1 research outputs found
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