demonstrated that nearly allphosphatidylserine and a minimum of 70% of phosphatidylethanolamine is on theinside surface of the human erythrocyte membrane, thus "presenting a strongevidence for an asymmetric arrangement of phospholipids" ().
First demonstration of an inhibition of sterol biosynthesis (HMG-CoA reductaseactivity) by oxygenated derivatives of cholesterol in cultured mammalian cells ().
For the first time, the existence of polyunsaturated fatty acids was reported inmarine bacteria ().
1961, 83, 3080) made the first total synthesis of arachidonic acid.
Imai J demonstrated that the oxidative desaturation of a saturated fatty acid (palmitic acid) is depressed in diabetic rats ().
Kennedy EP described the general pathways of the glycerolipid biosynthesis in animal cells ().
Bremer J et al.
Compared to bacterial infections, few drugs are available with which to treat fungal infections. This is largely attributable to the eukaryotic nature of fungal cells and the difficulty in identifying unique targets not shared with human hosts. Most therapies designed to treat fungal infections target the ergosterol biosynthetic pathway or its final product, ergosterol, a sterol cell membrane component that is unique to fungi (Fig. ). The most commonly used drug in both the treatment and prevention of candidiasis is fluconazole, a member of the azole family of drugs that targets the essential enzyme Erg11, lanosterol 14α-demethylase, in the ergosterol biosynthetic pathway (Fig. ) (, ). The standard therapy for the treatment of fluconazole-resistant fungal infections is amphotericin B, which binds ergosterol and permeabilizes the plasma membrane. These treatments are extremely effective against C. albicans, but resistant Candida strains and species are emerging and new treatments for systemic infections need to be developed (; reviewed in reference ). A particular difficulty with azole treatment is the inherent resistance, or rapid development of resistance, found in several non-C. albicans Candida species, such as C. glabrata and C. krusei (, , , , , , , , ). Therefore, there exists a clear demand for more effective treatment of infections caused by these emerging fungal pathogens.
Several classes of drugs have been developed that target enzymes other than Erg11 in the ergosterol biosynthetic pathway. Morpholines and allylamines inhibit specific ergosterol biosynthetic enzymes. For example, terbinafine is in the allylamine class of drugs and targets the enzyme squalene epoxidase (Erg1) (Fig. ) (). Terbinafine, available in both oral and topical forms as Lamisil, is used in the treatment of tinea pedis and onychomycosis but has not been effective as monotherapy for systemic infections. Fenpropimorph is a morpholine that targets two enzymes in the ergosterol biosynthetic pathway, sterol C8-C7 isomerase (Erg2) and sterol reductase (Erg24), and is used as a fungicide in agriculture (Fig. ) (). Fenpropimorph, like many other morpholines, mainly targets Erg2 but has been shown to inhibit Erg24 as well at higher concentrations (). Amorolfine is currently the only morpholine in clinical use and is only available in topical forms, primarily for the treatment of onychomycosis. Recent results have indicated that Erg24 is required for virulence of C. albicans in a murine model of infection (). However, to date, no drugs of the morpholine class have been developed for oral therapy in humans.
One drawback of azole drugs is that they are fungistatic rather than fungicidal. This characteristic probably contributes to the development of resistance seen in clinical isolates from immunocompromised patients. Since the cells are allowed to persist and immune function is not sufficient to clear residual fungal cells, a positive selection for drug-resistant mutants is established. A fungicidal drug with low toxicity would be the ideal treatment for these patients, but such therapy does not exist. It has recently been shown, however, that the calcineurin inhibitors cyclosporine A (CsA) and FK506 exhibit a potent fungicidal synergism with the azole class of drugs against C. albicans (, , , ). These results stimulated our interest in determining whether additional drugs targeting the ergosterol biosynthetic pathway also exhibit fungicidal synergism with calcineurin inhibitors, thus improving and expanding their antifungal properties. Here we show that both terbinafine and fenpropimorph exhibit a potent fungicidal synergism with calcineurin inhibitors in C. albicans, and we present evidence that L-685,818, a nonimmunosuppressive analog of FK506, can also participate in this drug synergy. Additionally, we establish that the molecular basis of synergism with FK506 involves inhibition of calcineurin by the FKBP12-FK506 complex. Importantly, we also illustrate that these synergistic drug combinations are effective against C. glabrata and C. krusei in vitro, which we previously demonstrated to be largely insensitive to the synergism between azoles and calcineurin inhibitors ().
For example, in fluconazole-susceptible C. albicans fluconazole only partially inhibits ergosterol and completely blocks obtusifoliol synthesis, whereas voriconazole completely inhibits both ergosterol and obtusifoliol synthesis 5. Itraconazole and fluconazole may also inhibit 3-ketoreductase, which catalyzes the reduction of the 3-ketosteroid obtusifolione to obtusifoliol in C. neoformans 6.
Fungicidal synergism between azoles and calcineurin inhibitors has previously been demonstrated in C. albicans (, , , ). We hypothesized that this synergy might also exist between calcineurin inhibitors and other antifungal drugs that inhibit ergosterol biosynthesis in C. albicans. To determine whether synergism between calcineurin inhibitors and fenpropimorph or terbinafine occurs, we employed disk diffusion halo assays. Wild-type C. albicans strain SC5314 was grown in the presence or absence of FK506 or CsA with either fenpropimorph or terbinafine (Fig. ). Fenpropimorph or terbinafine alone exhibited only modest growth inhibition. As previously demonstrated by Cruz et al. (), neither FK506 nor CsA alone exhibited any in vitro growth inhibition of the Candida strains used in our study (see Fig. ; data not shown).
We used FK506-resistant C. albicans strains (Table ) to demonstrate that the loss of calcineurin activity, and not a secondary effect of CsA and FK506, underlies their synergy with ergosterol biosynthesis inhibitors. The CNB1-1/CNB1 strain contains a two-amino-acid insertion that perturbs the FKBP12-FK506 binding site of the calcineurin B subunit, while the rbp1/rbp1 mutant lacks the FK506 binding protein FKBP12 (, ). FKBP12 is required for FK506 activity against calcineurin. FK506 must first complex with FKBP12 in order to bind and inhibit calcineurin, whereas CsA acts by first binding to cyclophilin A (, , -, ). The CNB1-1/CNB1 mutation prevents FKBP12-FK506 binding to calcineurin but has no effect on cyclophilin A-CsA binding (). The CNB1-1/CNB1 and rbp1/rbp1 mutant strains were modestly inhibited by fenpropimorph or terbinafine alone, and addition of FK506 had no further effect on growth inhibition, as determined by halo assay and the MICs and FICs based on NCCLS criteria (Fig. and Table ). When the CNB1-1/CNB1 and rbp1/rbp1 mutants were exposed to CsA in combination with fenpropimorph or terbinafine, enhanced inhibition with halo clearing comparable to that of wild-type cells under the same conditions was observed (Fig. ). These findings demonstrate that FK506 synergy with fenpropimorph and terbinafine is mediated via FKBP12-dependent inhibition of calcineurin.
Although the morpholine drugs target both Erg2 and Erg24, it has previously been demonstrated that Erg2 is the major target of fenpropimorph in C. albicans (). To investigate the synergistic potential of calcineurin inhibitors in combination with morpholine drugs that primarily target Erg24, an Erg24-deficient (erg24/erg24) mutant (Table ) was tested for sensitivity to CsA and FK506. The erg24/erg24 mutants grew more slowly than the wild type, even in the absence of a drug, and showed hypersensitivity to both CsA and FK506 compared to the wild type (Fig. ). As a positive control, the erg24/erg24 mutant was compared to a C. albicans Erg6-deficient (erg6/erg6) mutant strain previously found to be hypersensitive to CsA and FK506 (, ). The ERG6 gene encodes an enzyme responsible for methylating zymosterol and converting it to ergosterol (Fig. ). The erg24/erg24 mutant was as sensitive to both calcineurin inhibitors as the erg6/erg6 mutant (Fig. ). On the basis of these findings, morpholines or other drugs that target Erg24 could potentially exhibit synergistic activity with calcineurin inhibitors.
Azoles target the ergosterol biosynthetic enzyme lanosterol 14α-demethylase and are a widely applied class of antifungal agents because of their broad therapeutic window, wide spectrum of activity, and low toxicity. Unfortunately, azoles are generally fungistatic and resistance to fluconazole is emerging in several fungal pathogens. We recently established that the protein phosphatase calcineurin allows survival of Candida albicans during the membrane stress exerted by azoles. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are dramatically synergistic with azoles, resulting in potent fungicidal activity, and mutant strains lacking calcineurin are markedly hypersensitive to azoles. Here we establish that drugs targeting other enzymes in the ergosterol biosynthetic pathway (terbinafine and fenpropimorph) also exhibit dramatic synergistic antifungal activity against wild-type C. albicans when used in conjunction with CsA and FK506. Similarly, C. albicans mutant strains lacking calcineurin B are markedly hypersensitive to terbinafine and fenpropimorph. The FK506 binding protein FKBP12 is required for FK506 synergism with ergosterol biosynthesis inhibitors, and a calcineurin mutation that confers FK506 resistance abolishes drug synergism. Additionally, we provide evidence of drug synergy between the nonimmunosuppressive FK506 analog L-685,818 and fenpropimorph or terbinafine against wild-type C. albicans. These drug combinations also exert synergistic effects against two other Candida species, C. glabrata and C. krusei, which are known for intrinsic or rapidly acquired resistance to azoles. These studies demonstrate that the activity of non-azole antifungal agents that target ergosterol biosynthesis can be enhanced by inhibition of the calcineurin signaling pathway, extending their spectrum of action and providing an alternative approach by which to overcome antifungal drug resistance.