Mitotic spindle poisons

• What is the role microtubules?

  Key components of the cytoskeleton and essential in eukaryotic cells. They are crucial for:

  • Cell shape
  • Transport of vesicles
  • Play a role in cell signalling
  • Critical role in mitosis
• What is the structure of microtubules?

  Long, slender, filamentous tubes comprised of a-tubulin and B-tubulin heterodimers (units stuck together)

  • Tubulin heterodimers are connected by non-covalent bonds
  • Many tubulin heterodimers wrap around in a spiral structure (due to missteps)
  • Creates a hollow tube comprised of 13 filaments - the microtubule
  • Microtubule itself has general (+)- and (-)-ends
• What is the structure of a-tubulin and B-tubulin heterodimers?

  • Associate in solution as heterodimers (a-tubulin and B-tubulin)
  • An a-/B-tubulin heterodimer has 4 defined surfaces - (+)-end surface (top), (-)-end surface (bottom), H3 surface, and ML surface
  • Additionally, the a-tubulin unit and B-tubulin unit individually have (+)- and (-)-ends

  
• What are the four factors characterising dynamism?

  • Rate of microtubule growth
  • Rate of microtubule shrinkage
  • Catastrophe - frequency of transition from growth (or pause) to shrinking
  • Rescue - frequency of transition from shrinking to growth (or pause)
• What is treadmilling?

  Results from the combined effects of microtubule growth and shrinkage where heterodimer units add to one end and leave from the other. By doing so, looks like the microtubule is "moving" but actual individual heterodimers remain stationary

  
• What are the microtubule dynamics?

  
  

  Microtubules are in highly dynamic and constantly in flux, temporally and spatially

  • (+)-end dynamics are faster than (-)-end - rate at which tubulin heterodimers go on and come off is faster at (+)-end
  • Still, both ends can add/lose new heterodimer units
  • Tubulin heterodimer units are in equilibrium between soluble pool (solution) and being part of microtubule
  • (+)-end also associated with GTP cap - hydrolysing out the GTP units will compromise microtubule stability can cause it to fray and fall apart

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

  • Both α- and β-tubulin units have a GTP/GDP binding site at the (+)-end
  • Within the heterodimers, the α unit retains a GTP tightly bound (due to position of interaction with the β (-)-end) and is non-exchangeable
  • However, β (+)-end is accessible and hydrolysis of its bound GTP to GDP occurs ~same time/shortly after addition to the growing microtubule
  • Normally the ‘GTP cap’ has either GTP or GDP/Pi (stable)
  • If the cap is lost (e.g. hydrolysis), the ends fray resulting in catastrophe (change to depolymerisation)

  
• Where does the complexity of microtubules arise?

  Observations of microtubule dynamics in vitro was many orders of magnitude slower than in cells - implies something else controls it.

  In reality, large amount of functional diversity of microtubules achieve through:

  • Microtubule associated proteins (MAPs)
  • Soluble tubulin
  • Microtubule surfaces and ends
  • Isotypes, i.e. 7 α-tubulin and 9 β-tubulin isotypes - some ubiquitious, some tissue-specific
  • Post-translational modifications, e.g. polyglutamylation, polyglycylation, phosphorylation etc. when a-tubulin and B-tubulin are being produced
• What is the role of microtubules during interphase?

  • During interphase = microtubules turn over (i.e. exchange with soluble tubulin pool) slowly - heterodimers pop on and off without changing overall shape
  • At the onset of mitosis = entire network disassembles and is replaced by a new population of spindle microtubules which are 4-100 times more dynamic - rate of processes sped up, treadmilling occurs faster
  • For at least some cells this is a result of an increase in ‘catastrophe’ and a decrease in ‘rescue’ rather than any change in rates of shortening and growth
• What is the role of microtubules during prometaphase?

  • Nuclear envelope broken down
  • Needs timely and correct attachment of chromosome kinetochores to the spindles
  • During prometaphase, microtubules ‘hunt’ - begin at a spindle pole , rapidly elongating (adding heterodimers in one direction and shooting out into cytoplasm to reach and attach to a chromosome) and shrinking (retracting if they fail to attach to a chromosome)
  • Essentially, probing the cytoplasm until they find and locate all chromosome kinetochores
  • They grow and shrink up to 10 µm until successful attachment
• What is the role of microtubules during early metaphase?

  • Complex movement of chromosomes
  • Congression – movement to the equator to form the metaphase plate

  
• What is the role of microtubules during anaphase?

  • Duplicated chromosomes separate
  • Chromosomes move towards the two spindle poles and thus to from two new daughter cells

  
• What is the role of microtubules during telophase?

  • Duplicated chromosomes have reached new spindle poles
  • Cell is dividing

  
• What is the rationale for targeting microtubules?

  • Microtubules have a key role in organising the chromosomes and cleaving to daughter cells at the right time during mitosis
  • Want to target this key step in cell division since cancer is characterised by rapid replication
• General overview of microtubules therapy history

  • Microtubules = a favourite target for naturally occurring (presumably self-protective), toxins produced by a wide range of plants/animals - competition to survive in the world
  • Large scale screening of natural products - found significant number of compounds/leads for future
  • e.g. Vinca alkaloids first identified 50 years ago from periwinkle leaves - serendipitously shown to have bone marrow toxicity for diabetes
  • Overall success with the mitotic spindle drugs - thought that microtubules represent the best cancer target identified to date
• What are types of antimitotic drugs?

  The surfaces of the globular part of tubulin has six known MTA "binding sites" (allosteric) where agents can bind

  • Microtubule targetings agents (MTAs) alter the dynamics and ultimately lead to mitotic arrest and cell death
  • Two types overall - microtubule stabilising agents (MSAs) or microtubule destabilising agents (MDAs)
  • Many compounds (or at least lead compounds) from plants, fungi and invertabrates
  • Four binding sites on β-tubulin (taxane, laulimalide/peloruside, vinca and maytensine) and two on α-tubulin (colchicine and pironetin)

  
• What does a semi-synthetic route mean?

  
  

  • Uses chemical compounds isolated from natural sources as the starting material and modifies it with extra synthesis to produce novel compounds
  • These isolated natural molecules are very close in structure to the final desired compound - a precursor
  • Isolated molecules can be obtained in larger yield than making the final desired compound from scratch - easier to just convert the isolated molecule
• What is the mode of action of taxane ligands?

  • Taxanes bind poorly to soluble pool of tubulin but with high affinity to the taxane pocket on the B subunit near the ML surface on ‘inside’ of the tubulin (facing lumen)
  • Access through small gaps, or fluctuations in microtubule lattice
  • Conformational change acts to strengthen lateral contacts/binding affinity between adjacent protofilaments - leads to microtubule stabilisation
  • One site per heterodimer so in principle 1:1 stoichiometry - but just 1 paclitaxel per several hundred reduces rate or extent of shortening by 50%

  
• What are some unknowns about taxane ligands' mode of action?

  • The exact mode of binding is compound specific - but all taxanes act in the same way
  • Its possible paclitaxel stabilises microtubules via. an allosteric mechanism (preventing dimer compaction)
• What are taxane ligands?


  • Paclitaxel – tetracyclic diterpenoid
  • Isolated from Taxus brevifolia in 1960s
  • Approved in 1992 by FDA for ovarian cancer
  • Currently produced by semi-synthetic route from 10-deacetylbaccatins III from European yew Taxus baccata
  • Used in diverse cancer therapies - around 30+ years
  • Side effects – myelosuppression (reversible) as these cells also rapidly divide, peripheral neuropathy

  
• What are laulimalide/peluroside ligands and their mode of action?


  • Laulimalide and peloruside A derive from sea sponges
  • X-ray and cryo-EM reveal a new, non-taxane site
  • Laulimalide/peluroside site is opposite side of ML surface (with respect to taxane), i.e. ‘outside’ MT wall
  • Microtubule stabilised by strengthening lateral contacts between protofilaments
  • Evidence that the microtubule stabilising agents can interact with both its host tubulin and the adjacent tubulin
  • Biological investigation ongoing

  
• What are vinca alkaloids?

  • Natural vinca alkaloids vincristine and vinblastine isolated from Periwinkle in the 50s
  • As 1st generation, achieved significant clinical success
  • Notable against childhood leukemia
  • Led to diverse, semi-synthetic analouges including vinorelbine
  • Similar to taxanes, significant side effect profile, i.e. myelosuppression (reversible) and peripheral neuropathy

  
• What is the mode of action of vinca alkaloids?

  • Vinca site is located at the (+)-end surface of β-tubulin
  • Drug binding alters the surface of the (+)-end, forming a ‘wedge'
  • This interferes with incorporation of new heterodimers
  • The (+)-ends remain curved, and the smooth microtubule wall cannot form
  • In addition, vinca site ligands can cause tubulin oligomers, decreasing the free tubulin pool
  • At high concentrations (e.g. 10-100 nM in HeLa cells) microtubules depolymerise - but really only need one or two ligands at the end of microtubule to reduce dynamics by 50%

  
• What is key factor should be targeted to stop biological activity of microtubules?

  Suppression of dynamics
• What is the maytansine site?

  • Maytansine and derivatives first isolated from Maytenus ovatus
  • Compounds currently under clinical evaluation
  • Maytansine site is close to the vinca site, but formed from other structures
  • In growing microtubules, the maytansine binding pocket of the (+)-end accommodates the (-)-end of the next α-tubulin unit
  • Incorporation of a ligand blocks this, impeding elongation

  
• What is eribulin?

  • Eribulin is a fully synthetic analogue of a marine natural product (halichondrin B)
  • Both compounds show a unique mechanism of action
  • Mainly binds with high affinity to the (+)-end of existing microtubules - prevents future heterodimer binding
  • Used in metastatic breast cancer and for unresectable liposarcoma
  • Also shows non-mitotic effects – e.g. vascular remodelling which reduces tumour hypoxia and also a decreased metastasis and invasion
  • Being investigated in a range of solid tumours – breast, NSCLC, prostate, brain, cervical, etc.

  
• What is the colchicine site?

  • Colchicine isolated from autumn crocus
  • Has been used clinically for treating gout
  • Severe toxicity at doses required for anti-tumour effects
  • Lower toxicity compounds, such as combretastatin, are currently in clinical trials

  
• What is the mode of action of the colchicine binding site?

  • Binding site is located between the α- and β-tubulin units
  • Slow binding, practically irreversible
  • Binding to the heterodimer stabilises it in the curved conformation
  • During polymerisation, the ends transition from curved at the tip, to straight in the body
  • Presence of a colchicine type ligand prevents this, inhibiting polymerisation
  • Likely that it doesn’t bind to the microtubule tip, rather it binds to the soluble pool first and is then incorporated
  • Presence of tubulin/colchicine units slows but doesn’t prevent polymerisation

  
• Why do these drugs work for only selected cancers?

  • Paclitaxel is very effective for ovarian, mammary and lung tumours - but essentially ineffective against solid tumours such as colon and kidney carcinomas
  • Vinca alkaloids are effective for many haematological cancers - but usually ineffective against solid tumours

  Possible modes of resistance

  • Overexpression of membrane transport proteins (ABC-transporters)
  • Microtubule associated proteins (MAPs)
  • Post translational modification
  • Up and/or down regulation of different tubulin isotypes