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Pharm Decks > Lecture 10 - Spindle Mitosis Poisons > Flashcards

Lecture 10 - Spindle Mitosis Poisons Flashcards

1
Q

What are microtubules?

A
  • components of the cytoskeleton
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2
Q

Microtubule structure overview

A
  • Alpha and Beta Tubulin heterodimers connected by a non-covalent bond
  • Have a + end associated with GTP cap
  • Have - end surface
  • ML surface
  • H3 surface
  • 13 filaments in spiral structure
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3
Q

Overview of dynamics of microtubules

A
  • Dynamics at positive end faster than dynamics at negative end
  • Rate of addition/loss faster at + end
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4
Q

How do microtubules expand?

A
  • Heterodimers free floating in proximity can be added on to either end
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5
Q

What is treadmilling and what effect does this give?

A
  • add heterodimers at one end whilst the are being lost from the other end
  • gives effect of microtubule moving in space
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6
Q

4 factors which characterise dynamism of microtubules

A
  • Rate of MT growth
  • Rate of MT shrinkage
  • Catastrophe
  • Rescue
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7
Q

What is catastrophe?

A

Frequency of transition from growth (or pause) to shrinking

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8
Q

What is rescue?

A

Frequency of transition from shrinking to growth (or pause)

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9
Q

What is the role of GTP/GDP in microtubule stability?

A
  • Both α and β units have a GTP/GDP binding site at the (+) end
  • Within heterodimers the α+ retains GTP tightly bound (due to interaction to the β (-)-end), and is non-exchangeable
  • At β (+)-end, Tubulin-bound GTP hydrolysed to tubulin–GDP and inorganic phosphate (Pi ) at the time or after tubulin adds to the microtubule ends
  • The Pi dissociates from the microtubule, leaving a microtubule core consisting of tubulin with stoichiometrically bound GDP.
  • MT end containing tubulin-bound GTP or GDP–Pi is stable, or ‘capped’, against depolymerization.
  • Hydrolysis of tubulin-bound GTP and release of Pi induces conformational changes in the tubulin molecules –> destabilize the microtubule polymer –> CATASTROPHE and shortening of MT
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10
Q

What is difference between MT dynamics in vivo and in vitro?

A

Slower in vitro

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11
Q

How is functional diversity achieved in microtubules?

A
  • Microtubule associated proteins (MAPs)
  • Soluble tubulin
  • Microtubule surfaces and ends
  • Isotypes
  • Post-translational modification
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12
Q

Role of MT during onset of mitosis?

A
  • onset of mitosis –> network of MT replaced by new population which are more dynamic
  • result of an increase in catastrophe and decrease in rescue
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12
Q

What is the process of hunting in prometaphase by MT?

A

microtubules ‘hunt’ by rapidly elongating and shrinking, probing the cytoplasm until they find the chromosome kinetochore

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13
Q

Why would we target microtubules?

A
  • play role in mitosis
  • …by organising the chromosomes, and cleaving daughter cells
  • cancer characterised by rapid replication
  • naturally occurring toxic molecules by plants which target them
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14
Q

What are the 2 groups of agents which bind to microtubules?

A

Surfaces of the globular part of tubulin has several binding sites that allow binding of:

  • Microtubule Stabilizing Agent (MSA’s)
  • Microtubule Destabilizing Agents (MDA’s)
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15
Q

What do the microtubule targeting agents affect?

A

MT dynamics, leading to mitotic arrest and cell death

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16
Q

How many MTA binding sites are known?

A
  • 6

4 on β-tubulin and two on α tubulin

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17
Q

What are the MTAs which bind to beta tubulin?

A
  • taxane
  • laulimalide/peloruside
  • vinca
  • maytensine
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18
Q

What are the MTAs which bind to alpha tubulin?

A

colchicine and pironetin

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19
Q

What are the microtubule stabilizing agents?

A
  • Taxane ligands
  • Laulimalide/peluroside ligands
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20
Q

What are the microtubule destabilising agents?

A
  • Vinca ligands
  • Maytensine ligands
  • Colchicine ligands
  • Pironetin ligands
21
Q

What is the name of the drug containing taxane ligands?

A

Paclitaxel

22
Q

What is the mode of action of paclitaxel?

A
  • Acts to strengthen lateral contacts between adjacent protofilaments – leads to MT stabilisation
  • Taxane pocket in MT is near ML surface on inside of tubulin
  • Bind poorly to soluble pool of tubulin
  • Bind high affinity to β subunit
23
Q

What binds well/not well to taxanes?

A

Heterodimers in solution don’t bind well to taxanes (poorly to soluble tubulin)

Bind high affinity to β subunit

24
Q

What is the stoichiometry of paclitaxel to MT dimer?

A

1:1

however typical microtubule (10,000 tubulin units) requires 5,000 paclitaxel molecules

25
Q

How does laulimadlide/peluroside ligands work?

A
  • binds to site
  • MT stabilised by strengthening lateral contacts between protofilaments
26
Q

How does vinca alkaloids work?

A
  • Vinca site is located at the + end surface of β-tubulin
  • Binding alters the surface of + end forming a ‘wedge’
    which interferes with incorporation of new heterodimers
  • The + ends remain curved and microtubule wall cannot form
  • cause tubulin oligomers, decreasing the free tubulin pool
  • depolymerise the MT
27
Q

How does vinca effect the dynamics of MT?

A
  • One or two ligands at the end of a microtubule will reduce dynamics by 50 %
28
Q

How does Maytansine work?

A
  • Maytansine site is close to the vinca site
  • In growing MT, maytansine binding pocket of the (+)-end accommodates the (-)-end of the next α-tubulin unit
  • forming a wedge
  • impedes elongation
29
Q

Problem with Colchicine

A
  • Very toxic
  • but looking in to combrestatin instead
30
Q

How does Colchicine work?

A
  • Colchicine binding site on ALPHA tubulin (between the α and β units)
  • Slow binding, practically irreversible
  • Binding stabilises in curved conformation
  • SLOWs but doesn’t prevent polymerisation
31
Q

What cancers does paclitaxel work in?

A

Ovarian, mammary and lung tumours

32
Q

What cancers does vinca work in?

A

Haematological cancers

33
Q

What are the possible modes of resistance against MTAs?

A
  • Over expression of ABC-transporters –> P-glycoprotein
  • Overexpression or binding of MT associated/regulatory proteins
  • Mutation in tubulin
  • Up and/or down regulation of different tubulin isotypes which change dynamics
  • Overexpression of multidrug resistance (MDR-1) gene
  • Changes in lipid composition
  • Over-expression of IL-6
  • Post translational modifications
34
Q

Most important thing to remember for MTAs and how they work

A

changes in dynamics of MT

35
Q

What are microtubules crucial for?

A
  • Cell shape
  • Transport of vesicles
  • Cell signalling
  • Mitosis
36
Q

What are the side effects of Vinca ligands and Taxane ligands?

A
  • Reversible Myelosuppression
  • Peripheral Neuropathy
37
Q

Overview of study of paclitaxel and resistance…

A
  • A549 lung cancer cells became dependent on paclitaxel
  • Withdrawal lead to faster dynamics
  • Cell adapted to slow effects of drug by making everything else go faster
  • Cells resistant and dependent on drug
38
Q

In paclitaxel what else might favour resistance?

A
  • Expression of endogenous microtubule-depolymerizing factors
39
Q

Where do Vincas originate from?

A

Periwinkle leaves

40
Q

Where do maytensine ligands originate from?

A

Maytenus ovatus

41
Q

Where do colchicine ligands originate from?

A

Autumn crocus

42
Q

Where do Taxane ligands (paclitaxel) orginate from?

A

Taxus Brevifolia

43
Q

Where do Laulimalide/Peluroside ligands originate from?

A

Sea sponges

44
Q

What other hallmark of tumour have antimitotic drugs been investigated in as a treatment and an example of this?

A
  • Angiogenesis –> acting as vascular-targeting agents
  • rapidly depolymerize microtubules of newly formed vasculature to shut down blood supply to tumours

Example: Combrestatin drugs in clinical trail

45
Q

Name of APC transporter which causes resistance

A
  • P-glycoprotein

(ATP-dependent drug efflux pumps)

46
Q

Where is the taxane pocket located?

A

Near ML surface on inside of tubulin

47
Q

Where is the Laulimalide/Perluroside binding pocket located?

A

Opposite side of ML surface with respect to taxane outside the MT wall

48
Q

Where is the Vinca Ligand binding site located?

A

At + end of BETA tubulin

49
Q

Where is the Maytensine site located?

A

Close to the Vinca site

50
Q

How does Colchicine bind to the MT?

A
  • likely it doesn’t bind to the microtubule tip
  • binds to the soluble pool first and is then incorporated

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