**8. Conclusions**

54 Dehydrogenases

These properties suggested that thioctoids might be promising anti-cancer agents. This possibility is further supported by their behavior in human tumor xenograft mouse models. Specifically, CPI-613 produces strong tumor growth inhibition in several tumor types. For example, the growth of BxPC-3 human pancreatic tumor xenografts implanted subcutaneously on posterior flanks is largely or entirely suppressed by intraperitoneal dosing with CPI-613 at 25-75mg/kg. Moreover, ca. 40% of these animals survive for more than 9 months (termination of study) without regrowth of tumors after an initial three week treatment regime. [A large fraction of the 60% mortality in this study is attributable to normal attrition in these genetically immunocompromised *nude* mice. Thus, cancer-free

post-treatment survival in these animals is apparently substantially higher than 40%.]

613 breakdown products are unlikely to pose a barrier to clinical development [70].

**7. The central role of lipoate as a metabolic regulatory signal, other** 

In view of the capacity of thioctoids to attack tumor cell PDH it may be of value to consider the potential of these agents to address tumor metabolism more generally. Indeed, the properties of lipoate suggest that it may act as a global mitochondrial regulatory signaling molecule, possibly addressing the entire flow of carbon through this compartment. On this view, lipoate analogs may address multiple regulatory pathways, some or all altered in

Specifically, two other enzyme complexes are recognizably homologous to PDH and catalyze analogous reactions. One of these is the branched chain oxoacid dehydrogenase complex (BCDH) as reviewed in [74]. The other is the KGDH complex (above). BCDH governs the entry of carbon from a series of amino acids into the TCA cycle. KGDH governs the entry of glutamine-derived carbon (Figure 1). Together with PDH control of carbohydrate derived carbon, these three enzymes directly control initial access of all carbon to the TCA cycle, except for that derived from fatty acids. Moreover, even fatty acid-derived

investigation of these investigational drugs.

**potential thioctoid tumor targets** 

tumor cells analogously to the well-understood PDH case.

Titration studies demonstrate that CPI-613 produces very substantial tumor growth inhibition in xenograft model systems at doses (0.1-1mg/kg) very substantially lower than the maximum tolerated dose (ca. 100mg/kg)[2; our unpublished results]. This indicates a very high possible therapeutic index. Large-animal toxicology studies further corroborate the very low toxicity of CPI-613 [69]. Moreover, drug metabolism studies indicate that CPI-

Based on this favorable mechanistic and pre-clinical animal data, Phase I/II clinical trials are currently underway at several locations (see comments at http://clinicaltrials.gov/ ct2/show/NCT01520805). These studies indicate that adverse events of CPI-613 in humans are low and mostly mild (well tolerated to at least 3,000mg/m2, when infused over one hour) and suggest efficacy against several advanced cancers, including refractory/relapsed AML, in several patients [71-73]. It will be of interest to continue to pursue the clinical

> The central role of lipoate-dependent dehydrogenases in governing carbon flow through mammalian mitochondria is reflected in their extensive regulation. Moreover, both empirical evidence and theoretical considerations indicate that the regulation of these

complexes is strongly controlled by the dynamic status of their lipoate residues, reflecting a moment-to-moment polling of the mitochondrial matrix. Finally, evidence and theory also indicate that some or most of these lipoate-dependent regulatory processes are very significantly altered in support of the substantial repurposing of tumor cell mitochondrial metabolism relative to its normal cell condition.

The Pyruvate Dehydrogenase Complex in Cancer:

Implications for the Transformed State and Cancer Chemotherapy 57

[4] Nealson, K H, Conrad, P G (1999) Life: Past, Present and Future Philos trans r. soc Lond

[5] Raymond, J, Segre, D (2006) The Effect of Oxygen on Biochemical Networks and the

[6] Garrett, R, Grisham, C M (2010) Biochemistry (4th ed) Belmont, CA: Brooks/Cole,

[7] Patel, M S, Korotchkina, L G (2006) Regulation of the Pyruvate Dehydrogenase

[8] Roche, T E, Hiromasa, Y (2007) Pyruvate Dehydrogenase Kinase Regulatory Mechanisms and inhibition in Treating Diabetes, Heart Ischemia, and Cancer. Cell. mol.

[9] Hiromasa, Y, Hu, L Y, Roche, T E (2006) Ligand-Induced Effects on Pyruvate

[10] Perham, R N (2000) Swinging Arms and Swinging Domains in Multifunctional Enzymes: Catalytic Machines for Multistep Reactions Annu. rev.biochem., 69: 961-1004. [11] Garland, P B, Randle, P J (1964) Control of Pyruvate Dehydrogenase in Perfused Rat Heart by Intracellular Concentration of Acetyl-Coenzyme. A. biochem. j. 91: C6-C12. [12] Kanzaki, T, Hayakawa, T, Hamada, M, FukuyoshY, Koike, M (1969) Mammalian Alpha-Keto Acid Dehydrogenase Complexes. IV. Substrate Specificities and Kinetic Properties of Pig Heart Pyruvate and 2-Oxoglutarate Dehydrogenase Complexes. J.biol. chem. 244:

[13] Linn, T C, Pettit, F H, Reed, L J (1969) Alpha-Keto Acid Dehydrogenase Complexes X Regulation of Activity of Pyruvate Dehydrogenase Complex From Beef Kidney Mitochondria by Phosphorylation and Dephosphorylation. Proc. nat. acad. sci. USA 62:

[14] Baker, J C, Yan, X H, Peng, T, Kasten, S, Roche, T E (2000) Marked Differences Between Two Isoforms of Human Pyruvate Dehydrogenase Kinase. J. biol. chem. 275: 15773-

[15] Roche, T E, Baker, J C, Yan, Y H, Hiromasa, Y, Gong, X M, Peng, T, Kasten, S A (2001) Distinct Regulatory Properties of Pyruvate Dehydrogenase Kinase and Phosphatase

[16] Kato, M, Chuang, J L, Tso, S C, Wynn, R M, Chuang, D T (2005) Crystal Structure of Pyruvate Dehydrogenase Kinase 3 Bound To Lipoyl Domain 2 of Human Pyruvate

[17] Knoechel, T R, Tucker, A D, Robinson, C M, Phillips, C, Taylor, W, Bungay, P J, Brown, D G (2006) Regulatory Roles of the N-Terminal Domain Based on Crystal Structures of Human Pyruvate Dehydrogenase Kinase 2 Containing Physiological and Synthetic

[18] Boulatnikov, I, Popov, K A (2003) Formation of Functional Heterodimers by Isozymes 1 and 2 of Pyruvate Dehydrogenase Kinase. Biochim. biophys. acta 1645: 183-192.

Isoforms. Prog. nucleic acid res. mol. biol., 70: 33-75.

Dehydrogenase Complex. Embo j. 24: 1763-1774.

Ligands. Biochemistry, 45: 402-415.

Dehydrogenase Kinase Isoform 2. J. biol. chem. 281: 12568-12579.

B biol sci, 354: 1923-1939.

Cengage Learning

life sci. 64: 830-849.

1183-1187.

234-241.

15781.

Evolution of Complex Life. Science, 311:, 1764-1767.

Complex. Biochem. soc. trans., 34: 217-222.

Collectively, these results indicate that the regulatory reprogramming of these dehydrogenases may represent a target-rich environment for developing new anti-tumor drugs that have the crucial properties that may be required to impart new efficacy to cancer chemotherapy – targets that are both essential to the malignant condition and nonredundant in their function. The preclinical and early clinical properties of agents directed at these targets support the possibility of useful promise in this therapeutic domain.
