New insights into the mechanisms of action of microtubule targeting agents that are effective...

New insights into the mechanisms of action of microtubule

targeting agents that are effective against metastatic breast cancer

Susan L. Mooberry, Ph.D.Professor of Pharmacology

Cancer Therapy & Research CenterUniversity of Texas Health Science Center at San Antonio

San Antonio, TX, 78229

Microtubules are Essential for Normal Cell Functions

• Microtubules are involved in:Separation of the chromosomes during cell division

Cell motility

Cell shape and cellular junctions

Intracellular trafficking and secretion

Signal transduction.

Jordan and Wilson, Nat Rev Cancer 2004, 4:253-265; Dumontet and Jordan, Nat Rev Drug Discov. 2009, 9:790-802; Komlodi-Pasztor et al., 2011, Nat Rev Clin Oncol; 8:244-250.

Polymerization and Structure of Microtubules

αβ tubulin microtubuleheterodimers nucleus


αβ tubulin microtubule growing heterodimers nucleus microtubule


Adapted from Risinger et al., Cancer Treat Rev. 2009, 35: 255-261.

αβ tubulin microtubule growing heterodimers nucleus microtubule 13 protofilaments

Microtubules are Intrinsically Dynamic

Elongation-growth at + end

Catastrophe-rapid change from growth to shrinkage

Pause-no change in length

Dynamics are essential for microtubule functions

Dynamic Instability- switching between growth and shortening

Jordan and Wilson, Nat Rev Cancer 2004, 4:253-265.

Dynamic Instability

GTP cap-stabilizes + end

+

-

Dynamic Instability

Lose GTP cap-destabilize microtubule

Microtubule Depolymerization

Microtubule Depolymerization

Microtubule targeting agents disrupt normal microtubule dynamics in diverse ways

Measure effects of microtubule targeting drugs on microtubule dynamics

Microtubule targeting drugs suppress microtubule dynamics thereby perturbing microtubule function

Jordan and Wilson, Nat Rev Cancer 2004, 4:253-265.

Microtubule Targeted Agents

Important drugs used for the treatment of metastatic breast cancer

Two classes: • Microtubule Depolymerizers- inhibit polymerization,

cause loss of cellular microtubules (Vinca alkaloids; vinblastine, vincristine, vinorelbine: halichondrins; eribulin; maytansines;DM1)

• Microtubule Stabilizers –stimulate polymerization, increase density of cellular microtubules (taxanes; paclitaxel, docetaxel, nab-paclitaxel: epothilones; ixabepilone)

Jordan and Wilson, Nat Rev Cancer 2004, 4:253-265. Dumontet and Jordan, Nat Rev Drug Discov. 2009, 9:790-802.


• The effects of these drugs on interphase microtubules are dramatic, but at the lowest concentrations these drug inhibit mitosis and are classified as antimitotics

• Mitotic spindle is highly dynamic-leading to increased susceptibility to microtubule targeting agents

Jordan and Wilson, Nat Rev Cancer 2004, 4:253-265. Dumontet and Jordan, Nat Rev Drug Discov. 2009, 9:790-802.

Mitotic spindle microtubules are targets for anti-cancer drugs

interphase

Microtubule dynamics increase 4-100 fold in mitosis

metaphase anaphase

Suppress microtubule dynamicsFormation of aberrant mitotic spindlesUnable to organize DNA

MicrotubuleBinding agent

Mitotic arrest

Cell Death

Successfulcell division

Jordan and Wilson, Nat Rev Cancer 2004, 4:253-265

S.L. Mooberry

S.L. Mooberry

S.L.M.

S.L.M.S.L.M.

New data suggests that interphase microtubules are also important targets

Interphase Microtubules

Komlodi-Pasztor et al., 2011, Nat Rev Clin Oncol; 8:244-250;

Polarized array with + end at periphery

Key role in cellular metabolism

Pathways for intracellular trafficking

S.L. Mooberry

Microtubule Targeted Agents Are Not the Same

• Bind to different binding sites on tubulin and on microtubules

• Suppress microtubule dynamics by subtly different mechanisms

• These differences may help explain lack of cross resistance

Microtubule Targeted Agents Are Not the Same

• Bind to different binding sites on tubulin and on microtubules

Microtubule Targeted Agents Bind to different sites on microtubules

Microtubule Depolymerizer

VinblastineOutside surface and + end

Microtubule Targeted Agents Bind to different sites on microtubules

Microtubule Depolymerizer Microtubule Stabilizer

Vinblastine Paclitaxel Outside surface and + end Interior surface

Microtubule Stabilizing Agents

Taxane site Binding pocket is in the interior of the microtubule

Multiple drug orientations possible

Taxane site agents:paclitaxeldocetaxel ixabepilone

Bind differently within the taxane binding pocket

Microtubule Stabilizing Agents Paclitaxel

Stabilizes both longitudinal dimer interactions and lateral protofilament interactions

Xiao H et al., PNAS 2006, 103: 10166-73.



Changes lateral interactions resulting in microtubule with 12 protofilaments

Khrapunovich-Baine M et al., J Biol Chem 2011, 286:11765-78. Matesanz R. et al., Biophysical J 2011, 101: 2970-80




Binding within β-tubulin transduces changes in structure of α-tubulin and site of motor and microtubule associated protein (MAP) binding

Khrapunovich-Baine M et al., J Biol Chem 2011, 286:11765-78 . Xiao H. et al., 2012, ACS Chem Biol. 7:744-52.

Microtubule Stabilizing Agents Docetaxel



Difference in orientation as compared to paclitaxel

Matesanz R. et al., Biophysical J 2011, 101: 2970-80


Ixabepilone

Stabilizes longitudinal dimer interactions and but less effect on lateral protofilament interactions

Difference in orientation as compared to paclitaxel engaging different protein moieties in β-tubulin

Khrapunovich-Baine M. et al., J Biol Chem 2011, 286:11765-78


Bind on interior surface of formed microtubule Stabilize longitudinal and lateral interactions

Drugs bind with different poses within the site

Contacts with different peptides in β-tubulin and distinct poses may initiate different interactions with microtubule associated proteins

Taxane site

Xiao H et al., PNAS 2006, 103: 10166-73. Matesanz R. et al., Biophysical J 2011, 101: 2970-80Khrapunovich-Baine M et al., J Biol Chem 2011, 286:11765-78 . Xiao H. et al., 2012, ACS Chem Biol. 7:744-52.

Microtubule Destabilizing Agents

Vinca DomainLocated on β-tubulin

Binding site on exterior of microtubules

Multiple drug orientations possible

Vinca domain binding agents:Vinca alkaloids: vinblastine, vinorelbine halichrondrins: eribulinmaytansines: maytansine, DM-1

Bind with distinct orientations in binding site


Vinca Alkaloids

• Bind at + end and along length of microtubule

• Can also bind heterodimersin solution at higher concentrations


Eribulin

• Eribulin binds only to the ends of microtubules

14.7 eribulin molecules bind per microtubule

• Few molecules of eribulin are needed to inhibit microtubule growth

At the concentration that inhibits growth 50%, only 0.5 molecules of eribulin are bound per microtubule

Smith et al. Biochemistry 2010;19:1331-1337.


DM1

• DM1 is an “end poison” binds preferentially to the ends of microtubules

37 molecules bind per microtubule

• Inhibits both microtubule shrinking and growth

.Lopus M et al. Mol Cancer Ther. 2010;99:2689-99.


Vinca Domain Binding Drugs

Suppress microtubule dynamics

Bind with distinct orientations in binding site

Subtle differences in how they inhibit microtubule dynamics

Lopus M et al. Mol Cancer Ther. 2010;99:2689-99 Smith et al. Biochemistry 2010;19:1331-1337.


• Suppress normal microtubule dynamics• Disrupt microtubules and • Prevent normal microtubule functions• Resulting in apoptosis

• They all are mechanistically distinct

In Conclusion: Microtubule Targeting Agents

● Effective drugs used in the treatment of breast cancer

● Mechanistic differences among microtubule targeting drugs

● All suppress microtubule dynamics and lead to mitotic arrest and ultimately apoptosis

● A recent hypothesis suggests that interruption of interphase microtubules might also be important

● New opportunities for the future by targeting microtubule disrupting agents to tumor-directed antibodies, T-DM1

New insights into the mechanisms of action of microtubule targeting agents that are effective...

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