Microtubule Stabilizers

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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.

Microtubule Targeted Agents

• 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.

Microtubule Targeted Agents

• 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

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