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Peptidomimetic InhibitorsPeptidomimetic Inhibitors : :An Integrated Synthetic and Theoretical An Integrated Synthetic and Theoretical
Approach to their DesignApproach to their Design
Everardo Macias, Patrick Tomboc
Eamonn F. Healy,
Chemistry Department,
St. Edward’s University,
Austin TX 78704
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AbstractAbstractPeptidomimetics represent a powerful approach to pharmaceutical treatments based on enzymatically controlled reactions. Peptidomimetics are simply small organic molecules that serve to mimic the transition state of the natural substrate and thus serve to competitively inhibit the enzyme process. We are focusing on the design and synthesis of inhibitors for the serine protease thrombin. Thrombin plays a critical role in the formation of insoluble fibrin that can lead to life threatening medical conditions. Our synthetic scheme utilizes hydroxy-aldehydes in the synthesis of polypeptide isoteres for active site inhibition. QSAR studies aid in the understanding of the steric and hydrophobic requirements of the enzymaticbinding sites.
EnzymeEnzyme Thrombin, a sub-class of the
hydrolyases is a serine protease that promotes blood clotting
Thrombin has an active site consisting of the catalytic triad: Ser 195, His 57 and Asp 102
Enzyme BindingSiteEnzyme BindingSite
In addition thrombin has three binding sites, labelled as S1, S2 and S3, that determine the strength and specificity of binding
The lipophilicity of S3 has been well determined
PeptidomimeticsPeptidomimetics Small peptide-like
molecules that mimic transition state of substrate and work by competitive inhibiting binding of the natural substrate
Peptide analog must be stable
Drug must be a reversible inhibitor of the enzyme but can be irreversible if the enzyme is unique to
the disease
NNH
O NH2
O
O
NH
Ph
N
NH-BuO
OH
R
HYDROXYETHYLAMINE ISOTERE NATURAL PEPTIDE
MIMICS
O
NH
Saquinavir
Project DesignProject Design Design a polypeptide
isotere based on a natural thrombin substrate (Phe-Pro-Arg tripeptide), shown on the right
Optimize a generalized scheme for isotere synthesis
Model the S2 and S3 steric and hydrophobic requirements
H2N
O
N
HN
NH
NH2
NH
O
O
Gly216
Ser195
Asp189
S3
S2
S1
His57
Asp102
ProjectProject
Use Quantitative Structure-Activity Relationships (QSAR) to identify optimum R2 and R3 binding fragments
Synthesize the isotere, shown in red on the right, designed to mimic the serine-195 mediated transition state
Gly216
Ser195
Asp189
S3
S2
S1
His57
Asp102
H2N
O
R3
NH
R2
R1
NH
O
OH
N
Retrosynthesis &Retrosynthesis &R NR2
NH2
OH
generalized isotere
R
NH2
OH
O
H
R
NH2
O
HR
NH2
O
OH
aminoacid
R
NH2
O
H
R
NH2
O
OH [H] +
S
N
Me3Si
1. BuLi
2. Me3SiCl
2-TST
R
NH2
OH
S
N1. CF3SO3Me
2. NaBH4
R
NH2
OH
N
S
Me
CuCl2
R
NH2
OH
O
H
+ HNR2
NaBH3CN
R
NH2
OH
NR2
S
N
Br
SynthesisSynthesis
ResultsResults
O
HR
S
N
Me3Si
1. BuLi
2. Me3SiCl
2-TST
R
OH
S
N
1. CF3SO3Me
2. NaBH4
3. CuCl2
S
N
Br
YIELD
IR
NMR
1.
2.
R = CH3
+ 2-TSTBu4NF
CH2Cl2
YIELD
IR
NMR
R
OH
S
N
3.R = CH3
R = CH3
R
OH
H
O
R = CH3
YIELD
IR
NMR
>70%
3100 cm-1, sharp; 2950 cm-1 ; 1610 cm -1, weak
7.7-7.8, multiplet, 2H ; 0.4, singlet, 9H
7.8-7.9, multiplet, 2H ; 3.6,multiplet,1H
1.2-1.5, multiplet, 4H
Structure-Activity Results from Ref 2Structure-Activity Results from Ref 2
R3 R2 Ki (nm)
CH3 benzyl (S) 17
CH3 phenethyl (R) 550
CH3 phenethyl (S) 235
CH3 phenylpropyl (R) 100
CH3 phenylpropyl (S) 4
H benzyl (R) 1112
H benzyl (S) 8
S3
S2
S1
HN
NH
NH2
NH
O
OX
S
N
O
R2
R3
QSARQSARQuantitative structure-activity relationships (QSAR) represent an attempt to correlate structural or property descriptors of compounds with their activities. These physicochemical descriptors, including parameters describing hydrophobicity, topology, electronic effects and steric effects, can be determined empirically or by computational methods. Once a correlation between structure and activity/property is found, new compounds can be screened to select those with the desired properties. Activities in which QSAR has found wide application include biological assays, chemical measurements, environmental risk assessment and de novo drug design.
QSAR MethodologyQSAR MethodologyLinear regression analysis of the predicted versus observed activity/property is most commonly used to develop the QSAR relationship. The higher the r2 value the better the fit. The technique of leave-one-out cross-validation, quantified as rCV, is used to assess the predictive power of the QSAR model.
Two parameters, molecular connectivity and valence connectivity, developed by Kier and Hall were used extensively in this study. While these are fundamentally topological parameters they have been shown to contain electronic as well as structural information.
Connectivity Indices
ResultsResults
Preliminary results, summarized by the plot of the predicted (y-axis) versus experimental (x-axis) binding constants, clearly show that a functional predictive model of the steric and electronic requirements of the S2 and S3 binding sites can be constructed. Initial results seem to indicate the energetic descriptors are more useful as predictors than simple topological properties. This work continues.
ResultsResultschemical sample connecti
vity
index 0
log P energy
steric
(kcal/mo
molar
refracti
vity
Binding
Constant
Ki (nM)
(S)CH3-benzyl-BenzoThia
zole(S)
23.001 2.901 10.201 131.883 18.000
(R)CH3-phenylethl-BenzoT
hiazole
23.709 3.001 22.865 136.599 550.000
(R)CH3-phenylpropyl-Thia 21.847 2.610 40.858 125.072 100.000
(S)CH3-phenylpropyl-Thia 21.847 2.610 35.824 125.072 6.000
(R)CH3-3Fquin-Thiazole 26.372 3.188 29.088 135.393 150.000
(S)CH3-3Fquin-BenzoThia
zole(S)
28.941 4.272 21.630 151.406 18.000
(S)CH3-3Fquin-Thiazole 26.372 3.188 30.545 135.393 10.000
(S)CH3-benzyl-Thiazole2(
S)
20.433 1.817 23.388 115.870 16.000
F = -47.976*B - 1605.799*C + 13.368*D
+ 123.102*E - 10621.018 r^2 = 0.925
rCV^2 = 0.119
-10.954
544.609
83.202
15.910
50.041
58.192
69.520
57.480
Graph 3. Ki-QSAR-W/O-Outlier3
Binding Constant Ki (nM)
F = -47.976*B - 1605.799*C + 13.368*D + 123.102*E - 10621.018 r^2 = 0.925 rCV^2 = 0.119
-100 0 100 200 300 400 500 600
-100
0
100
200
300
400
500
600
1
2
3
4
567
8
F = -47.976*B - 1605.799*C + 13.368*D + 123.102*E - 10621.018 r^2 = 0.925 rCV^2 = 0.119 =0.925 *Binding Constant Ki (nM)+ 8.128; r ^ 2 = 0.925
F = -47.976*B - 1605.799*C + 13.368*D + 123.102*E - 10621.018 r^2 = 0.925 rCV^2 = 0.119
1 (S)CH3-benzyl-BenzoThiazole(S)
2 (R)CH3-phenylethl-BenzoThiazole
3 (R)CH3-phenylpropyl-Thia
4 (S)CH3-phenylpropyl-Thia
5 (R)CH3-3Fquin-Thiazole
6 (S)CH3-3Fquin-BenzoThiazole(S)
7 (S)CH3-3Fquin-Thiazole
8 (S)CH3-benzyl-Thiazole2(S)
ResultsResultschemical sample connecti
vity
index 0
log P energy
steric
(kcal/mo
molar
refracti
vity
Binding
Constant
Ki (nM)
(S)CH3-benzyl-BenzoThia
zole(S)
23.001 2.901 10.201 131.883 18.000
(R)CH3-phenylethl-BenzoT
hiazole
23.709 3.001 22.865 136.599 550.000
(S)CH3-phenylethl-BenzoT
hiaz.58
23.709 3.001 23.749 136.599 235.000
(R)CH3-phenylpropyl-Thia 21.847 2.610 40.858 125.072 100.000
(S)CH3-phenylpropyl-Thia 21.847 2.610 35.824 125.072 6.000
(R)CH3-3Fquin-Thiazole 26.372 3.188 29.088 135.393 150.000
(S)CH3-3Fquin-BenzoThia
zole(S)
28.941 4.272 21.630 151.406 18.000
(S)CH3-3Fquin-Thiazole 26.372 3.188 30.545 135.393 10.000
(S)CH3-benzyl-Thiazole2(
S)
20.433 1.817 23.388 115.870 16.000
F = -29.160*B - 1116.702*C + 9.582*D
+ 84.612*E - 7350.105 r^2 = 0.731
rCV^2 = -0.247
-3.367
384.872
393.343
72.782
24.551
55.110
53.507
69.072
53.128
Graph 2. Ki-QSAR-all
Binding Constant Ki (nM)
F = -29.160*B - 1116.702*C + 9.582*D + 84.612*E - 7350.105 r^2 = 0.731 rCV^2 = -0.247
-100 0 100 200 300 400 500 600
-100
0
100
200
300
400
500
1
23
4
5
67
8
9
F = -29.160*B - 1116.702*C + 9.582*D + 84.612*E - 7350.105 r^2 = 0.731 rCV^2 = -0.247 =0.731 *Binding Constant Ki (nM)+ 33.028; r ^ 2 = 0.731
F = -29.160*B - 1116.702*C + 9.582*D + 84.612*E - 7350.105 r^2 = 0.731 rCV^2 = -0.247
1 (S)CH3-benzyl-BenzoThiazole(S)
2 (R)CH3-phenylethl-BenzoThiazole
3 (S)CH3-phenylethl-BenzoThiaz.58
4 (R)CH3-phenylpropyl-Thia
5 (S)CH3-phenylpropyl-Thia
6 (R)CH3-3Fquin-Thiazole
7 (S)CH3-3Fquin-BenzoThiazole(S)
8 (S)CH3-3Fquin-Thiazole
9 (S)CH3-benzyl-Thiazole2(S)
ReferencesReferences Alessandro Dondoni, et al.; Synthesis of TSTs and
Reactions with Carbonyl Compounds; J. Org. Chem. 1988, 53, 1748-1761
Benoit Bachand , et al.; Synthesis and Structure-Reactivity of Potent Bicyclic Lactam Thrombin Inhibitors; Bioinorg. & Med. Chem. 1999, 9, 913-918
Acknowledgements We gratefully acknowledge the support of the Welch
Foundation in the form of a Departmental Research Grant