Sterols synthesis inhibitors

Mevalonate ((118), Figure 6.27) is a key intermediate, and mevalonate kinase is a key early enzyme, in isoprenoid and sterol synthesis. Inhibitors of this enzyme have potential applications for treatment of cardiovascular disease and cancer. Mevalonate kinase activity is controlled post-transcriptionally via competitive inhibition at the ATP site by prenyl phosphates, such as geranyl diphosphate (119). A bifunctional inhibitor with micromolar IC50 values against mevalonate kinase and mevalonate 5-diphosphate decarboxylase (a... 

Figure 5. Structures of sterol synthesis inhibitors used and arranged to indicate some structural relationships.

Ergosterol is the major sterol. It decreases in treated cultures while obtusifoliol and 24-methylene-dihydrolanosterol accumulates. This shows that hexaconazole works as a sterol synthesis inhibitor. 

FIGURE 2 Examples of the variety of structures obtained in natural product screens. I, zaragozic acid A, is an inhibitor of mammalian and fungal sterol synthesis, obtained from fungi (48) II, L-696,474, is an inhibitor of the HIV protease, obtained from fungi (51) III, dehydrosoyasaponin I, is an agonist of the calcium-activated potassium channel, obtained from a medicinal plant (58) IV, tetrandrine, is an inhibitor of L-type calcium channels, obtained from a plant (78). 

In research for new fungicides and antimycotics, one has to look for a concept for pathogen-specific inhibitors. This means that inhibition of sterol synthesis should not take place at any common step the aim is to only inhibit pathogen-specific steps in biosynthesis. The reason for this is to minimize the risk of human toxicity. If one compares the biosynthesis of mammalian cholesterol to that of ergosterol, the main sterol of pathogenic fungi, it becomes obvious that there are at least four pathogen-specific steps to be inhibited. 

Another target, that at first seems to be unfavorable since it is principally common for all organisms, is the enzyme HMG-CoA-reduc-tase which is the regulatory enzyme in terpenoid biosynthesis. Results from trials with naturally produced inhibitors for that enzyme, such as Compactine and Mevinoline, indicate that these compounds are able to lower the cholesterol content in mammals, but not markedly depress sterol synthesis in fungi U3). 

Research groups in Japan [1] have screened over 8000 microbial extracts for their ability to produce an inhibitor of sterol synthesis in vitro. These studies led to the isolation of mevastatin from cultures of Penicillium citrinum. Mevastatin (Fig. 4.2) served as the lead-molecule for seven statins which currently are among the best-selling drugs. 

Herz, J. E., Swaminathan, S., Pinkerton, F. D., Wilson, W. K., Schroepfer, G. J., Jr. Inhibitors of sterol synthesis. A highly efficient and specific side-chain oxidation of 3 beta-acetoxy-5 alpha-cholest-8(14)-en-15-one for construction of metabohtes and analogs of the 15-ketosterol. J. Lipid Res. 1992, 55(4), 579-598. 

HMG-CoA reductase is the rate-determining enzyme of sterol synthesis, and its activity is regulated by competitive inhibition by compounds that bind to the same site as HMG-CoA. It is also regulated by substances that bind to other (allosteric) sites on the enzyme molecule. Inhibitors of this enzyme (e.g., simvastatin) are used as medicines to reduce cholesterol in patients whose cholesterol levels are too high. Through feedback inhibition, cholesterol is a strong inhibitor of the enzyme itself. No fungicides with this mode of action have yet been developed, but the possibility that they will be exists. 

The synthesis of fatty acids and sterols in the liver cytosol depends upon a common pool of acetyl-CoA. This was demonstrated by Decker and Barth in a series of experiments utilizing perfused rat liver [10]. Lipid synthesis was measured by incorporation of tritium from [ H]H20. They used (- )-hydroxycitrate to inhibit ATP-dependent citrate lyase and measured radioisotope incorporation into fatty acids and sterols as a function of the concentration of this inhibitor. A parallel decrease in incorporation into these two products was found as the concentration of (- )-hydroxycitrate in the perfusate was increased. Contrastingly, if radioisotopic acetate was used as the substrate in the perfusing medium, this inhibitor had relatively little effect on the rate of sterologenesis, a result that would be expected if the natural source of acetate was from the action of the cytoplasmic citrate lyase. Their experiments also demonstrated that the ratio of fatty acid synthesis to sterol synthesis in the liver of fed rats is about 10 1. 

Experimentally, the cholesterol content of cell surface membranes may be modified in vivo e.g. in the guinea pig by dietary means [131] or by modification of the culture media of micro-organisms [104]. In vitro, cholesterol modulation has also been achieved by the use of inhibitors of sterol synthesis [95] or liposomes [97,103,132-134]. 

Recently Kerkenaar and Kaars Sijpesteijn (1979) demonstrated that inhibition of respiration cannot be the primary cause of growth inhibition, and that the action of tridemorph is similar to that of known sterol biosynthesis inhibitors. Their study seems to favour in lipid biosynthesis, possibly ergosterol biosynthesis, as a primary mode of action of tridemorph, and would agree with the rather late interference of this compound with protein and RNA synthesis (Kerkenaar et al., 1979, 1981). 

Recent data indicate that ezetimibe inhibits a specific transport process in jejunal enterocytes, which take up cholesterol from the lumen. The putative transport protein is Niemann-Pick Cl-hke 1 protein (NPCILI). In wild-type mice, ezetimibe inhibits cholesterol absorption by about 70% in NPCILI knockout mice, cholesterol absorption is 86% lower than in wild-type mice, and ezetimibe has no effect on cholesterol absorption. Ezetimibe does not affect intestinal triglyceride absorption. In human subjects, ezetimibe reduced cholesterol absorption by 54%, precipitating a compensatory increase in cholesterol synthesis, which can be inhibited with a cholesterol synthesis inhibitor such as a statin. There is also a substantial reduction of plasma levels of plant sterols (campesterol and sitosterol concentrations are reduced by 48 and 41%, respectively), indicating that ezetimibe also inhibits intestinal absorption of plant sterols. 

However, there are other changes. There is an increase in sterol (chiefly ergosterol) and a shift in the relative proportions of carotenoids (8). These effects are prevented by the protein synthesis inhibitor cycloheximide (9). Since carotenoids and sterols share a common biosynthesis to farnesyl pyrc hosphate, the hormones must act by derepressing the synthesis of enzjnnes early in this pathway. Preliminary experiments have shown that mevalonate kinase activities are unaffected by trisporic acid, indicating that this is Unlikely to be the limiting enzjnae a more likely candidate is 3-hydroxy-3 ethylglutaryl CoA reductase. 

Tsujita Y, Kuroda M, Shimada Y, Tanzawa K, Aral M, et aL CS-SH. a competitive inhibitor of 3-hydroxy 3-methyl Jutaryl coenzyme A reductase Tissuc-selective inhibition of sterol synthesis and hypolipidemic effect cm various animal lecies. Biochim Biophys Acta 1986 ... 

In order to get experimental veiification of this proposal and also to interfere with sterol synthesis, we designed, first synthesized and evaluated novel azasteroid analogs of the presumptive carbocationic intermediate involved in the A -SR mechanism, based on the transition-state analog concept. These novel compounds were shown to be very powerful inhibitors of A -SR in vitro in a maize microsomal preparation (Figure 1)... 

Finally, this study yielded compounds which ai e powerful inhibitor of A -SR and of sterol synthesis, thus supporting the validity of our approach. In addition to their value to explore and manipulate sterol synthesis in plants, the novel compcunds described herein could be of pharmacological interest as inhibitors of A -SR in cholesterol synthesis in animals. 

Modulation of epidermal lipid biosynthesis has been reported to boost drag delivery. It has also been suggested that it is both the hydrophobic nature of the lipids as well as their tortuous, extracellular localization that are responsible for the restriction in the transport of most molecules across the stratum comeum. The function of this barrier depends on three key lipids cholesterol, fatty acid, or ceramides. Delays of synthesis ceramides in the epidermis have been reported as means of barrier perturbation. Inhibitors of lipid synthesis were used to enhance the trans-A cmaV dehvery of hdocaine or caffeine. Alteration of barrier function was produced by the fatty acid synthesis inhibitor S-(tetradecyloxy)-2-furancarboxylic acid, the cholesterol synthesis inhibitor fluvastatin, or the cholesterol sulfate, which resulted in a further increase in lidocaine absorption (38). The major components of sebaceous lipids in the skin are 45-60% TAGs, 25% wax and sterol esters, 12-15% squalene and 10% free fatty acids (39). Some fatty acids, especially unsaturated fatty acids, are well-known skin penetration enhancers. The addition of PC to dermal dosage forms has been reported to increase percutaneous absorption. Lipid disperse systems (LDSs) containing polar lipids, such as PC and glycosylceramide, are also useful for... 

The phase partition between polymers, in its version adapted to fungal material, is a very convenient and safe methcxi for the preparation of pure plasma membrane fractions.The lipids of Altemaria brassicicola, despite the lack of any form of linolenic acid, can be characterized by their high degree of unsaturation, even in the total lipid. This unsaturation can be increased, inside the DPG/PA fraction by the use of hexaconazole, which acts as an inhibitor of sterols synthesis. 

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