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RAS Insight

, by David Heimbrook

The structure of drug candidate L-778123 bound to the alpha subunit of human protein farnesyltransferase / geranylgeranyltransferase (Protein Data Bank 1S63; Reid TS, et al., Biochemistry 43, 9000, 2004)

I really enjoyed Mark Philips excellent review on opportunities to target RAS signaling by disrupting membrane localization of RAS. I worked at a major Pharma player in the RAS field during the height of efforts to block RAS translocation with FPTase inhibitors, and wanted to share this old war-story, because what is old sometimes becomes new again...Who reads scientific literature from the ‘90’s anymore?

As Mark notes, RAS has been an attractive target for cancer therapy since its discovery as a human oncogene in 19821. Soon thereafter, in the late 1980’s, I was working as an entry-level PhD scientist at Merck, in the Department of Virus and Cell Biology (there was no Cancer Research department at the time, because I think Merck leadership was unconvinced there was a market in Oncology which could match what was going on in cardiovascular medicine at the time – can you say Mevacor?).

The overall head of research at the time was Dr. Ed Scolnick, a renowned oncogene researcher who had recently come to Merck from NCI, bringing some of his research staff with him. Ed is one of the most thoughtful and analytical scientists I’ve ever met, and he started a program targeting RAS at Merck.Early efforts looked at blocking GTP binding, but were soon abandoned due to the extraordinarily high affinity for this nucleotide – there weren’t even good kinase inhibitors at this time, and RAS bound GTP orders of magnitude more tightly than most kinases bound ATP.There was a huge surge of interest when it was demonstrated that RAS proteins contained “CAAX” boxes that were sites for post-translational modification, including prenylation, that appeared to be important for membrane localization (summarized in reference 2).

With the many resources available, a significant drug discovery effort was quickly launched at Merck, as well as at other pharmaceutical companies. This was seen as a huge opportunity, and a race, and at Merck dozens of cell biologists, biochemists, and chemists were working on multiple hits and lead series simultaneously. There was a saying at Merck at the time – any program worth doing required at least a “Scolnick unit” of medicinal chemists (1 S.U. = 20 chemists); we had several S.U. on the RAS program. Prominent leaders of key program elements during the early days, world-class scientists all, included Drs. A. Oliff, J. Gibbs, N. Kohl, K. Koblan, S. Graham, and G. Hartman, but Ed also kept a close eye on the program, which was simultaneously invigorating and terrifying. Ironically, one of the biggest concerns was to identify selective inhibitors of FPTase that did not inhibit the closely related geranylgeranyl protein transferases (GGPTase I and II), because so many proteins are geranylgeranylated, it was believed that the toxicity resulting from inhibiting GGPTase would be overwhelming. Progress was rapid, with proof of concept FPTase inhibitors quickly demonstrating anti-proliferative effects in v-ras-transformed cells3. Much of the early cell biology work was done with Harvey or viral RAS, because the critical CAAX boxes were identical, all of the RAS isoforms are actually farnesylated in cells, and there was just more experience handling these isoforms.

Validation of targeting FPTase soon emerged in in vivo models. Although I was working on different programs under Dr. Oliff, we all shared the RAS team’s joy when transgenic H-RAS tumors rapidly melted away when treated with an FPTase inhibitor. They grew back, but could be retreated and again regressed. It was early days for transgenic mouse models, but I’ve never seen tumors respond so dramatically – before or since4. We thought we were really on to something important for cancer patients5.

However, warning signs soon emerged. Although K-RAS-driven tumor cells responded to FPTase inhibitors, they were somewhat less sensitive than H-RAS transformed cells. In addition, biomarker assays demonstrated a prominent shift in the electrophoretic mobility of H-RAS, but not K-RAS, in FPTase-inhibitor treated cells. The cause of this perplexing behavior was soon discovered to be cross-prenylation of K- and N-RAS by GGPTase-I, which only happened to any significant extent in FPTase-inhibitor treated cells. This was announced to the broader scientific community in a flurry of papers from Schering-Plough, Eisai, and academics in 1997 (refs. 6-8).

Undaunted, the strategy at Merck (and other pharmas, I would imagine) quickly shifted to exploration of either combinations of FPTase and GGPTase-I inhibitors, or a compound which would inhibit both. Since K-RAS is cross-prenylated, and it is the dominant mutated RAS isoform in human cancer, efforts were made to try to dial in the perfect combination or dose of these combo inhibitors to hit K-RAS without inducing massive non-specific toxicity. We soon discovered another curiosity – the potency of some compounds against GGPTase-I was dramatically affected by assay conditions. This effect was discovered by a biochemist in my lab, Dr. H. Huber, and so we became directly involved to identify biochemical assay conditions which could accurately predict GGPTase-I inhibition in vivo9.

We quickly abandoned the concept of a fixed drug combination to inhibit both enzymes, as variations in metabolism of the two compounds would give wildly different results in different patients. Instead, we went forward with a dual FPTase/GGPTase inhibitor, L-778123. This compound had sufficient FPTase and GGPTase inhibitory activity to inhibit prenylation of corresponding substrates in vitro and in vivo, and induced anti-tumor effects in tumor cell lines harboring either form of RAS, with acceptable toxicity – at least in mice. The compound moved into clinical development.

As a unique dual inhibitor of prenyl transferases, L-778123 was used in several clinical trials. Biomarker studies demonstrated inhibition of prenylation of both farnesylated and geranylgeranylated substrates in blood, but not K-RAS in tumors. Dose limiting toxicities were identified, which included both thrombocytopenia and QTc prolongation. Unfortunately, no clinical responses were observed in monotherapy10. The compound was also combined with radiation therapy, and the results were somewhat more promising, but ultimately the development of the compound was terminated11,12.

So, after over a decade of effort, Merck’s work on directly targeting RAS came to a halt. Was the failure of L-778123 a failure of the compound to hit the target, or just a reflection of intractable biology? Is it worth another go? From my personal perspective – I think the question has been asked and answered. If drug candidates don’t work robustly preclinically, don’t even bother in the clinic. It’s conceptually feasible a different dual inhibitor or a combination of selective FPTase and GGPTase-I inhibitors would work against K-RAS driven tumors, but it’s not such a likelihood that I would spend a couple hundred million to test it.

Selective FPTase inhibitors do inhibit proliferation of K-RAS transformed cells – apparently, just not by inhibition of prenylation of K-RAS. Is there more opportunity here? Compounds at other companies met a similar fate to L-778123, although several selective FPTase inhibitors continue to be used as both tool compounds and for exploratory clinical studies13. As of today, I’m unaware of any significant FDA-approved indications for FPTase inhibitors.

Merck and other players in the FPTase field moved on to other targets in the RAS pathway, leading to discoveries of RAF and MEK kinase inhibitors, some of which have been used to good effect in the clinic and have illuminated new biology through selective inhibition of their targets. Now, the NCI’s RAS program is circling back to some new ways to target RAS directly, working in collaboration with Pharma, academia, and even some familiar faces from the old days. Three decades after its discovery, time to knock RAS out.

About the Author

David Heimbrook is the President of Leidos Biomedical Research, Inc., which operates the Frederick National Laboratory for Cancer Research for the National Cancer Institute. Dr. Heimbrook came to the FNLCR in 2011 from Hoffman-LaRoche, where he was head of Global Oncology Discovery.


  1. Shih, C., and Weinberg, R.A. Cell 29 161 (1982).
  2. Lowy, D.R., and Willumsen, B.M. Nature 341 384 (1989)
  3. Kohl, N., et al., Science 260 1934 (1993).
  4. Kohl, N.E., et al. Nature Medicine 1 792 (1995).
  5. Gibbs, J.B., Oliff, A., and Kohl, N.E. Cell 77 175 (1994).
  6. Zhang, F.L, et al., J. Biol. Chem. 272 10232 (1997).
  7. Rowell, C.A., et. Al, J. Biol. Chem 272 14093 (1997).
  8. Whyte, D.B., et. Al., J. Biol. Chem. 272 14459 (1997).
  9. Huber, H.A., et al., J. Biol. Chem. 276 24457 (2001).
  10. Britten, CD, et. al., Clin. Cancer Res. 7 3894 (2001)
  11. Hahn, S.M., et al., Clin. Cancer Res. 8 1064 (2002).
  12. Martin, N.E., et al. Clin. Cancer Res. 15 5447 (2004).
  13. Moorthy, N.S., Curr. Med. Chem. 20 4888 (2013).
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