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 Center
University 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 microtubule heterodimers nucleus
Polymerization and Structure of
Microtubules
αβ tubulin microtubule heterodimers nucleus growing microtubule
Polymerization and Structure of
Microtubules
αβ tubulin microtubule heterodimers nucleus growing microtubule 13 protofilaments
Adapted from Risinger et al., Cancer Treat Rev . 2009, 35: 255-261.
Microtubules are Intrinsically Dynamic
Dynamics are essential for microtubule functions
Dynamic Instability- switching between growth and shortening
Elongation-growth at + end
Catastrophe-rapid change from growth to shrinkage
Pause-no change in length
Jordan and Wilson, Nat Rev Cancer 2004, 4:253-265.
Dynamic Instability
+
GTP cap-stabilizes
+ end
-
Dynamic Instability
Lose GTP capdestabilize 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 Depolymerizersinhibit 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
Microtubule dynamics increase
4-100 fold in mitosis
S.L. Mooberry interphase
S.L. Mooberry
S.L.M.
S.L.M.
metaphase anaphase
Microtubule
Binding agent
Successful cell division
Mitotic arrest
S.L.M.
Suppress microtubule dynamics
Formation of aberrant mitotic spindles
Unable to organize DNA Cell Death
Jordan and Wilson, Nat Rev Cancer 2004, 4:253-265
New data suggests that interphase microtubules are also important targets
S.L. Mooberry
Interphase Microtubules
Polarized array with + end at periphery
Key role in cellular metabolism
Pathways for intracellular trafficking
Komlodi-Pasztor et al., 2011, Nat Rev Clin Oncol; 8:244-250;
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
Vinblastine
Outside surface and
+ end
Microtubule Targeted Agents
Bind to different sites on microtubules
Microtubule Depolymerizer
Vinblastine
Outside surface and + end
Microtubule Stabilizer
Paclitaxel
Interior surface
Microtubule Stabilizing Agents
Taxane site
Binding pocket is in the interior of the microtubule
Multiple drug orientations possible
Taxane site agents: paclitaxel docetaxel 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.
Microtubule Stabilizing Agents
Paclitaxel
Stabilizes both longitudinal dimer interactions and lateral protofilament interactions
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
Microtubule Stabilizing Agents
Paclitaxel
Stabilizes both longitudinal dimer interactions and lateral protofilament interactions
Changes lateral interactions resulting in microtubule with 12 protofilaments
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
Stabilizes both longitudinal dimer interactions and lateral protofilament interactions
Changes lateral interactions resulting in microtubule with 13 protofilaments
Difference in orientation as compared to paclitaxel
Matesanz R. et al., Biophysical J 2011, 101: 2970-80
Microtubule Stabilizing Agents
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
Microtubule Stabilizing Agents
Taxane site
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
Xiao H et al., PNAS 2006, 103: 10166-73. Matesanz R. et al., Biophysical J 2011, 101: 2970-80
Khrapunovich-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 Domain
Located on β-tubulin
Binding site on exterior of microtubules
Multiple drug orientations possible
Vinca domain binding agents:
Vinca alkaloids: vinblastine, vinorelbine halichrondrins: eribulin maytansines: maytansine, DM-1
Bind with distinct orientations in binding site
Microtubule Destabilizing Agents
Vinca Alkaloids
•
Bind at + end and along length of microtubule
•
Can also bind heterodimers in solution at higher concentrations
Microtubule Destabilizing Agents
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.
Microtubule Destabilizing Agents
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.
Microtubule Destabilizing Agents
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