1.Study On Novel Molecular Mechanisms Of Action Of Nintedanib Based On Chemical Proteomics 2.Polymorphic Study And Pharmacokinetic Evaluation Of IMM-H007

Nintedanib,a triple angiokinase inhibitor of platelet-derived growth factor receptors(PDGFR),vascular endothelial growth factor receptors(VEGFR)and fibroblast growth factor receptors(FGFR),has been approved by FDA for three indications until now,including idiopathic pulmonary fibrosis(IPF),chronic fibrosing interstitial lung disease(ILDs)and systemic sclerosis-associated interstitial lung disease(SSc-ILD).Due to the lack of understanding of pathogenesis of IPF and the fact that other PDGFR,VEGFR and FGFR inhibitors do not have anti-IPF efficacy,the direct target of nintedanib against IPF remains unclear.In this project,based on the structure-activity relationship of nintedanib,we designed and synthesized five probes:P1-P5.The biological activity evaluations were performed to yield photoaffinity probes P2 and P3 with similar activities compared to nintedanib.The in-gel fluorescence imaging experiments were conducted to confirm the photolabeling ability of probes,and to optimize the concentration and incubation time of probes.Competitive binding experiments revealed that the probe and nintedanib bound to the same molecular targets.Using affinity-based protein profiling(AfBPP)technique,P3 was utilized to disclose the targets of nintedanib in human umbilical vascular endothelial cells(HUVECs),followed by target identification using protein mass spectrometry.Through analyzing mass spectrometry data,we first identified tripeptidyl-peptidase 1(TPP1)as a potential direct target of nintedanib.It was demonstrated by western blot experiment that direct interaction existed between nintedanib and TPP1.The confocal microscopy experiments revealed that the probe co-localized with TPP1.Molecular docking showed that nintedanib could occupy the active pocket of TPP1,TPP1-OB domain,where telomerase could be recruited,and interact with crucial residues in this binding pocket.Telomerase regulation was closely associated with cellular proliferation and formation of IPF.Nintedanib may affect the recruitment of telomerase by binding to TPP1,thereby to exert its anti-IPF efficacy.Additionally,using the same strategy and analyzing procedure,P2 was utilized to identify the binding proteins of nintedanib in human pulmonary fibroblast(HPF).The results demonstrated that discoidin domain-containing receptor 2(DDR2)and TANK-binding kinase 1(TBK1),with the least ratio of enrichment in the competition group,were high-confidence potential targets of nintedanib,and highly related to phenotypes of cellular proliferation and IPF,which was worthy of in-depth study.This project identified TPP1 as a new target,and DDR2,TBK1 as potential targets of nintedanib,beyond the angiokinase,by performing AfBPP technique.These newly-discovered molecular targets broadened our understanding of the mechanisms of action of nintedanib and provided new inspiration for the treatment of IPF,ILDs and SSc-ILD.Polymorphisms exist in most of therapeutic drugs,which greatly affects the quality and efficacy of drugs.2’,3’,5’-tri-O-acetyl-N6-(3-hydroxyphenyl)adenosine(IMM-H007)was modified from cordycepin,and clinical studies had demonstrated that it had significant hypolipidemic effect.In this article,we studied the polymorphism of IMM-H007.Through polymorphism screening,it was found that there were two crystal forms for IMM-H007,and H and L crystal form were prepared by appropriate methods.Subsequently,a variety of techniques were performed to characterize the crystal forms.The thermogravimetric analysis revealed that both crystal forms were non-solvent form.The two crystal forms could be effectively identified by powder X-ray diffraction,differential scanning calorimetry and infrared spectroscopy.By cultivating the single crystal,we determined the absolute configuration of IMM-H007 and the crystal parameters of H crystal form.Under the conditions of accelerated test,both crystal forms displayed good stability.Pharmacokinetic studies showed that there was big difference in the bioavailability of the two crystal forms,and H crystal form was significantly better than L.