Inhibitors of Sterol Biosynthesis and Amphotericin B Reduce the Viability of Pneumocystis carinii f. sp. carinii

Abstract

Pneumocystis carinii synthesizes sterols with a double bond at C-7 of the sterol nucleus and an alkyl group with one or two carbons at C-24 of the side chain. Also, some human-derived Pneumocystis carinii f. sp. hominis strains contain lanosterol derivatives with an alkyl group at C-24. These unique sterols have not been found in other pathogens of mammalian lungs. Thus, P. carinii may have important differences in its susceptibility to drugs known to block reactions in ergosterol biosynthesis in other fungi. In the present study, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, squalene epoxidelanosterol cyclase, lanosterol demethylase, ⌬ 8 to ⌬ 7 isomerase, and S-adenosylmethionine:sterol methyltransferase were tested for their effects on P. carinii viability as determined by quantitation of cellular ATP levels in a population of organisms. Compounds within each category varied in inhibitory effect; the most effective included drugs targeted at squalene synthase, squalene epoxide-lanosterol cyclase, and ⌬ 8 to ⌬ 7 isomerase. Some drugs that are potent against ergosterol-synthesizing fungi had little effect against P. carinii, suggesting that substrates and/or enzymes in P. carinii sterol biosynthetic reactions are distinct. Amphotericin B is ineffective in clearing P. carinii infections at clinical doses; however, this drug apparently binds to sterols and causes permeability changes in P. carinii membranes, since it reduced cellular ATP levels in a dose-dependent fashion.Organisms with parasitic lifestyles commonly scavenge host sterols for the formation of new membranes and for other cellular functions. Some parasites and other organisms can survive solely on the sterols taken up, or scavenged, from their environments, such as mammalian hosts and culture media. Cholesterol is abundant in lung surfactant (11), which is secreted by the epithelial type II cells into the alveolar lumen, in which Pneumocystis spp. proliferate extracellularly. The pathogen apparently forms the bulk of its membrane bilayers by scavenging host cholesterol, which accounts for about 75% of the organism's free sterols (17). Some microdomains within membranes contain complexes where sterol molecules with a specific and precise three-dimensional conformation must be present in order to function optimally. Thus, if the array of available host sterols does not include those that fulfill the stereochemical requirements for proper functioning of the parasite's membrane components, the organism must synthesize those particular sterols to remain viable. These parasite-synthesized sterols, distinct from those available from the host, have been described as metabolic sterols (12) because the organism needs them and continues expending energy to synthesize them.Antiergosterol agents have proved particularly useful for deeply invasive or systemic mycosis, but Pneumocystis carinii does not contain ergosterol, which is consistent with the unusual nature of this pathogen and its unique phylogenetic s...

Comprehensive and definitive structural identities of Pneumocystis carinii sterols

“…With the exception of some plant pathogenic rust fungi in the class Uredinales, production and accumulation of C 28 and C 29 ⌬ 7 24-alkylsterols are not typical of most fungi (33). The efficacy of the sterol ⌬ 8 -tosterol ⌬ 7 inhibitor AY 9944 in reducing P. carinii viability (21) indicates that this modification of the sterol nucleus may be another excellent target for the development of chemotherapeutic agents against this ubiquitous opportunistic pathogen.…”

“…20-Piperidin-2-yl-5 ␣ -pregnan-3 ␤ -20( R )-diol and 24,25-( R , S )-epiminolanosterol were reported to inhibit both the proliferation of P. carinii in vitro (static activity) and the accumulation of 24-alkylsterols, presumably as a consequence of SAM:SMT inhibition (17). In another study, it was shown that 24,25-epiminolanosterol did not kill P. carinii organisms; however, several other inhibitors that target different reactions in sterol biosynthesis had cidal activity against P. carinii in vitro (21).…”

Biofilm Formation by Pneumocystis spp

“…Biofilm formation was measured by a ATP bioluminescence assay used extensively by our lab to assess the viability of Pneumocystis (5,(7)(8)(9)27). The assay is similar in concept to the XTT reduction assay used for fungal biofilm growth, which assesses mitochondrial function, but is a more sensitive and rapid method.…”

Essential Eukaryotic Core

“…Drugs affecting genes in the essential eukaryotic core are likely to be broad spectrum in the fungal kingdom, for which there are few effective therapies (Georgopapadakou and Walsh 1994;Kaneshiro et al 2000). All known drug targets for Pneumocystis are associated with sterol metabolism, and resistance is already reported (Beard et al 2000) for the two major drugs for Pneumocystis pneumonia.…”