Antifungals Flashcards
LO
Outline the mechanisms of action of the major antifungal agents (griseofulvin, amphotericin, imidazole’s, fluorocytosine)
What are some types of fungi that make an enormous contribution to our everyday life?
If applicable, provide examples of the organisms
- edible mushrooms
- mould (e.g.,Penicillium roqueforti)
- Yeast (Saccharomyces cerevisiaw)
- Mould (Penicillium)
- Other toxins (A. Phalloides)
- Lichens (symbiotic)
- Mycorrhizia (plant growth)
>1.5 million species (but 300 associated with humans)
What type of organisms are fungi?
Eukaryotes (unicellular and multicellular, cell nucleus, etc)
What are fungi incapable of doing?
Incapable of producing food/ not self-sustaining (heterotrophs)-
Lichens are the exception to this as they are symbiotic
Where are fungi present?
They are naturally present in the environment
present as commensals (an organism that uses food supplied in the internal or external environment of the host, without establishing a close association with the host, for instance by feeding on its tissues)
What do fungi cause?
Give an example of this
Cause superficial (‘cosmetic’), (sub)cutaneous and systemic (tissues/organs) = mycoses (a diseases caused by infection with a fungus, such as ringworm or thrush)
How may some fungi infection occur?
Some occur as primary infections but in general only infectious opportunistically
What are the three problematic types of fungi?
Yeast
Moulds
Dermatophytes
What are dermatophytes and what do they cause?
Collection of fungi which share the ability to metabolise the subsist on keratin
Cause superficial infections names after the part of the body involved (in latin)
What is the most common dermatophyte and provide some examples
Most common is tineas
Tineas pedis - athletes foot
Tineas capitis – scalp
Tineas corporis – body
Tineas cruris – groin
Usually only a cosmetic problem (e.g., ‘ringworm’) but hard to eradicate
Fungi can be deadly, what type of people is infections from this found in?
Tell me some hospital stats to support this
Usually in immunocompromised (AIDS, immunosuppressants, radiotherapy, organ transplants, etc.)
And hospital infections: pregnancy, diabetes, antibiotic treatment
Invasive candidiasis mortality rate ~ 40%
Invasive cryptococcosis mortality ~ 30%
Invasive aspergillosis ~ 20%
Tell me about yeast, what type of organism it is, its appearance and how they reproduce?
Unicellular
Form smooth round colonies
Usually white in colour
Reproduce by budding/ fission
State two fungal infections caused by yeast
Candida
Crytococcus
Tell me about Candida
Give some examples
Candida are most clinically relevant and multiple species cause disease
Candida albicans most common- found in pharynx, GI and GU tract, vagina
Candida auris- multi-drug resistance first discovered in 2011
Tell me about cryptococcus
What are the two most common types?
Cryptococcus cause opportunistic infections e.g., meningitis, pneumonia, etc.)
Cryptococcus neoformans and gattii most common
Tell me about the structure of moulds, their appearance and how they reproduce?
Multicellular
long filaments/ colonies appear fuzzy
large variety of colours
Reproduce by spore formation (sexual or asexual)
Where is both mould and spores found?
In soil and decaying vegetation
How diverse are moulds?
Extremely diverse group of organisms, vast majority are non-pathogenic
What are the two major pathogenic groups of moulds?
Two major pathogenic groups
Aspergillus- some multi-drug resistance
Mucorales
What do moulds cause?
Cause rhinosinusitis and various forms of pulmonary infection in immunocompromised patients
The other problem of growing resistance to antifungal drugs ‘a global issue’
Timeline of antifungal development
What makes the fungi cell wall different to eukaryotes?
Cell wall containing glucans and chitin, as well as a cell membrane containing ergosterol
Why are fungal infections more difficult to treat than bacterial infections?
- Grow slowly
- Occur in poorly penetrated tissues (e.g., devitalise or avascular tissue)
What type of treatment do fungal infections usually require?
Whats the issue with this?
Usually require prolonged treatment (can require resistance due to long periods of treatment. Hence why we now use combination therapy to try and overcome this)
What are the two ways in which antifungals can work?
They can be fungistatic (stopping fungi from growing) or they can be fungicidal (killing fungi)
What are the main sites of antifungals
Cell wall
Membrane
other
What type of antifungals can work in the cell wall and provide an example for each
What type of antifungals can work in the membrane, provide an example(s) for each
Tell me some other types of antifungals and some examples
Antifungals
Tell me about the structure of the fungal cell walls
Comprises of an inner layer containing the polysaccharide beta-1,3-glucan, beta-1,6-glucan and chitin, and an outer layer containing mannose-proteins (N- and O-linked)
What does the structure of the cell wall provide?
Environmental protection force resistance (e.g., osmotic)
What is the fungal cell wall also involved in?
Morphogenesis (i.e., cell division, budding)
What does the fungal cell wall prevent?
Leakage of mannose-proteins by anchoring them
What are the mannose-proteins involved in?
Cell adhesion, structure, and immune-evasion e.g., dectin-1 in candida
What is the fungal cell wall essential for?
Fungal survival and reproduction
Structure and synthesis of fungal cell walls
Beta-1,3-glucan synthesis
What is the structue of beta-1,3-glucan?
A polysaccharide of glucose monomers linked by glycosific bonds (glycogen in humans/ cellulose in plants)
What is beta-1,3-glucan synthase responsible for?
What does it require and tell me about its subunits
Beta-1,3-glucan production from UDP-Glc
- Catalytic subunit(s) in the plasma membrane
- Requires GTP-binding for activity (Rho)
Tell me about UDP= uridine diphosphate
- Nucleotide diphosphate
- Ester of pyrophosphoric acid with the nucleoside uridine
- UDP consists of the pyrophosphate group, the pentose sugar ribose and the nucleobase uracil
- Important for the process of glycogenesis (process which stores glucose as glycogen in liver and muscles.
Give an example of a Beta-1,3-glucan synthesis inhibitor?
Echiocandina
Tell me about how Beta-1,3-glucan synthesis inhibitors work?
Act via un-competitive (binds to the non-catalytic subunit of the enzyme so not competing with substrate) inhibition of beta-1,3, glucan synthase (FKS1p subunit)
Echiocandins is the ‘penicillin of antifungals’ what does this mean?
They prevent the synthesis of fungal cell wall
What type of diseases is Echiocandins used to treat and how is it administered and why is this the case
Broad spectrum and used to treat Candida and Aspergillus infections
Poor bioavailability so administered intravenously
Beta-1,3-glucan synthesis inhibitors (Echiocandins)
What is chitin?
How is the polymer made?
A polysaccharide of N-acetylglucosamine monomwers linked by glycosidic bonds (1,4beta)
What is Chitin similar to?
Peptidoclycan
(structural component of bacterial cell walls)
Tell me about Chitin synthase
This is responsible for chitin production
Where is the active site of chitin?
On the inner membrane side
Chitin synthesis
Give an example of a chitin synthesis inhibitor
Nikkomycin Z
What does Nikkomycin Z inhibit?
The synthesis of chitin via competitive inhibition of chitin synthase
What is Nikkomycin Z structurally similar to?
UDP-GlcNAc (mimics)
How is Nikkomycin Z thought to enter the cells?
By peptide endocytosis mechanism
What is Nikkomycin Z used in?
Veterinary setting but currently undergoing clinical trials in humans (phase I)
(combination therapy?)
What is the main difference between fungal and other eukaryotic/ prokaryotic membranes?
The presence of ergosterol
Why are fungal cell membranes thought to be related to the climatic instabilites?
The highly varying humidity and moisture conditions
Encountered by fungi in their typical ecological niches (plant and animal surfaces, soil, etc.)
Drugs that bind to ergosterol or prevent its synthesis are what?
Selective antifungals
Name a membrane disrupting agent?
polyenes
How do polyenes disrupt membranes?
They bind reversibly with high affinity to ergosterol-containing membranes (sometimes weakly to cholesterol-containing)
What are polyenes used to treat?
Used to treat systemic infections of candida and cryptococcus
How are polyenes taken?
Taken orally/ applied topologically (can’t cross BBB) and sometimes delivered as a formulation with lipids/ liposomes (reduced toxicity)
How can polyene be used on objects?
Give an example of a large macrocyclic polyene
Lactone
Why is polyene used on objects?
Polyene kills fungus, hence why in image above mould doesn’t grow where that is present
Tell me about Amphotericin B and Nystatin, what both are used to treat, how they are taken and their polarities/ what this is the case
Give two examples of polyene antibiotics that are potent antifungal agents
Amphotericin B and nystatin are two polyene antibiotics that are potent antifungal agents. These drugs are active against most pathogenic fungi like Aspergillus and Candida
What are the two key interactions with polyenes and what does this lead to?
2 key interactions are hydrophobic and electrostatic interactions
Leads to pore formation and increases cell membrane permeability, particularly to monovalent cations (especially K+), leads to leaks, cell lysis and cell death
Can promote the entry of other anti-metabolites
Whats the relationship between cholesterol and ergosterol?
Competitive binding of cholesterol and ergosterol to the polyene antibiotic nystatin
Tell me about the binding of the anti-fungal amphotericin B to the fungi cell wall agents ergosterol and how does this compare with cholesterol?
Tell me some side effects of polyenes
Serious side effects e.g., shaking, vomiting, fever, anaphylaxis, nephrotoxicity (rapid deterioration in the kidney function due to medications and chemicals)- potentially lethal
How could we potentailly make polyenes more specific?
Can also increase prostaglandin synthesis
Lipid formulations- more soluble and less toxic (not perfect)
Name some antifungals and what processes they involved in?
Azoles/triazoles (e.g., ketoconazole)- Ergosterol synthesis
Allyamines (e.g., Terbinafine)- Ergosterol synthesis
Echinocandins (e.g., caspofungin)- Cell wall synthesis
Nikkomycin Z- Cell wall synthesis
Polyenes (e.g., amphotericin)- membrane disruption
Grizeofulvin- anti-mitotic
Flucytosine- nucleic acid synthesis
Tell me the simple steps to ergosterol synthesis
What happens if the amounts of ergosterol were reduced?
Reducing ergosterol disrupts the close packing of phospholipid acyl chains and impairs the functions of membrane-bound enzymes, such as ATPases and the electron transport system – slows growth, and fungi die!
What compounds are used as ergosterol synthesis inhibitors?
Allyamines
What are the stages to the inhibition of squalene epoxidation?
What does squalene epoxidase contain in its active site?
Squalene epoxidase contains FAD redox co-factor in its active site
How can you block ergosterol synthesis?
Block ergosterol synthesis by un-competitive inhibition of squalene epoxidase- bind region off active site of enzyme and therefore prevent enzyme activity
As mentioned previously, decreasing the levels of ergosterol are toxic for fungi, what other factor also is?
Decreases levels of ergosterol, as well as increasing squalene levels, also toxic for the fungi
How does the mammalian cholesterol biosynthesis pathway compare to the fungi ergosterol synthesis pathway?
Mammalian cells have the same enzyme in the cholesterol biosynthesis pathway with lower affinity for these inhibitors
What is the Terbinafine IC50 for squalene epoxidase in candida and rat liver?
Terbinafine IC50 (inhibitor concentration, half activity for enzyme) for squalene epoxidase in Candida is 0.03 μM and in rat liver is 77 μM (77x magnitude higher in rats)
[Note: IC50 is concentration of inhibitor where response (or binding) is reduced by half; lower it is, higher the affinity]
Narrow spectrum but effective against nail and skin dermatophytes (lots of side effects if taken orally)
Name another ergosterol synthesis inhibitor?
Thiocarbamates
Tell me the following about thiocarbamates…
- what they inhibit
- what they’re used to tread
- other names they are sold under
‘Probably’ inhibits squalene epoxidase
Used to treat jock itch, athlete’s foot and ringworm
Tolnaftate is sold under several brand names, most notably Tinactin and Odour Eaters
Name another ergosterol synthesis inhibitor?
Azoles
Tell me the stages to the inhibition of lanosterol C14 demethylase
What is the largest class of antifungals in clinical use and how can they be taken?
The azoles are the largest class of anti-fungals in clinical use
They can be taken orally/ intravenously
How do the azoles block ergosterol synthesis?
Block ergosterol synthesis by competitive inhibition of lanosterol C14 demethylase
What effect does decreasing the levels of ergosterol, as well as increasing lanosterol levels have on fungi?
It is toxic to fungi
Whats a typical IC50 of an azole for lanosterol C14 demethylase in Candida and in mammalian cells?
A typical IC50 of an azole for lanosterol C14 demethylase in Candida is 1 nM and in mammalian cells is 1 μM
What type of infections are azoles used to treat?
Candida infections
Among the azole group of antifungals there are compounds containig Imidazoles and Triazoles, name some examples for each
Tell me the requirements of the azole in order to inhibit ergosterol synthesis
- A basic imidazole or 1,2,4-triazole is essential for binding the iron atom via the ring nitrogen(s)
- Remainder of the azole molecule binds to the apoprotein in a manner dependent on the individual azole’s structure
- Most active have two or three aromatic rings, at least one of which is substituted with halogens or other nonpolar groups (most active have fluorine/chlorine in the structure)
- The large nonpolar part resembles the steroid molecule in binding to the enzyme
- Since it works by inhibition of cytochrome P450 (CYP 3A4) it may interact with other drugs that are metabolised by this enzyme (e.g., cyclosporine, an immunosuppressant)
What is the most recent Triazole?
Tell me about it
Name another ergosterol synthesis inhibitor?
Morpholines
What do morpholines inhibit?
C14 reductase and C8 isomerase
What do morpholines cause?
Causes accumulation of ignosterol containing a C14 double bond
Compare the IC50 of C14 reductase and C8 isomerase
IC50 for C14 reductase is 2.93 µM and for C8 isomerase is 1.8 nM - synergistic effect of inhibiting two steps in the same pathway
How are morpholines marketed?
Marketed as Curanail, Loceryl, Locetar or Odenil. Available over the counter as a nail lacquer
Can morpholines be used to target systemic infections?
Cannot be used to target systemic infections at the moment but it is good as a topical treatment for things like nails
Name a morpholine example
Amorolfine
What compound is involved in nucleuc acid synthesis?
Flucytosine
What are the stages of flucytosines involvement in nucleic acid synthesis?
What are flucytosines often used an?
Often used as an adjunctive or for systemic fungal infections e.g., Cryptococcal meningitis
Rarely used on its own (high resistance rate)
What are the two mechanisms of inhibition that flucytosine has?
2 mechanisms of inhibition: ribosome structures of tRNAs are disrupted as they don’t fold properly and therefore don’t get protein synthesis properly, also get inhibition of DNA synthesis
Irreversible inhibition of thymidylase synthase
Folic acid co-factor adds the methyl group –> intermediate step –> permanently bound enzyme intermediate via a covalent bond which doesn’t proceed any further
Suicide inhibitor and it inactivates the enzyme (thymidylate synthase) irreversibly
Name an antimitotic
Griseofulvin
Tell me about Griseofulvin
What does it interact with?
What does it target?
What does it have a similar effect to?
What does it bind to?
How is it administered?
First antifungal developed (natural product)
Interacts with tubulin, interfering with microtubule/spindle formation and mitosis (cells arrested in metaphase) and prevents growth (fungistatic)
Targets cytoskeleton
Similar effect to vinblastine (anti-cancer agent)
Binds to keratin / selectivity is thought to involve the energy-dependent uptake into fungal cells (100x greater than mammalian cells)
Administered orally (ineffective topically) and used to treat dermatophyte infections
Fungi can develop antifungal resistance (mainly non-transferable), for the following compounds how is this done?
- Echinocandims
- Polyenes
- Azoles
- Flucytosine
Antifungal resistance (mainly non-transferable)
Echinocandins (cell wall synthesis) (non-competitive)
- Point mutations to target site and/or overexpression of FKS1 and FKS2 genes (proteins)- helps to overcome
Polyenes (ergosterol membranes)
- Changes in ergosterol biosynthesis / use of different sterols to regulate membrane fluidity
Azoles (ergosterol synthesis) (non-competitive)
- Point mutations to target site and/or overexpression of lanosterol c14 demethylase
- Decreased intracellular drug concentration (activation of ABC efflux pumps or reduction of uptake mechanisms)
Flucytosine (nucleic acid synthesis)
- Changes in nucleic acid metabolism / increased synthesis of 5-UMP substrate
What can be used to identify new antifungal targets and how can these be used to do this?
Identifying new antifungal targets
Genetics: Identify the minimal genome: essential (prevent resistance) and non-conserved (selective) – screening of knock outs
Database (sequence) comparison: Database of Essential Genes (DEG), Online GEne Essentiality (OGEE), Essential Genes on Genomic Scale (EGGA) etc.
e. g., inositol phosphoryl ceramide synthase (AUR1) – sphingolipid synthesis (important in fungi lifecycle?)
e. g., Cryptococcus trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-phosphate phosphatase (TPS2) – glycolysis regulator in Cryptococcus however, not found in mammals
A new class? same old target: Triterpenoids
First new class in many (~20) years
Approved in June 2021 (US)
Same target as Echinocandins (targets glucan-synthase)
- different site target than echinocandins
Key advantage: oral administration
Active against resistant Candida strains (including C. auris)!
Will we get resistance?
Potential future targets?
summary
- Various antifungals exist that are selectively toxic for fungi (over other eukaryotes or prokaryotes)
- Some of these bind proteins unique to fungi (glucan synthase, chitin synthase, ergosterol, etc.); some exploit differences in drug affinities for similar enzymes (e.g., ergosterol synthesis); whilst others exploit differences in uptake/conversion mechanisms (e.g., cytosine deaminase)
- Drug resistance is a problem but might be solvable
- Consequently, researchers are exploiting methods to determine novel target genes (and their protein products) that may lack homologs in other eukaryotes or prokaryotes (via gene knockout and/or sequence analysis experiments)
