Transcript File

Introduction to GPCR’s
• GPCR’s are a superfamily of cell surface
receptors
• They are characterised by their 7TM
serpentine structure
• They are the target of over 50% of
pharmaceutical drugs known today
• The only know x-ray structure for a GPCR is
that for dark-state (inactive) bovine
rhodopsin
• Accurate 2 adrenergic homology models
may enable fast efficient drug screening,
and the potential development of potent
cardiovascular and anti-asthmatic drugs
Construction of the 2-AR models
•The 2-adrenergic receptor (2-AR) model was built using the
2.8 Å crystal structure of rhodopsin (pdb code 1F88) as a
template, using WHATIF (Vriend, 1990).
•The extracellular loops, intracellular loops and the TM bundle
cavity were hydrated using explicit water molecules, added
using PLIM (Harris et al., 1993).
•The undefined intracellular loop 3, Gln236-Glu239, was added
using a database search.
•Molecular dynamics simulations were used to generate a
“relaxed” model.
•Biochemically and biophysically derived distance restraints
were rigorously separated into those relevant to the inactive
and those relevant to the active structure (see tables).
Approach inspired by Herzyk et al. 1995.
The restraints, invoked during MD simulations as half or full
harmonic restraints were applied to generate the active and
inactive structures.
Interactions in the active site of the b2
active receptor
•
•
•
•
•
Figure 1. The interactions of
epinephrine with the residues of the
binding site in the active receptor
model
Asp 113
Leu 311
Asn293
Ser203,204 & 207
Trp 257
Interactions in the active site of the b2
inactive receptor
• Increased Trp257
interaction
• Asn312 α/β selectivity
• Almost no interaction with
the three serines from
TM5 which are
responsible for activation
Figure 2. The interactions of carteolol
Hydrochloride with the residues of the
binding site in the inactive receptor
model
Assessing the quality of the models
using ligand docking-scoring
• the selective potency of the active and inactive 2
adrenergic models were tested to ascertain whether
the receptors were well modelled
• the experimental hypothesis is that an active receptor
model should have a higher affinity for agonists than
antagonists, and vice versa for the inactive model
• Docking experiments were performed using LigandFit
as implemented in Cerius2 (Venkatachalam et al.,
1990) a high throughput virtual screening program
• The aim being to select adrenergic specific ligand
subtypes, from a ligand library sourced mainly from
the CDS
A Novel LIGSCORE-DOCKSCORE
Hybrid Method of Analysis
Figure. 1 The crystal structure of HIV protease (white, space filled) complete with the
active residues (grey, ball and stick) and the crystal structure location of the docked 4PHV
ligand (green, stick) and the top experimental conformer (gold, stick). A shows the best
conformer using the original (top DOCKSCORE) method of analysis, B shows the best
conformer using the new LIGSCORE-DOCKSCORE hybrid method.
Results for the active model
•
Table 1. Analysis of the selective potency results for
the active b2 receptor. Yield represents the number
of b2-agonists selected divided by the total number
of agonists (16), the enrichment factor is the
number of agonists selected divided by the
Table 2. Beta-2
active receptor
yield, enrichment
factor hit rateagonists
as derived from
the above
expected
number
of selected
and
the hit
graph
rate is the number of agonists selected over the
total number of ligands.
% of total ligands
•
Figure 2. The Selective Potency of the Active β2
Adrenergic Receptor as Defined by DOCKSCORE
Ranking. The black line is the traditional
DOCKSCORE line, the orange represents the
results obtained from a novel hybrid DOCKSCORELIGSCORE method. The dashed line represents
the expected behavior from a random set of ligands
and the dotted line shows the ideal selectivity where
all of the agonists are selected first prior to any of
the other ligands.
Yield
Enrichment Factor
Hit rate (%)
5%
37.50%
5.73
75.00%
10%
50.00%
4.37
50.00%
15%
62.50%
3.95
41.67%
20%
87.50%
4.12
43.75%
25%
Average
87.50%
3.42
4.32
35.00%
49.08%
Results for the inactive model
•
Table 1. Analysis of the selective potency results for
the active b2 receptor. Yield represents the number
of b2-agonists selected divided by the total number
of agonists (16), the enrichment factor is the
number of agonists selected divided by the
expected number of selected agonists and the hit
rate is the number of agonists selected over the
Table 2. Beta-2 active receptor yield, enrichment factor hit rate as derived from the above
total number of ligands.
graph
•
Figure 3. The Selective Potency of the Inactive β2
Adrenergic Receptor as Defined by DOCKSCORE
Ranking. The black line is the traditional
DOCKSCORE line, the orange represents the
results obtained from a novel hybrid DOCKSCORELIGSCORE method. The dashed line represents the
expected behavior from a random set of ligands and
the dotted line shows the ideal selectivity where all
of the agonists are selected first prior to any of the
other ligands.
% of total ligands
Yield
Enrichment Factor
Hit rate (%)
5%
3.57%
0.44
11.11%
10%
15.52%
1.28
31.25%
15%
35.71%
1.87
41.67%
20%
50.00%
2.06
45.16%
25%
Average
53.57%
1.78
1.49
39.47%
33.73%
Further experimentation
•
Two more b2 inactive models were produced with their active site ‘based’
chris help on a better explanation of this on the structure of the 2
propranolol stereoisomers (R-S with S being 100 times more active blocker
than R).
•
As expected the ‘R’ model produced gave a binding site with a higher
Number of B-AR Antagonists
selected
selectivity for adrenergic antagonists than its ‘S’ equivalent
40
35
30
S-isomer
R-isomer
Average
Ideal
25
20
15
10
5
0
1
13 25 37 49 61 73 85 97 109 121 133 145 157
Dockscore Ranking
.
Conclusions
• The results produced infer that the β2 models produced
are highly competent
• Potential for drug development / testing
References
1) Vriend, G. (1990). WHATIF: A molecular modelling and drug design
program. J. Mol. Graph., 8, 52 – 56.
1)Venkatachalam, C.M., X. Jiang, T. Oldfield, and M. Waldman (submitted
2002). LigandFit: A novel method for the shape-directed rapid docking of
ligands to protein active sites, J. Mol. Graphics Modeling,
Acknowledgements
•We would like to acknowledge the NFCR for the
valuable funds provided