Platinum-RNA Modifications Following Drug Treatment in <i>S. cerevisiae</i> Identified by Click Chemistry and Enzymatic Mapping

With the importance of RNA-based regulatory pathways, the potential for targeting noncoding and coding RNAs by small molecule therapeutics is of great interest. Platinum­(II) complexes including cisplatin (<i>cis</i>-diamminedichloroplatinum­(II)) are widely prescribed anticancer compounds that form stable adducts on nucleic acids. In tumors, DNA damage from Pt­(II) initiates apoptotic signaling, but this activity is not necessary for cytotoxicity (e.g., Yu <i>et al.</i>, 2008), suggesting accumulation and consequences of Pt­(II) lesions on non-DNA targets. We previously reported an azide-functionalized compound, picazoplatin, designed for post-treatment click labeling that enables detection of Pt complexes (White <i>et al.</i>, 2013). Here, we report in-gel fluorescent detection of Pt-bound rRNA and tRNA extracted from picazoplatin-treated <i>S. cerevisiae</i> and labeled using Cu-free click chemistry. These data provide the first evidence that cellular tRNA is a platinum drug substrate. We assess Pt­(II) binding sites within rRNA from cisplatin-treated <i>S. cerevisiae</i>, in regions where damage is linked to significant downstream consequences including the sarcin-ricin loop (SRL) Helix 95. Pt-RNA adducts occur on the nucleotide substrates of ribosome-inactivating proteins, as well as on the bulged-G motif critical for elongation factor recognition of the loop. At therapeutically relevant concentrations, Pt­(II) also binds robustly within conserved cation-binding pockets in Domains V and VI rRNA at the peptidyl transferase center. Taken together, these results demonstrate a convenient click chemistry methodology that can be applied to identify other metal or covalent modification-based drug targets and suggest a ribotoxic mechanism for cisplatin cytotoxicity