Farnesyltransferase-Mediated Delivery of a Covalent Inhibitor Overcomes Alternative Prenylation to Mislocalize K-Ras

Abstract

Mutationally activated Ras is one of the most common oncogenic drivers found across all malignancies, and its selective inhibition has long been a goal in both pharma and academia. One of the oldest and most validated methods to inhibit overactive Ras signaling is by interfering with its post-translational processing and subsequent cellular localization. Previous attempts to target Ras processing led to the development of farnesyltransferase inhibitors, which can inhibit H-Ras localization but not K-Ras due to its ability to bypass farnesyltransterase inhibition through alternative prenylation by geranylgeranyltransferase. Here, we present the creation of a neo-substrate for farnesyltransferase that prevents the alternative prenlation by geranylgeranyltransferase and mislocalizes oncogenic K-Ras in cells.

Figure 1.

Proposed method to overcome innate resistance to inhibition of K-Ras post translational modifications. Path a depicts the normal course of K-Ras post translational modification by FTase, which leads to membrane localization after several subsequent steps. Path b shows the scenario when the active site of FTase is blocked with an FTI: a second prenylation enzyme, GGTase, can catalyze geranylgeranylation of K-Ras, which still leads to properly localized and functional K-Ras. Path c represents our proposed approach: proper incorporation of a cysteine-reactive chemical electrophile (E) into an FTI could allow FTase to irreversibly transfer a covalent inhibitor to the normally prenylated cysteine, thereby blocking any other modification at the site and causing mislocalization of K-Ras.

Figure 2.

Design and in vitro activity of electrophilic FTI. a. The ternary crystal structure of a known reversible inhibitor 1 (green) complexed with FTase (gray surface) and the c-terminal tail of K-Ras (purple) (PDB:1D8D) suggests a possible site of attachment for an electrophile on a nitrogen in the inhibitor (blue) towards the target K-Ras cysteine (yellow). b. The known reversible inhibitor 1 was adapted into the general scaffold 2 to facilitate incorporation of an electrophile (E) and variation of the electrophile-charged phosphonate “warhead” (dashed box). The structures of electrophilic FTI 3–7 and non-electrophilic control 8 are shown along with their percent modification of K-Ras in an in vitro LC/MS assay (20 µM inhibitor, 10 µM K-Ras, 10 µM FTase, 4 hrs). c. Representative LC/MS chromatogram of the reaction between K-Ras and inhibitor 6. Peaks have been re-labeled for clarity.

Figure 3.

6* displaces K-Ras(G12C) from the plasma membrane. a. Representative images showing the localization of mCerulean-K-Ras (G12C) in 293 cells after 9 hours of treatment with DMSO, Lovastatin, 6* or 8* (25µM). Bar=10µm. b. Fluorescence intensity linescans were measured across the edge of cells as depicted by the arrows overlaid on the fluorescence images. Aligned and normalized intensity linescans (see Materials and Methods) are plotted for cells treated with DMSO or 6* (n=50, gray lines). The average linescan for each population (black line) and linescans corresponding to the cells shown on the fluorescence images (dashed red line) are highlighted in the graphs. c. The ratio of the maximum edge fluorescence to internal cellular fluorescence was calculated for each cell (see Materials and Methods) and summarized in box and whiskers plots for each population of cells (n=50). Whiskers correspond to min/max values. Statistically significant differences between DMSO- and drug-treated cells were calculated using the Mann-Whitney test (***p<0.0001).

Figure 4.

6* mislocalizes oncogenic K-Ras into the cytoplasm. a. PSN-1 cells (K-Ras G12R) were treated with the indicated inhibitors for 24 h and the lysates were separated into cytoplasmic (C) and membrane (M) fractions. K-Ras can bypass both the peptide competitive and farnesyl pyrophosphate competitive FTase inhibitors through GGTase in order to maintain its proper localization. In contrast, 6* induces the mislocalization of K-Ras to the cytoplasm. Lova = Lovastatin, inhibits FTase and GGTase by blocking cellular synthesis of their activated prenylation substrates, GG = GGTI 298, peptide competitive GGTase inhibitor, L744 = L744,832 peptide competitive FTase inhibitor, FPT3 = FPT III, farnesyl pyrophosphate competitive inhibitor, the pro-drug version of 1. b. 6* inhibits the membrane localization of K-Ras in a dose dependent manner at 24h. c. The mislocalization of K-Ras by 6* is time dependent. d. Cellular fractionation of PSN-1 cells treated with either L744, 6*, or 6* in the presence of increasing concentrations of L744 show the cellular activity of 6* is dependent on K-Ras binding to FTase.

Figure 5.

6* in combination with statins can inhibit K-Ras signaling and proliferation. a-c. PSN-1 cells were treated with the indicated inhibitors for 24h and whole cell lysates were analyzed by the specified antibodies. d. 72h proliferation of PSN-1 cells against a dose response of either 6* or 8* +/− 1 µM Lova (mean ± S.D., n=2).