Small molecule inhibitors of metabolic flux as potential antitumour agents


Session type:

Jonathan Williams1,2, Adrian Harris2, Angela Russell1,3, Carole Bataille1, Stephen Davies1, Gu Liu1, Andrew Lomas2,3, Anassuya Ramachandran2
1Chemistry Research Laboratory, University of Oxford, Oxford, UK, 2Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK, 3Department of Pharmacology, University of Oxford, Oxford, UK


The up-regulation of glycolysis, even under normoxic conditions, is a hallmark of most primary and metastatic cancers.1 A high aerobic glycolytic capacity (Warburg effect) is correlated with poor prognosis and increased tumour aggressiveness. 6-Phosphofructokinase-1 (PFK-1) catalyses the first irreversible step of glycolysis, namely the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, and is activated by fructose-2,6-bisphosphate, a product of four isoenzymes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB1-4).2 Selective targeting of these enzymes may provide opportunity for the development of antitumour agents.2


Target validation was performed in U87 glioblastoma and MCF-7 breast carcinoma cells, under normoxic (20% O) and hypoxic (0.1% O2) conditions, utilising small interfering RNAs and quantitative real time polymerase chain reaction (qPCR) techniques. The effect of the knockdown of PFK isoenzyme expression on metabolic flux towards glycolysis or the pentose phosphate pathway (PPP) has been investigated using fluorescence-activated cell sorting (FACS), confocal microscopy and metabolite assays. Our group has used in silico screening of a 25,000 member in-house medicinal chemistry database to identify potential small molecule inhibitors of PFKFB3 and PFKFB4, previously reported to be hypoxia-inducible.3,4


PFKFB3 and PFKFB4 were confirmed to be highly induced in both cell lines under hypoxic conditions, in an HIF-1α dependent manner. siRNA knockdown of both isoenzymes markedly reduced cell growth in vitro. Initial findings suggest differing roles for these isoenzymes on lipid metabolism, indicating an effect on the activity of the PPP. Computational modeling has been used to identify potential small molecule inhibitors of these enzymes, from which further analogues have been synthesised. A robust assay has been optimised and validated, to assess their inhibitory activity.


Small molecule inhibitors of PFKFB3 and PFKFB4 offer a potential targeted antitumour therapy. The expression of these isoenzymes is highly induced under hypoxic conditions and markedly affects cell growth. Work to further interpret these results, using mass spectrometry-based metabolomics, is ongoing.