Hypoglycemic Pharmacodynamic Evaluation And Mechanistic Study Of LXQ-87,A Bromophenolic Compound Targeting PTP1B

With the development of social economy and the improvement of people’s living standard,diabetes has become a "killer" that endangers human health.Type 2 diabetes mellitus(T2DM)is the most common type of diabetes,accounting for approximately 90%of the total diabetes cases.The main features of T2DM are hyperglycaemia,hyperinsulinemia and insulin resistance,of which insulin resistance is thought to be the main driver for the development of T2DM.Protein tyrosine phosphatase 1B(PTP1B)has been shown to be an effective drug target for the treatment of T2DM.PTP1B dephosphorylates the insulin receptor(IR)in the insulin signaling pathway,thereby weakening insulin signaling and causing hyperglycemia and insulin resistance in the body.Existing PTP1B inhibitors suffer from low oral utilization and poor targeting,so it is important to develop novel and efficient PTP1B inhibitors.Li in our group designed and synthesized a series of compounds targeting PTP1B,which are lead compounds such as the marine bromophenolic compounds BDDPM and BDB.We screened a compound LXQ-87(IC50=1.061 μM)with good PTP1B inhibition activity in vitro enzyme activity screening assay.It was evaluated for hypoglycemic pharmacodynamics in vitro enzyme level,in vitro cellular level and in vivo animal level experiments.In vitro enzyme level,we determined the type of inhibition of PTP1B by LXQ-87 through enzymatic reaction kinetics experiments;we used surface plasmon resonance(SPR)technique to confirm whether the affinity interaction between LXQ-87 and PTP1B could take place;and we used computer simulation docking to predict the possible binding mode of LXQ-87 and PTP1B.In vitro cellular level,we examined the cytotoxic activity of LXQ-87 using the CCK8 assay;we determined whether LXQ-87 and PTP1B could be effectively targeted at the cellular level using the cellular thermal shift assay(CETSA);we established a high concentration insulin-induced insulin resistance cell model and investigated whether LXQ-87 could improve insulin resistance in HepG2,primary hepatocytes,C2C12 and 3T3L1 metabolic cells using Western blot technique;we examined whether LXQ-87 could increase glucose consumption in HepG2,C2C12 and 3T3L1 cells using glucose oxidase assay.In vivo animal level,we evaluated the safety of LXQ-87 by acute toxicity assay;we investigated whether LXQ-87 could improve glucose metabolism disorders in db/db mice by chronic oral administration,including evaluation of hypoglycemic activity in animals and improvement of insulin resistance in metabolic tissues.In vitro enzyme level,we found that LXQ-87 was a non-competitive inhibitor of PTP1B and that it was capable of producing an effective affinity interaction with PTP1B(KD=6.12×10-8 M).LXQ-87 may inhibit PTP1B activity by inhibiting the formation of the active conformation of PTP1B and it was a potential metastable inhibitor.In vitro cellular level,we found that LXQ-87 can effectively bind to PTP1B target.LXQ-87 increased phosphorylation of IR and AKT in the insulin signaling pathway and improved insulin resistance in HepG2,primary hepatocytes,C2C12 and 3T3L1 cells induced by high insulin concentrations.LXQ-87 enhanced the glucose consumption capacity.In vivo animal level,we found that chronic oral administration of LXQ-87 significantly reduced fasting glucose,improved oral glucose tolerance and insulin resistance in db/db mice,and enhanced insulin sensitivity in peripheral tissues and organs such as liver,skeletal muscle and adipose.In summary,we have designed and synthesized an active PTP1B inhibitor using PTP1B as the target,and evaluated its efficacy at the cellular and animal levels,and elucidated the mechanism of action associated with it,which provides strong data support for the further development of PTP1B inhibitors in the future,and also provides an experimental basis for the development of new anti-diabetic drugs.