The Binding Protein Research Of Structurally Novel Antitumor Lead Compound Rasfonin

Bioactive small molecule compounds are an important source of novel drug discovery because of their varied biological activity, especially natural products isolated from plants, animals and marine life. Our preliminary study has evaluated the pharmacological activity of a structurally novel small molecule, Rasfonin(provided by the lab of Professor Che Yong-sheng), which is isolated from a fungus Doratomyces sp. on the Qinghai-Tibetan plateau at an altitude above 4,000 m. Studies found that Rasfonin had effects on ras-mutated tumor cells selectively, and it remarkably inhibited proliferation, clone formation, migration and invasion of K-ras mutant pancreatic cancer cells(Panc-1). Study in vivo demonstrated that Rasfonin had significant antitumor efficacy on CD1 nude mice xenograft model of Panc-1. Preliminary study of the mechanism of Rasfonin showed that it was able to inhibit Ras activation by down-regulating the expression of Ras activated protein Sos1, consequently affected the phosphorylation of Ras-MAPK downstream kinases. The results of radioactive ligand labeling experiments on 19 kinds of protein kinases in EGFR signaling pathway(including Ras signaling pathway) showed that Rasfonin could up-regulated p70S6 K activation( kinase of m TOR downstream).The antitumor effect of Rasfonin is remarkable, and the mechanism of Rasfonin is unique, which has no similar reports yet. Rasfonin is expected to become an antitumor lead compound, but its pharmacological mechanism and target are not clear. This research studies on the intracellular binding proteins of Rasfonin.Target discovery methods of bioactive small molecule generally used can be broadly divided into two categories: forward(phenotype-based) and reverse(target-based) approach. The former one can detect the changes of biochemical indicators of signaling pathway affected by bioactive small molecule, study on the potential binding proteins indirectly. The latter one can isolate the binding protein of natural state, obtain the information related to the potential targets directly. The latter one is more simple and direct. Among the various existing methods of targets discovery which can direct isolate the binding proteins, the traditional methods based on affinity chromatography are applied most widely. In this method, bioactive small molecules are immobilized on the solid matrix, fishing out the binding proteins. Affinity chromatography has been successfully used in many cases, such as the binding protein FKBP12 of immunosuppressant FK506, the binding protein cycolphilin of cyclosporine A(Cs A), and the new target protein glyoxalase 1(GLO1) of non-steroidal anti-inflammatory drug indomethacin, etc.However, there are still many limitations in the classical affinity chromatography, and some improved methods have been appeared in recent years. For example, Yamamoto launched a new method based on affinity chromatography named serial affinity chromatography, which could reduce the effects of high concentration of organic reagents helping dissolve the small molecules. This method does not need to set up negative control group and competitive control group. In this method, the protein mixture continuously react with multiple column which immobilized with bioactive small molecule, and the specific binding protein can be found by comparing the retained binding proteins on each column. In addition, binding proteins with weak affinity are easily eluted in the classical affinity chromatography. Aim at this problem, the introduction of photo-affinity labeling group can enhance the combination of small molecules and their target proteins.In traditional methods like affinity chromatography, small molecules are usually immobilized on solid phase, so they need to be modified firstly. But modification tends to affect the activity of small molecules. In 2009, a new target identification technique, Drug Affinity Responsive Target Stability(DARTS), was reported on Proceedings of the National Academy of Sciences(PNAS). DARTS is based on a finding that the conformation of binding proteins might be less susceptible to proteolysis by bound to small molecules than its unbound state, so that the binding proteins can be isolated from the complex protein mixtures. DARTS is based on the binding affinity of small molecules and their targets. The major advantage of DARTS is the use of unmodified compounds or even traditional Chinese medicine complex with unknown composition, providing a new thought and technique for the target discovery of small molecule compounds. There have been a few studies discovered the potential targets of small molecules using DARTS. For example, Randall’s research found that α-ketoglutarate, a tricarboxylic acid cycle intermediate, extended the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β was identified as a novel binding protein of α-ketoglutarate using DARTS. In Molly’s research, target identification of potential protein targets of grape seed extract(GSE) was carried out utilizing DARTS, and they found ten kinds of possible binding proteins of GSE.In order to find out binding proteins of Rasfonin and provide clues to antitumor target research of Rasfonin, we employed two techniques that can directly isolate the binding proteins of small molecules, affinity chromatography and DARTS.1. Research of binding proteins of antitumor compound Rasfonin based on affinity chromatography1.1. Modification and activity detection of RasfoninDue to the requirement of affinity chromatography method that the small molecule should be immobilized on solid substrates, we chose biotin-avidin system as the connective medium according to the structure-activity relationship analysis of Rasfonin. The lab of Professor Che Yong-sheng modified A304(Rasfonin) and its negative control small molecule A321. In order to investigate whether biotin modification changed the activity of these small molecules, we detected the effect of A304, A321 and their biotin derivatives, A304-B, A321-B, on proliferation of Panc-1 cells by CCK8. The results showed that A304 had obvious inhibitory on Panc-1 with the IC50 of 9.07 μM, which is consistent with the previous results, while A304-B inhibited Panc-1 cell proliferation with IC50 of 9.5 μM. The inhibitory of A321 on the proliferation of Panc-1 cells was not obvious with IC50 of 19.3 μM, while the IC50 of A321-B is 14.09 μM. Results indicated that the modification of A304 did not affect its activity and A304-B could be used in affinity chromatography, whereas the activity of A321 was enhanced a little after modified, which might affect the subsequent experiments.1.2. Study of binding protein of Rasfonin based on affinity chromatography1.2.1. Application of affinity chromatography for searching binding proteins of RasfoninThis research conducted the classical affinity chromatography in combination with SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and coomassie brilliant blue staining to separate the protein samples. In order to exclude non-specific binding proteins, we set negative control group and competitive control group. The results showed that there were three differential protein bands in the group of A304-B and A321-B within the molecular weight of 40- 70 k Da by comparison with the two control groups.Agarose beads can easily conceal non-specific binding proteins because of their large diameter(about 45- 165 microns), while magnetic beads are smaller and uniform and result in lower non-specific-binding background. In order to reduce the non-specific binding adsorption, we also utilized magnetic beads(Dynabeads ? M-280) as solid phase in the classic affinity chromatography. The results showed that there were five protein bands of significant difference in the similar position of above experiment.Besides, we also conducted serial affinity chromatography which need no negative control group to find the binding proteins of Rasfonin. The protein sample reacted with two column immobilized A304-B(A304-B-1 and A304-B-2) continuously. The results showed that there were four significant binding protein bands in the A304-B-1 group within the molecular weight 35- 70 k Da, by comparison with the solvent control group and A304-B-2 group. Besides the 35 k Da-band, other bands were of the similar molecular weight and sites compared with the result of the classic affinity chromatography.1.2.2. Identification and analysis of the differential protein bandsAccording to the results of the classical affinity chromatography and the serial affinity chromatography, we identified five groups of protein bands which were consistent and significant, using nano LC, mass spectrometry, and Mascot index based on NCBI nr Green Plants database. The score reflects the matching degree, the higher the score values, the higher matching degree is. Generally, score above 200 indicates that the protein is reliable. But when the score is less than 200, if there are at least two unique fragments, the protein can also be trusted. On this basis, we analyzed the results of each group and excluded the protein whose score was less than 200 or also appeared in the solvent control group, we found 30 kinds of differential proteins, which were contained in Rasfonin-treated group but not contained in solvent control group. Finally, we found out 10 kinds of proteins closely related to tumor or correlative signaling pathways based on literature, and they might be the binding proteins of Rasfonin.2. Study of binding protein of Rasfonin based on DARTS2.1. Validation of DARTS principleDARTS makes use of the characteristic that the conformation of the binding proteins may be more stable by binding to small molecule ligands than its unbound state, so we can find out the binding protein of small molecule by observing the protein after treatment of small molecule and protease. In order to verify the feasibility of DARTS principle, according to the literature, we chose rapamycin and its high-affinity target protein, FKBP12, for DARTS validation. The results showed that under the condition described in the literature, compared with the solvent control group, the proteolysis of FKBP12(human recombinant protein) bands in the rapamycin-treated group was significantly reduced. Results suggest that rapamycin can indeed protect its target protein FKBP12 from proteolysis. The results indicated that DARTS principle was feasible.2.2. Exploration of the experimental conditions of DARTSNow there have been several successful cases that found the binding proteins of small molecules by DARTS, but the specific procedure for DARTS is not in detail, and the experimental condition applied still needs to explore and optimization. In this study, we explored the key condition of DARTS, the proteases species, the concentration of protease and the duration of proteolysis.2.2.1. Comparison of the ability of different kinds of proteasesProteolysis plays an important role in DARTS. In order to investigate the ability of different species protease in DARTS, we chose three different kinds of protease(Subtilisin, Thermolysin and Pronase). We employed proteolysis on protein complex extracted from Jurkat cells of 15 min at room temperature. The concentration of each protease included five gradients: 1:500, 1:1000, 1:2500, 1:5000 and 1:10000. The results showed that the proteolysis of Subtilisin is so robust that could digest almost all protein complex of concentration 1:500. Thermolysin showed the weakest effect on the protein mixture. Pronase, which is obviously more moderate, could approximately hydrolyze the protein mixture by 30- 60 % at the range of 1:500- 1:5000. The results are consistent with the reports. The results showed that Pronase was suitable for DARTS at the concentration range of 1:500- 1:5000.2.2.2. Effect of different concentration of protease on DARTSAccording to the literature and our results, we chose Pronase to investigate the effect of different concentration on DARTS. We used different concentrations(1:500, 1:1000, 1:2500 and 1:5000) of Pronase on α-ketoglutarate at same proteolysis temperature and endurance. The results showed that, for α-ketoglutarate, the most significant differential protein bands could be observed under the condition of 1:1000 Pronase, and the result was consistent with the original literature. Other concentration condition were not suitable because of the ability of digestion is too weak or too strong. Results indicated that Pronase 1:1000 was more suitable for the presentation of binding proteins in DARTS.2.2.3. Effect of different duration of proteolysis on DARTSOn the basis of the optimal protease concentration of α-ketoglutarate(Pronase 1:1000), we examined the effect of different duration of proteolysis(5, 15, 30 min) on the DARTS of α-ketoglutarate. The results showed that there was no obvious influence on the protein bands when digested 5 min. When the protein mixture was digested 15 min, there were five differential bands. When digested 30 min, the protein could be observed no significant differential bands. The results showed that the condition of Pronase 1: 1000 for 15min-digestion might be suitable for the presentation of binding protein in DARTS.2.2.4. Verification of the optimized proteolysis conditionIn order to verify the feasibility of the optimized proteolysis condition above(Pronase 1:1000 and digesting for 15min), we chose mycophenolic acid(MPA), which was not verified by DARTS and with known target, and investigated in two levels, human recombinant protein level and protein mixture level.Results of DARTS of human recombinant protein level showed that under the condition of Pronase 1:1000 and digesting for 5 min, compared with the solvent control group, the rh IMPDH1 of MPA-treated group was significantly enhanced, indicated that MPA could indeed protect its target protein rh IMPDH1 from proteolysis. The results of DARTS of protein mixtures level showed that under the condition of Pronase 1: 1000 and digesting for 15 min, there were two distinct differential protein bands in molecular weight of 55- 70 k Da. Then we confirmed that IMPDH1 was contained in the differential protein band presented at molecular weight of 55 k Da by western blotting analysis.The results suggested that the optimized proteolysis condition(Pronase 1:1000 and digesting for 15 min) by previous study was practical, and might be more suitable for the initial DARTS exploration of small molecule compounds. The feasibility of this condition on more compounds remained to be further studied.2.3. Study of binding proteins of Rasfonin based on DARTS2.3.1. Application of DARTS for searching the binding proteins of RasfoninBased on the optimization for DARTS experimental conditions, we conducted DARTS using Pronase 1:1000 and digestion for 5 min to 30 min. As a result, compared with DMSO solvent control group, there were two differential protein bands between 40- 55 k Da both in 10 and 100 μM Rasfonin-treated group when digested for 5 min and 15 min. When the cell line was replaced with Panc-1, the result was consistent with the one mentioned above. Compared with the results of affinity chromatography, the differential protein bands appeared at a consistent molecular weight range, indicating that it was more likely that the binding protein was in the range of 40- 55 k Da.2.3.2. Identification and analysis of the differential protein bandsRefer to the identification and analysis of the differential protein obtained by affinity chromatography, and we conducted identification and Mascot analysis on the two differential protein bands of DARTS. After excluded the protein appeared in the solvent control group or with low matching degree, we found 12 kinds of differential proteins totally with high reliability. There was no overlapping of differential proteins found by affinity chromatography and DARTS. According to the investigation of these proteins, we found out 10 kinds of proteins closely related to tumor or correlative signaling pathways, which might be the binding proteins of Rasfonin. Conclusion1. This study established a technical platform for binding protein research of Rasfonin, including traditional technique affinity chromatography and new technique DARTS. Moreover, we optimized the experimental condition for DARTS.2. We found 30 kinds of differential proteins of Rasfonin using affinity chromatography. And 10 kinds of differential proteins were closely associated with tumor or related signaling pathway based on literature.3. We found 12 kinds of differential proteins of Rasfonin using DARTS. And 11 kinds of differential proteins were closely associated with tumor or related signaling pathway based on literature.In summary, this is the first report of binding protein research of Rasfonin, which is a lead compound against tumor, employing traditional affinity chromatography and new technique DARTS. And we found a total of 21 kinds of possible binding proteins of Rasfonin, providing important clues to further determination of its antitumor target. Further validation of these differential proteins is still going on.