AB - 2-Hydroxyestradiol (2-OH-E2) stimulates progestin secretion by granulosa cells, but the intracellular locus of the stimulatory effect has not been clarified. The objectives of the present studies were to 1) determine the role of de novo sterol synthesis in the effect of 2-OH-E2 on progestin biosynthesis, and 2) examine the effects of 2-OH-E2 on cholesterol side-chain cleavage (SCC) activity and the level of messenger RNA (mRNA) for P450scc. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase with lovastatin (5 μg/ml) or mevinolin (5 μg/ml) reduced FSH- and 2-OH-E2-stimulated (but not E2-stimulated) progesterone production. Mevalonate (20 mM) enhanced basal progesterone production and reversed the inhib-itory effect of lovastatin but did not affect progesterone biosynthesis in the presence of 2-OH-E2. As an index of the activity of cholesterol SCC enzyme, granulosa cells were exposed to 25-hydroxycholesterol (10 μg/ml) for 24 h and progesterone secretion monitored. Conversion of 25-hydroxycholesterol into progesterone was stimulated 2- to 3-fold by maximally effective concentrations of 2-OH-E2, E2, and FSH. 2-OH-E2 and/or E2 further enhanced 25-hydroxycholesterol conversion in the presence of FSH, LH, and epinephrine. Aminoglutethimide, an inhibitor of SCC, reduced 2-OH-E2- and 2-OH-E2 plus FSH-stimulated progesterone production by 97% and 95%, respectively. 2-OH-E2 also increased basal (by 2 to 3-fold) and FSH-stimulated (to 3.5-fold of FSH-treated controls) levels of mRNA for cytochrome P450scc. Collectively, our studies support the hypothesis that 2-OH-E2-enhanced progesterone biosynthesis by porcine granulosa cells is dependent on de novo cholesterol synthesis and is associated with increased levels of the mRNA encoding cytochrome P-450scc, which leads to increases in basal and gonadotropin-induced SCC activity.
DMIs are a commercially very successful group of systemic fungicides targeting cell membrane integrity by inhibiting C14 demethylation during sterol formation. Regardless of their site-specific mode of action, resistance development evolved in a typical stepwise manner leading to a prolonged effectiveness for more than four decades and rendering those fungicides a unique paradigm. A number of resistance mechanisms acting individually or in combination have been identified over the years to be associated with decreased sensitivity to these compounds in certain pathogen populations in the field including target-site modification, target gene (cyp51) overexpression, increased efflux, and multiple paralogues of the target gene. Various mutations in the coding region of the cyp51 gene usually confer different levels or no resistance to different members of DMIs contrary to ones leading to target gene overexpression which decrease the sensitivity of isolates to all members of the DMI group. Increased efflux mediated by drug transporters belonging to the ABC or MFS transporter families results in resistance to all DMI members as well as to other unrelated fungicides. Pleiotropic effects of resistance mutations on fitness parameters, variable levels, and lack of cross-resistance between members of the DMI group probably account for the delayed resistance development and can be partly attributed to a polygenic control of resistance. These unique characteristics and the large number of registered members make DMI indispensable components of control programs against important plant pathogens.
Cholesterol is present in calf serum added to the culture medium and is also abundant in the lung surfactant that lines the mammalian alveolus. Although P. carinii can scavenge cholesterol from its environment and incorporates it into new membranes, this sterol alone does not appear to be sufficient for maintaining cell viability. The experiments performed in this study demonstrate that the specific steps in sterol biosynthesis are necessary to maintain the integrity of the organisms and that their disruption leads to the loss of organism viability. The distinct 24-alkylsterols identified in the organism (, , , ) apparently fulfill the precise structural requirements for proper functioning of specific dynamic membrane microdomains. The cholesterol structure evidently cannot substitute at these sites. Thus, despite the presence of abundant cholesterol, which it can and does scavenge from the host or culture medium, the pathogen must synthesize its own distinctive sterol components. Hence, the sterols that are synthesized de novo by P. carinii can be considered the organism's metabolic sterols (). The results of this study indicate that reactions in sterol biosynthesis are attractive drug targets for treatment of P. carinii infections. Cotreatment with compounds targeting other metabolic pathways would seem a rational approach for PCP therapy.
The inhibitors of the reaction producing mevalonate had little or no adverse effect on P. carinii cellular ATP levels. In light of the effectiveness of other inhibitors of sterol biosynthesis in reducing organism viability, these results raise the question of whether P. carinii may have an operational Rohmer-Arigoni (pyruvate–glyceraldehyde 3-phosphate or 1-deoxy--xylulose 5-phosphate [DOXP]) pathway by which isopentenyl diphosphate is synthesized (, ). This suggestion is consistent with the observation that lovastatin failed to enhance the incorporation of exogenous radiolabeled mevalonate into P. carinii isoprenoid products (D. Sul and E. S. Kaneshiro, unpublished data), unlike its effect in many other cell types with only the acetate-mevalonate pathway (). Also, it has been reported that the P. carinii HMG-CoA reductase activity is sensitive to lovastatin (IC50 = 4 nM) (). However, the enzyme activity in this organism is only 100 pmol/mg of total P. carinii protein/min (), which is about a 1,000 times less than that found in other cell types such as rat liver (130 nmol/mg of total liver homogenate protein/min) (). The other drugs tested in the present study that are known to inhibit sterol biosynthesis are targeted to reactions subsequent to the formation of isopentenyl diphosphate. Thus, if isopentenyl units are synthesized via the alternative DOXP pathway in P. carinii, this could explain how the organism can continue synthesizing sterols and maintaining its cellular ATP pool with a block in the acetate-mevalonate pathway. The DOXP pathway may coexist with the acetate-mevalonate pathway as found in Scenedesmus obliquus (). Such a mevalonate-independent pathway was recently identified in Plasmodium falciparum () and was shown to provide a novel drug target for these parasites. Treatment of malarial parasites in vitro with fosmidomycin and FR-900098 showed reductions in viability as well as inhibition of the target enzyme, DOXP reductase. Fosmidomycin was well tolerated by human volunteers and patients with bacterial infections. The putative existence of the nonmevalonate DOXP pathway opens new potential chemotherapeutic strategies against P. carinii infections ().
Polyene antibiotics do not inhibit sterol biosynthesis but bind to sterols within biomembranes. The sterol-drug complexes aggregate and form large pores in the membranes, leading to collapse of chemical gradients and cell death. Amphotericin B, which is used clinically for deeply invasive mycosis, has a higher affinity for ergosterol (in fungal membranes) than for cholesterol (in mammalian-cell membranes). However, at sufficient concentrations, it will bind cholesterol and other membrane sterols as well. The effects on P. carinii ATP levels demonstrate a dose-dependent adverse effect of amphotericin B on the viability of the pathogen (Fig. ; Table ) at moderate levels from 24 to 48 h of exposure and at marked levels after 72 h.
Squalestatin, GR 105155X, belongs to a group of complex fungal metabolites named squalestatins by Glaxo and zaragosic acids by Merck (). Squalestatins inhibit squalene synthase activity at nanomolar concentrations and display broad-spectrum antifungal activity (), but even at 100 μg/ml GR 105155X had no effect on P. carinii viability. There are now a number of squalestatin and zaragosic acid derivatives, and it is known that compounds with similar enzyme-inhibitory activities can have very different effects in lowering sterol biosynthesis. Thus, it is possible that some derivatives of squalestatin not tested in this survey may be effective in reducing P. carinii viability.
In organisms that produce 24-alkylsterols, the addition of alkyl groups to the C-24 position of the sterol side chain is catalyzed by one or two methyltransferase reactions, which commonly occur after demethylation of the lanosterol nucleus. Of the putative SAM:SMT inhibitors tested, only 24(25)-epiminolanosterol exhibited any ability to reduce P. carinii ATP levels, and only at the highest concentration used (Table ). This compound also exhibited moderate inhibition of 24-alkylsterol biosynthesis in P. carinii and of proliferation of the organism in short-term cultures ().
In most organisms, the double bond in the lanosterol nucleus at C-8 is reduced and the bond at C-7 is desaturated. However, the sterols that eventually accumulate in fungi, plants, and animals are dominated by those with a double bond at C-5. Pneumocystis is unusual in that the sterols that accumulate in the organism are mainly those with a C-7 instead of a C-5 double bond. The only Δ8 to Δ7 isomerase inhibitor tested in this study was AY 9944, which exhibited dose- and time-dependent activity and had moderate activity against P. carinii ATP pools (Fig. ; Table ).
In most organisms, three methyl groups are removed from the lanosterol nucleus (two at C-4 and one at C-14), the double bond at C-8 is removed, and a double bond at C-5 is added. In some organisms, the double bond at C-24 of lanosterol is also reduced (e.g., cholesterol synthesis in mammals). In most organisms, lanosterol is the preferred substrate for enzymes, including the C-14 cytochrome P-540-dependent lanosterol demethylase activity. However, the methyl groups at C-4 and C-14 of sterol molecules other than lanosterol can be removed. Interestingly, the activity of lanosterol demethylase inhibitors tested in this study can be placed in two general groups. Two imidazoles (GR 40317A and GR 42539X) were moderately effective against P. carinii (Fig. ; Table ), whereas the triazoles, including fluconazole, were not effective. The results for fluconazole are consistent with the report that this drug was ineffective in clearing PCP in experimental animals and in inhibiting proliferation of short-term primary cultures of the organism ().
In contrast to GR 105155X, both the biphenyl and naphthalene alkylamine derivatives CCI 16543 and CCI 14993 exhibited time- and dose-dependent effects in reducing cellular ATP levels in this organism. These results indicate that the inhibition of sterol biosynthesis by blocking squalene synthase activity leads to a loss of P. carinii viability.
Squalene epoxide is converted to lanosterol (C30) by a cyclase enzyme and the migration of two methyl groups during the closure of rings in the molecule. Drugs tested within this category of inhibitors were among the most effective in reducing ATP levels of P. carinii populations. All five inhibitors tested showed mostly moderate activity in reducing the cellular ATP contents of P. carinii populations (Fig. ; Table ). After 3 days of exposure to GR 90525A, marked activity was observed, as indicated by the low IC50.