Anti-tumor Peptides Targeted At G3BP Protein

G3BP1(Ras GAP SH3-binding protein)is one of the proteins described to specific bind to Ras GAP(Ras GTPase activating protein,Ras GAP)SH3 domain. It is composed of 466 amino acid and located endochylema. G3BP1 is the important regulatory and target molecules in Ras signaling pathway. It was reported that G3BP1 has endoribonuclease activity and regulate the formulation of stress granule(SGs). Additionally, G3BP1 is involved in a variety of growth,differentiation-related signaling pathwasys, apoptosis and RNA metabolism. G3BP1 is an important protein in the tumor growth.The aim of this study was to design a new peptide, GAP162, based on the interaction model between Ras GAP and G3BP1 NTF2-like domain and to do research on the anti-tumor effect and potential mechanism of peptides in the molecular and cellular level. The in vivo anti-tumor effect and pharmacokinetics of peptides targeting G3 BP were studied.Part Ⅰ Design and Anti-Proliferative Effect Mechanism of Peptides Targeting G3BPBased on the interaction model between Ras GAP and G3BP1 NTF2-like domain, P160,which was proposed to have stronger affinity to G3 BP than natural peptide fragment of Ras GAP, was designed using amino acid mutation method. GAP161 and GAP162 were designed based on P160 and cell penetrating peptides.ATP method was used to determine the potential effect of P162 on the growth of HCT116 and A549 cell. The results showed that P162 significantly inhibit the proliferation of HCT116 and A549 cells. The effect was much stronger than GAP161 and independent to cell penetrating peptide P167.A serial of assays were performed to investigate the in vitro anti-proliferative activity mechanism of GAP162. Flow cytometric analysis and Annxin V/PI staining of GAP162-treated cell were used to quantify apoptosis. Western blot was used to measure the key protein Caspase-3 involved in apoptosis. SPR was used to measure the affinity between GAP162 and G3BP1. Co-immunoprecipitation assay was performed to investigate the affect of GAP162 on interaction bwteen G3BP1 and Ras GAP. Immunocytochemistry was used to test the ability of GAP162 to affect the SG formation. Western Blot was used to find the affect of GAP162 to G3BP1 protein, phospho-G3BP1(p Ser149) and C-myc.The results showed that GAP162 significantly induced apoptosis in HCT116 cells by activating the apoptotic signal pathway. GAP162 markedly induced the cleavage of caspase-3.The results of Biacore determination showed that GAP162 can specifically bind to G3BP1 protein with much stronger affinity than GAP161. In HCT116 cell after treated with GAP162,reciprocal co-IP experiment indicated that GAP162 decrease the binding of Ras GAP to G3BP1.This was consistent with the design of peptide GAP162 base on the interaction between G3 BP and Ras GAP. Western Blot was used to determine the effect of GAP162 to G3BP1 protein and phospho-G3BP1(p Ser149) in HCT116 cell. The result showed that GAP162 didn’t affect G3BP1 protein level, but increase phospho-G3BP1(p Ser149) protein level.Immunocytochemistry assay showed that GAP162 could significantly inhibit SG formation in a dose- dependent manner. Finally, Western blot showed that GAP162 decrease C-myc protein levels. In summary, the mechanism of GAP162 anti-tumor activity was supposed as follow:GAP162 markedly suppressed HCT116 cell growth through the induction of apoptosis. The peptide bound with G3 BPs and further affected the phospho-G3BP1(p Ser149) and suppressed SG formation and decrease C-myc protein.Part Ⅱ:In vivo Pharmacodynamic and pharmacokinetic study of peptides targeting G3BPGAP161 was the first peptide targeting G3 BP to be performed in vivo and in vitro efficacy study. The pharmacokinetics of GAP161 was studied firstly. An HPLC-MS/MS method was developed to determine the concentration of GAP161 in rat plasma. For the characteristic of peptide GAP161, the method was optimized as follow: 1) To avoid the non-specific adsorption,polypropylene(PE) vials were used instead of glass vials. Preparing calibration curves in the presence of low amount of blank plasma further improved the linearity, especially for the low-concentration range; 2) With no digestion procedure required, the peptide GAP161 was analysis integrated; 3)0.5% DMSO was added as supercharging reagent;4) A semi-automated96-well ion-exchange solid phase extraction(SPE) was developed; 5) A large-pore C4 column was employed for separation. In the LC-MS/MS-based method described in the present study,the calibration curves ranging from 5 to 2000 ng m L-1 were linear for the analysis of GAP162 in rat plasma. The lower limit of quantification(LLOQ) was 5 ng·m L-1. The retention time for GAP161 and GAP120(IS) were 1.51 and 1.50 min under the described conditions. Thechromatographic running time by LC-MS/MS was within 3.0 min. The SPE procedure was performed on an automated μElute 96-well extraction system, which permitted a high throughput. The recovery was above than 57%. The intra-, inter-run precision and accuracy were both within the acceptable range. The stability results showed that plasma GAP161 was stable at most two freeze/thaw cycles(-80?25°C), at 4°C over 12 h, and autosampler storage in the reconstitution solution for 24 h. However GAP161 was less stable under ambient temperature. Therefore sample preparation(including blood collection, plasma separation and handling etc) were all performed on ice. The validation of the LC-MS/MS method showed that the method were able to apply to pharmacokinetic study.The concentrations of GAP161 in plasma at different timepoints were determined after single intravenous dosing to male Sprague-Dawley rats at dose level of 5 mg·kg-1. The pharmacokinetic parameters were calculated using the non-compartmental analysis. The half-life of terminal elimination(T1/2) of GAP161 was calculated as 1.84±0.14 h and MRT0.258±0.053 h. The apparent distribution volume( Vd) was 35894 ± 1621 m L·kg-1. This data suggested a short half-life, easily degradation and poor stability in vivo. The PK results provided supportive evidence of mild anti-tumor effect of GAP161 in vivo.Refer to the bioanalysis method of GAP161, an HPLC-MS/MS method was developed and validated to determine the concentration of GAP162 in rat plasma. The pharmacokinetic study of GAP162 after single intravenous dosing to male Sprague-Dawley rats was conducted. The pharmacokinetic parameters were calculated using the non-compartmental analysis. The half-life of terminal elimination(T1/2) of GAP162 was calculated as 1.43± 0.34 h and MRT0.649± 0.053 h. The apparent distribution volume(Vd) was 7872 ± 1061 m L·kg-1. The results suggested a similar half-life with GAP161 and a longer MRT compared with GAP161.Radiolable by Iodogen method, TAC precipitation method and molecular sieve chromatography(sec HPLC) method were combined to investigate the tissue distribution of GAP162 in nude mice bearing HCT116 tumor xenografts. The amount of radioactivity was determined for 125I-GAP162 quantification. After a single intravenous administration to Blab/c nude mice bearing HCT116 tumor, the AUC of lung is the highest and brain is the lowest,which indicated that GAP162 can’t pass blood brain barrier. Although the concentration of P162 in tumor is not the highest, P162 reached Cmax at 2 min post dosing(the same as heart and blood) and accumulated significantly over more time within the tumor. The elimination rate is much lower than other organs..In order to assess the anti-tumor activity of P162, a C26 xenograft tumor model was used.After implant 24 h, GAP162 were dosed in two different treatments(daily) with or without cisplatin(every other day). The body weight and tumor weight were recorded for calculation.GAP162 showed tumor inhibition in C26 xenograft tumor model with the effective dosage of 40mg/kg for ip dosing and 160 mg/kg for sc dosing. When used in combination with a genotoxin(cisplatin), P162 could act as a chemosensitizer to improve the efficacy of chemotherapeutic agents. P162 showed much better tumor inhibition than GAP161 with 2-fold TGI increasing.