RAS The Good The Bad and The Ugly

We chose H-RAS as the first target protein for several reasons. RAS proteins are, perhaps, the most intensively studied and best understood signal transduction proteins from the perspective of structure and function [8]. The H-RAS crystal structure has been determined to the level of 1.5 A, and the domains involved in guanine nucleotide binding and hydrolysis, effector interactions, interactions with regulating proteins, and processing have been extensively characterized. RAS proteins are initially synthesized in the cytoplasm where they undergo a series of post-translational modifications at their carboxy-terminal sequence, the CaaX-box (where ''C'' is cysteine, ''a'' an aliphatic amino acid, and ''X'' either serine or methionine), resulting in their farnesylation, and subsequent cleavage of the terminal tripeptide and carboxy-methylation [9, 10]. The processed proteins become localized on the cell membrane, a step that is essential to their functioning [1113]. They are required for the transduction of signals from many membrane receptors, including tyrosine kinases and some G-protein linked receptors and the RAF protein kinase from mammalian cells, leading to activation of the MAP kinase cascade [14-16]. RAS proteins play a central role in normal cellular physiology, and point mutations that activate the oncogenic potential of RAS genes are commonly found in human tumors [17, 18].

Oncogenic RAS proteins have been considered to be a logical target for the development of cancer therapeutics. One approach has been to find agents that block RAS processing by inhibiting farnesyl transferase (FTase) [19]. Such agents can, in fact, induce morphological reversion of cells transformed by RAS at concentrations that do not arrest normal cell growth. The results with FTase inhibitors suggest that, in general, the level of an inhibitor needed to block the transforming activity of RAS will not necessarily reduce normal RAS activity below levels sufficient for normal cell growth [20]. Non-farnesylated oncogenic [12Val]-RAS has, moreover, been shown to sequester RAF to the cytosol, thus leading to a cytostatic effect in transformed cells whereas growth and proliferation are slower in untransformed cells [21]. FTase inhibitors (Figure 3.1.1) might, therefore, be generally useful as anti-cancer agents. One potential obstacle to using such agents as drugs is that they can block the farnesylation of other critical proteins in humans, for example the y-subunit of transducin and nuclear lamins.

On the other hand, a small molecule that binds to the carboxy terminus of a particular RAS might prevent FTase from acting upon this RAS without inhibiting the activity of the transferase on other substrates. Indeed, the precise CaaX-sequences and the two amino acid residues preceding them distinguish the mem-

inhibit the enzyme

Fig. 3.1.1. Approaches to prevention of farnesylation of RAS.

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