ANTIFUNGAL DRUGS Modes of Action Mechanisms of Resistance

ANTIFUNGAL DRUGS Modes of Action Mechanisms of Resistance Sevtap Arikan, MD Hacettepe University Medical School Ankara Turkey

MOST COMMON FUNGAL PATHOGENS • Dermatophytes • Candida • Aspergillus • Cryptococcus • Rhizopus • . . .

ANTIFUNGAL DRUGS --by structure • POLYENES • • Amphotericin B, nystatin AZOLES Imidazoles: Ketoconazole. . Triazoles: Fluconazole, itraconazole, voriconazole, posaconazole, ravuconazole ALLYLAMINES Terbinafine, butenafine MORPHOLINE Amorolfine FLUORINATED PYRIMIDINE Flucytosine • ECHINOCANDINS Caspofungin, anidulafungin, micafungin • PEPTIDE-NUCLEOSIDE Nikkomycin Z • TETRAHYDROFURAN DERIVATIVES Sordarins, azasordarins • OTHER Griseofulvin

MODES of ACTION

ANTIFUNGAL DRUGS --by mode of action • Membrane disrupting • • • agents Amphotericin B, nystatin Ergosterol synthesis inhibitors Azoles, allylamines, morpholine Nucleic acid inhibitor Flucytosine Anti-mitotic (spindle disruption) Griseofulvin • Glucan synthesis inhibitors Echinocandins • Chitin synthesis inhibitor Nikkomycin • Protein synthesis inhibitors Sordarins, azasordarins

TARGETS for antifungal activity • Ergosterol (Cell membrane) ü Drug-ergosterol interaction Inhibition of ergosterol synthesis • RNA/EF 3 (Nucleic acid/protein synthesis) Incorporation of 5 -FU in RNA Inhibition of EF 3 • Glucan/Chitin (Cell wall) Inhibition of glucan/chitin synthesis

AMPHOTERICIN B generates pores in the membrane Clin Microbiol Rev 1999; 12: 501

TARGETS for antifungal activity • Ergosterol (Cell membrane) Drug-ergosterol interaction ü Inhibition of ergosterol synthesis • RNA/EF 3 (Nucleic acid/protein synthesis) Incorporation of 5 -FU in RNA Inhibition of EF 3 • Glucan/Chitin (Cell wall) Inhibition of glucan/chitin synthesis

Ergosterol synthesis

Azoles, allylamines & morpholines inhibit specific ENZYMES Clin Microbiol Rev 1998; 11: 382

TARGETS for antifungal activity • Ergosterol (Cell membrane) Drug-ergosterol interaction Inhibition of ergosterol synthesis • RNA/EF 3 (Nucleic acid/Protein synthesis) ü Incorporation of 5 -FU into RNA Inhibition of EF 3 • Glucan/Chitin (Cell wall) Inhibition of glucan/chitin synthesis

FLUCYTOSINE (5 -fluorocytosine) Cytosine permease 5 -FU 5 -FC cytosine deaminase 5 -FU 5 -fluorodeoxyuridine monophosphate thymidylate synthase inhibitor inhibits DNA synthesis 5 -FU uracil phosphoribosyl 5 -fluorouridilic acid (FUMP) transferase (UPRTase) FUMP phosphorylation 5 -fluoro-UTP incorporated into RNA disrupts protein synthesis

TARGETS for antifungal activity • Ergosterol (Cell membrane) Drug-ergosterol interaction Inhibition of ergosterol synthesis • RNA/EF 3 (Nucleic acid/protein synthesis) Incorporation of 5 -FU into RNA ü Inhibition of EF 3 • Glucan/Chitin (Cell wall) Inhibition of glucan/chitin synthesis

SORDARINS, AZASORDARINS • EF 3: A target in protein synthesis machinery unique to FUNGI • GM 237354. . . (sordarins) GW 471558. . . (azasordarins) • Yet investigational

TARGETS for antifungal activity • Ergosterol (Cell membrane) Drug-ergosterol interaction Inhibition of ergosterol synthesis • RNA/EF 3 (Nucleic acid/protein synthesis) Incorporation of 5 -FU into RNA Inhibition of EF 3 • Glucan/Chitin (Cell wall) üInhibition of glucan / chitin synthesis

ECHINOCANDINS Caspofungin is licensed • Inhibition of β-(1 -3) • • • glucan synthesis (of glucan synthase ? ? ) Secondary reduction in ergosterol & lanosterol Increase in chitin Kills hyphae at their growth tips and branching points Buds fail to seperate from the mother cell Yields osmotically sensitive fungal cells

TARGETS for antifungal activity • Ergosterol (Cell membrane) Drug-ergosterol interaction Inhibition of ergosterol synthesis • RNA/EF 3 (Nucleic acid/protein synthesis) Incorporation of 5 -FU into RNA Inhibition of EF 3 • Glucan/Chitin (Cell wall) ü Inhibition of glucan / chitin synthesis

NIKKOMYCIN • Competitive inhibition of chitin synthase • Yet investigational

MECHANISMS OF RESISTANCE

RESISTANCE is. . CLINICAL IN VITRO MOLECULAR

A resistant strain may be present due to: • • • Intrinsic resistance Replacement with a more resistant species Replacement with a more resistant strain Transient gene expressions that cause temporary resistance (epigenetic resistance) Alterations in cell type (? ) Genomic instability within a single strain (population bottleneck)

Clinical Resistance is a Multifactorial Issue • FUNGUS • HOST Immune status Site of infection Severity of infection Foreign devices Noncompliance with drug regimen Initial MIC Cell type: Yeast/hyphae. . Genomic stability Biofilm production Population bottlenecks • DRUG Fungistatic nature Dosing Pharmacokinetics Drug-drug interactions

Resistance to Amphotericin B • Technical difficulties in detection of • resistance in vitro In vivo resistance is rare C. lusitaniae, C. krusei C. neoformans Trichosporon spp. A. terreus S. apiospermum Fusarium spp. .

Mechanisms of Amphotericin B Resistance • Reduced ergosterol content (defective ERG 2 • • • or ERG 3 genes) Alterations in sterol content (fecosterol, episterol: reduced affinity) Alterations in sterol to phospholipid ratio Reorientation or masking of ergosterol Stationary growth phase Previous exposure to azoles (? )

Resistance to Azoles • • • Well-known particularly for fluconazole Data available also for other azoles A significant clinical problem RESISTANCE TO FLUCONAZOLE PRIMARY C. krusei Aspergillus C. glabrata C. norvegensis. . . SECONDARY C. albicans C. dubliniensis. . .

Mechanisms of Resistance to Azoles • Alteration of lanosterol (14 -alpha) demethylase • Overexpression of lanosterol demethylase • Energy-dependent efflux systems a. Major facilitator superfamily (MFS) proteins (BENr =MDR 1 of Candida. . . ) b. ATP-binding cassette (ABC) superfamily proteins (MDR, CDR of Candida) • Changes in sterol and/or phospholipid composition of fungal cell membrane (decreased permeability)

Azole Resistance Molecular Aspects • Single point mutation of ERG 11 gene Altered lanosterol demethylase • Overexpression of ERG 11 gene Increased production of lanosterol demethylase • Alterations in ERG 3 or ERG 5 genes Production of low affinity sterols • Increase in m. RNA levels of CDR 1 or MDR 1 genes Decreased accumulation of the azole in fungal cell

If your fungus is susceptible to azoles. . Clin Microbiol Rev 1998; 11: 382

If it is azole-resistant. . Clin Microbiol Rev 1998; 11: 382

Secondary Resistance in C. albicans to Fluconazole CID 1997; 25: 908 -910

Resistance to Terbinafine • Very rare • Primary resistance to terbinafine in a T. rubrum strain (ICAAC 2001, abst. no. J-104) • Mechanism: (? ) CDR 1 -mediated efflux (possible)

Resistance to Flucytosine • PRIMARY non-albicans Candida C. neoformans Aspergillus (highest) • SECONDARY C. albicans C. neoformans Secondary resistance develops following flucytosine MONOtherapy.

Mechanisms of Resistance to Flucytosine • Loss of permease activity • Loss of cytosine deaminase activity • Decrease in the activity of UPRTase

Flucytosine Resistance Molecular Aspects • FCY genes (FCY 1, FCY 2) encode for UPRTase FCY/FCY homozygotes possess high UPRTase activity FCY/fcy heterozygotes possess low UPRTase activity fcy/fcy homozygotes possess barely detectable UPRTase activity

Resistance to Echinocandins PRIMARY C. neoformans Fusarium spp. SECONDARY (? ) The only licensed member is caspofungin (Jan 2001, USA). Resistant mutants due to therapy are not available.

Echinocandin Molecular Resistance Aspects • FKS 1 encodes glucan synthase • GNS 1 encodes an enzyme involved in fatty acid elongation Resistance is observed following laboratory derived mutations in FKS 1 or GNS 1 • Other mechanisms (? )

Future Directions to Avoid Development of Resistance • Proper dosing strategies • Restricted and well-defined indications for prophylaxis with azoles Fungi will continue to develop NEW resistance mechanisms!. .

Final word • Antifungal resistance is a complex, gradual and multifactorial issue • Several uncertainties remain • Molecular assays to detect resistance are not simple • The best way to improve the efficacy of antifungal therapy is to improve the immune status of the host