Application of secondary structure prediction in antisense drug design targeting protein kinase C-alpha mRNA and QSAR...

To optimize the design of antisense drug targeting protein kinase C-alpha (PKC-alpha) mRNA and obtain better antisense drugs than ISIS3521 that is undergoing clinical trials.

RNAstructure (version 3.21, 1999) was utilized to predict the optimal and suboptimal secondary structures of human PKC alpha mRNA (GenBank, X52479), and 29 antisense phosphorothioate oligodeoxynucleotides (S-ODN) targeting the secondary structural elements, 3 partly matched S-ODN and 1 scrambled 3521 were designed. ISIS3521 was set as positive control. Mean (n = 3-5) 50% inhibitory effects on proliferation of A549 cells (IC50) of S-ODN were evaluated. Free energies (delta G degree 37) relating to the target secondary structural elements were calculated according to the nearest neighbor model. The quantitative structure-activity relationship (QSAR) analysis through multiple regression was obtained by SPSS.

Three S-ODN; (5'-AGCCCA-GCCGCTTGGCTGGG-3', 5'-AGGAGTGCAGCTGC-GTCAAG-3', 5'-TCAGAGGG-ACTGATGACTTT-3') had lower IC50[(48 +/- 7), (50 +/- 4), (64 +/- 2.7) nmol.L-1, respectively] than that of ISIS3521 [(81 +/- 25) nmol.L-1]. The number of bases comprising the target secondary structural element bulge loop, internal loop, and knot, the free energy of S-ODN (delta G degree 37S), and reaction (delta G degree 37R) were important parameters in QSAR equation. In the multiple regression, R was 0.68, P = 0.0193. Not tally with the equation, two S-ODN (5'-TCAAATGGAGG-CTGCCCGGC-3', 5'-AAAACGTCAGCCATGGTCCC-3') with favorable target structures and delta G degree 37 did not behave good activities.

Computer aided design was helpful to obtain S-ODN with better in vitro effect than current positive drug. The degree of instability of secondary structural elements and delta G degree 37 were important factors for drug activity. Other important factors needed for further investigation.