Download Lecture 06, case study - Taxol - Cal State LA

Combined approaches to Drug Discovery
In lecture, we will be focusing at different points on natural products,
receptor-based design, and pharmacophore-based design of drugs
In reality, drug development may combine these elements into a
synthetic approach
This lecture will illustrate how a combination of approaches led to the
development of new anti-cancer therapeutics based around a natural
product, the molecule taxol
Taxol
Overview of Talk:
I.
Cancer & the Microtubule Cytoskeleton
II.
Cellular Target: the Protein Tubulin
III.
Structure of Taxol & Mechanism of Action
IV.
History & Development of Taxol
V.
Resistance & the Future of Taxane-based Therapies
The Search for Anti-Cancer Drugs
Cancer is caused by normal cells that acquire mutations causing
them to proliferate and eventually metastasize, spreading
throughout the body and causing inevitable death
Small molecules that are selectively toxic to dividing cells have
potential as anti-cancer drugs, by killing tumor cells but not
most cells of the body
The Microtubule Cytoskeleton
The microtubule cytoskeleton is a highly regulated system affecting:
- transport of materials within the cell
- progression through cell division (mitosis)
The cytoskeleton is dynamically restructured:
Molecules that block polymerization or stabilize microtubules
can stop mitosis, by preventing cytoskeletal reorganization
Tubules are therefore a logical target for anticancer drugs
- Stop microtubule disassembly = stop cell division
The Protein Tubulin
Microtubules are composed of the protein tubulin
(1) Tubulin forms dimers, which consist of an a and a b subunit
(2) Dimers stack together into protofilaments,
which are linear strings
(3) Protofilaments bind laterally
to form hollow, cylindrical
microtubules
Downing, 2000
Tubule
Structure of Tubulin
Tubulin protein exists as two 450 a.a. monomers, a and b
- Each binds a high-energy GTP molecule
An a - b dimer then forms (GTP-tubulin)
Dimers polymerize to form long protofilaments
Polymerization causes hydrolysis of the b-GTP, which
destabilizes the microtubule
- GDP-tubulin wants to relax into a new conformation,
which dissociates from the microtubule
GTP
tubulin
All that holds the microtubule
together is a fast-growing cap
of recently added GTP-tubulin
GDP
tubulin
Loss of GTP- tubulin
cap...
collapse
Karp, 1999
Structure of Taxol
Taxol (“Paclitaxel”) comprises:
(A) a diterpene core (taxane skeleton)
(B) 3 phenyl ring-bearing side chains
(C) 2 acetoxy moeities
Structure of Taxol
Taxol comprises:
(A) a diterpene core
(B) 3 side chains bearing aromatic rings
(C) 2 acetoxy moeities
Structure of Taxol
Taxol comprises:
(A) a diterpene core
(B) 3 phenyl ring-bearing side chains
(C) 2 acetoxy moeities
1) addition of b-phenylalanine
2) oxidation of side chain C-2’
3) addition of benzoyl group to
side chain
Taxol: Mechanism of Action
Taxol was discovered to have an unprecedented mechanism of
action: it stabilizes microtubules, preventing them from
de-polymerizing
- Microtubule scaffold normally positions chromosomes,
then collapses as the replicated chromosomes are pulled
apart during cytokinesis
- Effect of Taxol is to trap mitotic cells within a cage of
microtubules, preventing disassembly of the scaffold
Selectively kills dividing cancer cells
Crystal Structure of Tubulin Dimer
3.7-Å resolution
crystal structure
2 b-sheets
surrounded by
12 a helices
Taxol bound
to b-tubulin
Kd = 15 nM
Nogales et al., 1998
Caplow et al., 1994
GDP
Taxol bound to b-tubulin
B9-B10 loop region in a-tubulin
Taxol occupies a hydrophobic cleft in b-tubulin, which is filled by
an 8-residue extender connecting B9-B10 in a-tubulin
Snyder et al., 2001
Empty binding pocket is
very hydrophobic
Taxol fits neatly into
the available space
Snyder et al., 2001
Binding of Taxol to b-Tubulin
- explains why linking these
rings with a tether yields
inactive drugs: the rings
interact hydrophobically
with the protein, not with
each other
Snyder et al., 2001
Histidine 229 residue of b-tubulin is interposed between the
C-2 and C-3’ phenyl rings of bound taxol, preventing
hydrophobic collapse of the taxol rings against each other
H-bond between
C-2’OH & backbone
carbonyl of Arg-369
meta but not para
substitutions on this
ring are still active,
due to space in the
hydrophobic pocket
Mechanism of Action:
Lateral Interactions
Taxol binds very near the M loop of
b-tubulin, which makes lateral contacts
between adjacent protofilaments
Stabilizes microtubules by strengthening
lateral interactions between protofilments
- stabilizes a conformation of M loop
that favors lateral contacts
- or - counteracts destabilizing effects of GTP
hydrolysis w/ compensating structural
change (GTP-b = Taxol-b)
Nogales et al., 1999
Development of Microtubule-based Therapies
A drug like Taxol probably could not have been developed
through rational drug design
(1) Microtubules are structural proteins that do not normally
bind to small molecules
- no ligand, like a receptor would have
- no natural substrate, like an enzyme would have
Development of Microtubule-based Therapies
A drug like Taxol probably could not have been developed
through rational drug design
(2) Microtubules are so unstable, they cannot be crystalized
for structural studies - except by treating them with Taxol!
- the binding site of Taxol was thus defined by electron
crystallographic studies of tubulin, because it was there
in the crystals already
The Search for Anti-Cancer Drugs
The National Cancer Institute has long searched for natural
products with anti-cancer potential
Extracts of plants, animals, and microbes are screened against
a panel of cultured tumor cells
- extracts showing novel patterns of activity are then
investigated, often by university researchers
Ethnobotany is the study of how cultures use plants for
medicinal or other purposes
-
Taxol and Ethnobotany
Yew trees have long been recognized as toxic, or used medicinally
- Julius Ceasar noted a rival king killed himself with a yew potion
- Name “Taxus” is from Greek word toxon, or poison
- Pliny the Elder noted that people died after drinking wine stored
in casks made of yew wood
- Poisonous nature of Yew is noted in Hamlet & Macbeth
- Brewed by native Americans to treat fever, arthritis
- Sacred tree to Celtic druids
Pacific Yew tree
Taxus brevifolia
Isolation of Taxol
1951: As part of a National Cancer Institute (NCI) initiative to isolate
new anti-cancer drugs, 35,000 plants were screened for
anti-tumor bioactivity
- Screening means testing extracts against cell lines derived from
a diverse array of human tumors (breast, stomach, lung, etc)
1964: Extract of Pacfic Yew bark showed anti-tumor activity, but
limited supply of bark (endangered tree) delayed isolation of
the active component
1971: Taxol identified as the anti-tumor molecule in Yew bark, but
not explored as a drug for a further 12 years
Cabri & DiFabio, 2000
Development of Taxol as
an Anti-cancer Drug
1983: NCI-sponsored Phase 1 clinical trials delayed by allergic
reactions to solvent in which Taxol was dissolved
-- also, supply of Taxol remained a persistent problem
1989: Results showed 30% response among patients with advanced
ovarian cancer (an otherwise untreatable disease)
treatment
control
http://www.taxol.com
Development of Taxol
1991: Special partnership formed between NCI and the company
Bristol-Myers-Squibb through a Commercial Research &
Development Agreement (CRADA)
- BMS assumed full risk & responsibility for developing Taxol as a
drug, in exchange for full access to NCI’s medicinal data
1992: Taxol approved as second-line treatment for ovarian cancer
if previous chemotherapies have failed
- Taxol was not originally patented; BMS has relied on proprietary
rights to NCI clinical and scientific data to maintain its exclusive
market position
Cabri & DiFabio, 2000
Current use of Taxol
Currently approved as a treatment for:
(1) first-line treatment of ovarian cancer, in combination with the drug
Cisplatin (1998)
(2) first-line treatment of non-small cell lung cancer (1999)
(3) breast cancer, in combination with other chemotherapy (1999)
(4) second-line treatment of AIDS-related Kaposi’s sarcoma (1997)
Current use of Taxol
- Taxanes used in ~25% of
US cancer treatements
- Account for > $2 billion
in worldwide sales (2005)
Taxol Supply: Trees vs. Patients?
Taxus brevifolia is part of old-growth forests in the Northwest
- Requested to be put on Endangered Species list in 1990
by environmental groups (denied)
Took the bark of 6 trees, each 100 years old, to produce enough
Taxol for one treatment!
- 13,000 kg of bark per 1 kg of pure Taxol
Ethical issue: how to balance future supply against immediate
needs?
Taxol Supply: Semi-synthesis
Semi-synthetic Taxol approved for treatment in 1995, following
discovery that the related European Yew produced 10 DAB, a
biosynthetic intermediate in its needles (a renewable resource)
Taxol
10 DAB
Taxol Supply: Total synthesis
4 total syntheses reported:
1) Holton (Florida State), 1994
2) Nicolaou (Scripps), 1994
- initial assembly of diterpene skeleton, followed by derivatization
3) Danishefsky (Columbia), 1995
- attachment of functionalized segments
4) Mukiyama, 1999
Taxol Supply: Biotechnology
1) Plant cell culture: Phyton Catalytic Inc., Ithaca NY holds US rights
to taxol production by plant cell fermentation (75,000 L)
2) A fungus (1994) and a bacterium Erwinia sp. (1995) isolated from
Taxus tree produce taxol in culture (but low yield)
- lateral gene transfer from host plant to pathogen??
- culture of pathogens could be an alternative to harvesting trees
3) Taxadiene synthase and other genes important in taxol biosynthesis
isolated by Crouteau (Washinton State)
- could lead to large-scale fermentation by genetic engineering
(move genes into E. coli, yeast)
Roadblocks in the Development of Taxol
1. Supply of the natural product
2. Complexity of synthesis
3. Poor solubility in water (testing, delivery, reactions to solvent)
4. Weak correlation of in vitro activity in bioassays with in vivo
activity against tumor xenografts
Taxol Resistance by Human Tumors
4 mutations commonly confer Taxol resistance in human ovarian
cancer cell lines:
1) Phenyalanine-270
Valine
2) Alanine-364
Threonine
3) Threonine-274
Isoleucine
4) Arginine-282
Glutamine
Taxol Resistance by Human Tumors
4 mutations commonly confer Taxol resistance in human ovarian
cancer cell lines:
1) Phenyalanine-270
Valine
- loss of Phe here eliminates
strong hydrophobic packing
with methyl group of Taxol’s
C-4 acetate
Taxol Resistance by Human Tumors
4 mutations commonly confer Taxol resistance in human ovarian
cancer cell lines:
3) Threonine-274
Isoleucine
- loss of Thr here eliminates
H-bonding between Taxol’s
O-21 and side chain -OH of
Thr274
Future of Microtubule-based Therapies
Rational alteration of the Taxane skeleton has resulted in
improved drugs such as Taxotere (“docetaxel”)
References
Cabri, W. & Di Fabio, R. (2000) From bench to market: The evolution of chemical syntheses. Oxford
University Press, N.Y., 266 pp.
Caplow, M., Shanks, J. & Ruhlen, R. (1994) How Taxol modulates microtubule disassembly.
J. Biol. Chem. 269, 23399-23402.
Downing, K.H. (2000) Structural basis for the interaction of tubulin with proteins and drugs that affect
microtubule dynamics. Annu. Rev. Cell Devel. Biol. 16, 89-111.
Giannakakou, P., Gussio, R., Nogales, E., Downing, K.H., Zaharevitz, D., Bollbuck, B., Poy, G.,
Sackett, D., Nicolaou, K.C. & Fojo, T. (2000) A common pharmacophore for epothilone and
taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells.
Proc. Natl. Acad. Sci. USA 97, 2904-2909.
Nogales, E., Wolf, S. & Downing, K.H. (1998) Structure of the ab tubulin dimer by electron
crystallography. Nature 391, 199-203.
Nogales, E., Whittaker, M. Milligan, R., & Downing, K.H. (1999) High-resolution model of the
microtubule. Cell 96, 79-88.
Snyder, J.P., Nettles, J.H., Cornett, B., Downing, K.H. & Nogales, E. (2001) The binding conformation
of Taxol in b-tubulin: A model based on electron crystalographic density. Proc. Natl. Acad. Sci. USA
98, 5312-5316.
Data from clinical trials obtained from http://www.taxol.com, a website managed by Bristol-Myers
Squibb corporation.
- references should follow format of PNAS, but include full title
References
Cabri, W. & Di Fabio, R. (2000) From bench to market: The evolution of chemical syntheses. Oxford
University Press, N.Y., 266 pp.
Caplow, M., Shanks, J. & Ruhlen, R. (1994) How Taxol modulates microtubule disassembly.
J. Biol. Chem. 269, 23399-23402.
Downing, K.H. (2000) Structural basis for the interaction of tubulin with proteins and drugs that affect
microtubule dynamics. Annu. Rev. Cell Devel. Biol. 16, 89-111.
Giannakakou, P., Gussio, R., Nogales, E., Downing, K.H., Zaharevitz, D., Bollbuck, B., Poy, G.,
Sackett, D., Nicolaou, K.C. & Fojo, T. (2000) A common pharmacophore for epothilone and
taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells.
Proc. Natl. Acad. Sci. USA 97, 2904-2909.
Nogales, E., Wolf, S. & Downing, K.H. (1998) Structure of the ab tubulin dimer by electron
crystallography. Nature 391, 199-203.
Nogales, E., Whittaker, M. Milligan, R., & Downing, K.H. (1999) High-resolution model of the
microtubule. Cell 96, 79-88.
Snyder, J.P., Nettles, J.H., Cornett, B., Downing, K.H. & Nogales, E. (2001) The binding conformation
of Taxol in b-tubulin: A model based on electron crystalographic density. Proc. Natl. Acad. Sci. USA
98, 5312-5316.
Data from clinical trials obtained from http://www.taxol.com, a website managed by Bristol-Myers
Squibb corporation.
- primary literature citations (experimental studies)
References
Cabri, W. & Di Fabio, R. (2000) From bench to market: The evolution of chemical syntheses. Oxford
University Press, N.Y., 266 pp.
Caplow, M., Shanks, J. & Ruhlen, R. (1994) How Taxol modulates microtubule disassembly.
J. Biol. Chem. 269, 23399-23402.
Downing, K.H. (2000) Structural basis for the interaction of tubulin with proteins and drugs that affect
microtubule dynamics. Annu. Rev. Cell Devel. Biol. 16, 89-111.
Giannakakou, P., Gussio, R., Nogales, E., Downing, K.H., Zaharevitz, D., Bollbuck, B., Poy, G.,
Sackett, D., Nicolaou, K.C. & Fojo, T. (2000) A common pharmacophore for epothilone and
taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells.
Proc. Natl. Acad. Sci. USA 97, 2904-2909.
Nogales, E., Wolf, S. & Downing, K.H. (1998) Structure of the ab tubulin dimer by electron
crystallography. Nature 391, 199-203.
Nogales, E., Whittaker, M. Milligan, R., & Downing, K.H. (1999) High-resolution model of the
microtubule. Cell 96, 79-88.
Snyder, J.P., Nettles, J.H., Cornett, B., Downing, K.H. & Nogales, E. (2001) The binding conformation
of Taxol in b-tubulin: A model based on electron crystalographic density. Proc. Natl. Acad. Sci. USA
98, 5312-5316.
Data from clinical trials obtained from http://www.taxol.com, a website managed by Bristol-Myers
Squibb corporation.
- secondary literature citations (book, review article)