In search of human cytochrome P450 1A1 enzyme inhibitor
Human cytochrome P450 1 (CYP1) family consists of three members; they are CYP1A1, CYP1A2 and CYP1B1. The mRNAs of CYP1A1 and CYP1B1 genes are known to be expressed in extra-hepatic tissues while that of the CYP1A2 gene is found in the liver amongst other tissues. Under normal circumstances, rarely has CYP1A1 protein expression been seen in human tissues. All three CYP1A enzymes have been widely studied for different reasons. The CYP1A1 enzyme is known to be induced by xenobiotics and poly-aromatic hydrocarbons (PAHs). Induction of CYP1A1 results in its involvement in Phase 1 biotransformation of xenobiotics and in the metabolism of pro-carcinogens, such as PAHs, to carcinogenic substances that eventually lead to cancer. Natural products such as flavonoids, chalcones and stilbenes have attracted a great deal of attention since these compounds have an ability to modulate the activity of CYPs by inhibiting CYP-specific enzymatic activity. Several compounds have been found which potently inhibit CYP1 family of enzymes. They are resveratrol, rhaphontigenin, isorhaphontegenin, α-napthoflavone, tetramethoxystilbene. Some of these compounds are available over the counter in pharmacies in the UK, mainland European countries and the US with proclamation of health benefits. Until now, no potent CYP1A1-specific inhibitor has been identified. Compounds that inhibit CYP1A1 can potentially act as chemo-preventive agents in the treatment of cancer. In this thesis, at first, a yeast strain that co-expresses CYP1A1 with a genetically engineered variant of human P450 reductase (ΔhRDM) was successfully grown; microsomes containing active CYP1A1 enzyme were prepared. A 200-compound library (available at DMU’s School of Pharmacy) consisting of chemically synthesised compounds belonging to chalcone flavonoid and stilbene classes were screened on the CYP1A1 microsomal enzyme to find a potent inhibitor of CYP1A1; later, CYP1A2 and CYP1B1 microsomal enzymes were used to determine specificity towards CYP1A1. Various inhibitors were identified with differing degrees of potency and specificity. The lead inhibitory molecules obtained from the initial in vitro screen were assessed in live yeast cells that expressed active CYP1A1 enzyme. The results interestingly indicated that there is a strict correlation between IC50s obtained in microsomal and cellular assays. We were curious to know whether the activation of a CYP by the novel reductase ΔhRDM created an active site geometry which is identical to the active sites formed by coupling of the wild-type human and yeast P450 reductases (hRD and yRD). Since currently there is no way of determining the active site of a membrane-bound protein complex using X-ray crystallography or 2-D NMR, we decided to use chemical tools (i.e. inhibitors of the CYP1 enzymes) to find out if the IC50s of inhibitory molecules could be used instead as a way of determining active site geometry. It was quite fascinating to see that the IC50 of a compound with a CYP activated by ΔhRDM is more or less the same as a CYP activated by a wild-type reductase (yRD or a hRD) indicating that the active site geometries of a CYP are the same irrespective of the coupled reductase. DMU 2157 (a heterocyclic chalcone) and DMU 110 (a flavonoid) were found to be potent inhibitors of CYP1A1 enzyme (IC50, 72nM and 397nM, respectively). Structural analysis of DMU 2157 suggested that the positions of the methoxy groups in ring ‘A’ and the position of the <N in the pyrido ‘B’ ring play important roles in facilitating inhibition of CYP1A1 enzyme. Therefore DMU 2157 analogues, modelled on ‘chalcone’ and ‘stilbene’ scaffolds, were chemically synthesized to better understand the structural features of DMU 2157 that contribute to specific inhibition of CYP1A1 and with the aim that more potent inhibitors could ultimately be designed. Intriguingly, besides obtaining CYP1A1-specific inhibitors, these chemical studies have led to compounds that are also exquisitely specific to CYP1B1 and CYP1A2. In conclusion, the body of data generated in this thesis has created the basic foundations for further studies that could eventually lead to entities with strong anticancer potential.
- PhD