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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
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
Gleevec PD17
6 H-bonds 2 H-bondscontacts 21 residues contacts 11 residuesIC50 = 100 nM IC50 = 5 nM
Despite fewer interactions with Abl, the drug PD17 is a better inhibitor
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 Gleevec’s steric clash with the open conformation led to an improved drug
Improving PD17
PD17 loses the H-bond to threonine 319 that is essential for Gleevec’s 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?
PD17
Hydroxyl group contributes new H-bond even better binding (IC50 = 0.4 nM)
Better binding than Gleevec(IC50 = 5 nM)
PD17
PD166326
With further improvements:Dasatinib, first 2nd-generation kinase inhibitor
Gleevec
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)
With further improvements:Dasatinib, first 2nd-generation kinase inhibitor
Tokarski et al. Cancer Res. 2006
- dasatinib binds to both active and inactive conformations
Phe-382-stackswith Gleevecpyrimidinering, locksactivationloop in inactive conformation
Gleevec occupies a
hydrophobicpocket that
is otherwisefilled by Phe-382
Tokarski et al. Cancer Res. 2006
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)
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..
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
Disease: Cholera (caused by bacterium Vibrio cholerae) Traveler’s 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
Step 1: Design a small galactose mimic that binds the toxin as a single-site inhibitor, based on the receptor’s structure
Natural ligand of cholera toxin is an oligosaccharide ending in a terminal galactose sugar
Substitutions wouldn’t 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
GluLys
Trp
Asn
H2O
H-bond acceptor H-bond donor
Step 1: Design a small galactose mimic that binds the toxin as a single-site inhibitor, based on the receptor’s 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 IC50’s than galactose itself
GluLys
Trp
Asn
H2O
H-bond acceptor H-bond donor
Step 1: Design a small galactose mimic that binds the toxin as a single-site inhibitor, based on the receptor’s structure
Most potent inhibitor was m-nitrophenyl--D-galactoside (MNPG)
Step 1: Design a small galactose mimic that binds the toxin as a single-site inhibitor, based on the receptor’s structure
Most potent inhibitor was m-nitrophenyl--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 M, a 100-fold improvement over galactose alone...
- however, still much lower than the affinity for the natural ligand
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
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 aren’t such good binders
each low affinity high affinity strong binding
vs.
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
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
scaffold
flexible linker arm (R1)
each one of these arms is the same as the one shown above
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 (M)
Galactose-based finger, alone 5,000
Galactose-based pentavalent ligand 16
Step 3: Combine the 2 ways to improve binding: make a pentavalent ligand using the improved galactose derivative
Attach m-nitrophenyl--D-galactoside (MNPG) to a scaffold, with linkers to position the fingers over the 5 binding sites of the pentamer
Step 3: Combine the 2 ways to improve binding: make a pentavalent ligand using the improved galactose derivative
Attach m-nitrophenyl--D-galactoside (MNPG) to a scaffold, with linkers to position the fingers over the 5 binding sites of the pentamer
IC50 (M)
Galactose-based finger 5,000
Galactose-based 16 pentavalent ligand
MNPG finger alone 195
MNPG pentavalent ligand 1
pentavalent ligand shows ~200-fold improvement over the best single-site derivative
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 inhibitor’s 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
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)
Now design a drug that will bind to 2 toxin pentamers simultaneously
green = natural ligand (oligosaccharide w/ terminal galactose)
blue = 1 arm of pentavalent (5-armed) ligand
brown = 1 arm of decavalent (10-armed) ligand
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