Lectures 16/17/18 - Case Study 1

Question 1

What are Cytoskeletal Drugs?

Answer 1

Small molecules that interact with actin/tubulin and alter filament dynamics

Question 2

In what two ways can cytoskeletal drugs impact filament dynamics?

Answer 2

• Stabilising Filaments - prevent depolymerisation and/or cause polymerisation
• Destabilising Filaments - prevent polymerisation and/or cause depolymerisation

Question 3

(i) Where are cytoskeletal drugs commonly isolated from?
(ii) Why are they effective for this purpose?

Answer 3

• Often produced by sessile organisms (e.g., Plants, Fungi, Sponges) which require them as a toxin for defence
• Effective toxins as actin/tubulin are highly conserved, and eukaryotic cells rely on the correct balance of assembly/disassembly of cytoskeletal filaments to survive

Question 4

Give Two Examples of Actin/Microtubule Targeting Agents

Answer 4

Microtubule Targeting - Taxol, Vinka Alkaloids
Actin Targeting - Phallotoxins, Jasplakinolide

Question 5

How can Cytoskeletal Drugs Stabilise Actin Filaments?

(2 Points)

Answer 5

• Actin Stabilising - via:
  1. Binding to and Stabilising Actin Filament (e.g., Phalloidin)
  2. Enhancing nucleation of Actin Filaments (e.g., Jasplakinolide)

Question 6

How can Cytoskeletal Drugs destabilise actin filaments?

(2 Points)

Answer 6

• Actin destabilising - via:
  1. Binding to barbed end, preventing polymerisation (e.g., Cytochalasins)
  2. Sequestering Actin Monomers, enhancing rate of depolymerisation (e.g., Latrunculin)

Question 7

What Cytoskeletal Drugs can Stabilise MTs?

(2 Points)

Answer 7

• Taxanes
• Epothilones

Question 8

How can Cytoskeletal Drugs Destabilise MTs?

(1 Point, 3 Examples)

Answer 8

• MTs Destabilising - via:
  1. Binding to MTs/Tubulin, blocking polymerisation (e.g., Colchicine, Vinca Alkaloids Nocodazole)

Question 9

By what mechanism does Phalloidin stabilise Actin filaments?

Answer 9

• Binds simultaneously to 3 different actin monomers in two protofilaments, stabilising interactions along a/between protofilament(s)

Question 10

Describe how Actin-Destabilising compounds can bind to Actin, giving specific examples for both

Answer 10

Bind to either:
* ATP-binding cleft of G-actin e.g., Latrunculin
* Barbed end of F-Actin e.g., Kabiramide C (Macrolide)

Question 11

How does Latrunculin destabilise Actin filaments?

Answer 11

Binds in ATP Cleft, and restricts conformational changes required for formation of stable interactions between monomers, thereby sequestering G-actin

Question 12

How do Macrolides destabilise Actin Filaments?

Answer 12

Bind to G-actin monomer at end which becomes barbed end in similar fashion to capping regulatory protein gelsolin (between SDI and SDIII)

Question 13

Why are Actin targeting agents not widely used as therapeutics?

Answer 13

• Display High toxicity due to lack of specificity for actin isoforms, hence cause unacceptable off-target effects (e.g., cardiotoxicity)

Question 14

How many different drug-binding sites are present on the a/B tubulin heterodimer?

Answer 14

6 Distinct Binding sites of which:
* 2 - targeted by MT-stabilising agents
* 4 - targeted by MT-destabilising agents

Question 15

What MT-targeting drugs have been approved for use in cancer treatment?

(2 Points)

Answer 15

1. Vinca Alkaloids (MT-destabilising)
2. Taxanes (MT-stabilising)

Question 16

(i) How do MT-targeting drugs effectively kill cancer cells?
(ii) Why do these drugs produce side effects?

Answer 16

(i) Anti-cancer MT drugs suppress MT dynamics in the mitotic spindle, thereby blocking mitosis and leading to cell arrest/apoptosis
(ii) Target Rapidly dividing cells, hence kill cancer cells but also normal cells (Stem Cells, Hair Follicles)

Question 17

How do Taxanes Stabilise MTs?

Answer 17

Bind to pocket on B-tubulin on the lumenal side of MTs, stabilising them and subsequently suppressing their dynamics

Question 18

How do Vinca Alkaloids destabilise MTs?

(2 Points)

Answer 18

• Form wedge at B-tip of tubulin heterodimer, which prevents curved-to-straight transition of tubulin required for incorporation into MTs
• This may also sequester tubulin dimers into ring-like oligomers, preventing polymerisation

Question 19

Define the 3 Mechanisms by which cancer cells obtain chemoresistance to MT-targeting compounds

Answer 19

1. Drug Efflux Pumps
2. Alteration of Tubulin isotypes expressed, leading to decreased sensitivity to particular drug
3. Deficient Induction of Apoptosis

Question 20

Peptides derived from what MT-associated proteins are under investigation as anti-cancer therapies?

(2 Points)

Answer 20

1. Neurofilaments (IFs)
2. Neuronal Cortical Collapse Factors

Question 21

What are Neurofilaments?

(5 Points)

Answer 21

• Heteropolymers composed of four subunits:
  1. Neurofilament Heavy Chain (NFH)
  2. Neurofilament Medium Chain (NFM)
  3. Neurofilament Light Chain (NFL)
  4. a-Internexin

Question 22

Why could peptides derived from Neurofilaments be used as an effective cancer therapy?

(3 Points)

Answer 22

• Neurofilaments contain sites in N-terminal (head) domains that can bind unpolymerised tubulin
• Peptides derived from NF N-terminal domain have been shown to inhibit in-vitro polymerisation of MTs (e.g., NFL-TBS.40-63)
• Can be taken up by certain types of cultured cells (e.g., Neurones) with little/no effect on other cell types (i.e. greater specificity than other drug treatments)

Question 23

What are Axonal Cortical Collapse Proteins (Give Example)?

Answer 23

• Regulatory proteins in axon, which maintain the organisation of microtubule bundles
• E.g., Efa6 - inhibits MT growth at cell periphery, helping to maintain organised MT bundles

Question 24

What is the Structure of Efa6? How does it inhibit MT growth?

(2 Points)

Answer 24

• C-terminus - contains localisation sequence
• N-terminus - contains MTED peptide, a 20aa motif which directly binds tubulin to inhibit MT growth

Question 25

(i) What is Oxidative Stress? (ii) How does it affect MT network in Cardiomyocytes?

Answer 25

(i) Characteristic of Ischemic Heart disease (+Other Heart Pathologies), where narrowing of arteries and subsequent reduced blood flow leads to significant increase in H2O2 (due to lack of oxygen) (ii) Oxidative Stress remodels the MT network in cardiomyocytes, leading to increased MT density

Question 26

What observations were made in Cardiomyocytes exposed/treated with Hydrogen Peroxide (H2O2)? | (5 Points)

Answer 26

* Presence of H2O2 did not significantly affect: 1. Growth Rate 2. Shrinkage Rate 3. Catastrophe Frequency * Increasing H2O2 conc. exponentially increased rescue frequency of MTs

Question 27

How did H2O2 Exposure Influence MT-dynamics? | (2 Points)

Answer 27

* H2O2 treatment causes oxidation of Cys residues in the MTs, resulting in localised damage that is repaired via incorporation of new tubulin heterodimer * Shrinking MTs can be rescued at sites of GTP-tubulin islands, therefore greater H2O2 exposure leads to increased recovery and subsequently longer and more dense MT networks

Question 28

(i) How do more dense MT networks impact cardiomyocytes? (ii) How can this be treated?

Answer 28

(i) Increased density has been shown to increase cardiomyocyte stiffness and contractile dysfunction in heart disease (ii) Restoration of cardiomyocyte contractility can be achieved using MT-destabilising agents to depolymerise the MT network