Download Lecture 08, Receptor-based I - Cal State LA

Applying pharmacophore-based
approach to improving Taxol
Started with a natural product that was highly active
- can other molecules with the same mechanism of action be
isolated?
- can we determine the pharmacophore for tubulin binding?
- can we improve the drug with this knowledge?
Alternative Microtubule-based Therapies
Following the discovery of Taxol, efforts to develop analogous
drugs have employed tubulin polymerization assays
- “Assay” means a lab test in which crude extracts, fractions
from chromatographic separations, or pure compounds are
applied to a system (proteins, cells, or whole animals)
- An active compound (also called a “hit”) is one that has a
desired effect (it inhibits a protein, kills bacteria, cures a
mouse of cancer)
Now that we recognize stabilizing microtubules is an effective
way to treat cancer, no need to screen for generically toxic
molecules; we can be much more directed in our approach
Alternative Microtubule-based Therapies
In a tubulin polymerization assay, chemicals are tested to see if
they can inhibit depolymerization of microtubules
- assays are performed in vitro, meaning in a test tube, not in
a living cell
Add tubulin, GTP, and the chemical to be tested
- Wait 30 min
- Measure turbidity (cloudiness of solution); as tubulin
polymerizes, microtubules come out of solution, making it
turbid
- Look under electron microscope; measure length of
resulting microtubules
Alternative Microtubule-based Therapies
Using in vitro assays, 3 classes of natural molecules
have been discovered which also stabilize microtubules:
(1) Epothilones A and B (from soil bacteria)
- water-soluble (easier delivery)
- bacteria can be mass-cultured
(2) Eleutherobin (from a tropical soft coral)
(3) Discodermalide (from a tropical sponge)
All 3 types competitively exclude Taxol, indicating they bind to
the same site on tubulin and share one mechanism of action
Understanding this mechanism should make possible the design
of more potent synthetic drugs for cancer therapy
Microtubule Stabilizing Molecules
(+)-Discodermalide
(-)-Taxol
(-)-Eleutherobin
(-)-Epothilone B (R=Me)
3 are diterpene-based, but no obvious structural similarities
Alternative Microtubule-based Therapies
Taxol and Epothilone
Taxol and Discodermolide
Although flat structures look very different, 3D models show
how other drugs can fill the same space as Taxol in the tubulin
binding pocket
Giannakakou et al., PNAS 2000
Tubulin-binding pharmacophore
Structural overlap
Pharmacophore
6.95 Å
Taxol
Epothilone B
6.93 Å
Spatially conserved atoms shown in red
Tubulin-binding pharmacophore
Structural overlap
Taxol
Epothilones are especially prone to
resistance mutations costing an
H-bond to C-7 OH
Epothilone B
Spatially conserved atoms shown in red
Tubulin-binding pharmacophore
Structural overlap
Taxanes are more resistant, due to
nearby alternate H-bond acceptors
at C-10 and C-9
Taxol
Epothilones are especially prone to
resistance mutations costing an
H-bond to C-7 OH
Epothilone B
Spatially conserved atoms shown in red
Tubulin-binding pharmacophore
Structural overlap
Taxol
C-12 methyl group of Epothilone B
makes it 14 times more potent than
Epothilone A (missing this -CH3)
Epothilone B
thiazole ring packs against Phe-270
side chain
Can the pharmacophore model
explain the activity of eleutherobin?
Can the pharmacophore model
explain the activity of eleutherobin?
eleutherobin
C-7 methyl
epothilone B
- packs against –CH3 of Thr 274
- explains why eleutherobin works against
Thr 274  Isoleucine mutant cells
Receptor-Based Drug Design
Knowing a protein is critical for the etiology of a disease, you can...
1. Design an assay using that protein to identify drug candidates
2. Starting w/ the structure of a protein-ligand complex, design
synthetic analogues or make modifications to create an inhibitor
- Exploit unique features of the protein of interest to improve
activity and confer specificity
3. Based on crystal or solution structures of the protein of interest,
use molecular modeling to design a novel small molecule that
will fit a potential binding site (de novo rational drug design)
Receptor-Based Examples
1. Targeting a single protein essential for disease progression
Gleevec, a new anti-cancer drug
2. Taking advantage of unique features of a protein target
Treating chronic myeloid leukemia
Chronic myeloid leukemia (CML) is a form of cancer in which white
blood cells proliferate out of control
This form of cancer is characterized by the presence of the
Philadelphia Chromosome, a translocation (or swapping) of the
ends of chromosomes 9 and 22
- 1st chromosomal defect linked to cancer (1960)
- One chromosome is shortened, another becomes longer
Philadelphia Chromosome
Normal
Translocated
The shorter (missing DNA)
Philadelphia chromosome
The translocation chromosome, w/ extra DNA
c-ABL
c-BCR
BCR/ABL
BCR/ABL is a fusion protein, combining parts of 2 different genes
c-ABL can bind DNA, actin, other proteins
1987: shown BCR/ABL fusion protein has tyrosine kinase activity
- Many oncogenes, mutated genes that predispose a cell to become
cancerous, are now known to be tyrosine kinases
A kinase is an enzyme that phosphorylates another protein, by
attaching a phosphate group onto an -OH
- There are 2 kinds: (1) serine/threonine kinases
(2) tyrosine kinases
Many tyrosine kinases participate
in signaling cascades that trigger
cell division, in response to
signals like growth factors
- Are normally tightly regulated by
the cell to control proliferation
- Mutation can change kinases into
oncogenes (= cancer-causing
genes) that are turned on
constitutively (= constantly),
instead of just when cell gets the
signal to start dividing
Sebolt-Leopold & English, Nature 2006
Kinase Activity
- phosphorylated substrate protein assumes a new conformation,
thus changing from inactive to active form
Bcr/Abl fusion protein is switched permanently “on”, sending the
internal signals for cell division without the appropriate external
signals (like growth factors)
Bcr/Abl is the unique cause of CML, making it a perfect target for
rational drug design
Drugs that target this kind of modified protein represent a new form
of cancer treatment
- Such cytostatic drugs target cancer selectively, rather than
indiscriminately killing all dividing cells, unlike most earlier
chemotherapies (which are cytotoxic)
- Drug discovery can begin by designing, atom by atom, new
medicines specific for particular types of cancer
A bunch of synthetic molecules were made by the pharmaceutical
company Ciba-Geigy (now part of Novartis), to test for tyrosine
kinase inhibition
Sent to a university researcher (Druker) who had developed a specific
bioassay to test for activity of the BCR/ABL fusion protein
A drug called STI-571 (Gleevec®) was found to inhibit Bcr/Abl
effectively enough to work as a drug, and specific enough
to have minimal side effects on normal cells
Targets only a subset of all cellular kinases:
(1) BCR/ABL
(2) receptors for platelet-derived growth factor (PDGF)
and stem cell factor (SCF)
(3) the proto-oncogene c-Kit
Gleevec
Clinical success of Gleevec
FDA approved in 2001
Surprisingly, Gleevec binds to the
inactive conformation of ABL
Jams activation loop, which cannot
switch to “open” conformation
Locks protein into a permanently
inactive conformation
Mechanism of Action
Upon signal, ABL auto-phosphorylates Tyrosine 393, a residue on
the “activation loop”
- The activation loop is a conserved regulatory element in kinases,
controlling their catalytic activity by switching between
different activity states
Once phosphorylated, the activation loop swings out into its “open”
conformation
- In the open conformation, 3 residues (Asp 381-Phe 382-Gly 383)
are positioned to catalyze substrate phosphorylation
- Also, catalytic site is no longer blocked by the collapsed loop
Normally:
(1) signal to cell
(2) ABL auto-phosphorylates
(3) activation loop swings out:
- exposes catalytic site
- aligns 3 essential residues
on the loop w/ the catalytic core
(4) substrate protein binds, gets
phosphorylated on tyrosine
Mechanism of Action
The open conformation is similar in all kinases, but the inactive
(“closed”) conformation is different in each protein
Gleevec wedges into the ATP-binding pocket and sticks into the
hydrophobic core of the kinase, locking Abl into its inactive
conformation
- Gleevec cannot bind to the active form of Abl
- Gleevec cannot bind to the inactive form of other kinases
Illustrates a whole new approach to drug design: exploit the unique
features of the inactive shapes of specific kinases
- Prevent them from activating + driving cell proliferation
Cancer cell + Gleevec:
(1) BCR/ABL fusion is always “on”
(2) Gleevec binds, locks activation
loop into inactive conformation
(3) Catalytic site is thus blocked
(4) Substrate proteins cannot be
phosphorylated
(5) No out-of-control cell division
for BCR/ABL blood cells
(6) No effect on normal cells
Complex H-bond network holds Gleevec in ATP-binding site of ABL
Met290
Ile313
Glu286
Thr315
Lys271
Met318
Phe382
Val256
Asp381
Leu370
Tyr253
Brown residues form a hydrophobic cage;
promote binding through van der Waals interactions
Met290
Threonine 315 is a critical
residue
- responsible for
ABL-Gleevec
specificity
Ile313
Glu286
Thr315
Lys271
Met318
- related kinases
lack this residue,
so Gleevec can’t
bind to them
Phe382
Val256
Leu370
21 amino acid residues
contact the drug
- 6 H-bonds total
- mostly van der Waals
Tyr253
Asp381
Gleevec Resistance
Patients pass through distinct stages:
chronic phase
blast crisis
5-6 years
3-6 months
(BCR/ABL)
(additional genetic defects)
Gleevec treatment results:
remission
eventual resistance
Late-stage cancers continue to proliferate even with Gleevec
Found that all contain 1 of 2 types of mutations:
(1) extra copies of BCR/ABL oncogene (gene amplification)
(2) mutations that decrease drug binding, OR keep ABL locked
into its active conformation (to which Gleevec doesn’t bind)
BCR/ABL gene was sequenced for 6 patients who relapsed during
Gleevec treatment, and all had the same mutation:
Threonine 315
Isoleucine 315
- In what 2 ways does this change prevent Gleevec from binding?
A single amino acid change confers drug resistance, resulting in
rapid death of the patient