Disruption of Oncogenic K-Ras4B Processing and Signaling by a Potent Geranylgeranyltransferase I Inhibitor

Abstract

Prenylation of the carboxyl-terminal CAAX (C, cysteine; A, aliphatic acid; and X, any amino acid) of Ras is required for its biological activity. We have designed a CAAX peptidomimetic, GGTI-287, which is 10 times more potent toward inhibiting geranylgeranyltransferase I (GGTase I) in vitro (IC 50 ‫؍‬ 5 nM) than our previously reported farnesyltransferase inhibitor, FTI-276. In whole cells, the methyl ester derivative of GGTI-287, GGTI-286, was 25-fold more potent (IC 50 Ras is a small guanine nucleotide binding protein that cycles between its active (GTP-bound) and inactive (GDP-bound) forms to transduce growth and differentiation signals from receptor tyrosine kinases to the nucleus (1, 2). Binding of epidermal and platelet-derived growth factors to their receptor tyrosine kinases results in autophosphorylation and recruitment of key signaling proteins to the receptor. Among these proteins are the Ras exchange factors that activate Ras by catalyzing the exchange of GDP for GTP. GTP-bound Ras activates a cascade of mitogen-activated protein (MAP) 1 kinases by recruiting Raf to the plasma membrane. Raf, a serine/threonine kinase, phosphorylates MAP kinase kinase, which in turn activates MAP kinase by phosphorylating it on threonine and tyrosine. Hyperphosphorylated MAP kinase translocates to the nucleus where it phosphorylates transcription factors that are involved in the regulation of growth-related genes. The growth signal is terminated when Ras hydrolyzes GTP to GDP (1-3). However, mutations that lock Ras in its GTP-bound form result in an uninterrupted growth signal and are believed to contribute to the development of more than one-third of human cancers (4, 5).

Both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-Ras prenylation but each alone is sufficient to suppress human tumor growth in nude mouse xenografts

“…We have therefore made GGTase I inhibitors and used these compounds to give further support for this notion (Lerner et al, 1995b and Figure 1). More recently, K-and N-Ras have been shown to be geranylgeranylated in cells treated with FTase inhibitors (Whyte et al, 1997;Rowell et al, 1997).…”

“…For example, the CAAX peptidomimetic FTI-277 inhibited H-Ras processing and oncogenic signaling with an IC 50 of 0.1 mM whereas inhibition of K B -Ras function required 100-fold higher concentrations . We have subsequently designed an inhibitor to the closely related enzyme geranylgeranyltransferase I (GGTase I), which transfers a C 20 geranylgeranyl group to the cysteine of CAAX sequences that have a leucine at the carboxyl terminus, and shown that K B -Ras processing and oncogenic signaling was sensitive to this inhibitor (Lerner et al, 1995b). This was consistent with the work of which showed that K B -Ras can be both farnesylated and geranylgeranylated in vitro as well as with the more recent work of Whyte et al (1997) and Rowell et al (1997) in intact human tumor cell lines.…”

Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of anti-proliferative, pro-apoptotic and anti-angiogenic effects

“…FTIs preferentially inhibit the proliferation of H-Ras transformed cells, with K-Ras transformed cells either being resistant to these agents or being inhibited at higher drug doses (James et al, 1996;Lerner et al, 1995b;Manne et al, 1995;Nagasu et al, 1995;Zhang et al, 1997). This relative resistance of K-Rastransformed cell lines to FTIs is due to the observation that K-Ras and (to a lesser extent) NRas, but not H-Ras, can be post-translationally modi®ed by geranylgeranyltransferase I (GGTase I) (Lerner et al, 1995b;Whyte et al, 1997), a related enzyme which transfers a 20-carbon prenyl group from geranylgeranyl pyrophosphate to cysteine residues in proteins. Cells lacking oncogenic Ras mutations can be inhibited by FTIs, but these e ects, as in our cells, occur at higher drug doses than for H-, N-, or K-Ras transformed cells (Nagasu et al, 1995).…”

Inhibition of the prenylation of K-Ras, but not H- or N-Ras, is highly resistant to CAAX peptidomimetics and requires both a farnesyltransferase and a geranylgeranyltransferase I inhibitor in human tumor cell lines

“…We have designed CAAM peptidomimetics such as FTI-276 that are potent and selective inhibitors of FTase over GGTase I in vitro, and that selectively block the processing of farnesylated but not geranylgeranylated proteins in whole cells (Lerner et al, 1995a,b). We have also designed CAAL peptidomimetics, such as GGTI-287 and GGTI-297, that are selective for GGTase I over FTase (Lerner et al, 1995a;McGuire et al, 1996;Vogt et al, 1996). Furthermore, we have shown that while H-Ras processing was highly sensitive to FTI-277 and resistant to GGTI-286, KB-Ras was more sensitive to GGTI-286 than FTI-277 (Lerner et al, 1995a).…”

“…We have also designed CAAL peptidomimetics, such as GGTI-287 and GGTI-297, that are selective for GGTase I over FTase (Lerner et al, 1995a;McGuire et al, 1996;Vogt et al, 1996). Furthermore, we have shown that while H-Ras processing was highly sensitive to FTI-277 and resistant to GGTI-286, KB-Ras was more sensitive to GGTI-286 than FTI-277 (Lerner et al, 1995a). These results suggested that H-Ras is only farnesylated, while KB-Ras may be both farnesylated and geranylgeranylated in whole cells (Lerner et al, 1995a).…”