Cell-Permeating α-Ketoglutarate Derivatives Alleviate Pseudohypoxia in Succinate Dehydrogenase-Deficient Cells

Abstract

Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are components of the tricarboxylic acid (TCA) cycle and tumor suppressors. Loss of SDH or FH induces pseudohypoxia, a major tumor-supporting event, which is the activation of hypoxia-inducible factor (HIF) under normoxia. In SDH-or FHdeficient cells, HIF activation is due to HIF1␣ stabilization by succinate or fumarate, respectively, either of which, when in excess, inhibits HIF␣ prolyl hydroxylase (PHD). To reactivate PHD, we focused on its substrate, ␣-ketoglutarate. We designed and synthesized cell-permeating ␣-ketoglutarate derivatives, which build up rapidly and preferentially in cells with a dysfunctional TCA cycle. This study shows that succinate-or fumarate-mediated inhibition of PHD is competitive and is reversed by pharmacologically elevating intracellular ␣-ketoglutarate. Introduction of ␣-ketoglutarate derivatives restores normal PHD activity and HIF1␣ levels to SDH-suppressed cells, indicating new therapy possibilities for the cancers associated with TCA cycle dysfunction.Under normal oxygenation conditions (normoxia), hypoxiainducible factor alpha (HIF␣) proteins (HIF1␣ and/or HIF2␣), which are the formation-limiting units of the HIF transcription factor heteromer, are constantly both synthesized and degraded, a process that maintains them in high availability but at a very low steady state (4, 25). The high turnover of HIF␣ is mediated by HIF␣ prolyl hydroxylases (PHD1 to -3, also known as EglN1 to -3 and HPH1 to -3). PHDs hydroxylate proline residues on the oxygen-dependent degradation (ODD) domain of HIF␣, generating docking sites for pVHL-part of an E3 ubiquitin ligase complex that targets HIF␣ for degradation (21,22). To catalyze proline hydroxylation, PHDs convert molecular oxygen and ␣-ketoglutarate to carbon dioxide and succinate (22). Although PHDs depend on oxygen for activity, their affinity for oxygen is low (K m ϭ 230 to 250 M), making them good oxygen sensors (11). Under hypoxia, prolyl hydroxylation of HIF␣ and its consequent interaction with pVHL are prevented, and HIF␣ proteins are stabilized (21). Stabilized HIF␣ units bind to the HIF␤ unit, setting off HIF transcription activity. Thus, HIF is physiologically activated by hypoxia, and its downstream targets respond accordingly by increasing angiogenesis and glycolysis.The aberrant stabilization of HIF␣ under normoxic conditions is termed pseudohypoxia. Pseudohypoxia is probably a major cause of tumors associated with VHL mutations (15). Pseudohypoxia was also shown recently in tumor cells with mutations in fumarate hydratase (FH) or in any of three of the four subunits of succinate dehydrogenase (SDH), SDHB, SDHC, or SDHD (5,8,9,19,20,28). Both FH and SDH are mitochondrial proteins; SDH forms complex II of the electron transport chain, and both SDH and FH are enzymes of the tricarboxylic acid (TCA) cycle. In addition, SDH and FH are also bona fide tumor suppressors. Dysfunction of either of these TCA cycle enzymes causes pseudohypoxia, leading to the enhanced neovasculari...

Novel two-step derivation method for the synchronous analysis of inherited metabolic disorders using urine

“…These two biomarkers have also been reported in previous studies (56,57); however, they cannot be verified as biomarkers for mitochondrial metabolic disorders in the present study, due to the limited number of cases (n=31). Therefore, they require further investigation.…”

Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death

“…Derivatized a-ketoglutarate destabilizes HIF1a and increases VHL binding under hypoxia We have previously reported that cell-permeating a-ketoglutarate derivatives efficiently reversed the pseudohypoxic phenotype present in SDH-deficient cells by reactivating PHDs (MacKenzie et al, 2007). Therefore we investigated the possibility that increasing intracellular levels of a-ketoglutarate would similarly reactivate HIF PHDs in hypoxic cells.…”

“…HCT116 cells were transfected and selected to stably express a previously described fusion protein containing the ODD domain of HIF1a fused to green fluorescent protein (GFP) (GFP-ODD; MacKenzie et al, 2007), herein called HCT116c18. As expected, PHD activity (which inversely correlated with GFP levels) was high in normoxia and decreased under hypoxia (Figure 1c, panels 1 and 2).…”

“…Synthesis of a-ketoglutarate derivatives 2-Oxopentanedioic acid 4-(1-trifluoromethylbenzyl) ester (TaKG) was synthesized as previously described (MacKenzie et al, 2007). 2-Oxopentanedioic acid 5-ethyl,4-(1-trifluoromethylbenzyl) ester (ETaKG) was synthesized using the same techniques by Domainex (Cambridge, UK).…”

“…We reported that cell-permeating a-ketoglutarate derivatives reversed succinate-mediated HIF stabilization by reactivating the PHDs in normoxic conditions (MacKenzie et al, 2007). These studies suggested that it may be possible to reverse the inhibition of the PHDs by low oxygen, resulting in low HIF expression in hypoxic conditions.…”