Biosynthesis of Amphotericin Derivatives Lacking Exocyclic Carboxyl Groups

Abstract

Amphotericin B is a medically important antifungal antibiotic that is also active against human immunodeficiency virus, Leishmania parasites, and prion diseases. The therapeutic use of amphotericin B is restricted by severe side effects that can be moderated by liposomal formulation or structural alteration. Chemical modification has shown that suppression of charge on the exocyclic carboxyl group of amphotericin B substantially reduces toxicity. We report targeted deletions of the amphN cytochrome P450 gene from the chromosome of the amphotericin-producing bacterium Streptomyces nodosus. The mutant strains produced amphotericin analogues in which methyl groups replace the exocyclic carboxyl groups. These compounds retained antifungal activity and had reduced hemolytic activity.There are increasingly urgent requirements for new antibiotics to treat infectious disease (1). The need is especially acute with systemic fungal infections that are increasing in incidence and are often fatal (2). Of the few antifungals available at present, the most reliable is amphotericin B (compound 1 in Fig. 1), a polyene macrolide synthesized by Streptomyces nodosus (3). Amphotericin B disrupts ergosterol-containing fungal membranes and has a broad spectrum of activity. Whereas resistance to most non-polyene antifungals has appeared rapidly, the most prevalent fungal pathogens of humans have been slow to develop resistance to amphotericin B, even after more than three decades of clinical use (4). The advantages of amphotericin B are offset by toxicity that results from low water solubility and interactions with cholesterol in mammalian membranes. Toxicity has also prevented full exploitation of the antiviral, antiprion, and antiparasitic properties of amphotericin B (3). The adverse effects of the drug can be reduced by liposomal formulation, heat-induced superaggregation, or structural alteration (5). Chemical modification has shown that suppression of charge on the exocyclic carboxyl group reduces toxicity and improves antifungal specificity (6). Further improvements have been made by derivatization with additional sugars (7).Substantial reduction in amphotericin B toxicity would minimize adverse side effects on patients and also allow high dose treatment of infections by emerging fungal pathogens that are sensitive only to high concentrations of the drug. Non-toxic amphotericin analogues and formulations may also be useful in other therapeutic areas. Polyene antibiotics delay the onset of prion diseases in animal models (8). These effects are thought to result from interactions of polyenes with cholesterol-rich membrane microdomains that contain glycosylphosphatidylinositolanchored prion proteins (3, 9). Amphotericin B formulations are gaining favor for treatment of cutaneous and visceral leishmaniasis, particularly as resistance to other anti-Leishmania drugs continues to increase (10). Polyenes may also become important in treatment of viral infections. Polyenes interfere with the sterol-rich membranes of enveloped viruses suc...

Engineered biosynthesis and characterisation of disaccharide-modified 8-deoxyamphoteronolides

“…Haemolytic activity was tested against horse erythrocytes as described previously (Carmody et al, 2005).…”

Polyene macrolide biosynthesis in streptomycetes and related bacteria: recent advances from genome sequencing and experimental studies

“…Of the biosynthetically engineered polyene analogues, the most promising are those in which the free carboxyl group is replaced by a methyl group. One example is 16-descarboxyl-16-methyl-amphotericin B, which is produced in high yields (100 mg/litre) by S. nodosus amphNM (Carmody et al, 2005;Murphy et al, 2010). The same compound has been synthesised by chemical modification of amphotericin B, but this approach is less efficient (Palacios et al, 2007).…”

Streptomyces nodosusHost Strains Optimized for Polyene Glycosylation Engineering

“…The polyenes discussed have been partially characterized in previous work. 6 The gel shows amphDI region DNA amplified from S. nodosus amphDI-DII-NM (lanes 1 and 3) and from parent strain S. nodosus amphDII-NM (lanes 2 and 4). Samples analyzed on lanes 1 and 2 were untreated controls, those in lanes 3 and 4 were treated with Sph I.…”

“…6,7) In this study, we took a step towards further improvement by glycosylation engineering, a developing technology that is being applied to other natural products. Altering sugar residues has enhanced the properties of doxorubicin, glycopeptide antibiotics, and several other bioactive compounds.…”