We report the analysis of CPI-613, the first member of a large set of analogs of lipoic acid (lipoate) we have investigated as potential anticancer agents. CPI-613 strongly disrupts mitochondrial metabolism, with selectivity for tumor cells in culture. This mitochondrial disruption includes activation of the well-characterized, lipoate-responsive regulatory phosphorylation of the E1α pyruvate dehydrogenase (PDH) subunit. This phosphorylation inactivates flux of glycolysis-derived carbon through this enzyme complex and implicates the PDH regulatory kinases (PDKs) as a possible drug target. Supporting this hypothesis, RNAi knockdown of the PDK protein levels substantially attenuates CPI-613 cancer cell killing. In both cell culture and in vivo tumor environments, the observed strong mitochondrial metabolic disruption is expected to significantly compromise cell survival. Consistent with this prediction, CPI-613 disruption of tumor mitochondrial metabolism is followed by efficient commitment to cell death by multiple, apparently redundant pathways, including apoptosis, in all tested cancer cell lines. Further, CPI-613 shows strong antitumor activity in vivo against human non-small cell lung and pancreatic cancers in xenograft models with low side-effect toxicity.

In platelets, agonists that stimulate phosphoinositide turnover cause the rapid phosphorylation of a protein of apparent relative molecular mass (Mr) 40-47,000, called P47, by protein kinase C (PKC). Diverse identities have been ascribed to P47 including lipocortin, inositol 1,4,5-trisphosphate 5-phosphomonoesterase, pyruvate dehydrogenase alpha subunit and an actin regulatory protein. We have isolated human P47 clones by immunological screening of a lambda gt11 complementary DNA library from HL-60 cells, a human promyelocytic leukaemia cell line. P47 recombinants thus identified hybridized to a 3.0 kilobase (kb) messenger RNA in mature white blood cell lines; the same mRNA was induced in HL-60 cells during differentiation. A 1,050 base pair (bp) open reading frame that could encode a protein of Mr40,087 was confirmed by comparison with peptide sequences from platelet P47, and by expression of the putative recombinant P47 in E. coli and in vitro. The P47 sequence appears to have been conserved throughout vertebrate evolution, and is not similar to any other known sequence including human lipocortin and the alpha subunit of pyruvate dehydrogenase. The P47 protein contains a potential Ca2+-binding 'EF-hand' structure and a region that strongly resembles known PKC phosphorylation sites.

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Preincubation of turkey erythrocytes with catecholamines desensitizes the fi-adrenergic receptor-adenylate cyclase complex in the plasma membranes of these cells. Photoaffinity labeling of the (3-adrenergic receptors with "SI-labeled pazidobenzylcarazolol (l25I-pABC) and subsequent analysis by NaDodSO4/polyacrylamide gel electrophoresis demonstrates an altered mobility of receptor peptides from desensitized cells compared to controls [Stadel, J. M., Nambi, P., Lavin, T. N., Heald, S. L., Caron, M. G. & Lefkowitz, R. J. (1982)J. BioL Chem. 257,[9242][9243][9244][9245]. The time course of alteration in 3-adrenergic receptor mobility correlates with that for desensitization of isoproterenol-stimulated adenylate cyclase activity. The altered mobility of the receptor peptides from desensitized cells is also observed if the receptors are first purified and then photoaffinity labeled with '25I-pABC. The cyclic nucleotide analog 8-bromoadenosine 3',5'-cyclic monophosphate partially mimics catecholamines in promoting desensitization of the adenylate cyclase and modification of the receptor. Phosphorylation of the fi-adrenergic receptor in intact turkey erythrocytes was assessed by preincubating the cells with [3P]orthophosphate, desensitizing them with catecholamine, purifying the receptors, and then subjecting them to NaDodSO4/ polyacrylamide gel electrophoresis. Desensitization is associated with a 2-to 3-fold increase in 32p incorporation into the receptor, which also demonstrates the characteristic alterations in mobility.These effects are blocked by the 13-adrenergic antagonist propranolol. Purified turkey erythrocyte 13-adrenergic receptors could be phosphorylated by incubation with [y-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase. The mobility of the phosphorylated receptor peptides on NaDodSO4/polyacrylamide gel electrophoresis appears to correspond to that of the desensitized receptors. These data show that catecholamine-induced desensitization of adenylate cyclase in turkey erythrocytes correlates with a stable modification of the 13-adrenergic receptor and is associated with agonist-promoted phosphorylation of ,ireceptor peptides.Prolonged exposure of target tissues to hormones or neurotransmitters attenuates the responsiveness of the tissue to subsequent challenge by these agents. This regulatory process termed "desensitization" or "refractoriness" has been studied in a wide variety of cell types (1-7). Investigations of adenylate cyclase-coupled ,B-adrenergic receptor systems indicate the existence of multiple mechanisms for desensitization (5,8,9).However, the molecular events that underlie the processes of desensitization remain to be elucidated. Recently we reported that catecholamine-induced desensitization of adenylate cyclase in turkey erythrocytes promotes a modification of the structure of /&adrenergic receptor peptides that results in decreased mobility of these peptides on NaDodSO4/polyacrylamide gel electrophoresis compared to controls (10). ,B-Adrenergic receptor pep...