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BIOL3027 > Antifungals > Flashcards

Antifungals Flashcards

1
Q

LO

A

Outline the mechanisms of action of the major antifungal agents (griseofulvin, amphotericin, imidazole’s, fluorocytosine)

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2
Q

What are some types of fungi that make an enormous contribution to our everyday life?

If applicable, provide examples of the organisms

A
  • 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)

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3
Q

What type of organisms are fungi?

A

Eukaryotes (unicellular and multicellular, cell nucleus, etc)

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4
Q

What are fungi incapable of doing?

A

Incapable of producing food/ not self-sustaining (heterotrophs)-

Lichens are the exception to this as they are symbiotic

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5
Q

Where are fungi present?

A

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)

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6
Q

What do fungi cause?

Give an example of this

A

Cause superficial (‘cosmetic’), (sub)cutaneous and systemic (tissues/organs) = mycoses (a diseases caused by infection with a fungus, such as ringworm or thrush)

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7
Q

How may some fungi infection occur?

A

Some occur as primary infections but in general only infectious opportunistically

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8
Q

What are the three problematic types of fungi?

A

Yeast

Moulds

Dermatophytes

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9
Q

What are dermatophytes and what do they cause?

A

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)

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10
Q

What is the most common dermatophyte and provide some examples

A

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

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11
Q

Fungi can be deadly, what type of people is infections from this found in?

Tell me some hospital stats to support this

A

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%

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12
Q

Tell me about yeast, what type of organism it is, its appearance and how they reproduce?

A

Unicellular

Form smooth round colonies

Usually white in colour

Reproduce by budding/ fission

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13
Q

State two fungal infections caused by yeast

A

Candida

Crytococcus

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14
Q

Tell me about Candida

Give some examples

A

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

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15
Q

Tell me about cryptococcus

What are the two most common types?

A

Cryptococcus cause opportunistic infections e.g., meningitis, pneumonia, etc.)

Cryptococcus neoformans and gattii most common

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16
Q

Tell me about the structure of moulds, their appearance and how they reproduce?

A

Multicellular

long filaments/ colonies appear fuzzy

large variety of colours

Reproduce by spore formation (sexual or asexual)

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17
Q

Where is both mould and spores found?

A

In soil and decaying vegetation

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18
Q

How diverse are moulds?

A

Extremely diverse group of organisms, vast majority are non-pathogenic

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19
Q

What are the two major pathogenic groups of moulds?

A

Two major pathogenic groups

Aspergillus- some multi-drug resistance

Mucorales

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20
Q

What do moulds cause?

A

Cause rhinosinusitis and various forms of pulmonary infection in immunocompromised patients

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21
Q

The other problem of growing resistance to antifungal drugs ‘a global issue’

Timeline of antifungal development

A
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22
Q

What makes the fungi cell wall different to eukaryotes?

A

Cell wall containing glucans and chitin, as well as a cell membrane containing ergosterol

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23
Q

Why are fungal infections more difficult to treat than bacterial infections?

A
  • Grow slowly
  • Occur in poorly penetrated tissues (e.g., devitalise or avascular tissue)
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24
Q

What type of treatment do fungal infections usually require?

Whats the issue with this?

A

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)

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25
Q

What are the two ways in which antifungals can work?

A

They can be fungistatic (stopping fungi from growing) or they can be fungicidal (killing fungi)

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26
Q

What are the main sites of antifungals

A

Cell wall

Membrane

other

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27
Q

What type of antifungals can work in the cell wall and provide an example for each

A
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28
Q

What type of antifungals can work in the membrane, provide an example(s) for each

A
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29
Q

Tell me some other types of antifungals and some examples

A
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30
Q

Antifungals

A
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31
Q

Tell me about the structure of the fungal cell walls

A

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)

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32
Q

What does the structure of the cell wall provide?

A

Environmental protection force resistance (e.g., osmotic)

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33
Q

What is the fungal cell wall also involved in?

A

Morphogenesis (i.e., cell division, budding)

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34
Q

What does the fungal cell wall prevent?

A

Leakage of mannose-proteins by anchoring them

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35
Q

What are the mannose-proteins involved in?

A

Cell adhesion, structure, and immune-evasion e.g., dectin-1 in candida

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36
Q

What is the fungal cell wall essential for?

A

Fungal survival and reproduction

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37
Q

Structure and synthesis of fungal cell walls

A
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38
Q

Beta-1,3-glucan synthesis

A
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39
Q

What is the structue of beta-1,3-glucan?

A

A polysaccharide of glucose monomers linked by glycosific bonds (glycogen in humans/ cellulose in plants)

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40
Q

What is beta-1,3-glucan synthase responsible for?

What does it require and tell me about its subunits

A

Beta-1,3-glucan production from UDP-Glc

  • Catalytic subunit(s) in the plasma membrane
  • Requires GTP-binding for activity (Rho)
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41
Q

Tell me about UDP= uridine diphosphate

A
  • 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.
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42
Q

Give an example of a Beta-1,3-glucan synthesis inhibitor?

A

Echiocandina

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43
Q

Tell me about how Beta-1,3-glucan synthesis inhibitors work?

A

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)

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44
Q

Echiocandins is the ‘penicillin of antifungals’ what does this mean?

A

They prevent the synthesis of fungal cell wall

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45
Q

What type of diseases is Echiocandins used to treat and how is it administered and why is this the case

A

Broad spectrum and used to treat Candida and Aspergillus infections

Poor bioavailability so administered intravenously

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46
Q

Beta-1,3-glucan synthesis inhibitors (Echiocandins)

A
47
Q

What is chitin?

How is the polymer made?

A

A polysaccharide of N-acetylglucosamine monomwers linked by glycosidic bonds (1,4beta)

48
Q

What is Chitin similar to?

A

Peptidoclycan

(structural component of bacterial cell walls)

49
Q

Tell me about Chitin synthase

A

This is responsible for chitin production

50
Q

Where is the active site of chitin?

A

On the inner membrane side

51
Q

Chitin synthesis

A
52
Q

Give an example of a chitin synthesis inhibitor

A

Nikkomycin Z

53
Q

What does Nikkomycin Z inhibit?

A

The synthesis of chitin via competitive inhibition of chitin synthase

54
Q

What is Nikkomycin Z structurally similar to?

A

UDP-GlcNAc (mimics)

55
Q

How is Nikkomycin Z thought to enter the cells?

A

By peptide endocytosis mechanism

56
Q

What is Nikkomycin Z used in?

A

Veterinary setting but currently undergoing clinical trials in humans (phase I)

(combination therapy?)

57
Q

What is the main difference between fungal and other eukaryotic/ prokaryotic membranes?

A

The presence of ergosterol

58
Q

Why are fungal cell membranes thought to be related to the climatic instabilites?

A

The highly varying humidity and moisture conditions

Encountered by fungi in their typical ecological niches (plant and animal surfaces, soil, etc.)

59
Q

Drugs that bind to ergosterol or prevent its synthesis are what?

A

Selective antifungals

60
Q

Name a membrane disrupting agent?

A

polyenes

61
Q

How do polyenes disrupt membranes?

A

They bind reversibly with high affinity to ergosterol-containing membranes (sometimes weakly to cholesterol-containing)

62
Q

What are polyenes used to treat?

A

Used to treat systemic infections of candida and cryptococcus

63
Q

How are polyenes taken?

A

Taken orally/ applied topologically (can’t cross BBB) and sometimes delivered as a formulation with lipids/ liposomes (reduced toxicity)

64
Q

How can polyene be used on objects?

A
65
Q

Give an example of a large macrocyclic polyene

A

Lactone

66
Q

Why is polyene used on objects?

A

Polyene kills fungus, hence why in image above mould doesn’t grow where that is present

67
Q

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

A
68
Q

Give two examples of polyene antibiotics that are potent antifungal agents

A

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

69
Q

What are the two key interactions with polyenes and what does this lead to?

A

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

70
Q

Whats the relationship between cholesterol and ergosterol?

A

Competitive binding of cholesterol and ergosterol to the polyene antibiotic nystatin

71
Q

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?

A
72
Q

Tell me some side effects of polyenes

A

Serious side effects e.g., shaking, vomiting, fever, anaphylaxis, nephrotoxicity (rapid deterioration in the kidney function due to medications and chemicals)- potentially lethal

73
Q

How could we potentailly make polyenes more specific?

A

Can also increase prostaglandin synthesis

Lipid formulations- more soluble and less toxic (not perfect)

74
Q

Name some antifungals and what processes they involved in?

A

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

75
Q

Tell me the simple steps to ergosterol synthesis

A
76
Q

What happens if the amounts of ergosterol were reduced?

A

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!

77
Q

What compounds are used as ergosterol synthesis inhibitors?

A

Allyamines

78
Q

What are the stages to the inhibition of squalene epoxidation?

A
79
Q

What does squalene epoxidase contain in its active site?

A

Squalene epoxidase contains FAD redox co-factor in its active site

80
Q

How can you block ergosterol synthesis?

A

Block ergosterol synthesis by un-competitive inhibition of squalene epoxidase- bind region off active site of enzyme and therefore prevent enzyme activity

81
Q

As mentioned previously, decreasing the levels of ergosterol are toxic for fungi, what other factor also is?

A

Decreases levels of ergosterol, as well as increasing squalene levels, also toxic for the fungi

82
Q

How does the mammalian cholesterol biosynthesis pathway compare to the fungi ergosterol synthesis pathway?

A

Mammalian cells have the same enzyme in the cholesterol biosynthesis pathway with lower affinity for these inhibitors

83
Q

What is the Terbinafine IC50 for squalene epoxidase in candida and rat liver?

A

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)

84
Q

Name another ergosterol synthesis inhibitor?

A

Thiocarbamates

85
Q

Tell me the following about thiocarbamates…

  • what they inhibit
  • what they’re used to tread
  • other names they are sold under
A

‘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

86
Q

Name another ergosterol synthesis inhibitor?

A

Azoles

87
Q

Tell me the stages to the inhibition of lanosterol C14 demethylase

A
88
Q

What is the largest class of antifungals in clinical use and how can they be taken?

A

The azoles are the largest class of anti-fungals in clinical use

They can be taken orally/ intravenously

89
Q

How do the azoles block ergosterol synthesis?

A

Block ergosterol synthesis by competitive inhibition of lanosterol C14 demethylase

90
Q

What effect does decreasing the levels of ergosterol, as well as increasing lanosterol levels have on fungi?

A

It is toxic to fungi

91
Q

Whats a typical IC50 of an azole for lanosterol C14 demethylase in Candida and in mammalian cells?

A

A typical IC50 of an azole for lanosterol C14 demethylase in Candida is 1 nM and in mammalian cells is 1 μM

92
Q

What type of infections are azoles used to treat?

A

Candida infections

93
Q

Among the azole group of antifungals there are compounds containig Imidazoles and Triazoles, name some examples for each

A
94
Q

Tell me the requirements of the azole in order to inhibit ergosterol synthesis

A
  • 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)
95
Q

What is the most recent Triazole?

Tell me about it

A
96
Q

Name another ergosterol synthesis inhibitor?

A

Morpholines

97
Q

What do morpholines inhibit?

A

C14 reductase and C8 isomerase

98
Q

What do morpholines cause?

A

Causes accumulation of ignosterol containing a C14 double bond

99
Q

Compare the IC50 of C14 reductase and C8 isomerase

A

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

100
Q

How are morpholines marketed?

A

Marketed as Curanail, Loceryl, Locetar or Odenil. Available over the counter as a nail lacquer

101
Q

Can morpholines be used to target systemic infections?

A

Cannot be used to target systemic infections at the moment but it is good as a topical treatment for things like nails

102
Q

Name a morpholine example

A

Amorolfine

103
Q

What compound is involved in nucleuc acid synthesis?

A

Flucytosine

104
Q

What are the stages of flucytosines involvement in nucleic acid synthesis?

A
105
Q

What are flucytosines often used an?

A

Often used as an adjunctive or for systemic fungal infections e.g., Cryptococcal meningitis

Rarely used on its own (high resistance rate)

106
Q

What are the two mechanisms of inhibition that flucytosine has?

A

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

107
Q

Irreversible inhibition of thymidylase synthase

A

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

108
Q

Name an antimitotic

A

Griseofulvin

109
Q

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?

A

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

110
Q

Fungi can develop antifungal resistance (mainly non-transferable), for the following compounds how is this done?

  • Echinocandims
  • Polyenes
  • Azoles
  • Flucytosine
A

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
111
Q

What can be used to identify new antifungal targets and how can these be used to do this?

A

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

112
Q

A new class? same old target: Triterpenoids

A

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?

113
Q

Potential future targets?

A
114
Q

summary

A
  • 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)

BIOL3027 (8 decks)

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