Pneumocystis carini pneumonia is a major cause of death in AIDS patients in the United States. The presently available treatments have limited use due to a high incidence of adverse reactions. Therefore, there is an urgent need for a safer method for treatment and prevention of this disease. Recent evidence has suggested that P. carinn is related to fungi and that the wall ofthe cyst form contains 1,3-13-glucan as a major constituent. Based on this, several proposed 1,3-f3-glucan synthesis inhibitors were evaluated for their ability to control P. carinu pneumonia in vivo. Compounds from two classes of 1,3-13-glucan synthesis inhibitors, the echinocandins and papulacandins, were found to be effective against P. carinii.Pneumocystis carinii pneumonia is the most prevalent opportunistic infection and a frequent cause of death in AIDS

The pneumocandins are natural lipopeptide products of the echinocandin class which inhibit the synthesis of 1,3-p-D-glucan in susceptible fungi. The lack of a corresponding pathway in mammalian hosts makes this mode of action an attractive one for treating systemic infections. Substitution by an aminoethyl ether at the hemiaminal and dehydration and reduction of the glutamine of pneumocandin Bo produced a semisynthetic compound (L-733,560) with intrinsic water solubility, significantly increased potency, and a broader antifungal spectrum. To evaluate the mechanism for the improved antifungal efficacy, we determined that L-733,560 was a more potent inhibitor of glucan synthase activity in vitro, did not affect the other membrane-bound enzymes tested, conferred susceptibility to lysis in the absence of osmotic support, and did not disrupt currents in liposomal bilayers or 86Rb+ fluxes from liposomes. In AspergiUus species L-733,560 also produced the same morphological alterations as pneumocandin Bo. A stereoisomer of L-733,560 with poor antifungal activity was a weak inhibitor of glucan synthase. All of these results support the notion that the enhanced antifungal activity of L-733,560 is achieved by superior inhibition of glucan synthesis and not by nonspecific membrane effects or a second mode of action.The echinocandins, pneumocandins, and papulacandins are antifungal agents which inhibit the synthesis of 1,3-,3-D-glucan in susceptible organisms (3,26,27,35,(38)(39)(40)42). Because the likelihood of mechanism-based toxicity is reduced, inhibition of the synthesis of a fungus-specific structure is an attractive mode of action for antifungal drug candidates. In addition to inhibition of in vitro glucan synthase activity, several observations on whole cells support the notion that these compounds act to inhibit cell wall synthesis in intact fungi. First, osmotic support prevents the lysis of cells treated with drug at the MICs (13, 45). Since the intact fungal cell wall counteracts the high turgor pressure of the protoplast, an agent which disrupts cell wall integrity produces membrane swelling and 'ultimately causes lysis in the absence of osmotic support. Second, susceptible organisms undergo gross morphological changes after treatment with these agents, consistent with cell wall alterations (10,13,16,22,28,33,44). Third, whole-cell labeling experiments which monitor macromolecular synthesis demonstrate the preferential inhibition of cell wall synthesis (3, 13). Lastly, Douglas et al. (21) recently showed that a mutation responsible for pneumocandin resistance in Saccharomyces cerevisiae cosegregates with enzyme activity resistant to inhibition by the pneumocandins.Originally, semisynthetic derivatives of the natural products were screened for reduced erythrocyte lysis, and this led to the development of the echinocandin B-based analog cilofungin (26,27 cantly alter the in vitro potency of the lipopeptide against Candida species compared with that of the natural product (17). Newer side chain analogs of the echi...

The pneumocandins are potent antifungal agents of the echinocandin class which are under development for use as broad-spectrum antimycotic therapy. One important consideration for any new therapeutic class for treating serious fungal infections is the potential for drug resistance development. In this study we have isolated and characterized four independent spontaneous Candida albicans mutants resistant to the potent semisynthetic pneumocandin L-733,560. These mutants have many of the properties of FKS1/ETG1 echinocandin-resistant mutants of Saccharomyces cerevisiae, including (i) cross-resistance to other 1,3-␤-D-glucan synthase inhibitors, such as papulacandin and echinocandins, but no change in sensitivity to other antifungal agents; (ii) in vitro glucan synthase activity that is more resistant to pneumocandins than the wild-type parent enzyme; and (iii) semidominant drug resistance in spheroplast fusion strains. The mutants were compared with C. albicans echinocandin-resistant mutants isolated by mutagenesis by L. Beckford and D. Kerridge (mutant M-2) (abstr. PS3.11, in Proceedings of the XI Congress of the International Society for Human and Animal Mycology, Montreal, Canada, 1992) and by A. Cassone, R. E. Mason, and D. Kerridge (mutant CA-2) (Sabouraudia 19:97-110, 1981). All of the strains had resistant enzyme activity in vitro. M-2 grew poorly and had low levels of enzyme activity. In contrast, CA-2 and the spontaneous mutants grew as well as the parents and had normal levels of glucan synthase activity. These results suggest that these resistant mutants may have alterations in glucan synthase. CA-2 was unable to form germ tubes, an ability retained by the spontaneous mutants. The virulence of the spontaneous mutants was unimpaired in a mouse model of disseminated candidiasis, while M-2 and CA-2 were 2 orders of magnitude less virulent than their parent strains. Significantly, mice challenged with the spontaneous mutant CAI4R1 responded therapeutically to lower levels of L-733,560 than would be predicted by the increase in in vitro susceptibility.

The in vivo anti-Candida activities of 1,3-13-D-glucan synthesis inhibitors L-671,329, L-646,991 (cilofungin), L-687,901 (tetrahydroechinocandin B), and L-687,781 (a papulacandin analog) were evaluated by utilizing a murine model of disseminated candidiasis that has enhanced susceptibility to Candida albicans but increased sensitivity for discriminating antifungal efficacy. DBA/2 mice were challenged intravenously with 1 x 104 to 5 x 104 CFU of C. albicans MY1055 per mouse. Compounds were administered intraperitoneally at concentrations ranging from 1.25 to 10 mg/kg of body weight twice daily for 4 days. At 6 h and 1, 2, 3, 4, 7, and 9 days after challenge, five mice per group were sacrificed and their kidneys were homogenized and plated for enumeration of Candida organisms (CFU per gram). Progressiveness of response trends and no-statisticalsignificance-of-trend doses were derived to rank compound efficacy. 1,3-13-D-Glucan synthesis 50% inhibitory concentrations were determined by using a C. albicans (MY1208) In the last decade, the incidence of serious fungal infections has reached record levels. This increase is due primarily to the AIDS epidemic, an expanding number of immune deficiency syndromes, and modern medical techniques that predispose a much larger population to opportunistic fungal infections. The number of current therapies available to counter the mycoses has been developed at approximately the same rate since the inception of antifungal drug therapy. Amphotericin B (AMB), developed in the 1950s, still remains the drug of choice for most fungal diseases mainly because it is broad spectrum and fungicidal, but it is considered to be relatively toxic (36, 37). The broad-spectrum azole antifungal agents developed more recently, although considered to be safer and less toxic than AMB, are fungistatic, which has limited their utility in many clinical settings (28,36). Many of these new antifungal agents are ineffective against deep-seated, life-threatening mycoses. Therefore, the critical need for new fungicidal agents which are safe and effective is evident.Our efforts toward identification of a promising antifungal agent have focused on 1,3-p-D-glucan synthesis inhibitors.

The echinocandins are a well-known class of lipopeptides characterized by their potent antifungal activity against Candida species. The mechanism of action of the echinocandins is generally thought to be the inhibition of beta-1,3-glucan synthesis, an important structural component in the cell wall of Candida species. Extensive structure-activity studies on the fatty acid side chain of echinocandin B (1) led to the preparation of the clinical candidate cilofungin (4). However, little is known about the cyclic peptide. We now report the preparation, by solid-phase synthesis, of a series of simplified analogs of cilofungin in which the unusual amino acids found in the echinocandins were replaced with more readily accessible natural amino acids. The solid-phase approach to the total synthesis of these analogs allowed us to conveniently explore structural modifications that could not be accomplished by chemical modification of the natural product. The simplest analog 5 showed no biological activity. Structural complexity was then returned to the system in a systematic fashion so as to reapproach the original cilofungin structure. Antifungal activity and the inhibition of beta-1,3-glucan synthesis were monitored at each step of the process, thereby revealing the basic structure-activity relationships of the amino acids and the minimal structural requirements for biological activity in the echinocandin ring system. The results suggests that the 3-hydroxy-4-methylproline residue enhances activity but the L-homotyrosine residue is crucial for both antifungal activity and the inhibition of beta-1,3-glucan synthesis.