1. ANTIFUNGAL DRUGS Modes of Action Mechanisms 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

4. MODES of ACTION

5. 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

6. 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

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

8. 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

9. Ergosterol synthesis

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

11. 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

12. FLUCYTOSINE (5-fluorocytosine)
Cytosine permease FC cytosine deaminase FU
5-FU
5-FU uracil phosphoribosyl 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

13. 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

14. SORDARINS, AZASORDARINS
EF3: A target in protein synthesis machinery unique to FUNGI
GM (sordarins)
GW (azasordarins)
Yet investigational

15. 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

16. 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

17. 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

18. NIKKOMYCIN Competitive inhibition of chitin synthase
Yet investigational

19. MECHANISMS OF
RESISTANCE

20. RESISTANCE is..
CLINICAL
IN VITRO
MOLECULAR

21. 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)

22. 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

23. 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.
...

24. 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
(?)

25. 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...

26. 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)

27. Azole Resistance Molecular 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

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

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

30. Secondary Resistance in C. albicans to Fluconazole
CID 1997; 25:

31. 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)

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

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

34. Flucytosine Resistance Molecular 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

35. 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.

36. Echinocandin Resistance Molecular 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 (?)

37. 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!..

38. 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