Discovery of fused heterocyclic pyrazine based novel anti-mitotic agents for metastatic melanoma - PROJECT SUMMARY/ABSTRACT. Malignant melanoma is known to be the most aggressive and deadliest form of skin cancer. Prognosis is poor in the advanced metastatic stage with less than 10% rate of survival. Thus, new therapeutic approaches are needed for the treatment of metastatic melanoma. Tubulin inhibitors such as paclitaxel, docetaxel, or nab-paclitaxel are widely used to treat cancers either as single agents or in combination. However, their clinical efficacy is often limited by the development of multidrug resistance, dose limiting hematopoietic toxicity, and cumulative neurotoxicity. Working to overcome these limitations, we have recently discovered a new scaffold of tubulin inhibitors, fused heterocyclic pyrimidine (FHP), that are highly potent against both taxane sensitive and resistant melanoma cell lines in vitro as well as in vivo. High-resolution X-ray crystal structures confirmed that FHPs directly bind at the colchicine site (CBS) in tubulin to destabilize microtubule networks, thereby enabling efficient molecular modeling guided structural optimization based on the existing co-crystal structures. According to our preliminary studies, our central hypothesis is that the novel FHP scaffolds can be further optimized to form the basis for the development of a new generation of tubulin inhibitors targeting the tubulin CBS for the treatment of multidrug resistant metastatic melanoma. Our objective in this application is to use molecular modeling guided synthetic chemistry based on the existing co-crystal structures and cancer biology to optimize the FHP scaffold for enhanced therapeutic potential. The objectives of this application will be accomplished by three specific aims: (1) Develop quinoxaline-based FHPs through rational lead optimization, cell line screening, and mode of action studies. Quinoxaline-based FHPs represent a novel approach to optimizing FHP-based inhibitors. Our preliminary data shows that quinoxaline-based FHPs potently inhibit proliferation of human A375 melanoma cells. The working hypothesis is that the modifications will improve their binding with tubulin, improve metabolic stability, improve water solubility, and reduce potential toxicity. Also, comprehensive screening against diverse sensitive and resistant cell lines will give feedback to the iterative lead optimization. We hypothesize that quinoxaline-based FHP analogues will maintain their (a) mode of action, (b) efficacy against diverse multi drug resistant cancer cell lines, (c) ability to dose dependently inhibit colony formation, and (d) ability to dose dependently introduce vascular disruption. (2) Develop imidazopyrazine-based FHPs through rational lead optimization, cell line screening, and mode of action studies. Our preliminary data shows that imidazopyrazine-based FHPs potently inhibit cancer cell proliferation, inhibit tubulin polymerization, and introduce mitotic arrest. To develop this new series of CBSIs we will perform similar analyses as in Aim 1, to include (A) molecular modeling guided iterative lead optimization, (B) screening against multiple normal and resistant cancer cell lines, and (C) mode of action studies. Our working hypothesis is that these modifications will lead to new imidazopyrazine-based FHPs with enhanced efficacy and improved therapeutic utilities. (3) we will determine MTD, and in vivo efficacy as well as toxicity for selected new FHPs. The working hypothesis is that the new FPH analogues will maintain strong in vivo efficacy with significantly improved therapeutic utilities. This study is innovative because (1) FHP platform represents a validated scaffold that interacts with the CBS of tubulin, (2) FHP scaffold can effectively overcome taxane resistance which is clinically innovative, and (3) FHP scaffold has better drug like properties and lower toxicities than CBS inhibitors that are in clinical trial.
