DF-003 is a selective, orally active, ATP-competitive ALPK1 inhibitor capable of crossing the blood-brain barrier. It effectively inhibits human ALPK1 and ALPK1[T237M] with IC50 values of 1.5 nM and 16 nM, respectively. The compound exhibits selectivity over the nearest kinase by more than 860-fold. DF-003 suppresses the upregulation of TNF, CXCL10, or CXCL8 in HEK-293 cells. It has applications in the study of retinopathy, optic nerve edema, splenomegaly, anhidrosis, and headaches associated with ROSAH syndrome.

DF-003 demonstrates dose-dependent inhibition of ALPK1 kinase activity (IC₅₀ = 1.5 nM) when applied at concentrations of 1-50.8 pM over 60 hours. Additionally, DF-003 (20 μM-0.763 nM, 2 hours) shows non-ALPK1 kinase inhibition against TAOK2/TAO1 (IC₅₀ = 1.29 µM), CAMK2g (IC₅₀ = 3.10 µM), and CAMK1a (IC₅₀ = 4.60 µM). It effectively inhibits DF-006-induced TIFAsome formation in HEK-293 cells within 1 hour at concentrations of 10-1000 nM, indicating intracellular ALPK1 inhibition. DF-003 also suppresses DF-006-induced upregulation of TNF (IC₅₀ = 8 nM) and CXCL8 (IC₅₀ = 6.5 nM) in THP-1 cells over a 2-hour period at 0.3-1000 nM. It dose-dependently inhibits UDP-mannose-enhanced ALPK1[T237M] activity (IC₅₀ = 16 nM) when administered from 1-50.8 pM over 60 hours. Without external stimuli, DF-003 (0.64-20 μM, 48 hours) reduces NF-κB reporter gene activity in ALPK1[T237M] OE HEK293 cells. Furthermore, at 0.64-20 μM over 30 hours, it inhibits cytokine upregulation in HEK-293 cells overexpressing ALPK1[T237M], while having no impact on the basal expression of TNF, CXCL10, or CXCL8 in cells overexpressing wild-type ALPK1. Lastly, DF-003 (0.64-20 μM, 30 hours) reduces enhanced CXCL8 secretion in HEK-293 cells overexpressing ALPK1[T237M] (IC₅₀ = 125 nM).

DF-003 (3-5 mg/kg, administered orally, once daily for a total of 10 days) effectively penetrates the blood-retina and blood-brain barriers, suppressing multi-tissue inflammatory responses induced by ALPK1[T237M] mutation. This includes the infiltration of retinal microglia, astrocyte activation, and cytokine expression in hALPK1[T237M]-KI C57BL/6N mice.