Inhibitors of farnesylation of Ras from a microbial natural products screening program.

Mutant ras oncogenes are associated with various human tumors such as pancreas, colon, lung, thyroid, bladder and several types of leukemia. Prenylation of Ras proteins plays a major role in cell proliferation of both normal and cancerous cells. Normal and oncogenic Ras proteins are posttranslationally modified by a farnesyl group that promotes membrane binding. Inhibitors of farnesyl protein transferase (FPTase), the enzyme that catalyzes the prenylation of Ras proteins, inhibit growth of tumor cells. In an effort to identify structurally diverse and unique inhibitors of FPTase, a program devoted to screening of natural products was initiated. This effort led to the identification of 10 different families of compounds, all of which selectively inhibit FPTase with a variety of mechanisms that are reviewed in this manuscript. These compounds originated from the fermentations of a number of microorganisms, either actinomycetes or fungi, isolated from different substrates collected in tropical and temperate areas. A chemotaxonomic discussion on the distribution of each compound among single or different types of microorganisms, either phylogenetically related or unrelated species, is included.

Clavaric acid: a triterpenoid inhibitor of farnesyl-protein transferase from Clavariadelphus truncatus.

Farnesyl-protein transferase (FPTase) catalyses the specific transfer of farnesyl to Ras-peptides that is essential for oncogenic activity in oncogene-mediated tumors. Specific inhibition of FPTase activity has been shown to reduce tumor development in nude mice challenged with oncogenic forms of ras, thereby establishing FPTase as a viable therapeutic target. Our continued efforts to discover inhibitors of FPTase has led to the discovery of a triterpenoidal inhibitor, clavaric acid (1). This compound inhibits rHFPTase with an IC50 value of 1.3 microM. Structure elucidation, structure modifications, and biological activity of clavaric acid are herein described.

Clavaric acid and steroidal analogues as Ras- and FPP-directed inhibitors of human farnesyl-protein transferase.

We have identified a novel fungal metabolite that is an inhibitor of human farnesyl-protein transferase (FPTase) by randomly screening natural product extracts using a high-throughput biochemical assay. Clavaric acid [24, 25-dihydroxy-2-(3-hydroxy-3-methylglutaryl)lanostan-3-one] was isolated from Clavariadelphus truncatus; it specifically inhibits human FPTase (IC50 = 1.3 microM) and does not inhibit geranylgeranyl-protein transferase-I (GGPTase-I) or squalene synthase activity. It is competitive with respect to Ras and is a reversible inhibitor of FPTase. An alkaline hydrolysis product of clavaric acid, clavarinone [2,24,25-trihydroxylanostan-3-one], lacking the 3-hydroxy-3-methylglutaric acid side chain is less active as a FPTase inhibitor. Similarly, a methyl ester derivative of clavaric acid is also inactive. In Rat1 ras-transformed cells clavaric acid and lovastatin inhibited Ras processing without being overtly cytotoxic. Excess mevalonate reversed the effects of lovastatin but not of clavaric acid suggesting that the block on Ras processing by clavaric acid was due to inhibition of FPTase and not due to inhibition of HMG-CoA reductase. Despite these results, the possibility existed that clavaric acid inhibited Ras processing by directly inhibiting HMG-CoA reductase. To directly examine the effects of clavaric acid and clavarinone on HMG-CoA reductase, cholesterol synthesis was measured in HepG2 cells. No inhibition of HMG-CoA reductase was observed indicating that the inhibition of Ras processing by this class of compounds is due to inhibition of FPTase. To date, clavaric acid is the second reported nitrogen-free compound that competes with Ras to inhibit FPTase activity. A series of related compounds derived from computer-based similarity searches and subsequent rational chemical synthetic design provided compounds that exhibited a range of activity (0.04 --> 100 microM) against FPTase. Modest changes in the structures of these inhibitors dramatically change the inhibitory activity of these inhibitors.

Kampanols: novel Ras farnesyl-protein transferase inhibitors from Stachybotrys kampalensis.

Farnesyl-protein transferase (FPTase) is a critical enzyme that participates in the post-translational modification of the Ras protein. Inhibitors of this enzyme have the potential of being novel anticancer agents for tumors in which the ras oncogene is found mutated and contributes to cell transformation. Continued screening of natural product extracts led to the isolation of kampanols, which are novel and specific inhibitors of FPTase. The most active kampanols exhibited IC50 values between 7 to 13 microM against human recombinant FPTase. The isolation, structure determination, and biological activity of these compounds are described.

Oreganic acid, a potent inhibitor of ras farnesyl-protein transferase.

A sulfated tricarboxylic acid fungal metabolite is an inhibitor of human farnesyl-protein transferase (FPTase). The compound, designated as oreganic acid, has a molecular weight of 494, an empirical formula of C22H38O10S and inhibits FPTase with an IC50 value of 14 nM. Oreganic acid is a selective inhibitor of FPTase because it does not inhibit human geranylgeranyl-protein transferase type I (GGPTase-I). It is not a time-dependent inhibitor, reversibly inhibits FPTase, is competitive with respect to farnesyl diphosphate and non-competitive with respect to the Ras acceptor peptide. The structure of oreganic acid resembles that of farnesyl diphosphate and most likely inhibits FPTase by mimicking farnesyl diphosphate at the active site of the enzyme.

Barceloneic acid A, a new farnesyl-protein transferase inhibitor from a Phoma species.

Three new diphenyl ethers, barceloneic acids A, B, and barceloneic lactone [1, 2, and 3, respectively] were isolated from a fermentation extract of a fungus of the genus Phoma. The structures of compounds 1-3 were determined by a combination of spectroscopic and single-crystal X-ray diffraction methods. The effect of these compounds on the inhibition of farnesyl-protein transferase (FPTase) was evaluated and results are presented. Barceloneic acid A [1] is a novel and modest inhibitor of FPTase with an IC50 value of 40 microM.

L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. III. Biological activity.

L-696,474, an inhibitor of the HIV-1 protease, was discovered in extracts of the fungal culture Hypoxylon fragiforme (MF5511; ATCC 20995). L-696,474 is a novel cytochalasin with a molecular weight of 477 and an empirical formula of C30H39NO4. L-696,474 inhibited HIV-1 protease activity with an IC50 of 3 microM and the mode of inhibition was competitive with respect to substrate (apparent Ki = 1 microM). Furthermore, L-696,474 was not a slow-binding inhibitor. The inhibition due to L-696,474 was also independent of the HIV-1 protease concentration. L-696,474 was inactive against pepsin, another aspartyl protease; stromelysin, a zinc-metalloproteinase; papain, a cysteine-specific protease or human leucocyte elastase, a serine-specific protease. Two other novel cytochalasins (L-697,318 and L-696,475) isolated from the same culture were inactive against the HIV-1 protease. Commercially available cytochalasins B, C, D, E, F, H and J were inactive while cytochalasin A was as active as L-696,474 against the HIV-1 protease.