1.D.20 The Pore-forming Polyene Macrolide Antibiotic/fungal Agent (PMAA) Family

Polyene macrolide antifungal agents include amphotericin (see also TC# 1.D.31), nystatin (the two most commonly used agents), natamycin, filipin, etc. (Ng et al., 2003). They bind sterols such as ergosterol, a major component of the fungal cell membrane. When present in sufficient concentrations, they form pores in the membrane that lead to K⁺ leakage and death of the fungus. Pore-formation is inflluenced by anaesthetics (Efimova et al. 2018).  Ergosterol is fairly unique to fungi, so the drugs do not have such catastrophic effects on animals. Cholesterol can replace ergosterol, but it is substantially less effective. In fact, a sterol is not absolutely required for pore formation (Bolard, 1986). The killing effect of Amphotericin B on fungi may be attributable to ergosterol binding instead of pore formation (Gray et al., 2012).  In general, sterols (cholesterol and esgosterol) affect membrane binding, partitioning and pore-formation by nystatin (Kristanc et al. 2014). The mechanisms of polyene action at the membrane level have been reviewed, focusing on their pore-forming activities (Kristanc et al. 2019). The disordering of membrane lipids in the presence of benzylamines and piperine plays a decisive role in the regulation of the conductance induced by the antifungal polyene macrolide antibiotic, nystatin (Efimova et al. 2020).

The biosynthesis of several polyene macrolides have been studied (Brautaset et al., 2000). Amphotericin B and other polyene antiobiotics form co-pores with the sterol and allow passage of monovalent ions such as Na⁺and Cl^-+(Gruszecki et al., 2003; Resat and Baginski, 2002). The formation of aqueous pores by amphotericin B (AmB) is at the basis of its fungicidal and leishmanicidal action. However, other types of nonlethal and dose-dependent biphasic effects that have been associated with the AmB action in different cells, including a variety of survival responses, are difficult to reconcile with the formation of a unique type of ion channel by the antibiotic. There is evidence that AmB forms nonaqueous, cation-selective channels at concentrations below the threshold at which aqueous pores are formed. Cohen (2010) provides a summary of the evidence supporting the formation of these channels and aqueous pores by AmB in lipid membranes. The influence of membrane parameters such as thickness fluctuations, the type of sterol present and the existence of sterol-rich specialized lipid raft microdomains in the formation process of such channels is discussed.

Amphotericin B (AmB) forms aqueous pores. Membrane potential and ion permeability measurements were used to demonstrate that AmB can form two types of selective ion channels in human erythrocytes, differing in their interaction with cholesterol. Romero et al. (2009) showed that AmB induced cation efflux (negative membrane polarization) across cholesterol-containing liposomes and erythrocytes at low concentrations (%u2264 1 μM), but reversal of such polarization was observed at concentrations > 1⁺μM. Cation-selective AmB channels are also formed across sterol-free liposomes, but aqueous pores are only formed at AmB concentrations 10 times greater. The effect of temperature on the AmB-mediated K⁺ efflux across erythrocytes revealed that the energies of activation for channel formation are negative and positive at AmB concentrations that lead predominantly to the formation of cation-selective channels and aqueous pores, respectively. The two types of AmB channels formed in human erythrocytes differ in their interactions with cholesterol and other membrane components.

The role of membrane components, sterols, phospholipids and sphingolipids in the formation and functioning of ion-permeable nanopores formed by antifungal macrolide antibiotics, amphotericin B, nystatin and filipin in planar lipid bilayers has been studied (Coutinho et al. 2004; Efimova et al. 2014). Dipole modifiers, flavonoids and styryl dyes were used as a tool to study the molecular mechanisms of polyene channel-forming activity. The introduction of dipole modifiers into the membrane bathing solutions changed the conductance of single channels and the steady state transmembrane current induced by polyene antibiotics in the sterol-containing phospholipid-bilayers. The conductance of single amphotericin B channels was found to depend on the dipole potential of the membrane. The experiments with various phospholipids, sterols, and polyenes led to the assumption that the shape of a phospholipid molecule, the presence of double bonds at the positions 7 and 22 of a sterol molecule, the number of conjugated double bonds, and the presence of an amino sugar in the polyene antibiotic molecule are important factors impacting the stability of polyene-lipid complexes forming ion-permeable pores. The channel-forming activity of polyene antibiotics is also affected by the physicochemical properties of polyene-enriched ordered membrane domains (Efimova et al. 2014).