CHEM2: Anthelmintic Avermectin Ligand-gated Ion Channel Agonists

Question 1

What are avermectins?

Answer 1

Macrolides

Question 2

Macrolide ring is __ - membered

Answer 2

16

Question 3

Where is the L-oleandrose disaccharide attached to the aglycone?

Answer 3

13-alpha-position

Question 4

Where do avermectins vary?

Answer 4

C-5 with hydroxy or medroxy groups

C-23 with B-hydroxyl group or 22/23-alkene

C-25 with isopropyl or sec-butyl groups

Question 5

Avermectins, macrolides, tetracycline antibiotics, statins + other natural products are Polyketides.

What are Polyketides?

What do all polyketides possess in their structure?

Answer 5

Compounds derived from multiple condensations of carbonyl compounds

Question 6

What are Polyketide synthases?

Answer 6

Enzyme complexes that produce polyketides.

Large class of 2^ndary metabolites in fungi, bacteria + other plants

Question 7

Avermectin PKS genes are organised into 2 blocks of open reading frames (ORFS).

What are they + function?

Answer 7

aveA1-aveA2

aveA3-aveA4

They encode for 12 sets of enzyme activity (modules), each containing distinctive active-site domains, required for one cycle of polyketide elongation.

Total of 55 active site domains possessing fatty acid synthase-like activities constitute giant multifunctional enzyme complex

Question 8

For the polyketide synthesis of avermectin B₁, name the active sites required for condensation

Answer 8

• Acyltransferase (AT)
• Acyl carrier protein (ACP)
• Beta-ketoacyl synthase (KS)

Each condensation cycle results in a beta-keto group

Question 9

For the polyketide synthesis of avermectin B₁, following each condensation, the beta-keto group is reduced and the hydroxy group is dehydrated.

What is it reduced to + the other dehydrated to?

Answer 9

Reduced to a hydroxy group by ketoreductase

Dehydrated to produce a double bond by dehydratase

Question 10

For the polyketide synthesis of avermectin B1, the end modules encodes for a specific domain.

What is this domain + it function?

Answer 10

Thioesterase (TE) domain

Releases the complete polyketide from PKS

Question 11

What do enolisable carbonyl compounds + all carbonyl compounds act as?

Answer 11

Enolisable = nucleophiles

All = electrophiles

Question 12

What is the simplest form of an aldol reaction?

Answer 12

Self-condensation

Question 13

Aldol reactions are either ______ catalysed

Answer 13

Base or acid - catalysed

Question 14

What type of reactions are enolisation + aldol formation?

Answer 14

Equilibrium reactions

If the aldol adduct has at least 1 alpha-hydrogen, it will dehydrate to an alpha-beta-unsaturated carbonyl compounds

Question 15

Dehydration is irreversible.

How does this help the overall condensation reaction?

Answer 15

It provides the driving force for the overall condensation reaction

Question 16

In nature, aldol reactions are _____-catalysed.

Question 17

In chemical synthesis, crossed aldol condensations under equilibrium conditions give mixtures of products.

What are the 2 solutions to this?

Answer 17

1. Use a symmetrical nucleophile that cannot enolise
2. Use non-equilibrium conditions

Question 18

What is the claisen Reaction?

Answer 18

Involves an enolate nucleophile + carbonyl electrophile but the carbonyl group is an ester (or thioester)

Results in a good leaving group, retaining the carbonyl compound

Question 19

A key feature of polyketide synthases = modularity

Protein consists of several modules with distinct functionality

Module configuration determines what polyketide is made

Modular approach leads to diversity in polyketide production.

What key domains (core modules) are there?

Answer 19

• Acyltransferase
• Acyl carrier protein
• Ketoacylsynthase
• Ketoreductase
• Dehydratase
• Thioesterase

Question 20

Acyltransferase

Answer 20

Question 21

Acyl carrier protein

Answer 21

Question 22

Ketoacylsynthase

Answer 22

Question 23

How can KS create new C-C bond?

Answer 23

KS uses a claisen condensation

Question 24

Ketoreductase

Answer 24

Question 25

Dehydratase

Answer 25

Question 26

Thioesterase

Answer 26

Question 27

Describe the polyketide biosynthesis of avermectin B₁

Answer 27

Question 28

In nature, what synthesises avermectins?

Answer 28

Streptomyces avermitillis

Question 29

During the original studies on avermectinSARs, compounds of the Β series were observed generally to be more potent than those of the A series.

What is enchancing the activity>

Answer 29

Unsubstituted hydroxy group at the 5-position

Question 30

In avermectin SARS, what was the effect on reduction of the 22,23-alkene?

Answer 30

Little effect on activity but further reduction caused a substantial decrease in activity

Question 31

In avermectin SARS, what was the effect of acetylation @ the 4’ position?

Answer 31

No change in activity

However acetylation @ 5-/23- position caused a decrease in activity

Question 32

Which avermectin is more active via oral admin?

Answer 32

Avermectin B₁ over B₂

Question 33

Why did avermectin B1 + B2 have different activity profiles against different nemotode species?

Why?

Answer 33

Linked to the different conformations of the rings containing C22-C23 and 22,23-dihydroavermectin B1 was synthesised and tested

Compound was selected for development as a broad-spectrum anti-parasitic agent on the basis of its overall efficacy by oral and parenteral routes and for its improved safety profile

22,23-Dihydroavermectin B1, containing ≥ 80% of the dihydro-B1a and ≤ 20% of the dihydro-B1b compounds was assigned the non-proprietary name ivermectin.

Question 34

Avermectins are ion channel agonist.

How?

Answer 34

Avermectins were proposed to be agonists for neurotransmitter function.

This causes an influx of chloride ions into the cells, leading to hyperpolarisation and subsequent paralysis of the neuromuscular systems.

Experimental studies showed that inhibition occurred via glutamate-gated chloride ion channels and γ-aminobutyric acid (GABA)-related chloride ion channels. Avermectin sinteract with these channels, preventing their closure.

As a consequence, synapse membranes become increasingly permeable to chloride ions, which leads to hyperpolarisation of the neuronal membrane and decreases, or prevents, neuronal transmission.

This, in turn, leads to paralysis of the somatic muscles, particularly the pharyngeal pump, causing the death of the parasite.

Question 35

Describe Ivermectin’s mode of action

Answer 35

This shows that ivermectin binds at the subunit interfaces stabilising the open (conducting) form of GluClα by preventing inward movement of the helices making up the ion pore.

Ivermectin potently activates GluClα while rendering the receptor susceptible to further activation by glutamate. Hence, at GluClα, ivermectinis a partial allosteric agonist

Question 36

If avermectins block the transmittance of electrical activity in nerves and muscle cells by stabilising glutamate and GABA-related chloride channels, which are expressed both in the host and the parasite, why are avermectins safe?

Answer 36

In mammals, glutamate- and GABA-related chloride channels, as well as neurons, are found predominantly in the central nervous system, whereas in arthropods and nematodes they are located in the peripheral nervous system, specifically at the neuromuscular junctions and the central ventral cords.

Ivermectin and other avermectins do not readily cross the blood-brain barrier in mammals at therapeutic doses.

This, coupled with the relatively low dose concentrations needed, ensures that mammals can ingest ivermectin with a high degree of safety.