The KRAS gene encodes for a small GTPase that influences cell growth and apoptosis. Single point mutations cause KRAS to be constitutively active, triggering oncogenesis. Mutations in KRAS are found in roughly 30% of all cancers. While targeting the KRAS protein to perturb function has been the focus of much effort, it has proven difficult for its small size and lack of ‘druggable’ pockets. An attractive alternative route is the down regulation of KRAS expression via small molecule-mediated stabilization of the G-quadruplex (G4) present within its promoter region. However, there are only few examples of biologically active small molecules that bind to nucleic acids has been the identification of selective interactions. Here, we demonstrate that screening a library of 3,609 FDA approved drugs and other chemical diverse compounds using a Small Molecule Microarray (SMM) against the KRAS G4 and seven other G4s in parallel to profile binding selectivity. We found the bisbiguanide drug chlorhexidine (CLX), an oral disinfectant, to preferentially bind to the KRAS G4. A series of biophysical experiments confirm that CLX binds reversibly with single digit micromolar affinity. CLX was demonstrated to down regulate KRAS protein expression in cells. When cancer cells were transiently transfected with HA-tagged KRAS plasmid driven by a CMV promoter (lacking a G4), CLX had no effect on HA-tagged KRAS protein expression. In these same cells, the native KRAS protein decreased. Furthermore, cancer cell lines harboring oncogenic mutations in the KRAS gene exhibit increased sensitivity to CLX. Through NMR studies that utilize a 13C-labeled version of CLX and docking studies, we propose a binding mode of CLX to the KRAS G4. This work provides a useful method to investigate nucleic acid targets for known drugs, and suggests that considering nucleic acid-binding events when studying off-target pharmacology in drug discovery could be valuable.

Small molecule microarray profiling reveals FDA approved drugs that bind to structured nucleic acids

256th National Meeting (2018)

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