Cell experiment:

A673 and MiaPaCa-2 cellsA673 and MiaPaCa-2 cells are cultured and plated in 1536-well white solid tissue culture plates. A 1536-well pintool dispenser outfitted with 20 nL pins is used to transfer 23 nL of LDH-IN-1 in DMSO to the 1536-well assay plates. After a 48 h incubation at 37℃, 2.5 μL of CellTiter-Glo is dispensed into each well using a BioRAPTR FRD dispenser. Plates are incubated at room temperature for 10 min, transferred to a ViewLux microplate imager, and the ATP-coupled luminescence is measured using a 1 s exposure[1].

Animal experiment:

Mice[1]Male CD1 mice approximately 6–8 weeks of age and a weight of approximately 20–30 g, are dosed with LDH-IN-1 at 2 mg/ kg (iv) and 50 mg/kg (po). Each cohort had three mice, and plasma is collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24h postdose for iv and 15 min 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h for po. Approximately 0.025 mL of blood is collected via the dorsal metatarsal vein at each time point. Blood samples are then transferred into plastic microcentrifuge tubes containing heparin–Na as anticoagulant. Samples are then centrifuged at 4000g for 5 min at 4 ℃ to obtain plasma. Plasma samples are then stored in polypropylene tubes, quickly frozen, and kept at –75 ℃ until analyzed by LC/MS/MS[1].

LDH-IN-1 is a novel pyrazole-based inhibitor of human lactate dehydrogenase (LDH) with IC50s of 32 and 27 nM for LDHA and LDHB, respectively.

LDH-IN-1 exhibits low nM inhibition of both LDHA and LDHB (IC50=32, 27 nM, respectively), submicromolar inhibition of lactate production, and inhibition of lactate in MiaPaCa2 pancreatic cancer and A673 sarcoma cells (IC50=0.517, 0.854μM, respectively). LDH-IN-1 inhibits the growth of MiaPaCa2 pancreatic cancer and A673 sarcoma cells with IC50s of 2.23 and 1.21 μM). Dose-response treatment of MiaPaCa-2 cells with LDH-IN-1 shows effects on cellular proliferation at concentrations as low as 250 nM and with nearly complete arrest of cell growth at 20 μM[1].

LDH-IN-1 shows clearance values that far exceed hepatic blood flow (HBF) in mouse species (90 mL/min/kg), with in vivo clearances of 227 mL/min/kg[1].

[1]. Rai G, et al. Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH). J Med Chem. 2017 Nov 22;60(22):9184-9204.