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

AIM: To optimize the design of antisense drug targeting protein kinase C-al pha (PKC-alpha) mRNA and obtain better antisense drugs than ISIS3521 that i s undergoing clinical trials. METHODS: RNAstructure (version 3.21, 1999) wa s utilized to predict the optimal and suboptimal secondary structures of hu man PKC alpha( mRNA (GenBank, X52479), and 29 antisense phosphorathioate ol igodeoxynucleotides (S-ODN) targeting the secondary structural elements, 3 partly matched S-ODN and 1 scrambled 3521 were designed. ISIS3521 was set a s positive control. Mean (n = 3 - 5) 50 % inhibitory effects on proliferati on of A549 cells (IC50) of S-ODN were-evaluated. Free energies (Delta G deg rees(37)) relating to the target secondary structural elements were calcula ted according to the nearest neighbor model. The quantitative structure-act ivity relationship (QSAR) analysis through multiple regression was obtained by SPSS. RESULTS: Three S-ODN: (5'-AGCCCAGCCGCTTGGCTGGG-3', 5'-AGGAGTGCAGC TGCGTCAAG-3' , 5'-TCAGAGGG-ACTGATGACTTT-3') had lower IC50[(45(+/-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 structu ral element bulge loop, internal loop, and knot, the free energy of S-ODN ( Delta G degrees 37S), and reaction (Delta G degrees(37R)) were important pa rameters in QSAR equation. Tn the multiple regression, R was 0.68, P=0.0193 . Not tally with the equation, two S-ODN ( 5'-TCAAATGGAGG-CTGCCCGGC-3', 5'- AAAACGTCAGCACCATGGTCCC-3') with favorable target structures and Delta G de grees(37) did not behave good activities. CONCLUSION: Computer aided design was helpful to obtain S-ODN with better in vitro effect than current posit ive drug. The degree of instability of secondary structural elements and De lta G degrees(37) were important factors for drug activity. Other important factors needed for further investigation.