TEPP-46 (ML265)

TEPP-46 (ML265) potently activates PKM2 in vitro with an AC50 = 92 nM and shows a high degree of selectivity over the other 3 pyruvate kinase isoforms. It binds at the dimer-dimer interface of the PKM2 homotetramer and is capable of activating PKM2 in cell lysate of pervanadate treated cells, which is a condition known to inhibit PKM2 activity through accumulation of phosphotyrosine peptides. This compound significantly increased the doubling time of H1299 cells under hypoxic conditions, but interestingly showed no effect under normoxia[1].

In a 7-week mouse xenograft model (H1299 mouse xenograft), activation of PKM2 with TEPP-46 (ML265) significantly reduced tumour size and occurrence without signs of acute toxicity. This compound also gave superior plasma concentrations that persisted at higher levels over the 24 hour study, and displayed good oral bioavailability, low clearance, a long half-life and good volume of distribution. [1].

• PKM2 activity assay

  Pyruvate kinase activity is measured by monitoring pyruvate-dependent conversion of NADH to NAD+ by lactate dehydrogenase (LDH). Briefly, for cell line experiments, the medium is replaced with fresh medium 1 hr prior to the start of treatment with DMSO or TEPP-46 (ML265). Also, where indicated, 100 μM pervanadate is added 10 min prior to cell lysis. Cells are lysed on ice with RIPA buffer containing 2 mM DTT and protease inhibitors and clarified by centrifugation at 21,000 × g. 5 μL of the supernatant is used to assess pyruvate kinase activity. Pyruvate kinase activity was subsequently normalised for total protein content.

• Cell Lines

  H1299 cells

• Concentrations

  50 μM

• Incubation Time

  1 h

• Method

  H1299 cells incubated with 50 μM TEPP-46 (ML265) for 1 hour, lysed followed by 2-D electrophoresis, Western blot and PGAM1 antibody staining.

• Animal Models

  Male Balb/c mice

• Dosages

  IV: 1 mg/kg, PO: 10 mg/kg; IP: 10 and 50 mg/kg

• Administration

  IV, IP and PO