Sequence-Selective Covalent CaaX-Box Receptors Prevent Farnesylation of Oncogenic Ras Proteins and Impact MAPK/PI3 K Signaling

Abstract

Oncogenic Ras proteins are implicated in the most common life-threatening cancers. Despite intense research over the past two decades, the progress towards small-molecule inhibitors has been limited. One reason for this failure is that Ras proteins interact with their effectors only via protein-protein interactions, which are notoriously difficult to address with small organic molecules. Herein we describe an alternative strategy, which prevents farnesylation and subsequent membrane insertion, a prerequisite for the activation of Ras proteins. Our approach is based on sequence-selective supramolecular receptors which bind to the C-terminal farnesyl transferase recognition unit of Ras and Rheb proteins and covalently modify the essential cysteine in the so-called CaaX-box.

Keywords: CaaX-box; K-Ras4B; Ras protein; covalent inhibitor; molecular recognition.

Figure 1

Structure of K‐Ras4B(GTP).

Figure 2

Structures of Nestler‐receptor (1) and molecular forceps MFZ‐021 (2).

Figure 3

Incubation (LC−MS analysis) of C‐terminal undecapeptides with acrylamide modified receptor MFZ‐105 (7) and Thiol modified receptor MFZ‐115 (23). C‐terminal undecapeptides: Rheb, Ac‐ASQGKSSCSVM‐OH; H‐Ras, Ac‐PGCMSCKCVLS‐OH; K‐Ras, Ac‐KKKSKTKCVIM‐OH.

Figure 4

ESI‐MS spectra of a K‐Ras4B/MFZ‐148 (27) conjugate. The mass of the charged entities are labelled in black and the respective charge in red. A) Spectrum of unmodified K‐Ras4B protein. B) Spectrum of K‐Ras4B modified with MFZ‐148 (27).

Figure 5

Molecular dynamics simulation of MFZ‐021 (2) with full‐length Rheb. A) Protein‐ligand interaction diagram. B) Minor cluster with reorientation of the H‐domain and interactions with G‐domain residues. Magenta backbone: switch 2. The stacked bar charts are normalized over the course of the trajectory: for example, a value of 0.7 suggests that 70 % of the simulation time, the specific interaction is maintained. Values over 1.0 are possible as some protein residues may make multiple contacts of same subtype with the ligand.

Figure 6

Dynamic simulation of K‐Ras4B (PDB: 4DSO)/MFZ‐148 (27) protein‐ligand complex. A) Protein‐ligand interaction diagram. B) Interactions of MFZ‐148 (27) with K‐Ras4B CaaX‐box. C) Interactions of MFZ‐148 (27) with switch 1 residues.

Figure 7

Thiol‐based CaaX‐box inhibitors reduce tumor cell viability. A) Dose response curves of HCT116 after 72 h incubation with MFZ‐112 (20, no sigmoidal curve) MFZ‐115 (23, IC₅₀=538±58 μM), MFZ‐117 (25, IC₅₀=445±50 μM) and MFZ‐148 (27, IC₅₀=413±10 μM); Dose response curves of NCI−H358 after 72 h incubation with MFZ‐112 (20, no sigmoidal curve fit), MFZ‐115 (23, IC₅₀=1,741±0,39 mM), MFZ‐117 (25, IC₅₀=488±50 μM) and MFZ‐148 (27, IC₅₀=312±36 μM). B, C) Western Blot analysis of RAS‐dependent Erk phosphorylation of serum starved HCT116 cells after 2 h and 20 h incubation with potential CaaX‐box inhibitors. Data is represented as mean ± SD, n=3.

Figure 8

CaaX‐box inhibitors modify RAS‐dependent signaling cascades. A) Treatment of NCI−H358 cells with thiol‐based inhibitors MFZ‐115 (23) and MFZ‐117 (25) for 20 h, followed by 10 min. stimulation von 100 ng/ml EGF under serum‐free conditions. B) Western blot analysis of NCI−H358 and HCT116 cells after 20 h treatment with 4‐fluoroproline derivate MFZ‐148 (27). n=3.