Abstract
The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3Cpro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3Cpro, we measured cleavage kinetics of predicted SVV-001 3Cpro substrates. An efficient substrate, L/VP4 (kcat/KM = 1932 ± 183 M-1s-1), was further optimized by a P2’ N→P substitution yielding L/VP4.1 (kcat/KM = 17446 ± 2203 M-1s-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3Cpro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3Cpro, with direct implications for anti-cancer therapeutic development.
Highlights
Oncolytic viruses are replication competent viruses that selectively infect and lyse cancer cells while causing little to no harm to normal tissues [1]
Once we identified a substrate with high turnover efficiency, we confirmed the ability of the substrate, L/VP4.1, to be recognized and cleaved in a cellular assay by the 3Cpro produced during an active SVV-001 infection in the context of both a FRET fusion protein and a fluorogenic peptide
As the endogenous sequences were expected to have the highest probability of being efficiently cleaved by the SVV-001 3Cpro, our initial kinetic studies focused on the ten intrinsic substrates to identify the substrate with the highest turnover efficiency
Summary
Oncolytic viruses are replication competent viruses that selectively infect and lyse cancer cells while causing little to no harm to normal tissues [1]. Because of their inherent selectivity, the majority of oncolytic viruses are well tolerated by patients even at high doses. One such virus, Seneca Valley Virus (SVV-001), is an oncolytic picornavirus that infects tumors with neuroendocrine features, including small cell lung cancer (SCLC) and pediatric neuroendocrine tumors, with high selectivity [2].
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