**6. Thioctoid lipoate analogs exaggerate cancer-specific, PDK-mediated repression of PDH to shut down tumor cell mitochondria and induce cell death**

The PDH complex includes a dense cloud of lipoate residues making up the functional catalytic co-factor domain of the E2 activity (above). Acyl groups and reducing potential can be back-transferred to this lipoate cloud from the surrounding mitochondrial matrix pool as well as being generated by the conventional PDH forward reaction (above). Moreover, these acyl groups and reducing potential can apparently be rapidly shuttled between these residues before ultimately being transferred to CoA and NAD+[28]. Finally, the oxidation/acylation state of the elements of the lipoate cloud strongly effect the activity of the PDK regulatory kinases, with acylation/reduction stimulating PDK activity (above). Thus, each PDK homo- or heterodimer docked at any point on the PDH complex is apparently continuously exposed to the result of an ongoing, real-time poll of the

mitochondrial matrix energy status. Further, these PDKs are then able to act rapidly on all their E1 targets in the PDH complex in response to these polling results as a result of the combination of the highly flexible linkers surrounding the lipoyl domains to which they are bound (Figure 3) and their ability to move hand-over-hand through the complex.

The Pyruvate Dehydrogenase Complex in Cancer:

Implications for the Transformed State and Cancer Chemotherapy 53

effects is apparently to exploit tumor-specific PDH regulation, in part or entirely, to shut down tumor mitochondrial function. As continued mitochondrial function is required for the survival of tumor cells, even in the hypoxic solid tumor environment (above), these effects are expected to engender the observed response, rapid commitment to and execution

**Figure 4.** Structure of thioctoid CPI-613 compared to biogenic lipoates. The structure of the thioctoid CPI-613 is shown at bottom. This molecule is a non-redox active analog of lipoate designed to have some resemblance to the acylated form of lipoate as it occurs in the normal catalytic cycle of PDH (Figure 2). The corresponding biogenic lipoate intermediates in the PDH catalytic cycle are

illustrated at top.

of mitochondrially controlled cell death pathways.

*In vitro* studies demonstrate that free lipoate and lipoate-containing lipoyl domain fragments can interact productively with the intact PDH complex (above). This suggests that lipoate analogs might be able to perturb PDH regulation *in vivo*. Moreover, this perturbation might show strong selectivity for tumor cells in view of the substantial reconfiguration of PDK regulation in cancer (above). Based on the original PDH regulatory biochemistry and the presumption, subsequently confirmed (above), that PDH regulation was likely to be substantially altered in tumor cells, we began a systematic study of lipoate analogs as anticancer agents in the late 1990s (Patent # 6331559, filed 1999). The concept was that appropriately designed lipoate analogs might mimic the effects of specific intermediate states of lipoate (Figures 3 and 4) on PDK function and, thereby, misinform the PDH regulatory machinery in ways that were selectively toxic to tumor cells.

These studies initially demonstrated that lipoate analogs, designated thioctoids, had strong anti-cancer activity in cell culture [2; our unpublished results]. Many of our initial analyses were carried out using our current lead clinical investigational compound, CPI-613 (Figure 4). Indeed, these agents induce cell death in cancer cells extremely efficiently, with all cells dying within 24-48 hours by a mixture of apoptosis and necrosis-like mechanisms. This is in contrast to DCA which typically induces cell death in only a small fraction of tumor cells in culture [1; our unpublished results]. Each of the many human tumor cell lines we have examined is comparably sensitive to thioctoids, with tumor genotype at oncogenes and multidrug resistance loci having little or no effect on response [2].

Though the precise biochemical mechanism of action of thioctoids remains to be established, the *in vivo* behavior of these compounds demonstrates that they modulate PDK regulation of PDH [2; our unpublished results]. Moreover, these tumor effects are diametrically opposite to the effects of DCA and of other small molecule PDK inhibitors. Specifically, DCA induces *de-phosphorylation* of PDH E1 *in vivo* [55] while thioctoids induce *hyperphosphorylation in vivo* [2; our unpublished results]. This thioctoid-induced PDH hyperphosphorylation is selective for tumor cells and correlates with inactivation of PDH activity as assessed by analysis of the oxidative flux of pyruvate carbon through the complex. Coincident with this inhibition of PDH activity, thioctoids induce rapid shut down of mitochondrial ATP synthesis, typically reducing mitochondrial ATP production from pyruvate and glutamine carbon sources to less that 10% of controls within 15-30 minutes [2]. Finally, the capacity of CPI-613 to kill tumor cells is significantly attenuated in cell culture systems by RNAi knockdown of regulatory PDKs, supporting a role for PDKs in the response to these agents.

Collectively, these results indicate that thioctoid lipoate analogs kill tumor cells by addressing tumor-specific PDK regulation of PDH. Moreover, the consequence of these effects is apparently to exploit tumor-specific PDH regulation, in part or entirely, to shut down tumor mitochondrial function. As continued mitochondrial function is required for the survival of tumor cells, even in the hypoxic solid tumor environment (above), these effects are expected to engender the observed response, rapid commitment to and execution of mitochondrially controlled cell death pathways.

52 Dehydrogenases

mitochondrial matrix energy status. Further, these PDKs are then able to act rapidly on all their E1 targets in the PDH complex in response to these polling results as a result of the combination of the highly flexible linkers surrounding the lipoyl domains to which they are

*In vitro* studies demonstrate that free lipoate and lipoate-containing lipoyl domain fragments can interact productively with the intact PDH complex (above). This suggests that lipoate analogs might be able to perturb PDH regulation *in vivo*. Moreover, this perturbation might show strong selectivity for tumor cells in view of the substantial reconfiguration of PDK regulation in cancer (above). Based on the original PDH regulatory biochemistry and the presumption, subsequently confirmed (above), that PDH regulation was likely to be substantially altered in tumor cells, we began a systematic study of lipoate analogs as anticancer agents in the late 1990s (Patent # 6331559, filed 1999). The concept was that appropriately designed lipoate analogs might mimic the effects of specific intermediate states of lipoate (Figures 3 and 4) on PDK function and, thereby, misinform the PDH

These studies initially demonstrated that lipoate analogs, designated thioctoids, had strong anti-cancer activity in cell culture [2; our unpublished results]. Many of our initial analyses were carried out using our current lead clinical investigational compound, CPI-613 (Figure 4). Indeed, these agents induce cell death in cancer cells extremely efficiently, with all cells dying within 24-48 hours by a mixture of apoptosis and necrosis-like mechanisms. This is in contrast to DCA which typically induces cell death in only a small fraction of tumor cells in culture [1; our unpublished results]. Each of the many human tumor cell lines we have examined is comparably sensitive to thioctoids, with tumor genotype at oncogenes and

Though the precise biochemical mechanism of action of thioctoids remains to be established, the *in vivo* behavior of these compounds demonstrates that they modulate PDK regulation of PDH [2; our unpublished results]. Moreover, these tumor effects are diametrically opposite to the effects of DCA and of other small molecule PDK inhibitors. Specifically, DCA induces *de-phosphorylation* of PDH E1 *in vivo* [55] while thioctoids induce *hyperphosphorylation in vivo* [2; our unpublished results]. This thioctoid-induced PDH hyperphosphorylation is selective for tumor cells and correlates with inactivation of PDH activity as assessed by analysis of the oxidative flux of pyruvate carbon through the complex. Coincident with this inhibition of PDH activity, thioctoids induce rapid shut down of mitochondrial ATP synthesis, typically reducing mitochondrial ATP production from pyruvate and glutamine carbon sources to less that 10% of controls within 15-30 minutes [2]. Finally, the capacity of CPI-613 to kill tumor cells is significantly attenuated in cell culture systems by RNAi knockdown of regulatory PDKs, supporting a role for PDKs in the

Collectively, these results indicate that thioctoid lipoate analogs kill tumor cells by addressing tumor-specific PDK regulation of PDH. Moreover, the consequence of these

bound (Figure 3) and their ability to move hand-over-hand through the complex.

regulatory machinery in ways that were selectively toxic to tumor cells.

multidrug resistance loci having little or no effect on response [2].

response to these agents.

**Figure 4.** Structure of thioctoid CPI-613 compared to biogenic lipoates. The structure of the thioctoid CPI-613 is shown at bottom. This molecule is a non-redox active analog of lipoate designed to have some resemblance to the acylated form of lipoate as it occurs in the normal catalytic cycle of PDH (Figure 2). The corresponding biogenic lipoate intermediates in the PDH catalytic cycle are illustrated at top.

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%.]

The Pyruvate Dehydrogenase Complex in Cancer:

Implications for the Transformed State and Cancer Chemotherapy 55

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 lipoate-

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

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

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

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

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

containing enzymes.

reprogramed in malignancy.

remains to be determined.

mitochondrial consumers of regulatory information.

explore this possibility in detail going forward.

**8. Conclusions** 

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

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 investigation of these investigational drugs.
