**7. The central role of lipoate as a metabolic regulatory signal, other potential thioctoid tumor targets**

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 tumor cells analogously to the well-understood PDH case.

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 carbon flow is implicitly governed by lipoate-dependent dehydrogenases because no fatty acid carbon can be oxidatively released without first passing through the KGDH complex [6]. Thus, all carbon flow through mitochondria is ultimately controlled by lipoatecontaining enzymes.

The role of lipoate residues in controlling PDH function and the unique capacity of these residues to poll and reflect the mitochondrial energy status strongly suggests that lipoate residues will play a role in regulating BCDH and KGDH function. While this possibility remains to be investigated, it is noteworthy that the BCDH complex is regulated by kinases homologous to PDKs. It is plausible that these regulatory kinases are also responsive to lipoate redox/acylation state. Moreover, in view of the rather dramatic reprogramming of tumor mitochondrial metabolism, it is also plausible that BCDH regulatory kinases may be reprogramed in malignancy.

KGDH is apparently not regulated by PDK-like kinases and the details of its metabolic regulation remain to be investigated. However, the complex shows strong allosteric regulation by end products, consistent with important regulatory objectives [75, 76]. Thus, it is likely that KGDH lipoates also participate in this regulation in some way that largely remains to be determined.

As reviewed in section 3 above, the lipoate residues of PDH, BCDH and KGDH exist as a cloud polling crucial features of the mitochondrial energy state, including the aggregate levels of free reducing potential (NADH) and of their respective acyl-CoA products. Moreover, these details of the mitochondrial energy state are likely to be among the state variables most informative about the moment-to-moment metabolic needs of the mitochondrion. Further, these regulatory goals of mitochondria are so substantially altered in tumor cells that we anticipate that all these potential lipoate-sensitive regulatory targets might be substantially altered in tumor cells, as they clearly are in the PDH case. Finally, the long boom-like structure of the enzyme linked lipoamide moieties (Figures 2) and their attachment to highly mobile E2 enzyme subdomains (Figure 3) raises an additional important regulatory possibility. It is conceivable that these actively polling lipoate residues are not merely reporting their results to their individual complexes but also to other mitochondrial consumers of regulatory information.

Collectively, these considerations suggest that thioctoid lipoate analogs may achieve their dramatic inhibition of tumor cell mitochondrial metabolism by addressing multiple, essential metabolic regulatory processes in a tumor-specific fashion. The promising preclinical and early clinical properties of thioctoid CPI-613 indicate that it will be of interest to explore this possibility in detail going forward.
