Session: Current Topics in Biological Chemistry:

The goal of this research is to characterize the human cytochrome P450 enzyme 4X1 (CYP4X1). The expression pattern of rat CYP4X1 suggests that this protein may be involved in the neurovascular function in the brain. One study also indicates that substrate binding site inhibition with a chalcone (CH625) led to a reduction in tumor angiogenesis. Thus, our foremost plan is to investigate the binding affinity of reported and likely substrates and developing a selective inhibitor based on the structure of CH625. We have CYP4X1 protein purified using NINTA (nickel) beads affinity column chromatography made possible by a 6-residue histidine tag. The purified protein was characterized using UV/VIS absorption spectroscopy. Namely, binding affinity and carbon monoxide difference assays. Binding affinity assays were obtained for the two previously published substrates, arachidonic acid, and anandamide. Additionally, initial steps towards specific CYP4X1 inhibitor design were accomplished by synthesizing different chalcones analogous to CH625 via a solvent-free method that gives a better yield than using ethanol. Synthesized compounds were characterized using Thin layer chromatography and NMR spectroscopy. We are currently working on determining the binding affinity with purified CYP4X1 enzyme for each of these compounds plus additional potential ligands like Eicosapentaenoic acid, Docosahexaenoic, Oleic acid, and Linoleic acid. This will enable us to identify features that are important for a good inhibitor.

Mehwish Khokhar

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