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CYP24A1 (25-hydroxyvitamin D-24-hydroxylase) is a useful enzyme target for a range of medical conditions including cancer, cardiovascular and autoimmune disease, which show elevated CYP24A1 levels and corresponding reduction of calcitriol (the biologically active form of vitamin D). Calcitriol has antiproliferative and pro-differentiating properties, however use of calcitriol as a therapeutic drug is limited by hypercalcaemia. An alternative approach is the use of CYP24A1 inhibitors to prevent the metabolism of calcitriol. The aim of this research is to design and synthesise novel inhibitors of CYP24A1 to enhance the endogenous levels of circulating calcitriol. Furthermore, it is important to develop compounds that are selective for CYP24A1 over CYP27B1 so that the generation of calcitriol itself is not blocked. In order to understand the requirements of inhibitor binding to the enzyme-active site, it would be useful to have a 3D structure of both human CYP24A1 and CYP27B1. However, to date, no human crystal structures are available for either of these enzymes. Therefore, a homology model for CYP24A1 has been developed and published. A CYP27B1 homology model was developed, using a combination of homology modelling, molecular dynamics simulations, and molecular docking to understand the satisfactory explanation of the binding selectivity of the CYP27B1 model with the natural substrate and with selective inhibitor complexes. Docking results for CYP27B1 showed amino acids Arg107, Asn387 and Asp320 have an important role in binding interactions to form hydrogen bonds with inhibitors. The development of potent and selective inhibitors from three azole series was investigated. Development of series one using pyridine, imidazole and triazole as the haem binding group was synthesised successfully. The compounds exhibited weak potency and IC50 ranging between 10.2 to 28.4 μM against CYP24A1. Owing to the low CYP24A1 inhibitory activity the compounds were not evaluated against CYP27B1. The series two bis(3-methyl-1-phenyl-1H-pyrazol-5ol) was synthesised successfully. Two compounds were the moderate CYP24A1 inhibitors and so were further evaluated against CYP27B1. However, these compounds showed enzymatic inhibition (IC50 = 0.57 μM and 0.41 μM) against CYP27B1, that is they were more selective for CYP27B1 which could be rationalised from docking experiments. A series of (E)-N-(2-(1H-imidazol-1- yl)-2-(phenylethyl)-3/4-styrylbenzamides have been synthesised using an efficient synthetic route and shown to be potent inhibitors of CYP24A1 (IC50 0.11 - 0.35 μM) compared with the standard ketoconazole. Molecular modelling using our CYP24A1 homology model showed the inhibitors to fill the hydrophobic binding site, forming key transition metal interaction between the imidazole nitrogen and the haem Fe3+ and multiple interactions with the active site amino acid residues. |
