Improving Gleevec: Insight from the Receptor Structure

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Improving Gleevec Insight from the Receptor
Structure
Gleevec cannot bind to the open (active) form of
the Abl kinase - would collide with open
conformation of the activation loop
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Remove portion of molecule causing steric clash
with the open (active) conformation of the
activation loop
Arrived at new class of drug, PD17, predicted to
still bind competitively at ATP-binding site of
the Abl kinase
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Gleevec
PD17
6 H-bonds 2 H-bonds contacts 21
residues contacts 11 residues IC50 100
nM IC50 5 nM
Despite fewer interactions with Abl, the drug
PD17 is a better inhibitor
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Inactive Abl PD17 Active Abl PD17
Despite making fewer contacts with the target
protein, PD17 is a better inhibitor because it
binds to both conformations of Abl - Thus,
losing an H-bond but removing Gleevecs steric
clash with the open conformation led to an
improved drug
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Improving PD17
PD17
PD17 loses the H-bond to threonine 319 that is
essential for Gleevecs activity however,
this residue remains available for H-bonding
near the end of PD17 Can PD17 be improved by
engineering a new H-bond to Thr319?
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PD17
Better binding than Gleevec (IC50 5 nM)
PD166326
Hydroxyl group contributes new H-bond even
better binding (IC50 0.4 nM)
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With further improvements Dasatinib, first
2nd-generation kinase inhibitor
Gleevec
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With further improvements Dasatinib, first
2nd-generation kinase inhibitor
- 325 times more effective than Gleevec against
normal CML cancer - effective against tumors
expressing 14 out of 15 resistance mutations
(all but the dreaded Thr-315 ? Isoleucine)
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Tokarski et al. Cancer Res. 2006
With further improvements Dasatinib, first
2nd-generation kinase inhibitor
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Tokarski et al. Cancer Res. 2006
Gleevec occupies a hydrophobic pocket that is
otherwise filled by Phe-382
Phe-382 p-stacks with Gleevec pyrimidine ring,
locks activation loop in inactive conformation
- dasatinib binds to both active and inactive
conformations
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Receptor-Based Design
Knowing that BCR/ABL fusion protein is the
specific cause of CML... (1) Identify a small
molecule that selectively inhibits this kinase
(Gleevec) (2) Perform structural studies to
understand mechanism of action -
discover new mode of drug action selective
binding to inactive kinase structure
(varies from kinase to kinase) (3) Use
structural information to make a drug that binds
either conformation (PD17) (4) Through a
second round of structural studies, add H-bonding
interactions to optimize the inhibitor
(PD166326)
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Receptor-Based Design
Knowing that BCR/ABL fusion protein is the
specific cause of CML... (5) Create 2nd
generation drug Dasatinib More effective than
Gleevec because a) binds both active and
inactive forms.. b) causes few distortions
of protein, compared to ATP-bound
form.. c) makes fewer interactions
with P-loop other parts of ABL..
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Receptor-Based Examples
1. Targeting a single protein essential for
disease progression Improving Gleevec, a new
anti-cancer drug 2. Taking advantage of unique
features of a protein target Prophylactic
Inhibition of Cholera Toxin
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Disease Cholera (caused by bacterium Vibrio
cholerae) Travelers diarrhea (E. coli) -
combined, kill over 1 million people per
year Target pentameric protein toxins The
pathogenic bacteria V. cholerae and E. coli
affect humans by producing a protein toxin that
forms a pentamer - toxin has 5 identical
subunits that come together in a star-shape -
released in lumen of the intestine - each of
the 5 units binds to an oligosaccharide on
epithelial cell surfaces, gaining entry
into the cell Strategy design inhibitors to
block binding of receptors to natural
ligand on cell surface, thus preventing
toxin from entering
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Step 1 Design a small galactose mimic that binds
the toxin as a single-site
inhibitor, based on the receptors structure
Natural ligand of cholera toxin is an
oligosaccharide ending in a terminal galactose
sugar Substitutions wouldnt work at O3, O4
each acts as H-bond donor acceptor with
protein side chains Also, no substitutions at
O6, which is bonded to 2 waters
Glu
Lys
Asn
Trp
H2O
H-bond acceptor H-bond donor
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Step 1 Design a small galactose mimic that binds
the toxin as a single-site
inhibitor, based on the receptors structure
Substitutions would work at O1, O2 - only lose
1 H-bond, to a displaceable H2O 35 galactose
analogues purchased tested to see if they
could inhibit binding of natural ligand to the
toxin protein 7 had lower IC50s than galactose
itself
Glu
Lys
Asn
Trp
H2O
H-bond acceptor H-bond donor
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Step 1 Design a small galactose mimic that binds
the toxin as a single-site
inhibitor, based on the receptors structure
Most potent inhibitor was m-nitrophenyl-a-D-galact
oside (MNPG)
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Step 1 Design a small galactose mimic that binds
the toxin as a single-site
inhibitor, based on the receptors
structure Most potent inhibitor was
m-nitrophenyl-a-D-galactoside (MNPG) -
retains favorable binding interactions of the
natural ligand - nitrophenyl group
displaces a water molecule -
structure-based design came up with an inhibitor
Kd of 10 mM, a 100-fold improvement over
galactose alone... - however, still much
lower than the affinity for the natural ligand

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Options for designing high affinity protein
inhibitors (1) Make a drug that binds
tightly to the binding site - 5
molecules must bind per toxin pentamer,
independently (2) Make a penta-valent
inhibitor, that is, one molecule with 5
inhibitory fingers linked to a central core
- 1 molecule binds per toxin pentamer,
but fingers bind semi- cooperatively
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In multivalent binding, binding of 1 finger
aligns other fingers with their receptor
sites - this increases the overall binding
affinity, by decreasing entropic costs
associated with multiple ligands binding
independently - linkers can also make
favorable contacts with the protein surface,
further promoting binding allows
you to make a potent inhibitor even if the
fingers on their own arent such good
binders
vs.
each low affinity high affinity strong
binding
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Step 2 Determine whether making a pentavalent
ligand improves
binding Multi-valent drug design is a strategy
to get higher binding affinity by exploiting
the presence of multiple, identical binding sites
on a target protein - for instance, many
proteins are multimeric, meaning composed
of several identical subunits - design a
single, large molecule which presents multiple
copies of an inhibitor, arranged to jam
all binding sites on the target

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Step 2 Determine whether making a pentavalent
ligand improves binding to
cholera toxin Attach galactose to a scaffold,
using flexible linkers to space out 5 sugar
residues joined to a central core
galactose
flexible linker arm (R1)
scaffold
each one of these arms is the same as the one
shown above
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Step 2 Determine whether making a pentavalent
ligand improves binding Attach
galactose to a scaffold, using flexible linkers
to space out 5 sugar residues joined to a
central core IC50 (mM) Galactose-based
finger, alone 5,000 Galactose-based
pentavalent ligand 16
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Step 3 Combine the 2 ways to improve binding
make a pentavalent ligand using
the improved galactose derivative Attach
m-nitrophenyl-a-D-galactoside (MNPG) to a
scaffold, with linkers to position the fingers
over the 5 binding sites of the pentamer
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Step 3 Combine the 2 ways to improve binding
make a pentavalent ligand using
the improved galactose derivative Attach
m-nitrophenyl-a-D-galactoside (MNPG) to a
scaffold, with linkers to position the fingers
over the 5 binding sites of the pentamer
IC50 (mM) Galactose-based finger 5,000 Galactos
e-based 16 pentavalent ligand MNPG
finger alone 195 MNPG pentavalent
ligand 1 pentavalent ligand
shows 200-fold improvement over the
best single-site derivative
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Yellow MNPG ligand Green 1 arm of
pentavalent ligand
Red a water molecule that forms
hydrogen bonds w/ natural galactose
protein amide - displaced by an oxygen of
the inhibitors nitrophenyl ring
Pentavalent ligand fills the toxin pocket in
similar manner as the free MNPG inhibitor, but
with the higher binding affinity that comes
with multivalency
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Step 4 Continue to improve binding affinity
change scaffold (1) Improve fit of linkers -
make more rigid less conformations, binding is
more entropically favored -
enhance interactions with protein surface -
present linker makes van der Waals contacts w/
side chains glu, tyr, his, lys, arg (2)
Increase valency go from penta-valent (5
ligands) to deca-valent (10 ligands)
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Now design a drug that will bind to 2 toxin
pentamers simultaneously
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green natural ligand (oligosaccharide w/
terminal galactose) blue 1 arm of pentavalent
(5-armed) ligand brown 1 arm of decavalent
(10-armed) ligand