ASK1 (IC50 = 2.87 nM)
ASK1-IN-1 (GS-444217) targets MAP Kinase Kinase Kinase 5 (ASK1, MAP3k5), a redox-sensitive serine threonine kinase; the in vivo EC50 for inhibition of the renal ASK1 pathway in rats is 1.6 μM [1]
ASK1-IN-1 (GS-444217) targets ASK1 (MAP3k5), which activates p38 and JNK MAPKs[2]

ASK1-IN-1 is a potent inhibitor of the apoptosis signal regulating kinase 1 (ASK1) inhibitor (Compound 4 from patent WO2016025474A1). In an in vivo pulmonary hypertension (PH) model, it reduces right ventricular (RV) hypertrophy and pulmonary arterial pressure in a dose-dependent manner. The hallmarks of pulmonary vascular disease, such as reduced pulmonary vascular resistance (PVR), improved pulmonary pressure, decreased pulmonary vascular remodeling, improved vascular function, decreased maladaptive RV hypertrophy, and improved RV function, can all be lessened by inhibiting ASKl. The pathological changes linked to PH may also be slowed, avoided, and/or reversed by inhibiting ASKl signaling.

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1. In HEK293T cells with adenoviral overexpression of human ASK1 (AdASK1), treatment with ASK1-IN-1 (GS-444217) at concentrations ranging from 0.001 to 10 μM for 2 hours dose-dependently inhibited phosphorylation of ASK1 and its downstream substrates (MKK3/6, MKK4, p38, JNK) as measured by Western blot; treatment with 1 μM ASK1-IN-1 (GS-444217) for durations from 1 minute to 4 hours showed time-dependent inhibition of ASK1 activity, and washout of the drug followed by serum-free media replacement (0–240 minutes) demonstrated reversibility of ASK1 inhibition [1]
2. In a KINOME scan binding assay against 442 kinases, 1 μM ASK1-IN-1 (GS-444217) exhibited high selectivity for ASK1, with minor affinity for ribosomal s6 kinase-4 (RSK4) and dual-specificity tyrosine phosphorylation–regulated kinase-1A (DYRK1A) (sphere radius in the assay corresponds to inhibitor affinity, with ASK1 having the largest dot) [1]
3. In rat cardiomyocytes with adenoviral overexpression of ASK1, ASK1-IN-1 (GS-444217) reduced phosphorylation of p38 and JNK induced by ASK1 activation [2]
4. In primary mouse cardiac fibroblasts and human pulmonary adventitial fibroblasts derived from PAH patients, ASK1-IN-1 (GS-444217) reduced activation and migration of these fibroblasts [2]

ASK1-IN-1 dose-dependently decreases pulmonary arterial pressure and right ventricular (RV) hypertrophy in an in vivo model of pulmonary hypertension (PH).

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1. In Sprague-Dawley rats challenged with auranofin (30 mg/kg, i.p.) to induce oxidative stress (OS)-mediated ASK1 pathway activation, a single oral dose of 30 mg/kg ASK1-IN-1 (GS-444217) reduced renal p-ASK1, p-p38, and p-JNK levels (measured by Western blot), and decreased renal mRNA expression of inflammatory cytokines (Il1b, Ccl2, Cxcl2) and caspase activity [1]
2. In a rat renal ischemia/reperfusion (I/R) injury model, oral administration of 30 mg/kg ASK1-IN-1 (GS-444217) before 30 minutes of bilateral renal ischemia reduced serum creatinine and blood urea nitrogen concentrations, and decreased renal tubular necrosis (H&E staining) and apoptosis/necrosis (TUNEL stain) scores after 24-hour reperfusion [1]
3. In a rat unilateral ureteral obstruction (UUO) model, oral administration of 30 mg/kg ASK1-IN-1 (GS-444217) (1 hour before surgery, twice daily for 7 days) reduced renal p-p38 and p-JNK levels (Western blot), decreased collagen IV deposition, cortical interstitial α-SMA-positive myofibroblasts, renal epithelial cell apoptosis/necrosis (TUNEL), and Col1a1 mRNA expression [1]
4. In 10-week-old db/db eNOS⁻/⁻ mice (a diabetic kidney disease (DKD) model) treated with 0.3% ASK1-IN-1 (GS-444217) in chow for 8 weeks, the drug halted glomerular filtration rate (GFR) decline, reduced proteinuria (urinary albumin to creatinine ratio), glomerulosclerosis score, collagen IV deposition, and podocyte loss (WT-1 staining) [1]
5. In Sprague-Dawley rats after 5/6 nephrectomy (chronic glomerular injury model), combination treatment with ASK1-IN-1 (GS-444217) (0.3% in chow plus 30 mg/kg once daily p.o.) and enalapril (50 mg/l in drinking water) for 4 weeks led to greater reduction in proteinuria and regression of glomerulosclerosis compared to monotherapy; monotherapy with ASK1-IN-1 (GS-444217) also reduced systolic blood pressure and glomerulosclerosis progression [1]
6. In two rat models of pulmonary arterial hypertension (PAH: monocrotaline and Sugen/hypoxia), oral administration of ASK1-IN-1 (GS-444217) dose-dependently reduced pulmonary arterial pressure and right ventricular (RV) hypertrophy; efficacy was observed even when administered to animals with established disease, associated with reduced ASK1 phosphorylation, pulmonary artery muscularization, and fibrotic gene expression in the RV [2]
7. In a murine model of RV pressure overload induced by pulmonary artery banding, ASK1-IN-1 (GS-444217) directly reduced cardiac fibrosis and improved cardiac function [2]

ASKl inhibition improves vascular function and decreases the signs and symptoms of pulmonary vascular disease, such as pulmonary vascular resistance (PVR), pulmonary pressure, remodeling of the pulmonary arteries, maladaptive RV hypertrophy, and RV function. The pathological changes linked to PH may be slowed, avoided, or even reversed by inhibiting ASKl signaling. Both of the ASK1-IN-1 doses—0.1+0.05 for 0.1% and 0.1±0.1 ng/mL for 0.2%—reduce plasma levels of B-type natriuretic peptide (BNP). ASK1-IN-1 treatment reduced the number of completely muscularized arterioles in Su/Hx rats by 35.6% or 32.6%, respectively.

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1. Enzymatic competition assay: The interaction between ASK1-IN-1 (GS-444217) and ATP was measured by assessing ASK1 enzymatic activity in the presence of increasing concentrations of ASK1-IN-1 (GS-444217) (0–24 nM) and ATP (0–2,400 μM), confirming that ASK1-IN-1 (GS-444217) acts as an ATP-competitive inhibitor of ASK1 [1]
2. Surface plasmon resonance (SPR) assay: The kinetics and binding affinity of ASK1-IN-1 (GS-444217) to ASK1 were determined using SPR; experimental data (black traces) were fitted to a simple 1:1 interaction model (red traces) to analyze binding characteristics [1]
3. KINOME scan binding assay: The selectivity of 1 μM ASK1-IN-1 (GS-444217) was evaluated against a panel of 442 kinases, covering major kinase families (TK, TKL, STE, CK1, AGC, CAMK, CMGC, and Other); the assay quantitatively mapped interaction patterns, with sphere radius indicating inhibitor affinity and dot size representing relative affinity for ASK1, RSK4, and DYRK1A [1]

1. ASK1 inhibition assay in HEK293T cells: HEK293T cells were transfected with adenoviral vectors overexpressing human ASK1 (AdASK1); cells were treated with serial 1:3 dilutions of ASK1-IN-1 (GS-444217) (0.001–10 μM) for 2 hours, then cell lysates were prepared and Western blot was performed to measure phosphorylation levels of ASK1, MKK3/6, MKK4, p38, and JNK, with GAPDH as a loading control [1]
2. Time-course assay in HEK293T cells: AdASK1-overexpressing HEK293T cells were treated with 1 μM ASK1-IN-1 (GS-444217) for durations ranging from 1 minute to 4 hours; Western blot was used to assess ASK1 pathway activation markers to determine the time-dependent effect of the drug [1]
3. Reversibility assay in HEK293T cells: After treatment of AdASK1-overexpressing HEK293T cells with ASK1-IN-1 (GS-444217), the drug was washed out and replaced with serum-free media; at time points from 0 to 240 minutes post-washout, cell lysates were analyzed by Western blot to evaluate the reversibility of ASK1 inhibition [1]
4. Cardiomyocyte assay: Rat cardiomyocytes were transfected with adenoviral vectors to overexpress ASK1, then treated with ASK1-IN-1 (GS-444217); phosphorylation levels of p38 and JNK were measured to assess the drug’s effect on ASK1 downstream signaling [2]
5. Fibroblast activation/migration assay: Primary mouse cardiac fibroblasts and human pulmonary adventitial fibroblasts from PAH patients were cultured and treated with ASK1-IN-1 (GS-444217); the activation status and migration capacity of these fibroblasts were evaluated using relevant cellular functional assays [2]

in vivo model of pulmonary hypertension (PH

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1. Rat auranofin-induced ASK1 activation model: Sprague-Dawley rats were administered a single oral dose of 30 mg/kg ASK1-IN-1 (GS-444217) or vehicle (equal volume); 30 minutes later, rats were challenged with auranofin (30 mg/kg, i.p.) to induce OS-mediated ASK1 activation. Kidney cortex samples were collected 30 minutes after auranofin administration for Western blot, mRNA expression, and caspase activity analysis (n=8 for drug group, n=5 for vehicle group) [1]
2. Rat renal I/R injury model: Sprague-Dawley rats received oral administration of 30 mg/kg ASK1-IN-1 (GS-444217) or vehicle just before 30 minutes of bilateral renal ischemia. After a 24-hour reperfusion period, serum samples were collected to measure creatinine and blood urea nitrogen, and kidney tissues were harvested for pathological scoring of tubular necrosis (H&E) and apoptosis/necrosis (TUNEL) (n=5–8 per group) [1]
3. Rat UUO model: Sprague-Dawley rats underwent sham or UUO surgery; ASK1-IN-1 (GS-444217) (30 mg/kg) or vehicle was orally administered 1 hour before surgery and continued twice daily for 7 days. Kidney lysates were analyzed by Western blot for p-p38 and p-JNK, and renal sections were stained for collagen IV, α-SMA, and TUNEL to assess fibrosis and cell death; Col1a1 mRNA was measured by RT-PCR (n=4 for sham, n=8 for UUO group) [1]
4. Mouse DKD model (db/db eNOS⁻/⁻): 10-week-old db/db eNOS⁻/⁻ mice were fed standard chow (vehicle) or chow containing 0.3% ASK1-IN-1 (GS-444217) for 8 weeks; a baseline group was euthanized at 10 weeks. GFR was measured by inulin-FITC clearance, proteinuria by urinary albumin to creatinine ratio (UACR) at weeks 10, 14, 18, and kidney tissues were analyzed for glomerulosclerosis (PAS staining), collagen IV deposition, and podocyte loss (WT-1 staining) (n=9–12 per group) [1]
5. Rat 5/6 nephrectomy model: 8 weeks after 5/6 nephrectomy, Sprague-Dawley rats were randomized by sclerosis index (SI) scores and assigned to control (standard chow), ASK1-IN-1 (GS-444217) (0.3% in chow plus 30 mg/kg once daily p.o.), enalapril (50 mg/l in drinking water), or combination groups for 4 weeks. Systolic blood pressure, UACR, and glomerulosclerosis severity (PAS staining) were assessed (n=12–14 per group) [1]
6. Rat PAH models (monocrotaline and Sugen/hypoxia): ASK1-IN-1 (GS-444217) was administered orally to rats with established PAH ; pulmonary arterial pressure, RV hypertrophy, ASK1 phosphorylation, pulmonary artery muscularization, and RV fibrotic gene expression were measured [2]
7. Murine RV pressure overload model (pulmonary artery banding): ASK1-IN-1 (GS-444217) was administered orally to mice with RV pressure overload; cardiac fibrosis and cardiac function were evaluated [2]

[1]. ASK1 contributes to fibrosis and dysfunction in models of kidney disease. J Clin Invest. 2018 Oct 1;128(10):4485-4500.

[2]. ASK1 Inhibition Halts Disease Progression in Preclinical Models of Pulmonary Arterial Hypertension. Am J Respir Crit Care Med. 2018 Feb 1;197(3):373-385.

1. ASK1-IN-1 (GS-444217) is a potent and selective small molecule ASK1 inhibitor that optimizes biochemical potency and lipophilic ligand efficiency (LLE) while maintaining low lipophilicity through structure-based drug design [1]. 2. ASK1 activation was observed in both glomerular and tubular compartments in renal biopsies of patients with diabetic nephropathy (DKD); ASK1-IN-1 (GS-444217) reduced ASK1 pathway activation in a rodent model of kidney injury/fibrosis simulating DKD pathology, and its combination with enalapril enhanced proteinuria reduction and glomerular sclerosis regression [1]. 3. Oxidative stress drives pathological remodeling in PAH through MAPK activation; ASK1-IN-1 (GS-444217)By inhibiting ASK1 (a redox-sensitive top MAPK), the progression of PAH is prevented by reducing pulmonary vascular and right ventricular remodeling, representing the first evidence of a causal role for ASK1 in the pathogenesis of PAH [2]

C₂₃H₂₁N₇O

411.46

411.18

C, 67.14; H, 5.14; N, 23.83; O, 3.89

1262041-49-5

57504987

White to off-white solid powder

2

1

5

6

31

654

0

C1CC1C2=CN(C=N2)C3=CC(=NC=C3)C(=O)NC4=CC=CC(=C4)C5=NN=CN5C6CC6

ZGCMQKWOUIMBEP-UHFFFAOYSA-N

InChI=1S/C23H21N7O/c31-23(20-11-19(8-9-24-20)29-12-21(25-13-29)15-4-5-15)27-17-3-1-2-16(10-17)22-28-26-14-30(22)18-6-7-18/h1-3,8-15,18H,4-7H2,(H,27,31)

4-(4-cyclopropylimidazol-1-yl)-N-[3-(4-cyclopropyl-1,2,4-triazol-3-yl)phenyl]pyridine-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.08 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.08 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)