A new class of small molecules that bind the HCV RNA IRES IIA subdomain with sub-micromolar affinity is reported. The benzimidazole 'hit' 1 with a KD approximately 100 microM to a 29-mer RNA model of Domain IIA was identified from a 180000-member library using mass spectrometry-based screening methods. Further MS-assisted SAR (structure-activity relationships) studies afforded benzimidazole derivatives with sub-micromolar binding affinity for the IIA RNA construct. The optimized benzimidazoles demonstrated activity in a cellular replicon assay at concentrations comparable to their KD for the RNA target.

A technique for lead discovery vs RNA targets utilizing mass spectrometry (MS) screening methods is described. The structure-activity relationships (SAR) derived from assaying weak binding motifs allows the pharmacophores discovered to be elaborated via "SAR by MS" to higher affinity ligands. Application of this strategy to a subdomain of the 23S rRNA afforded a new class of compounds with functional activity.

Two types of oligonucleotides were synthesized with linker groups attached at the 5'-end. Both were repeating dimers of deoxyribocytidine and deoxyriboadenosine. A 20-mer was prepared with a thiol-containing linker, masked as a disulfide, and a 50-mer was prepared with a vicinal diol-containing linker. A tetraiodoacetylated poly(ethylene glycol) (PEG) derivative was synthesized and reacted with the thiol-containing 20-mer to provide an oligonucleotide PEG conjugate of precisely four oligonucleotides on each PEG carrier. The vicinal diol on the 50-mer was oxidized to an aldehyde and conjugated to keyhole limpet hemocyanin (KLH) to provide an oligonucleotide-KLH conjugate by reductive alkylation. The conjugates were annealed with complementary (TG)n strands. While the double-stranded oligonucleotide-KLH conjugate is an immunogen, eliciting the synthesis of antibodies against oligonucleotides, the PEG conjugate has the biological property of specifically suppressing (tolerizing) B cells which make antibodies against the immunizing oligonucleotide.

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