ANTIFUNGAL DRUGS Modes of Action Mechanisms of Resistance

1
ANTIFUNGAL DRUGSModes of ActionMechanisms of
Resistance

• Sevtap Arikan, MD
• Hacettepe University Medical School
• Ankara Turkey

2
MOST COMMON FUNGAL PATHOGENS

• Dermatophytes
• Candida
• Aspergillus
• Cryptococcus
• Rhizopus
• ...

3
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

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MODES of ACTION
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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

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TARGETS for antifungal activity

• Ergosterol (Cell membrane)
• Drug-ergosterol interaction
• Inhibition of ergosterol synthesis
• RNA/EF3 (Nucleic acid/protein synthesis)
• Incorporation of 5-FU in RNA
• Inhibition of EF3
• Glucan/Chitin (Cell wall)
• Inhibition of glucan/chitin synthesis

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AMPHOTERICIN B generates pores in the membrane

• Clin Microbiol Rev 1999
  12 501

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TARGETS for antifungal activity

• Ergosterol (Cell membrane)
• Drug-ergosterol interaction
• Inhibition of ergosterol synthesis
• RNA/EF3 (Nucleic acid/protein synthesis)
• Incorporation of 5-FU in RNA Inhibition of
  EF3
• Glucan/Chitin (Cell wall)
• Inhibition of glucan/chitin synthesis

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Ergosterol synthesis
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Azoles, allylamines morpholines inhibit
specific ENZYMES

• Clin Microbiol Rev 1998 11
  382

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TARGETS for antifungal activity

• Ergosterol (Cell membrane)
• Drug-ergosterol interaction
• Inhibition of ergosterol synthesis
• RNA/EF3 (Nucleic acid/Protein synthesis)
• Incorporation of 5-FU into RNA Inhibition of
  EF3
• Glucan/Chitin (Cell wall)
• Inhibition of glucan/chitin synthesis

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FLUCYTOSINE (5-fluorocytosine)

• Cytosine permease 5-FC cytosine deaminase
  5-FU
• 5-FU
• 5-FU uracil phosphoribosyl
  5-fluorouridilic acid (FUMP)
• transferase (UPRTase)
• FUMP phosphorylation

5-fluorodeoxyuridine monophosphate thymidylate
synthase inhibitor inhibits DNA synthesis
5-fluoro-UTP incorporated into RNA disrupts
protein synthesis
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TARGETS for antifungal activity

• Ergosterol (Cell membrane)
• Drug-ergosterol interaction
• Inhibition of ergosterol synthesis
• RNA/EF3 (Nucleic acid/protein synthesis)
• Incorporation of 5-FU into RNA
• Inhibition of EF3
• Glucan/Chitin (Cell wall)
• Inhibition of glucan/chitin synthesis

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SORDARINS, AZASORDARINS

• EF3 A target in protein synthesis machinery
  unique to FUNGI
• GM 237354... (sordarins)
• GW 471558... (azasordarins)
• Yet investigational

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TARGETS for antifungal activity

• Ergosterol (Cell membrane)
• Drug-ergosterol interaction
• Inhibition of ergosterol synthesis
• RNA/EF3 (Nucleic acid/protein synthesis)
• Incorporation of 5-FU into RNA
• Inhibition of EF3
• Glucan/Chitin (Cell wall)
• Inhibition of glucan / chitin synthesis

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ECHINOCANDINSCaspofungin is licensed

• Inhibition of ß-(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

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TARGETS for antifungal activity

• Ergosterol (Cell membrane)
• Drug-ergosterol interaction
• Inhibition of ergosterol synthesis
• RNA/EF3 (Nucleic acid/protein synthesis)
• Incorporation of 5-FU into RNA
• Inhibition of EF3
• Glucan/Chitin (Cell wall)
• Inhibition of glucan / chitin synthesis

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NIKKOMYCIN

• Competitive inhibition of chitin synthase
• Yet investigational

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MECHANISMS OF RESISTANCE
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RESISTANCE is..
CLINICAL
IN VITRO
MOLECULAR
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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)

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Clinical Resistance is a Multifactorial Issue

• FUNGUS
• Initial MIC
• Cell type Yeast/hyphae..
• Genomic stability
• Biofilm production
• Population bottlenecks

• HOST
• Immune status
• Site of infection
• Severity of infection
• Foreign devices
• Noncompliance with drug regimen

• DRUG
• Fungistatic nature
• Dosing
• Pharmacokinetics
• Drug-drug interactions

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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.
• ...

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Mechanisms of Amphotericin B Resistance

• Reduced ergosterol content (defective ERG2 or
  ERG3 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
• (?)

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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...

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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 MDR1 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)

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Azole ResistanceMolecular Aspects

• Single point mutation of ERG11 gene
• ?Altered lanosterol demethylase
• Overexpression of ERG11 gene
• ?Increased production of lanosterol demethylase
  
• Alterations in ERG3 or ERG5 genes
• ?Production of low affinity sterols
• Increase in mRNA levels of CDR1 or MDR1 genes
• ?Decreased accumulation of the azole in fungal
  cell

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If your fungus is susceptible to azoles..

• Clin Microbiol Rev 1998 11 382

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If it is azole-resistant..

• Clin Microbiol Rev 1998 11 382

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Secondary Resistance in C. albicans to
Fluconazole

• CID 1997 25 908-910

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Resistance to Terbinafine

• Very rare
• Primary resistance to terbinafine in a
• T. rubrum strain (ICAAC 2001, abst. no. J-104)
• Mechanism (?)
• CDR1-mediated efflux (possible)

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Resistance to Flucytosine

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

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Mechanisms of Resistance to Flucytosine

• Loss of permease activity
• Loss of cytosine deaminase activity
• Decrease in the activity of UPRTase

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Flucytosine ResistanceMolecular Aspects

• FCY genes (FCY1, FCY2) 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

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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.

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Echinocandin ResistanceMolecular Aspects

• FKS1 encodes glucan synthase
• GNS1 encodes an enzyme involved in fatty acid
  elongation
• Resistance is observed following laboratory
  derived mutations in FKS1 or GNS1
• Other mechanisms (?)

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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!..

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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