Bendiabdellah, Yassine; (2010) Discovery of novel natural product based HIF-1α:HIF-1β inhibitors as potential anticancer agents. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Hypoxia-inducible factor 1 (HIF-1), a heterodimeric protein comprising of the HIF-1α and β sub-units, is a key regulator of angiogenic and glucose metabolism processes utilised by hypoxic tumour cells for survival and growth. The inhibition of heterodimeric protein-protein interactions (PPIs) such as the HIF-1α/HIF-1 interaction is a viable approach for novel chemotherapeutic strategies. Using an ELISA-based screen, rolitetracycline was originally identified by the Melillo laboratory (NCI) as a selective inhibitor of the HIF-1α:HIF-1β interaction, although this particular lead molecule has very poor cellular penetration properties. The main goal of this project was to synthesise libraries of rolitetracycline mimetics to explore SAR, and to try to obtain more-potent and more-selective analogues that might be progressed into pre-clinical studies. The design of the first focused library of tetracyclic mimetics involved retention of the core molecular framework and spatial conformation of rolitetracycline, while changing perimeter functional groups. These molecules were synthesized by employing consecutive Diels-Alder cycloadditions and Indium-mediated chemistry. An alternative synthetic approach to other libraries involved asymmetric Diels-Alder cycloadditions employing various chiral catalysts to prepare enantiomerically pure tetracyclic mimetics. BINOL-ate catalysts were found to be the most effective, and a series of novel polyketide heterocyclic frameworks were prepared using highly regio- and diastereoselective one-pot reactions via a domino process to produce tetracyclic mimetics containing oxygen and nitrogen heterocycles. A selection (40 molecules) of the total (54 molecules) synthesized during this project were screened in a luciferase-based plasmid reporter assay using U251-HRE cells that contained a HIF Response Element (HRE) and in U251-pGL3 control cells that lacked a HRE. This identified seven "hit" molecules and distinct structure activity relationships (SARs). The two best "hits", YB036 and YB039, selectively inhibited HIF-1 dependent luciferase expression with EC50 values of 1.75 μM and 1.45 μM, respectively. These molecules were progressed into a screening cascade which showed that they selectively reduced the level of HIF-1 protein in cells while not affecting the levels of constitutively expressed HIF-1. They also selectively inhibited down-stream genes in the HIF-1 signalling pathway such as VEGF. Crucially, YB036 and YB039 had good cellular penetration characteristics compared to rolitetracycline. Although a suitable candidate for progression into pre-clinical studies was not identified during the course of this project, these studies have laid the foundations for further studies in the future that might achieve this goal.

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