Structural Basis For Selective Inhibition Of The Insulin-like Growth Factor 1 Receptor Kinase Domain By Picropodophyllotoxin

The insulin-like growth factor receptor(IGF-1R)is a disulfide-linked homodimer belonging to the receptor tyrosine kinase(RTKs)family.IGF-1R is widely found in various cell membranes and is highly expressed in tumor cells.The IGF-1R-mediated signaling pathway has a facilitative effect on tumor formation,cell proliferation and migration,which makes it a prospective tumor immunotherapy drug target.The intracellular tyrosine kinase region of IGF1R(IGF-1RK)is a potent site for IGF-1 R inhibition.As a natural small molecule compound,Picropodophyllotoxin(PPP)is a tautomer with Podophyllotoxin(PTOX),an antitumor lead compound,and they only have conformational differences at the C-ring 2 position,but only picropodophyllotoxin has been reported to target IGF-1R and has no inhibitory effect on the highly homologous insulin receptor(IR).Investigating the molecular mechanism of IGF-1R targeting by picropodophyllotoxin is important for the development of novel antitumor drugs targeting IGF-1R.In this paper,we attempted to investigate the mechanism of IGF-1R inhibition by crystallography,and obtained the first IGF1RK-PPP complex crystals with a resolution of 2.5 A.The specific mechanism of IGF-1 R inhibition by picropodophyllotoxin was also described at the molecular level.Firstly,the results of in vitro antitumor activity evaluation,cell cycle blocking validation and in vitro kinase activity function evaluation revealed that the cellular activity and cell cycle blocking ability of picropodophyllotoxin were lower than that of podophyllotoxin,but its cytotoxicity and phosphorylation of IGF-IRK were better than that of podophyllotoxin.In summary,the results indicated that picropodophyllotoxin has the characteristics of low toxicity and great targeting.Then,attempts were made to achieve soluble expression of humanderived IGF-1RK protein monomers in a variety of protein expression systems.Although the soluble expression of TrxA-IGF1RK and MBP-IGF1RK proteins was achieved in the E.coli expression system,their states were highly aggregated,and although the IGF-1RK monomeric protein was successfully expressed in the Saccharomyces cerevisiae expression system,the protein expression was unstable and easily degraded under this system,and high purity IGF1RK protein could not be obtained.The IGF-1RK monomeric protein was successfully expressed in an insect cell expression system by preparing high-titer baculovirus-infected cells,and the activity and high purity could be maintained in a long purification step,which provided the prerequisite for subsequent culturing of IGF1RK-PPP complex crystals.Finally,an attempt was made to resolve the specific mechanism of action of picropodophyllotoxin with IGF-1RK by using the method of culturing IGF1RK-PPP complex crystals.After the initial screening of the protein crystallization kit and empirical screening,the conditions of 20%(w/v)PEG 8000,100 mM Tris-HCl(pH 8.5),and 200 mM MgCl2 were successfully screened,and the IGF1RKPPP complex crystals were grown simultaneously using the Soaking method and the cocrystallization method.Under these conditions,the crystals were mostly in the shape of clusters,squares or short rods.By optimizing the crystal growth conditions,Square-shaped IGF1RKPPP complex crystals with a resolution of up to 2.5 ? were obtained by co-crystallization for the first time,and the molecular mechanism of their interaction was elucidated by structural analysis.In this study,we achieved soluble expression of IGF-1RK monomer in an insect cell expression system,attempted to resolve the mechanism of selective targeting of PPP to inhibit IGF-1RK using a co-crystallization approach,and obtained crystals of IGF1 RK-PPP dimeric complex for the first time,resolved the binary complex structure of IGF1RK-PPP,and elucidated the mechanism of their interaction.However,some amino acids are missing in the structure,probably due to the low quality of the crystal.We are still advancing the crystal diffraction and resolution of IGF1RK-PPP complex,trying to obtain a more complete structure of the complex and reveal the structural basis of the selective inhibition of IGF-1RK by PPP.The results of the study will provide a theoretical basis for selective targeting of IGF-1R inhibitors.