Study On The Interaction Of Tyrosine Kinase With Small Molecules Using SMMs

Receptor tyrosine kinase is one of the important modulators which involved in various normal cellular processes including embryo growth, development, metabolism and cell apoptosis. Dysregulation of the metabolism of tyrosine kinase in normal cells, genes mutation, or overexpression can induce tumor, inflammation, as well as diabetes. Recently, receptor tyrosine kinases have become attractive therapy targets for many diseases especially cancer therapy. Kinase c-Met belongs to one of the subfamily of receptor tyrosine kinases. Recent study showed that c-Met overexpression occurs in lung, breast, colon, prostate and other cancers and it gradually becomes an important drug target, particularly as anti-cancer drug target.The small molecule microarrays(SMMs) is a library of compounds that is immobilized on a two-dimensional surface, such as a glass slide, and is spatially addressable and miniaturized. SMMs have recently been used to identify ligands for several proteins, explore the interactions of macromolecule and their ligands and study enzyme function.The aim of this paper is to establish a primary method using SMMs technique to study the interactions of tyrosine kinase c-Met with small molecules, and focus on exploring the validation of the probability of this method.The main work of this paper and results are as follows:a. Small molecules T9b-T9f, designed by crystal information of complex SU11274 with c-Met, that methy piperazine group of SU11274 did not participate in the binding with c-Met kinase domain, were synthesized. Their structures were verified by 1H-NMR and ESI-MS.b. The inhibition activities of T9b-T9f for c-Met tyrosine kinase were evaluated by biochemical methods. The results showed that, for tissue source receptor tyrosine kinases, the inhibition activites of T9d and T9e were 79% and 68% respectively, both higher than positive compound SU11274. The tyrosine kinase inhibition activities from high to low were T9d, T9e, T9b, SU11274 and T9c. For recombinant source tyrosine kinase, the inhibition activites of all these five compounds were lower than SU11274. The results of these two evaluation methods were basicly consistent, and molecules T9b, T9d, T9e and T9f, whose inhibiton activities for other tyrosine kinase beside c-Met may exist, were non-specific kinase inhibitors, however, SU11274 is the specific c-Met tyrosine kinase inhibitor.c. Small molecules T9b-T9f were immobilized on the isocyanated glass slide. Using SMMs to evaluate the interaction of c-Met and small molecules T9b-T9f, the results showed that for any molecule fluorescence intensity was dose dependent and even in the same concentration, the corresponding fluorescence intensity of different molecules differed. When all the five molecules concentration is 100 mM, the fluorescence intensity of molecules from high to low is T9d, T9e, T9f, T9c and T9b. When in 50 mM, the fluorescence intensity of molecules from high to low is T9f, T9d, T9e, T9c and T9b. The KD for c-Met kinase of T9d, T9e and T9f are 0.88, 3.699 and 1.333 respectively. Compared with the biochemical evaluation results, we can see that the binding activity of molecule T9f with c-Met increased related to molecules T9b, T9c, T9d and T9e, and was only lower than molecule T9d, this probably because of the spatially expanding of T9f structure on the glass surface which is advantageous to form conformation for kinase binding. The binding activity of T9d and T9e are consistent with the results of biochemical evaluation, however, T9b is relatively decreased probably because of limitation of the spatial spread on glass surface. We can conclude that molecule T9d could be used as positive probe for further SMMs screening research. Combined with the docking results that the binding mode of T9f with c-Met kinase on the glass surface is similar with the binding mode of SU111274 with c-Met in the free surrounding, we could attempt to add a six-carbon linker to the current isocyanate surface for the perfection of SMMs technique method and eliminate the spatial limitation of compound immobilization.d. The molecular docking study was done by AUTODOCK Vina. And the docking results exhibited that no matter in free surrounding or immobilized on the glass surface, the binding mode of each molecule was the same, which can be a good validation for the probability of using SMMs to study the interaction of tyrosine kinase and small molecules.