Angiogenesis is an important hallmark of cancer and a well-established target for anti-tumor treatment. Recent concept of tumor vessel normalisation (TVN) suggests a new approach to affect tumor microenvironment and opens novel therapeutic opportunities. PFKFB3, a rate-limiting enzyme, which can boost glycolysis through PFK-1 by production of its allosteric activator fructose-2,6-bisphosphate (F2,6BP), was shown to play an important role in endothelial cells (EC) metabolism. It was demonstrated that PFKFB3 knockdown resulted in 2-fold reduction of the total sprout length in human umbilical vein endothelial cells (HUVECs) (De Bock K. 2013) as well as inhibition of PFKFB3 in vivo could be beneficial for TVN, reduction of metastasis and promotion of chemotherapy (Cantelmo A.R. 2016).
Enzymatic activity was measured in cell free assay using recombinant full-length human PFKFB3. Relative F2,6BP level was measured in A549 cell extracts. Absolute level of fructose-1,6-bisphosphate (F1,6BP) in A549 cells was estimated by LCMS/MS and F1,6BP with isotopic tag for quantitative analysis. For in vitro studies of angiogenesis we used human EC spheroids. VEGF-A was added after polymerization as a stimulation factor. EC sprouting was quantified by measuring the sprout length cumulatively. In vivo studies are performed on syngeneic and xenograft models. Immunohistochemistry staining is used to describe angiogenesis together with standard tumor assessment.
Using our proprietary molecular modelling tool, we designed a series of novel small molecule PFKFB3 inhibitors to target the F2,6BP binding pocket, rather than occupying the ATP-pocket, which leads to highly selective inhibition and strong reduction of potential off-target binding. Lead seria has IC50 of 1-10 nM range in cell free assay, completely suppresses cellular level of F2,6BP (direct product of PFKFB3) and reduces the cellular level of F1,6BP (product of PFK-1) twice at EC50 < 1 µM. Inhibitors reduced HUVEC sprouting in vitro to basic level with EC50 1-10 µM in a dose-dependent manner. The additional results of tumor angiogenesis in vivo will be presented.
Pharmacological PFKFB3 inhibition using novel small molecule compounds impairs angiogenesis and has potential in anti-tumor treatment.Legal entity responsible for the study
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K. Khodova: Employee and management team member in Gero LLC, T. Eremeeva, D. Shishov: Employee and patent co-author in Gero LLC, P. Fedichev: Co-founder, employee, management team member and patent co-author in Gero LLC