Amphotericin B: Structure, Mechanism of Action, Drug Resistance, Toxicity & Clinical Uses

Table of Contents

• Introduction to Amphotericin B
• Structure of Amphotericin B
• Mechanism of Action of Amphotericin B
• Drug Resistance of Amphotericin B
• Toxicity of Amphotericin B
• Conclusion
• References

Introduction to Amphotericin B

• Amphotericin B is a polyene antifungal agent belonging to the polyene class of broad-spectrum antifungal drugs.
• Polyene antifungals are biochemically characterized by a macrolide structure, which consists of a lactone ring containing multiple conjugated double bonds and is attached to one or more sugar moieties.
• Polyenes also contain multiple hydroxyl (-OH) groups, contributing to their amphiphilic nature—having both hydrophilic and hydrophobic regions.
• This amphiphilic structure enables polyene antifungals to interact with the fungal cell membrane and specifically target ergosterol, the major sterol component of the fungal membrane.
• Amphotericin B binds to ergosterol and disrupts the fungal cell membrane by forming pores, leading to leakage of intracellular components and ultimately fungal cell death.
• Amphotericin B is a naturally derived antifungal compound produced by Streptomyces species, which are filamentous bacteria belonging to the Actinobacteria.
• Other important polyene antifungal agents include nystatin and natamycin.

Structure of Amphotericin B

• Discovery and origin: Amphotericin B (AmB) was originally discovered in the fermentation broth of Streptomyces nodosus culture M4575. The organism was isolated from a soil sample collected from the Orinoco Basin in Venezuela.
• Macrocyclic lactone ring: The molecular structure of AmB contains a large macrocyclic lactone ring. One side of this ring contains a series of conjugated double bonds, forming the characteristic polyene region.
• Hydrophobic polyene tail: The region containing the conjugated double bonds is predominantly hydrophobic and is commonly described as the hydrophobic “tail” or polyene tail.
• Hydrophilic region: On the opposite side of the molecule is a polyhydroxyl chain, which contains multiple hydroxyl groups. AmB also contains a characteristic mycosamine sugar moiety, contributing to its hydrophilic region or “head.”
• Amphipathic nature: The presence of both hydrophobic and hydrophilic regions gives Amphotericin B its amphipathic (amphiphilic) character. However, this structure also makes AmB poorly soluble in water, creating challenges for its formulation and administration.
• Amphotericin B deoxycholate: To formulate AmB for intravenous administration, it was combined with sodium deoxycholate, a bile salt that helps form a micellar preparation. The formulation is administered in a 5% glucose solution and maintains the drug in a suitable form for intravenous delivery.
• Fungizone: The conventional sodium deoxycholate formulation of Amphotericin B is known as Amphotericin B deoxycholate, commercially associated with the name Fungizone.
• Clinical applications: Amphotericin B deoxycholate has been used for the treatment of serious systemic fungal infections (systemic mycoses), including disseminated histoplasmosis and mucormycosis. It has also been used in combination with flucytosine for cryptococcal meningitis.
• Toxicity: Despite its important antifungal activity, conventional AmB deoxycholate is associated with significant host toxicity, particularly because its interaction with sterols is not completely exclusive to fungal membranes.
• Low resistance: Amphotericin B has retained considerable clinical importance because resistance to the drug remains relatively uncommon compared with many other antifungal agents, making it an important option for severe and potentially life-threatening fungal infections.

Mechanism of Action of Amphotericin B

Amphotericin B (AmB) exerts its antifungal activity primarily by interacting with ergosterol in the fungal cell membrane. Its mechanism is more complex than simple pore formation, and several models have been proposed to explain its fungicidal activity.

1. Pore Formation Model

• Targeting ergosterol: Amphotericin B has a high affinity for ergosterol, the major sterol present in fungal cell membranes. Its interaction with ergosterol disrupts normal membrane organization and permeability.
• Membrane permeabilization: After binding to ergosterol, AmB can organize into membrane-associated complexes that create transmembrane channels or pores.
• Leakage of cellular components: These pores allow the uncontrolled movement of ions such as K⁺ and Mg²⁺, as well as other small cellular contents, across the membrane. The resulting loss of ionic balance and essential intracellular components contributes to fungal cell death.
• Why ergosterol is targeted: AmB preferentially interacts with ergosterol because structural differences between ergosterol and cholesterol, the principal sterol in mammalian membranes, influence their binding affinity for AmB. This selectivity contributes to its antifungal activity, although it is not absolute and helps explain the drug's host toxicity.
• Role of the polyene and hydrophilic regions: The hydrophobic polyene region of AmB interacts with the membrane environment and ergosterol, while its hydrophilic polyol region contributes to the formation of an aqueous pathway through the membrane.
• Stabilization of the pore: Intermolecular interactions, including hydrogen bonding involving the polar groups of adjacent AmB molecules, help stabilize the membrane-associated complex.
• Pore dimensions: In the classical pore model, AmB–ergosterol complexes can form relatively large membrane-spanning channels. Earlier structural models proposed a pore approximately 0.46 nm in diameter, consisting of an organized complex of polyene molecules and sterol.

2. Molecular Interactions Between Amphotericin B and Ergosterol

The interaction between AmB and ergosterol is supported by several types of molecular forces:

• Van der Waals interactions: These interactions can occur when AmB and ergosterol adopt closely aligned or approximately parallel orientations within the membrane.
• Hydrogen bonding: A key polar interaction occurs between the 3β-hydroxyl (3β-OH) group of ergosterol and the mycosamine moiety of Amphotericin B.
• π-related interactions: Interactions involving the conjugated polyene system of AmB and the hydrocarbon region of ergosterol contribute to their association within the membrane.

3. Importance of Ergosterol in Fungal Cells

• Ergosterol is an essential component of fungal membranes and contributes to several important cellular processes.
• It helps regulate membrane fluidity and organization and influences the function of membrane-associated proteins.
• Ergosterol-dependent membrane organization is also involved in processes such as cell signaling, cell division, and endocytosis.
• Consequently, sequestration or disruption of ergosterol can have effects extending beyond simple membrane permeabilization.

4. Surface Adsorption Model

• The surface adsorption model proposes that AmB does not necessarily need to form transmembrane pores to exert antifungal activity.
• According to this model, the high affinity of AmB for ergosterol allows the drug to bind and adsorb onto ergosterol-containing membrane regions.
• This interaction alters the organization and physical properties of the fungal membrane, ultimately impairing membrane function without requiring the formation of classical transmembrane pores.

5. Sterol Sponge Model

• The sterol sponge model, described by Anderson and colleagues, provides another explanation for AmB-mediated membrane disruption.
• In this model, AmB molecules form aggregates parallel to the membrane surface.
• These aggregates function like a “sterol sponge,” sequestering ergosterol from the fungal membrane.
• Removal of ergosterol from its normal membrane environment disrupts membrane organization and interferes with essential cellular functions.
• This model helps explain why ergosterol sequestration itself can contribute to AmB's fungicidal activity, independently of conventional pore formation.

6. Reactive Oxygen Species and Oxidative Stress

• In addition to membrane-related mechanisms, evidence suggests that AmB can induce oxidative stress in fungal cells.
• AmB-associated oxidative stress can increase the production or accumulation of reactive oxygen species (ROS).
• Excessive ROS can cause:
• Protein carbonylation
• Lipid peroxidation
• DNA damage
• Disruption of cellular functions
• Ultimately, fungal cell death
• Studies in Candida albicans have provided evidence of an oxidative-stress response during AmB exposure. The fungus can increase antioxidant defenses, including enzymes such as catalase and superoxide dismutase (SOD), in an attempt to neutralize ROS.
• C. albicans can also increase the production of polyamines such as spermidine, spermine, and putrescine, which may contribute to cellular protection against oxidative stress.
• Some studies have proposed that oxidative damage may partly originate from the auto-oxidation of Amphotericin B itself, potentially generating reactive species that contribute to cellular damage.

Overall Mechanism

Amphotericin B → Ergosterol binding/sequestration → membrane disruption → ion and cellular-content leakage + membrane dysfunction → oxidative stress and cellular damage → fungal cell deatbh

Key point: Amphotericin B was traditionally explained mainly through ergosterol-dependent pore formation, but current mechanistic models also recognize ergosterol sequestration (“sterol sponge”) and oxidative stress as important contributors to its fungicidal activity.

Drug Resistance of Amphotericin B

Although Amphotericin B (AmB) has a relatively low frequency of resistance compared with many other antifungal agents, resistance can develop through several mechanisms. These mechanisms can reduce the interaction of AmB with its target, increase fungal tolerance to cellular stress, or limit drug access to the fungal membrane.

1. Sterol Alteration

• One of the recurrent mechanisms of AmB resistance is alteration of fungal sterol composition through mutations in ERG genes, which are involved in the ergosterol biosynthetic pathway.
• In Candida albicans, loss-of-function mutations in ERG11 and ERG3 can disrupt ergosterol biosynthesis and lead to the accumulation of alternative sterols, including:
• Lanosterol
• 4,14-Dimethylzymosterol
• Eburicol
• Substitutions in ERG11 and reduced activity of ERG5 have also been associated with altered sterol profiles in AmB-resistant C. albicans.
• In Cryptococcus neoformans, AmB resistance has been associated with inactivation of ERG2, resulting in changes in the fungal sterol composition.
• Importantly, sterol-independent mechanisms of AmB resistance have also been observed in C. neoformans, indicating that resistance is not exclusively dependent on modifications of ergosterol biosynthesis.

2. Increased Catalase Production

• Amphotericin B can trigger stress-response mechanisms in fungal cells, including a strong oxidative burst and increased production of reactive oxygen species (ROS).
• AmB-resistant Aspergillus terreus has been reported to exhibit a marked increase in catalase production.
• Increased catalase activity enhances the fungus's ability to detoxify hydrogen peroxide and counteract oxidative stress, thereby reducing cellular damage caused by AmB-associated ROS.
• This enhanced antioxidant defense can improve fungal survival and contribute to AmB resistance or tolerance.

3. Cell Wall Alteration

• Changes in fungal cell wall composition and structure can also contribute to reduced susceptibility to Amphotericin B.
• In Aspergillus flavus, an increased proportion of the 1,3-α-glucan fraction in the cell wall has been described as a factor associated with increased AmB resistance.
• In Candida tropicalis, an enlarged cell wall associated with excessive 1,3-β-glucan has been reported.
• These structural changes may reduce the ability of AmB to reach the fungal plasma membrane, thereby limiting its access to membrane ergosterol and contributing to resistance.

4. Role of Molecular Chaperones in AmB Resistance

• Molecular chaperones, particularly heat-shock proteins (HSPs), help fungal cells respond to environmental and antifungal-induced stress and may contribute to the development or maintenance of AmB resistance.

Hsp90

• Hsp90 is a central stress-response protein that assists in the folding, stabilization, and functional maintenance of other proteins, particularly under stressful conditions.
• Hsp90 may contribute to antifungal resistance through several mechanisms:
• Disruption of Hsp90 function can increase the expression or emergence of genetic and phenotypic variation, increasing the possibility that highly tolerant fungal variants will arise.
• Hsp90 can stabilize otherwise unstable mutant proteins, allowing some resistance-associated mutations to retain their function.
• By maintaining stress-response pathways, Hsp90 can help fungal cells adapt and survive under antifungal pressure.

Hsp70

• Elevated expression of the Hsp70 gene has been associated with increased Amphotericin B resistance in Aspergillus flavus.
• Conversely, inhibition of Hsp70 can increase the susceptibility of A. flavus to AmB, supporting its role in fungal stress adaptation and resistance.

Toxicity of Amphotericin B

• Amphotericin B (AmB) has a relatively high selectivity for ergosterol, the major sterol of fungal cell membranes. However, this selectivity is not absolute.
• At high concentrations or with prolonged exposure, AmB can also interact with cholesterol in mammalian cell membranes. This interaction can disrupt mammalian cell membranes and contributes to the drug's dose-limiting toxicity.
• The major toxic effects of Amphotericin B can be broadly divided into acute infusion-related toxicity and chronic toxicity, particularly renal toxicity (nephrotoxicity).

1. Acute Toxicity

• Amphotericin B can produce acute infusion-related reactions shortly after intravenous administration.
• Its microbial origin and interaction with immune-recognition pathways can stimulate TLR2 and CD14, leading to activation of innate immune responses and the release of proinflammatory cytokines.
• This inflammatory response contributes to common infusion-related adverse effects, including:
• Fever
• Chills and rigors
• Nausea
• Vomiting
• Headache
• Hypertension or hypotension
• These reactions are particularly associated with conventional Amphotericin B deoxycholate formulations.

2. Chronic Toxicity: Nephrotoxicity

• Renal toxicity is one of the most clinically important adverse effects of Amphotericin B, particularly during prolonged or high-dose therapy.
• AmB can interact with cholesterol-containing membranes of renal tubular cells, contributing to cellular injury.
• Renal exposure to AmB can also involve low-density lipoprotein (LDL) receptor-mediated uptake/endocytosis, which facilitates the accumulation of the drug in renal cells and contributes to nephrotoxicity.
• Amphotericin B also causes vasoconstriction of the afferent renal arterioles, reducing renal blood flow and glomerular filtration.
• These effects can lead to increased serum creatinine, reduced kidney function, electrolyte abnormalities, and other manifestations of renal injury.

Conclusion

• Despite its significant host toxicity, Amphotericin B (AmB) remains an important antifungal agent for the clinical treatment of severe systemic mycoses, particularly because of its broad antifungal activity and the relatively low incidence of resistance.
• The development of lipid-based formulations has helped improve the safety profile of Amphotericin B:
• Amphotericin B lipid complex (ABLC) is associated with lower nephrotoxicity compared with conventional AmB deoxycholate.
• Liposomal Amphotericin B (L-AmB) generally produces fewer and less severe infusion-related reactions and has improved tolerability.
• Amide conjugates of Amphotericin B have also demonstrated enhanced antifungal activity and lower acute toxicity in vivo, making them promising candidates for further development.
• These newer AmB derivatives and formulations have the potential to expand the clinical applications and therapeutic safety of Amphotericin B while retaining its important antifungal properties.
• Overall, Amphotericin B continues to have a valuable role in antifungal therapy because its potent activity against serious fungal infections and relatively low frequency of resistance remain major therapeutic advantages.  

References

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