ANTIFUNGAL DRUGSANTIFUNGAL DRUGSModes of ActionModes of ActionMechanisms of Mechanisms of

ResistanceResistance

ANTIFUNGAL DRUGSANTIFUNGAL DRUGSModes of ActionModes of ActionMechanisms of Mechanisms of

ResistanceResistance

Sevtap Arikan, MDSevtap Arikan, MDHacettepe University Medical School Hacettepe University Medical School

Ankara TurkeyAnkara Turkey

MOST COMMON FUNGAL MOST COMMON FUNGAL PATHOGENSPATHOGENS

• DermatophytesDermatophytes

• CandidaCandida

• AspergillusAspergillus

• CryptococcusCryptococcus

• RhizopusRhizopus

• ......

ANTIFUNGAL DRUGSANTIFUNGAL DRUGS--by structure--by structure

• POLYENESPOLYENES

Amphotericin B, Amphotericin B, nystatinnystatin

• AZOLESAZOLES

Imidazoles: Imidazoles: Ketoconazole..Ketoconazole..

Triazoles: Fluconazole, Triazoles: Fluconazole, itraconazole, voriconazole, itraconazole, voriconazole, posaconazole, ravuconazoleposaconazole, ravuconazole

• ALLYLAMINESALLYLAMINES

Terbinafine, butenafineTerbinafine, butenafine

• MORPHOLINEMORPHOLINE

AmorolfineAmorolfine

• FLUORINATED PYRIMIDINEFLUORINATED PYRIMIDINE

FlucytosineFlucytosine

• ECHINOCANDINSECHINOCANDINS

Caspofungin, Caspofungin, anidulafungin, anidulafungin, micafunginmicafungin

• PEPTIDE-NUCLEOSIDEPEPTIDE-NUCLEOSIDE

Nikkomycin ZNikkomycin Z

• TETRAHYDROFURAN TETRAHYDROFURAN DERIVATIVESDERIVATIVES

Sordarins, Sordarins, azasordarinsazasordarins

• OTHEROTHER

GriseofulvinGriseofulvin

MODES of ACTIONMODES of ACTION

ANTIFUNGAL DRUGSANTIFUNGAL DRUGS--by mode of action--by mode of action

• Membrane disrupting Membrane disrupting agents agents

Amphotericin B, nystatinAmphotericin B, nystatin

• Ergosterol synthesis Ergosterol synthesis inhibitorsinhibitors

Azoles, allylamines, Azoles, allylamines, morpholinemorpholine

• Nucleic acid inhibitorNucleic acid inhibitor

FlucytosineFlucytosine

• Anti-mitotic (spindle Anti-mitotic (spindle disruption)disruption)

GriseofulvinGriseofulvin

• Glucan synthesis Glucan synthesis

inhibitorsinhibitors

EchinocandinsEchinocandins

• Chitin synthesis Chitin synthesis

inhibitorinhibitor

NikkomycinNikkomycin

• Protein synthesis Protein synthesis inhibitorsinhibitors

Sordarins, azasordarinsSordarins, azasordarins

TARGETS TARGETS for antifungal activityfor antifungal activity

• Ergosterol Ergosterol (Cell membrane) (Cell membrane) Drug-ergosterol interactionDrug-ergosterol interaction

Inhibition of ergosterol synthesisInhibition of ergosterol synthesis

• RNA/EF3 RNA/EF3 (Nucleic acid/protein synthesis) (Nucleic acid/protein synthesis) Incorporation of 5-FU in RNAIncorporation of 5-FU in RNAInhibition of EF3Inhibition of EF3

• Glucan/Chitin Glucan/Chitin ((Cell wall) Cell wall) Inhibition of glucan/chitin synthesisInhibition of glucan/chitin synthesis

AMPHOTERICIN B AMPHOTERICIN B generates pores in the generates pores in the

membranemembrane

Clin Microbiol Clin Microbiol Rev Rev 1999; 12: 1999; 12: 501501

TARGETS TARGETS for antifungal activityfor antifungal activity• Ergosterol Ergosterol ((Cell membrane) Cell membrane)

Drug-ergosterol interactionDrug-ergosterol interaction Inhibition of ergosterol synthesisInhibition of ergosterol synthesis

• RNA/EF3RNA/EF3 (Nucleic acid/protein synthesis) (Nucleic acid/protein synthesis)

Incorporation of 5-FU in RNAIncorporation of 5-FU in RNAInhibition of EF3Inhibition of EF3

• Glucan/ChitinGlucan/Chitin (Cell wall) (Cell wall)

Inhibition of glucan/chitin synthesisInhibition of glucan/chitin synthesis

TERB

Ergosterol synthesisErgosterol synthesis

Azoles, allylamines & Azoles, allylamines & morpholines inhibit specific morpholines inhibit specific

ENZYMESENZYMES

Clin Microbiol Rev Clin Microbiol Rev 1998; 11: 382 1998; 11: 382

TARGETS TARGETS for antifungal activityfor antifungal activity

• Ergosterol Ergosterol (Cell membrane) (Cell membrane) Drug-ergosterol interactionDrug-ergosterol interactionInhibition of ergosterol synthesisInhibition of ergosterol synthesis

• RNA/EF3RNA/EF3 (Nucleic acid/Protein synthesis) (Nucleic acid/Protein synthesis) Incorporation of 5-FU into RNAIncorporation of 5-FU into RNA

Inhibition of EF3Inhibition of EF3

• Glucan/ChitinGlucan/Chitin (Cell wall) (Cell wall)Inhibition of glucan/chitin synthesisInhibition of glucan/chitin synthesis

FLUCYTOSINE FLUCYTOSINE (5-fluorocytosine)(5-fluorocytosine)

Cytosine permease Cytosine permease 5-FC cytosine deaminase 5-FC cytosine deaminase 5-FU5-FU

5-FU 5-FU

5-FU uracil phosphoribosyl 5-fluorouridilic acid 5-FU uracil phosphoribosyl 5-fluorouridilic acid (FUMP) (FUMP)

transferase (UPRTase)transferase (UPRTase)

FUMPFUMP phosphorylationphosphorylation

5-fluorodeoxyuridine 5-fluorodeoxyuridine monophosphatemonophosphate

thymidylate synthase inhibitorthymidylate synthase inhibitorinhibits DNA synthesisinhibits DNA synthesis

5-fluoro-UTP5-fluoro-UTPincorporated into RNAincorporated into RNA

disrupts protein synthesisdisrupts protein synthesis

TARGETS TARGETS for antifungal activityfor antifungal activity

• ErgosterolErgosterol (Cell membrane) (Cell membrane)Drug-ergosterol interactionDrug-ergosterol interactionInhibition of ergosterol synthesisInhibition of ergosterol synthesis

• RNA/EF3RNA/EF3 (Nucleic acid/protein synthesis) (Nucleic acid/protein synthesis) Incorporation of 5-FU into RNAIncorporation of 5-FU into RNA

Inhibition of EF3Inhibition of EF3

• Glucan/ChitinGlucan/Chitin (Cell wall) (Cell wall) Inhibition of glucan/chitin synthesisInhibition of glucan/chitin synthesis

SORDARINS, SORDARINS, AZASORDARINSAZASORDARINS

• EF3EF3: A target in protein synthesis : A target in protein synthesis machinery unique to FUNGImachinery unique to FUNGI

• GM 237354... GM 237354... (sordarins)(sordarins)GW 471558...GW 471558... (azasordarins)(azasordarins)

• Yet investigationalYet investigational

TARGETS TARGETS for for antifungalantifungal activity activity

• Ergosterol Ergosterol (Cell membrane) (Cell membrane) Drug-ergosterol interactionDrug-ergosterol interactionInhibition of ergosterol synthesisInhibition of ergosterol synthesis

• RNA/EF3RNA/EF3 (Nucleic acid/protein synthesis) (Nucleic acid/protein synthesis) Incorporation of 5-FU into RNAIncorporation of 5-FU into RNAInhibition of EF3Inhibition of EF3

• Glucan/ChitinGlucan/Chitin (Cell wall) (Cell wall) Inhibition of Inhibition of glucanglucan / chitin synthesis / chitin synthesis

ECHINOCANDINSECHINOCANDINSCaspofungin is licensedCaspofungin is licensed

• Inhibition of Inhibition of ββ-(1-3) -(1-3) glucan synthesis (of glucan synthesis (of glucan synthase ??)glucan synthase ??)

• Secondary reduction in Secondary reduction in ergosterol & lanosterolergosterol & lanosterol

• Increase in chitinIncrease in chitin• Kills hyphae at their Kills hyphae at their

growth tips and growth tips and branching pointsbranching points

• Buds fail to seperate Buds fail to seperate from the mother cellfrom the mother cell

• Yields osmotically Yields osmotically sensitive fungal cells sensitive fungal cells

TARGETS TARGETS for antifungal activityfor antifungal activity

• Ergosterol Ergosterol (Cell membrane) (Cell membrane) Drug-ergosterol interactionDrug-ergosterol interactionInhibition of ergosterol synthesisInhibition of ergosterol synthesis

• RNA/EF3RNA/EF3 (Nucleic acid/protein synthesis) (Nucleic acid/protein synthesis) Incorporation of 5-FU into RNAIncorporation of 5-FU into RNAInhibition of EF3Inhibition of EF3

• Glucan/ChitinGlucan/Chitin (Cell wall) (Cell wall) Inhibition of glucan / Inhibition of glucan / chitinchitin synthesis synthesis

NIKKOMYCINNIKKOMYCIN

• Competitive Competitive inhibition of chitin inhibition of chitin synthasesynthase

• Yet investigationalYet investigational

MECHANISMS OF MECHANISMS OF RESISTANCERESISTANCE

RESISTANCE is..RESISTANCE is..

IN VITROIN VITRO

CLINICALCLINICAL

MOLECULARMOLECULAR

A resistant strain may be A resistant strain may be present due to:present due to:

• Intrinsic resistanceIntrinsic resistance

• Replacement with a more resistant speciesReplacement with a more resistant species

• Replacement with a more resistant strainReplacement with a more resistant strain

• Transient gene expressions that cause Transient gene expressions that cause temporary resistance (epigenetic temporary resistance (epigenetic resistance)resistance)

• Alterations in cell type (?)Alterations in cell type (?)

• Genomic instability within a single strain Genomic instability within a single strain (population bottleneck)(population bottleneck)

Clinical Resistance is a Clinical Resistance is a Multifactorial IssueMultifactorial Issue

• FUNGUSFUNGUSInitial MICInitial MIC

Cell type: Yeast/hyphae..Cell type: Yeast/hyphae..

Genomic stabilityGenomic stability

Biofilm productionBiofilm production

Population bottlenecksPopulation bottlenecks

• DRUGDRUGFungistatic natureFungistatic nature

DosingDosing

PharmacokineticsPharmacokinetics

Drug-drug interactionsDrug-drug interactions

• HOSTHOSTImmune statusImmune status

Site of infectionSite of infection

Severity of infectionSeverity of infection

Foreign devicesForeign devices

Noncompliance with Noncompliance with drug regimendrug regimen

Resistance to Resistance to Amphotericin BAmphotericin B• Technical difficulties in detection of Technical difficulties in detection of

resistance in vitroresistance in vitro• In vivo resistance is rareIn vivo resistance is rare

C. lusitaniae, C. kruseiC. lusitaniae, C. kruseiC. neoformansC. neoformansTrichosporonTrichosporon spp. spp.A. terreusA. terreusS. apiospermumS. apiospermumFusarium Fusarium spp.spp.......

Mechanisms of Mechanisms of Amphotericin B Amphotericin B

ResistanceResistance• Reduced ergosterol content (defective Reduced ergosterol content (defective

ERG2 or ERG3 genes)ERG2 or ERG3 genes)• Alterations in sterol content (fecosterol, Alterations in sterol content (fecosterol,

episterol: reduced affinity)episterol: reduced affinity)• Alterations in sterol to phospholipid ratioAlterations in sterol to phospholipid ratio• Reorientation or masking of ergosterolReorientation or masking of ergosterol• Stationary growth phaseStationary growth phase• Previous exposure to azolesPrevious exposure to azoles• (?)(?)

Resistance to AzolesResistance to Azoles• Well-known particularly for fluconazoleWell-known particularly for fluconazole

• Data available also for other azolesData available also for other azoles

• A significant clinical problemA significant clinical problem

RESISTANCE TO FLUCONAZOLERESISTANCE TO FLUCONAZOLE

PRIMARYPRIMARY C. krusei C. krusei

Aspergillus Aspergillus

C. glabrata C. glabrata

C. norvegensis...C. norvegensis...

SECONDARYSECONDARY C. albicansC. albicans

C. dubliniensis...C. dubliniensis...

Mechanisms of Mechanisms of Resistance to AzolesResistance to Azoles

• Alteration of lanosterol (14-alpha) demethylase Alteration of lanosterol (14-alpha) demethylase

• Overexpression of lanosterol demethylase Overexpression of lanosterol demethylase

• Energy-dependent efflux systemsEnergy-dependent efflux systems

a. Major facilitator superfamily (MFS) a. Major facilitator superfamily (MFS) proteins proteins (BEN(BENrr =MDR1 of =MDR1 of CandidaCandida...)...)

b. ATP-binding cassette (ABC) superfamily b. ATP-binding cassette (ABC) superfamily proteins (MDR, CDR of proteins (MDR, CDR of CandidaCandida))

• Changes in sterol and/or phospholipid composition Changes in sterol and/or phospholipid composition of fungal cell membrane (decreased permeability)of fungal cell membrane (decreased permeability)

Azole ResistanceAzole ResistanceMolecular AspectsMolecular Aspects

• Single point mutation of ERG11 geneSingle point mutation of ERG11 geneAltered lanosterol demethylase Altered lanosterol demethylase

• Overexpression of ERG11 gene Overexpression of ERG11 gene Increased production of lanosterol demethylase Increased production of lanosterol demethylase

• Alterations in ERG3 or ERG5 genesAlterations in ERG3 or ERG5 genes Production of low affinity sterols Production of low affinity sterols

• Increase in mRNA levels of CDR1 or MDR1 genesIncrease in mRNA levels of CDR1 or MDR1 genes Decreased accumulation of the azole in fungal cellDecreased accumulation of the azole in fungal cell

If your fungus is If your fungus is susceptible susceptible to azoles..to azoles..

Clin Microbiol Rev 1998; 11: 382Clin Microbiol Rev 1998; 11: 382

If it is azole-resistant..If it is azole-resistant..

Clin Microbiol Rev 1998; 11: 382Clin Microbiol Rev 1998; 11: 382

Secondary Resistance in Secondary Resistance in C. albicansC. albicans to to FluconazoleFluconazole

CID 1997; 25: 908-910CID 1997; 25: 908-910

Resistance to Resistance to TerbinafineTerbinafine

• Very rareVery rare

• Primary resistance to terbinafine in a Primary resistance to terbinafine in a T.T. rubrumrubrum strain strain (ICAAC 2001, abst. no. J-104)(ICAAC 2001, abst. no. J-104)

• MechanismMechanism: : (?)(?)CDR1-mediated efflux (possible)CDR1-mediated efflux (possible)

Resistance to Resistance to FlucytosineFlucytosine

• PRIMARYPRIMARY non-non-albicansalbicans CandidaCandidaC. neoformansC. neoformans

AspergillusAspergillus (highest) (highest)

• SECONDARYSECONDARY C. albicansC. albicans

C. neoformansC. neoformans

Secondary resistance develops following Secondary resistance develops following flucytosine MONOtherapy. flucytosine MONOtherapy.

Mechanisms of Mechanisms of Resistance to Resistance to FlucytosineFlucytosine

• Loss of permease activityLoss of permease activity

• Loss of cytosine deaminase activityLoss of cytosine deaminase activity

• Decrease in the activity of UPRTaseDecrease in the activity of UPRTase

Flucytosine ResistanceFlucytosine ResistanceMolecular AspectsMolecular Aspects

• FCY genes (FCY1, FCY2) encode for UPRTaseFCY genes (FCY1, FCY2) encode for UPRTase

FCY/FCY homozygotes possess high UPRTase FCY/FCY homozygotes possess high UPRTase activityactivity

FCY/fcy heterozygotes possess low UPRTase activityFCY/fcy heterozygotes possess low UPRTase activity

fcy/fcy homozygotes possess barely detectable fcy/fcy homozygotes possess barely detectable UPRTase activityUPRTase activity

Resistance to Resistance to EchinocandinsEchinocandins

PRIMARYPRIMARY C. neoformansC. neoformans

Fusarium spp.Fusarium spp.

SECONDARYSECONDARY (?) (?)

The only licensed member is caspofungin The only licensed member is caspofungin (Jan 2001, USA). Resistant mutants due to (Jan 2001, USA). Resistant mutants due to therapy are not available. therapy are not available.

Echinocandin ResistanceEchinocandin ResistanceMolecular AspectsMolecular Aspects

• FKS1 encodes glucan synthase FKS1 encodes glucan synthase • GNS1 encodes an enzyme involved in fatty GNS1 encodes an enzyme involved in fatty

acid elongationacid elongation

Resistance is observed following Resistance is observed following laboratory derived mutations in FKS1 or laboratory derived mutations in FKS1 or GNS1 GNS1

• Other mechanisms (?)Other mechanisms (?)

Future Directions to Future Directions to Avoid Development of Avoid Development of

ResistanceResistance• Proper dosing strategies Proper dosing strategies

• Restricted and well-defined indications for Restricted and well-defined indications for prophylaxis with azolesprophylaxis with azoles

Fungi will continue to develop NEW Fungi will continue to develop NEW resistance mechanisms!..resistance mechanisms!..

Final wordFinal word• Antifungal resistance is a complex, gradual and Antifungal resistance is a complex, gradual and

multifactorial issuemultifactorial issue

• Several uncertainties remainSeveral uncertainties remain

• Molecular assays to detect resistance are not Molecular assays to detect resistance are not simplesimple

• The best way to improve the efficacy of The best way to improve the efficacy of antifungal therapy is to improve the immune antifungal therapy is to improve the immune status of the host status of the host