The complestatins as HIV-1 integrase inhibitors. Efficient isolation, structure elucidation, and inhibitory activities of isocomplestatin, chloropeptin I, new complestatins, A and B, and acid-hydrolysis products of chloropeptin I.

From the screening of a microbial extract library, isocomplestatin (1), a new axial-chiral isomer of complestatin (2) which is a known rigid bicyclic hexapeptide, was identified as a potent natural product inhibitor of HIV-1 integrase, a unique enzyme responsible for viral replication. Isocomplestatin showed inhibitory activities (IC(50)) in coupled 3'-end processing/strand transfer (200 nM), strand transfer (4 microM), and HIV-1 replication (200 nM) in virus-infected cells. Attempted large-scale isolation of 1 by the literature method, used for the isolation of complestatin, led to lower yield and limited availability. We have developed several new, two-step, high-yielding absorption/elution methods of isolation based on reverse-phase chromatography at pH 8 that are applicable to scales from one gram to potential industrial quantities. We have also discovered and determined the structure of two new congeners of 1, namely, complestatins A (4) and B (5), with almost equal HIV-1 integrase activity. They differ from 1 at C2' and C3' of the tryptophan moiety (residue F). Selective acid hydrolysis of chloropeptin I (3), itself a known acid-catalyzed rearranged isomer of 1 and 2 (8'- vs 7'-substitution in tryptophan residue F, respectively), an isomer of complestatin, and isocomplestatin resulted in a number of fragments (6-10) with retention of most of the HIV-1 integrase activity. The structure-activity relationship as revealed by these compounds could possibly lead to the design of better inhibitors or understanding of the HIV-1 integrase target.

Efficient syntheses, human and yeast farnesyl-protein transferase inhibitory activities of chaetomellic acids and analogues.

Chaetomellic acids are a class of alkyl dicarboxylic acids that were isolated from Chaetomella acutiseta. They are potent and highly specific farnesyl-pyrophosphate (FPP) mimic inhibitors of Ras farnesyl-protein transferase. We have previously described the first biogenetic type aldol condensation-based total synthesis of chaetomellic acid A. Modification of the later steps of that synthesis resulted in the efficient syntheses of chaetomellic acids A and B in three steps with 75-80% overall yield. In this report, details of the original total syntheses of chaetomellic acids A, B and C, the new syntheses of acids A and B and structure-activity relationship of these compounds against various prenyl transferases including human and yeast FPTase and bovine and yeast GGPTase I are described. Chaetomellic acids are differentially active against human and yeast FPTase. Chaetomellic acid A inhibited human and yeast FPTase activity with IC50 values of 55 nM and 225 microM, respectively. In contrast, chaetomellic acid C showed only a 10-fold differential in inhibitory activities against human versus yeast enzymes. In keeping with molecular modeling-based predictions, the compounds with shorter alkyl side chains (C-8) were completely inactive against FPTase.

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.

Resorcylic acid lactones: naturally occurring potent and selective inhibitors of MEK.

A resorcylic acid lactone, L-783,277, isolated from a Phoma sp. (ATCC 74403) which came from the fruitbody of Helvella acetabulum, is a potent and specific inhibitor of MEK (Map kinase kinase). L-783,277 inhibits MEK with an IC50 value of 4 nM. It weakly inhibits Lck and is inactive against Raf, PKA and PKC. L-783,277 is an irreversible inhibitor of MEK and is competitive with respect to ATP. L-783,290, the trans-isomer of L-783,277, was isolated from the same culture and evaluated together with several semi-synthetic resorcylic acid lactone analogs. A preliminary structure-activity relationship is presented. Several independent cell-based assays have been carried out to study the biological activities of these resorcylic acid lactone compounds and a brief result summary from these studies is presented.

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.

Quinoxapeptins: novel chromodepsipeptide inhibitors of HIV-1 and HIV-2 reverse transcriptase. I. The producing organism and biological activity.

Quinoxapeptin A and B are novel chromodepsipeptides which were isolated from a nocardioform actinomycete with indeterminant morphology. Quinoxapeptins A and B are potent inhibitors of HIV-1 and HIV-2 reverse transcriptase and almost equally active against two single mutants forms as well as a double mutant form of HIV-1 reverse transcriptase. Quinoxapeptin A and B are specific inhibitors of HIV-1 and HIV-2 reverse transcriptase because they did not inhibit human DNA polymerase alpha, beta, gamma and delta. Quinoxapeptin A and B are structurally similar to luzopeptin A which was also active against HIV-1 and HIV-2 reverse transcriptase.

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.

Chaetomella acutiseta produces chaetomellic acids A and B which are reversible inhibitors of farnesyl-protein transferase.

Chaetomellic acids A and B, isolated from Chaetomella acutiseta, are specific inhibitors of farnesyl-protein transferase that do not inhibit geranylgeranyl transferase type 1 or squalene synthase. Chaetomellic acids A and B are reversible inhibitors, resemble farnesyl diphosphate and probably inhibit FPTase by substituting for farnesyl diphosphate. Chaetomellic acid production appears to be widespread within the genus Chaetomella.

Microbial transformation of L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease.

The microbiological transformation of L-696,474 [1], a novel cytochalasin that is an inhibitor of HIV-1 protease, was investigated using Actinoplanes sp. ATCC 53771. Six hydroxylated metabolites 2-7 of 1 were isolated and purified using reversed-phase hplc. All six metabolites were found to have undergone hydroxylation at the C-16 methyl group (C-22) of 1. Three of the compounds, 3, 4, and 5, were further hydroxylated at the para (C-29), the meta (C-28), and both the para and the meta, positions of the phenyl ring, respectively. Metabolites 6 and 7 were shown to result from vicinal dihydroxylation on both C-16 and its attached Me (C-22). The metabolite 7 was further hydroxylated on the meta position of the phenyl ring. The structures of the metabolites were established using spectroscopic techniques including ms, 1H nmr, 13C nmr, and various 2D nmr spectroscopy experiments.

Selective inhibition of farnesyl-protein transferase blocks ras processing in vivo.

The ras oncogene product, Ras, is synthesized in vivo as a precursor protein that requires post-translational processing to become biologically active and to be capable of transforming mammalian cells. Farnesylation appears to be a critical modification of Ras, and thus inhibitors of the farnesyl-protein transferase (FPTase) that catalyzes this reaction may block ras-dependent tumorigenesis. Three structural classes of FPTase inhibitors were identified: (alpha-hydroxyfarnesyl)phosphonic acid, chaetomellic acids, and zaragozic acids. By comparison, these compounds were weaker inhibitors of geranylgeranyl-protein transferases. Each of these inhibitors was competitive with respect to farnesyl diphosphate in the FPTase reaction. All compounds were assayed for inhibition of Ras processing in Ha-ras-transformed NIH3T3 fibroblasts. Ras processing was inhibited by 1 microM (alpha-hydroxyfarnesyl)phosphonic acid. Neither chaetomellic acid nor zaragozic acid were active in this assay. These results are the first demonstration that a small organic chemical selected for inhibition of FPTase can inhibit Ras processing in vivo.

L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. II. Isolation and structure.

A novel HIV-1 protease inhibitor, L-696,474 (C30H39NO4, 477), was isolated from the fermentations of the fungus Hypoxylon fragiforme (ATCC 20995, MF5511) and purified by silica gel chromatography followed by crystallization. Spectroscopic studies have shown the competitive inhibitor L-696,474 to be a novel cytochalasin. Two related novel cytochalasins were also isolated and had no effect on the enzyme.

L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. I. The producing organism and its fermentation.

A novel cytochalasin, L-696,474, (18-dehydroxy cytochalasin H) that inhibits HIV-1 protease was discovered in fermentations of a bark-inhabiting Ascomycete, Hypoxylon fragiforme. The product was first identified from extracts of an agar medium. Fermentation studies on a number of media indicated that the product can be made on several solid and liquid media. Optimum production was obtained from growth in a complex medium composed of glycerol, glucose, citrate, Ardamine, soybean meal, tomato paste, and inorganic salts. Other Hypoxylon spp., related species of Xylariales, and other fungi known to produce cytochalasins, were also surveyed for their ability to make L-696,474. Only one other Hypoxylon fragiforme isolate was found to make this novel cytochalasin; none of the other cultures surveyed made L-696,474 or any other compounds which inhibit HIV-1 protease.

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.

HIV-1 protease inhibitory activity of L-694,746, a novel metabolite of L-689,502.

L-689,502 is a potent inhibitor of HIV-1 protease activity in vitro. Microbial biotransformations of L-689,502 by cultures belonging to the genus Streptomyces sp. were performed. Extracts of culture broths were examined for the production of metabolites of L-689,502 that could inhibit HIV-1 protease activity. One culture, MA 6804 (Streptomyces lavendulae, ATCC 55095), produced L-694,746 that, while being structurally related to L-689,502, is a novel metabolite and a potent inhibitor of HIV-1 protease.

Prenatal ethanol exposure impairs lesion-induced plasticity in a dopaminergic synapse after maturity.

This study examined the consequences of alcohol (ethanol) exposure during fetal life on lesion-induced dopaminergic synapse responsiveness (plasticity) in the olfactory tubercle of the adult rat. Normally, in the olfactory tubercle, olfactory bulbectomy elicits alterations in pre- and postsynaptic dopaminergic markers, including, respectively, (1) increased tyrosine hydroxylase activity and immunoreactivity, which is associated with dopaminergic axon sprouting, and (2) increased dopaminergic receptor density and potentiated dopamine activation of adenylate cyclase. We have utilized biochemical and quantitative immunocytochemical methodology to examine these synaptic markers in olfactory bulbectomized or sham-operated adult rats. These animals were offspring of dams which were administered one of the following diets during pregnancy: (1) liquid diet containing 35% ethanol-derived calories ad libitum; (2) liquid diet containing an isocaloric amount of maltose-dextrin instead of ethanol, pair-fed; or (3) unaltered liquid diet ad libitum. The results show that prenatal alcohol exposure leads to suppression of the lesion-elicited dopaminergic synapse responsiveness in the olfactory tubercle. There were no significant differences between offspring born to control and pair-fed animals, indicating that the observed abnormalities were not due to alterations in their nutritional status. In conclusion, the present data are a biochemical and quantitative immunocytochemical demonstration of impaired lesion-induced synaptic responsiveness. This renders a new dimension in support of previous evidence indicating that prenatal alcohol exposure leads to altered neuroanatomical, neuroendocrinological and behavioral responsiveness to various challenges. Such impaired synaptic responsiveness may underlie brain functional abnormalities characteristic of fetal alcohol syndrome.

Estrogen regulation of epidermal growth factor receptor messenger ribonucleic acid.

Previous studies have demonstrated that 17 beta-estradiol (E2) causes a 3-fold increase in epidermal growth factor (EGF) receptors in uterine membranes. We now report that the increase in uterine EGF receptor levels is due to an increase in the steady-state levels of EGF receptor mRNA. After a single E2 injection, EGF receptor mRNA levels, as determined by RNA blots, increase 3- to 4-fold between 1 and 3 h, remain elevated at 6 h, and decline between 12 and 18 h. The effect is specific for E2 since the nonestrogenic hormones progesterone, dexamethasone, 5 alpha-dihydrotestosterone, and the inactive stereoisomer of E2, 17 alpha-estradiol, are without effect. E2-Mediated increases in EGF receptor mRNA levels are blocked by actinomycin D but not by puromycin. Taken together, these results indicate that E2 regulates the level of EGF receptor by increasing the steady-state concentration of EGF receptor mRNA in vivo.