GS-444217 is a selective ATP-competitive inhibitor of apoptosis signal-regulating kinase 1 (ASK1, IC50: 2.87 nM).

ASK1:2.87 nM (cell free)

GS-444217 demonstrated high selectivity for binding to ASK1 versus the other kinases in the panel. The affinity of GS-444217 for ASK1 (KD = 4.1 nM) was 53-fold greater than the affinity for DYRK1A (KD = 220 nM) and 104-fold greater than the affinity for RSK4 (KD = 430 nM). Treatment with GS-444217 reduced ASK1 phosphorylation and prevented the phosphorylation of MKK3/6, MKK4, p38, and JNK at concentrations of 0.3 μM and above with full suppression of ASK1 activity at 1 μM. GS-444217 reduced ASK1 activity within 5 minutes of addition to the cultures, reaching a maximum level of inhibition by 30 minutes. Removal of GS-444217 from the cultures resulted in reactivation of ASK1 autophosphorylation within 10 minutes and near-complete recovery 2 hours after drug washout [1].

Treatment with GS-444217 abrogated p38 MAPK activation in diabetic kidneys but had no effect upon hypertension in Nos3(-/-) mice. Early intervention with GS-444217 significantly inhibited diabetic glomerulosclerosis and reduced renal dysfunction but had no effect on the development of albuminuria. Late intervention with GS-444217 improved renal function and halted the progression of glomerulosclerosis, renal inflammation, and tubular injury despite having no effect on established albuminuria [2]. One dose of GS-444217 (30 mg/kg) given 30 minutes before administration of auranofin (30 mg/kg) suppressed the activation of ASK1, p38, and JNK in renal cortex. Auranofin administration induced mRNA expression of inflammatory cytokines (Il1b, Ccl2, and Cxcl2) and increased caspase activity in the kidney, and these downstream effects of ASK1 activation were inhibited by GS-444217. Comparing plasma concentrations of GS-444217 with the corresponding phosphorylated p38 (p-p38) signal in kidney, GS-444217 had an in vivo EC50 of approximately 1.6 μM for inhibiting the ASK1 pathway in rodent kidney [1].

Human embryonic kidney cells (HEK293T) were infected with full-length human ASK1 adenovirus or with an inactive truncated ASK1 adenovirus (K709R mutant with N-terminally truncated protein) as a negative control using the following conditions: (a) dose response: cells were infected for 24 hours followed by incubation for 2 hours with 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM GS-444217; (b) kinetics: after cells were infected for 24 hours, 1 μM GS-444217 was added to cells for 1, 5, 10, or 30 minutes or 1, 2, or 4 hours; (c) off-rate kinetics: cells were infected for 24 hours followed by incubation with GS-444217 for 30 minutes. After 30 minutes, medium was replaced with serum-free medium without compound and incubated for 0, 10, or 30 minutes or 1, 2, or 4 hours [1].

Male Sprague-Dawley rats (176–200 g, 7–8 weeks old) were randomly assigned to weight-matched treatment groups: (a) sham procedure, n = 8; (b) ischemia 30 minutes, n = 8; (c) ischemia 30 minutes plus GS-444217 (30 mg/kg, p.o.), n = 8. Bilateral renal occlusion was performed on anesthetized rats held at 37°C for 30 minutes. After recovering from anesthesia, rats were placed in metabolic cages for a 24-hour collection of urine. Sham rats underwent midline incision with surgery duration of 30 minutes but were not subjected to occlusion. Necropsy was performed on all rats 24 hours after surgery to collect kidneys and blood. Renal I/R studies were performed at Physiogenix Inc. Serum was analyzed for creatinine and blood urea nitrogen concentrations on a clinical chemistry analyzer. The right kidney was fixed in formalin and stained with H&E to assess tubular necrosis by pathology and with TUNEL to detect apoptotic cells [1].