In this report, we have demonstrated a unique approach to cancer treatment by inhibiting FAK and VEGFR-3 through targeting the site of their protein-protein interaction. This study has demonstrated that we can inhibit the function of these tyrosine kinases by targeting their binding site. Moreover, our computational approach for molecular docking has identified a small molecule that not only decreased activity of both VEGFR-3 and FAK, but had anti-tumor effects that were synergistic with chemotherapy in vivo.
We selected the VEGFR-3 binding site on the FAT domain of FAK as a template for our in silico
studies because of the importance of both of these kinases in cancer cell survival and tumor progression. We virtually docked potential small molecules and identified compound 1
(Chloropyramine hydrochloride). It was functionally equivalent to the FAK-inhibiting peptide from the VEGFR-3 7
, decreased cell proliferation and caused apoptosis in breast cancer cells. To prove that this small molecule affects interaction of VEGFR-3 with FAK, we analyzed FAK-VEGFR-3 co-localization and co-precipitation in immunohistochemical and biochemical experiments. We have shown that treatment with 1
decreased co-localization and FAK-VEGFR-3 complex formation. Thus, in silico
modeling demonstrated that peptide binding sites of FAK are appropriate targets for non-peptide small drug-like molecule binding.
Studies with peptide inhibitors already have indicated that blockade of specific protein–protein interactions have therapeutic promise for treating a variety of human cancers 35–37
. The major advantage of protein-protein inhibitors is their high selectivity. For example, the nutlins inhibitors of the p53-MDM2 interaction activated apoptosis in cells expressing wild-type p53 and showed a 10–20 fold selectivity for cells with active versus mutated p53 38
. In the present study, targeting the site of FAK-VEGFR-3 protein-protein interaction represents a novel approach to targeting tyrosine kinases that can potentially be used to disrupt their “interactome” and inhibit specific downstream signaling. Until now, the main approach to target FAK was to inhibit the catalytic activity of the tyrosine kinase by interfering with the binding of ATP. Three such inhibitors have been reported by Novartis 22
and Pfizer 23,24
. All of them inhibit FAK kinase activity, but have varying degrees of crossreactivity with other tyrosine kinases 39
. Similarly, the only known inhibitor for VEGFR-3 is MAZ-51, which suppressed mammary tumor growth in rats 40
, but not having a broad clinical utility. Clinically, broad range tyrosine kinase inhibitors are being used to target the VEGFR family in addition to other receptor tyrosine kinases with varying degrees of success 41
. In this study, we have shown the specificity of 1
for FAK and VEGFR-3 whereby it changed the phosphorylation and activation status of VEGFR-3 and FAK by disrupting their interaction and did not have a demonstrable effect on the activity of other receptor and non-receptor protein kinases.
The small molecule 1
that targeted the FAK-VEGFR-3 binding site was chloropyramine hydrochloride, belonging to the class of antagonists of histamine receptor H1. This small molecule was analyzed in mouse ascites tumor experiments of Honti and Puntoky over 40 years ago 42
when the hypothesis that histamine might be involved in carcinogenesis was proposed 43
, but the results were inconclusive. In our experiments, we have shown a unique biological specificity of this drug for the FAK-VEGFR-3 interaction. In our breast cancer xenograft models, we have shown that treatment with 1
reduced tumor burden more than 80%, and this effect was not related to its antihistamine properties when compared to the histamine blocker 2
One of the most significant aspects of our findings relates to the ability of 1
to sensitize breast cancer cells to chemotherapy. When 1
was administered with the standard chemotherapeutic for breast cancer, doxorubicin, we saw a pronounced synergistic effect, and this effect was still significant when we reduced the dose of both drugs. Because FAK is a survival signal, and has been directly implicated in chemoresistance 44
, we hypothesize that the decreased phosphorylation of FAK and VEGFR-3 caused by 1
results in a greater sensitivity of the cancer cells to chemotherapy.
In summary, our data suggest that the FAK-VEGFR-3 protein-protein interaction is an excellent site to develop small molecule inhibitors to provide the basis for highly specific novel cancer therapeutic agents. By targeting this interaction, survival signaling in the tumors can be interrupted, and this may provide a useful method of augmenting the effects of chemotherapy in breast cancer.