Ki16425 (Debio 0719) is a competitive, potent and reversible antagonist to LPA1, LPA2 and LPA3 with Ki of 0.34 μM, 6.5 μM and 0.93 μM, respectively.

LPA2:6.5 μM(Ki), LPA1:0.34 μM(Ki), LPA3:0.93 μM(Ki)

Ki16425主要抑制LPA1和LPA3介导的反应，但对LPA2的影响较为适中。在THP-1细胞、3T3成纤维细胞和A431细胞中，Ki16425能够抑制由LPA引起的Ca(2+)反应，但在PC-12细胞和HL-60细胞中的影响较小，这表明Ki16425似乎是评估特定LPA受体在LPA短期反应中作用的有用工具。Ki16425能够抑制由LPA在Swiss 3T3成纤维细胞中诱导的长期DNA合成和细胞迁移。[1] Ki16425减少了LPA诱导的p42/p44促分裂原激活蛋白激酶（MAPK）激活，同时自身作为p42/p44 MAPK的弱刺激剂，这是典型的蛋白质激动剂特性。Ki16425还显著减少了NGF对p42/p44 MAPK的刺激作用，并抑制了NGF在PC-12细胞中诱导的神经突起生长。[2] Ki16425显著抑制了滑膜液诱导的COX-2蛋白表达。LPA增强IL-1作用对COX-2表达的影响也被Ki16425抑制。[3]

Ki-16425 (30 mg/kg, i.p.) 在注射溶磷脂酸30分钟前给药时，能完全阻断由LPA引起的神经病理性疼痛样行为，而在90分钟前给药则无效，这表明Ki-16425是一种短效抑制剂。Ki-16425还能抑制神经损伤引起的背根神经节中Caα2δ-1的上调和脊髓背角SP免疫反应性的减少。[4]

High-throughput screening: Screening is conducted at the ICCB-Longwood screening facility. 10 μL of recombinant USP14 protein are dispensed into each well of a 384-well low volume plate in duplicate, using a Wellmate plate dispenser. 33.3?nL of compound from the library are pin-transferred into the wells using a Seiko pin transfer robotic system, followed by pre-incubation for about 30?min. The last two columns of each plate are used for positive and negative controls for the assay. To initiate the enzyme reaction, 10?μL of VS-proteasome plus Ub-AMC mixture are added to each well, using a Wellmate dispenser. Samples are then incubated for another 45?min. Ub-AMC hydrolysis is measured at Ex355/Em460 using an Envision plate reader. The final concentrations of USP14, VS-proteasome and Ub-AMC are 15?nM, 1?nM and 0.8 μM, respectively. The final concentration of test compound is approximately 17?μM. Enzymes and substrates are prepared in Ub-AMC assay buffer (50?mM Tris-HCl (pH?7.5), 1?mM EDTA, 1?mM ATP, 5 mM MgCl2, 1?mM DTT, and 1 mg/Ml ovalbumin).