Post on 13-Dec-2015
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