Bio-engineering of RGD Derivatized Dihydrolipoamide Dehydrogenase as a Novel Anti-Cancer Drug

Dihydrolipoamide dehydrogenase (DLDH) catalyzes the NAD⁺-dependent oxidation of dihydrolipoic acid (or amide) into lipoic acid (or amide). Interestingly, NAD⁺ dependent metabolic and signaling pathways are highly altered in cancer cells. The enzyme activity is critical for energy and redox balance in the cell and is often associated with elevated levels of Reactive Oxygen Species (ROS) production.

ROS production and DNA binding render DLDH with pro-apoptotic properties. However, in order to exercise of its cytotoxicity in cancer cells, DLDH must be specifically targeted to and incorporated into these cells. This was achieved by bio-engineering the human DLDH with RGD tails (DLDH^RGD) which are specifically recognized by integrins overexpressed on the surface of cancer cells. RGD moieties were located at the N and C termini of DLDH in order to avoid interference with enzyme activity and DNA binding.

The cytotoxic effect of DLDH^RGD was demonstrated with cutaneous melanoma (B16F10) cancer cell lines, while normal kidney (HEK293) remain unharmed. Confocal and FACS assays showed that in contrast to DLDH, DLDH^RGD incoporates into the cancer cells via interaction with integrins and induces apoptotic cell death. ROS production and DNA binding may be involved in this procces.

In in-vitro studys ROS production was found to be associated with enzymatic activity of DLDH^RGD. DNA binding was independent of the enzyme substrate presents and associated with the C terminal region of the protein.

In-vivo assay showed positive safety profile using IV, SC and IP (behavior, blood chemistry and histopathology assay) and high efficacy (murine B16F10 melanoma subcutaneous model in C57bL female mice).