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Antisense candidates against protein kinase C-alpha designed based on phylogenesis and simulant structure of mRNA
Hai-Feng Song1, Zhong-Ming Tang, Shou-Jun Yuan
1Department of Pharmacology and Toxicology, Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China. songhf@nic.bmi.ac.cn
Aim:
To optimize the antisense drug design by the combined method of phylogenetic analysis and secondary structure prediction and to get ideal candidates.
Methods:
The phylogenetic analysis and the secondary structure simulation were performed by computer. Oligodeoxynucleotides (ODN) were designed against the full-conserved blocks with low local reaction free energy of protein kinase C (PKC)-alpha mRNA. The in vitro effects of ODN were evaluated by human A549 lung carcinoma cells and mouse B16-BL6 melanoma cells, the expression of target mRNA was detected by in situ hybridization and RT-PCR. The in vivo effects of ODN were also evaluated by models of A549 xenografts in nude mice and B16 melanoma in mice.
Results:
Three ODN had significantly lower IC50 values than that of ISIS3521, the positive control, on A549 cells in vitro. Five ODN inhibited the growth of B16-BL6 cells with IC50 <100 nmol/L, while IC50 of ISIS3521 was >200 nmol/L. In situ hybridization and RT-PCR showed that the best candidate AP1261 inhibited the expression of PKC-alpha at mRNA level in a dose-dependent manner. AP1261 inhibited the growth of A549 and B16 tumors in vivo at 0.005-0.5 mg.kg(-1).d(-1). The inhibitory rate of AP1261 on A549 tumors was greater than that of ISIS3521 at the same dose. ISIS3521 did not affect the growth of B16 tumors.
Conclusion:
AP1261 may be of value as an antitumor agent or adjuvant and the combined method of phylogenetic analysis and secondary structure prediction is a potential helpful tool for antisense drug design.