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Discovery of a PFKFB3 inhibitor for phase I trial testing that synergizes with the B-Raf inhibitor vemurafenib


In human cancers, loss of PTEN, stabilization of hypoxia inducible factor-1α, and activation of Ras and AKT converge to increase the activity of a regulator of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). This enzyme synthesizes fructose-2,6-bisphosphate (F2,6BP), which is an activator of 6-phosphofructo-1-kinase, a key step of glycolysis that is tightly controlled by multiple metabolic feedback mechanisms. We recently identified the first competitive small molecule inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), and have now sought to develop a more potent PFKFB3 inhibitor with improved PK properties for testing in clinical trials.

Materials and methods

Methods included recombinant PFKFB3 assays, PK studies using mass spectrometry, pre-clinical toxicity and efficacy studies, Western blot analyses for HIF-1α and PFKFB3 in A375 melanoma cells, F2,6BP assessments and flow cytometry for apoptosis.


We report the discovery of a novel 3PO-derivative, PFK158, that is more potent than 3PO, has improved PK properties and causes ~80% growth inhibition in several mouse models of human-derived tumors. We also demonstrate that PFK158 is well tolerated in rats and dogs providing key support for a phase 1 trial in cancer patients that will initiate in 2014. Once the MTD is established, we intend to conduct multiple phase 1/2 trials of PFK158 in combination with targeted agents given the ability of PFK158 to suppress glycolysis. 50% of melanomas harbor a BRAFV600E mutation that promotes glucose metabolism, survival and proliferation and BRAFV600E inhibitors are effective in ~50% of melanoma patients. Unfortunately, resistance to these agents develops within six months and most patients die within two years of diagnosis. Genetic amplifications of BRAFV600E are a common cause of resistance, and BRAFV600E stabilizes HIF-1α, an established promoter of PFKFB3. We thus hypothesized that PFKFB3 may be essential for intrinsic resistance to BRAFV600E inhibitors. In unpublished results, we demonstrate that 3 hours of exposure to 1 μM vemurafenib reduces HIF-1α, PFKFB3, and F2,6BP in BRAFV600E positive A375 melanoma cells and that PFK158 markedly sensitizes these cells to the apoptotic effects of vemurafenib.


In conclusion, PFK158 is the first PFKFB3 inhibitor to be examined in a phase I trial and may have significant clinical utility when combined with agents that target driver oncogenes. Importantly, our data provide rationale for the conduct of pre-clinical studies of PFK158 combined with vemurafenib in transgenic BRAFV600E melanoma mouse models which are in turn expected to justify a phase 1/2 trial of the combination in BRAFV600E-positive melanoma patients.

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Telang, S., O’Neal, J., Tapolsky, G. et al. Discovery of a PFKFB3 inhibitor for phase I trial testing that synergizes with the B-Raf inhibitor vemurafenib. Cancer Metab 2 (Suppl 1), P14 (2014).

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