Fig 2: Development of a PFKFB kinase activity assay and screening of potential modulators. (A) Schematic of the coupled enzyme activity assay protocol. Purple indicates the reaction mix containing purified PFKFB isoforms. Red indicates enzymes added for the coupled assay. The ultimate product is glycerol-3-phosphate and the assay measures the disappearance of NADH at 340nm. (B) Plate-reader screening of candidate compounds (all at 10 μM in DMSO; wells A3 and E10 (red boxes) were DMSO controls). (C) IC50 determination of candidate inhibitor B2 (structure on inset) for PFKFB2 (top) and PFKFB3 (lower) kinase activities. (D) IC50 determination of known PFKFB3 kinase inhibitor PFK158 (structure on inset) for PFKFB2 (top) and PFKFB3 (lower) kinase activities.

Fig 3: Recombinant PFKFB2 is phosphorylated in vitro. (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining proteins bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria (left lanes), lose phosphorylation after purification (middle lanes), but are re-phosphorylated upon treatment with PKA and ATP (right lanes). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with λ-phosphatase for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in Supplemental Figure 2.

Fig 4: Recombinant PFKFB2 is phosphorylated in vitro.(A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria (left lanes), lose phosphorylation after purification (middle lanes), but are re-phosphorylated upon treatment with PKA and ATP (right lanes). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with λ-phosphatase for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in S2 Fig.

Fig 5: Development of a PFKFB kinase activity assay and screening of potential modulators.(A) Schematic of the coupled enzyme activity assay protocol. Purple indicates the reaction mix containing purified PFKFB isoforms. Red indicates enzymes added for the coupled assay. The ultimate product is glycerol-3-phosphate and the assay measures the disappearance of NADH at 340nm. (B) Plate-reader screening of candidate compounds (all at 10 μM in DMSO; wells A3 and E10 (red boxes) were DMSO controls). (C) IC50 determination of candidate inhibitor B2 (structure on inset) for PFKFB2 (top) and PFKFB3 (lower) kinase activities. (D) IC50 determination of known PFKFB3 kinase inhibitor PFK158 (structure on inset) for PFKFB2 (top) and PFKFB3 (lower) kinase activities.