration M Concentration M Dex 5 mpk2110 3 mpk peripheral blood mononuclear cells (hPBMCs) and whole blood for impact on LPS-, IL-1-, R848-, and/or CpG-me- 2110 10 2110mpk 30 mpk 2110 100 mpk 2110 PO BID diated signaling. The inhibitors were also tested in vivo in acute LPS challenge, chronic collagen-induced arthritis (CIA), imiquimod-induced psoriasis and MSU air pouch gout models. Figure 2: Human PBMC cultured serum free at a density of 100,000 cells per well (A) or 200,000 cells per well (B) were stimulated Figure 4: Human whole blood was diluted 1:1 with PBS and Figure 5: Female Lewis rats were administered drug PO 30 IRAK4 Inhibitors Block Collagen-Induced Arthritis in Mice with 1 μg/mL LPS (A) for 5hrs; or stimulated with 0.5 μM CpG for 20 hrs (B). Cell supernatant was analyzed for TNFα production treated with IRAK4 inhibitors for 30 minutes prior to R848 stim- minutes prior to stimulating with LPS IV. Serum samples col- BID IP Dosing by ELISA. Negative control ND-1659 lacks IRAK4 inhibitory activity. IC50 values are reported in Table 1. ulation at 1 μM. Plasma samples collected 24 hours post stim- lected at 1 hr post LPS administration, followed by TNFα ELISA Results: Here, we feature three highly potent, selective IRAK4 inhibitors, ND-346, ND-2110 and ND-2158. The Kis 2.0 ulation followed by TNFα ELISA analysis. IC50 values reported analysis. **** p < 0.001 5.0 Naïve 40 of ND-346, ND-2110, and ND-2158 for IRAK4 are 50, 7.5 and 1 nM, respectively. These compounds are highly in Table 1. A Vehicle ND-2110 35 selective against 334 kinases, and are potent inhibitors of IL-1-induced IRAK1 degradation in MRC5 cells, LPS-, Potent and Selective IRAK4 Inhibitors Identi ed 4.0 ND-2158 30 1.0 IL-1-, R848 (TLR-7 agonist)- and CpG (TLR-9 agonist)-induced cytokine production in hPBMCs and whole blood. IRAK4 Inhibitors Block IL-1β-induced IRAK1 Degradation in MRC5 Cells Dex Furthermore, these compounds are effi cacious in all in vivo murine models tested, showing effi cacy ≤30 mg/kg BID. 3.0 25

ND-346 IRAK4 Ki = 0.05 M ND-2110 IRAK4 Ki = 0.007 M ND-2158 IRAK4 Ki = 0.001 M IL-1 + compound @ 30 mM (3X dilution) 20 0.0 37% 39% Conclusion: Utilizing unique and innovative structure-based drug design, we have rapidly discovered potent and Ki M 2.0 15 MW Mock PBS IL-1 Figure 6: MRC5 cells were treated with IRAK4 inhibitor for 30 Clinical Score selective IRAK4 inhibitors as potential drug candidates for the treatment of chronic rheumatic diseases. 1 to 0.1 AUC Calculation ND-346 Clinical score with 10 -1.0 IRAK1 minutes prior to IL-1β stimulation for another 30 minutes. Cell 1.0 63% 0.1 to 0.01 lysates were subjected to western blot analysis for IRAK1, and 5 Body Weight Change (g) IRAK4 Plays an Essential Role in Mediating IL-1/TLR Signaling to Pro-in ammatory IRAK4 IRAK4 IRAK4 ND-2158 IRAK1 GAPDH to confi rm equivalent protein loading. Similar GAPDH 0.01 to 0.001 0 0 -2.0 signal was detected for all experiments, and representative is 0 1 2 3 4 5 6 7 8 9 10 11 Cytokine and Chemokine Production ND-2110 IRAK1 shown. Arthritis Day PO Dosing TLRs IL-1RI GAPDH IL-1 5.0 40 2.0 PAMPs CLK1, 2 CLK1, 2 Naïve B Vehicle CYTOPLASM 35 P ND-2110 and ND-2158 Reduce Clinical Symptoms in Murine IMQ-Induced ND-2110 QD LPS, CpG DNA, IL-1 family, 4.0 ND-2110 BID LTA, flagelin, including IL-18 30 1.0 IRAK4/ IRAK4/ IRAK4/ TIR Dex IP dsRNA, zymosan MyD88 IRAK1 IRAK1 MyD88 TIR and IL-33 Psoriasis Model in vivo 6% TIR IRAK1 TIR 3.0 25 Vehicle Naïve Dex 20 0.0 32% A 2.0

TRAF6 Clinical Score 15 IKK MAP3K AUC Calculation Clinical score with ERK Figure 7: IRAK4 inhibitors are effi cacious in 10 63% -1.0

1.0 Body Weight Change (g) p38 a Balb/c mouse model of imiquimod-induced 5 NF- B psoriasis (MD Biosciences). IP BID dosing NUCLEUSNUCLEUS JNK Figure 3: ND-2110 and ND-2158 are highly potent and selective IRAK4 inhibitors. Kinase inhibitory activity was evaluated using ra- 0 0 -2.0 2110, 3 mpk 2110, 10 mpk 2110, 30 mpk begins on day 0 following IMQ application. dioactive kinase assays at 10 μM ATP (Reaction Biology Corp.). Over 300 kinases were tested at 10 μM compound concentration 0 1 2 3 4 5 6 7 8 9 10 11 and based on the % inhibition results, the kinases that were inhibited >50% were evaluated in IC dose response assays. Kinase Ki A. Representative mice were photographed InflammatoryInnfflammammatory ResponseResponssee 50 Arthritis Day < 1 μM shown on kinome maps. to show degree of skin scaling. IRAK4 inhi- bition helps to resolve psoriatic skin compa- Figure 9: Male DBA mice, immunized with collagen on day 0 and 21, were randomly enrolled upon disease onset (n = 8 per group, Nimbus Approach Enables Rapid Discovery of Potent, Drug-like IRAK4 Inhibitors rable to dexamethasone (Dex). B. Animals except for n = 4 naïve controls). Day 1 is designated as the fi rst treatment day, and mice were evaluated daily for clinical scores till Table 1: Potency and Drug-Like Properties of IRAK4 Inhibitors were scored daily for clinical symptoms. Clin- day 11, shown as average scores on the left panels. Middle panels show the average clinical scores with AUC calculation, and % in- hibition relative to vehicle is indicated in the bar graph. Mice were weighed on alternating days, and change in body weight on day 11 2158, 3 mpk 2158, 10 mpk 2158, 30 mpk ical scores averages for days 5, 8, and 10 are Ideas 1.3 MM 3,000 60,000 shown. **** p < 0.0001; *** p < 0.001; ** p < from day 1 is shown in the right panels. A. 2110 and 2158 were dosed at 30 mg/kg IP BID, and dexamethasone at 0.1 mg/kg IP BID, ND-346 ND-2110 ND-2158 Total compounds 0.01; * p < 0.05 in vehicle 10% HPbCD. Left panel - IRAK4 inhibitors vs vehicle: p<0.05 days 2-11; dexamethasone (Dex) vs vehicle: p<0.05 day 1, Biochemical IRAK4 Kinase IRAK4 Ki µM 0.05 0.007 0.001 p<0.01 day 2, p < 0.001 days 3-5, p <0.0001 days 6-11. Middle panel - IRAK4 inhibitors vs vehicle: p<0.5; dex vs vehicle p<0.0001. 40 <300 Assay synthesized Right panel - 2158 and dex vs vehicle: p<0.05, 2110 vs vehicle IP BID p<0.01. B. 2110 was dosed at 30 mg/kg PO QD and BID, in THP1 cells (LPS -> TNF )1 1.0 0.59 0.13 vehicle 0.5% MC in saline. Left panel - 2110 BID vs vehicle: p<0.05 days 8-11. The control groups dexamethasone and naïve are Potency 8,000 nM 100 nM <10 nM the same as in A. All groups shown are part of the same study. PBMC (LPS-> TNF ) 2 6.5 0.40 0.10 ATP analog docked to IRAK4 showing Cytokine Production IC50 PBMC (R848 -> TNF ) 3 ND 0.67 0.09 3 Vehicle G kcal/mol high-energy hydration sites (spheres) (µM) SUMMARY 4 B 6.0 PBMC (CpG -> TNF ) ND 0.45 0.16 ND-2110 30 mpk • IRAK4 is a sought after target for the treatment of autoimmune diseases 5 Whole Blood (R848 -> TNF ) ND 0.54 0.77 2.5 ND-2158 30 mpk 5.0 * 6 Dex • Previous attempts to identify small molecule modulators of IRAK4 resulted in poor selectivity and inadequate drug- 4.0 Protein Binding Human PPB % Bound 63 73 82 *** Milestone achieved 2 like properties Clint (mL/min/kg) *** **** 3.0 Human in vitro Stability6 Microsomes 8 19 17 **** **** • Using a physics-based computational approach, Nimbus has uncovered previously unexploited drivers of potency 2.0 Hepatocytes 7 21 12 1.5 7 8 Rat PK (IV) 3mpk Clint (ml/min/Kg) 113 67 72 ** and selectivity. These insights were used to discover and design the fi rst truly selective IRAK4 inhibitors 1.0 Initiate HTL Lead Candidate In vivo PoC 8 **** T1/2 (hr) 1.2 1.3 1.8 8 1 *** C (µM) 1.7 1.4 0.5 Clinical Score 0 2 4 6 8 10 12 Rat PK (PO) 10 mpk7 max • Robust in vivo effi cacy was subsequently demonstrated in collagen-induced arthritis, psoriasis and mono-sodium F (%) 94 25 118 Months urate (MSU) gout mouse models AUC (µM hr) 1.4 1.5 1.58 0.5 Physical properties MW(Da), solubility ( M) 300 – 325, 830 400-425, 291 425-450, >300 Figure 1: Structure based modeling approaches were used to conduct a virtual screen of IRAK4 and led to the rapid optimization of • Potent in vitro inhibition of cytokine production was observed in cells and whole blood lead matter. The technology allows the generation of a large number of “virtual compounds” (ideas) that are docked into the model 0 1THP cells stimulated with 0.3 μg/mL LPS for 5 hrs; cell supernatant measured for TNFα production by ELISA; 2See Figure 2A; 3Human PBMC (100,000 cells per well) stimulated with 1 μM from which the most promising were selected for synthesis. Time from screening to in vivo PoC was accelerated compared to typical R848 for 5 hrs; cell supernatant measured for TNFα production by ELISA; 4See Figure 2B; 5See Figure 4; 6Standard Assay performed at Pharmaron; 7PK conducted in Sprague Dawley rat. IV 5 8 10 • Nimbus compounds have good drug-like properties and are candidates for further development for treating infl am- drug discovery programs. formulation 10% HP-β-CD in saline; oral formulation 0.5% MC in saline; 8PK conducted in female Lewis rat. Oral formulation ND-2158 (0.5% MC in saline) Study Days matory diseases

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