3rd annual international conference on pharmaceutical sciences
-
Upload
vivekanand-chatpalliwar -
Category
Health & Medicine
-
view
128 -
download
0
Transcript of 3rd annual international conference on pharmaceutical sciences
Design, Docking, Synthesis and in vitro Binding Studies of Few
Benzamide Derivatives with Glucokinase Enzyme
PHA2016/2662075
Dr. V. A. ChatpalliwarM Pharm. (Pharm. Chem.), Ph. D.
Prof., Pharm. Chem.Head, Dept. Pharm. Chem.
S. N. J. B. Coll. Pharm.Neminagar, Chandwad
Dist.: Nashik
Global Prevalence of Diabetes mellitus
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Facts about diabetes
In 2014 the global prevalence of diabetes * was estimated to be 9% among adults aged 18+ years (1).
In 2012, an estimated 1.5 million deaths were directly caused by diabetes .
More than 80% of diabetes deaths occur in low- and middle-income countries.
WHO projects that diabetes will be the 7th leading cause of death in 2030.
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Secondary complications
Prolonged exposure of tissues to sustained high levels of glucose in blood result in developing
secondary complications
Available Medicines and some new Strategies
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
No medicine guarantee normal life to diabetic patients
Single medicine is unable to provide strict glucose levels
β-cell apoptosis cannot be avoided
No medicine comes without a side-effect
• Sulphonylureas
• Glinides
• Biguanides
• Incretin mimetic (GLP-1)
• Biguanides
• Thiazolidinediones
(Pioglitazone, Rosiglitazone)
• α-Glucosidase inhibitor
• (Acarbose, Miglitol)
Search for novel strategies with safer drugs are required to provide near-to-normal life
Tripathi B. K., et al., Medical Science Monitoring, 12(7), 2006, 130-147Tahrani A. A., et al., New Horizons, 378, 2011, 182-197
Matschinsky F.M., et al., Medicine Reports, 2010, 1-5
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Dual role in glucose homeostasis
Matschinsky F.M. et al., Medicine Reports, 2010, 1-5
Sarabu R., et.al., J. Med. Chem., 55, 2012, 7021-7036
GK Protein Structure
Schematic Representation of for GK demonstrating active (closed) and inactive (super-open)
conformations
0.4 mM
≥ 15 mM
≥ 7.5 mM
T. O. Johnson, et al., Annual Reports in Med. Chem., 41, 2006, 141-154
Strategy
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Crystal Structure of Human Glucokinase
Filipski, K. J., et al., Bioorg. Med. Chem. Lett.,23, 2013, 4571-78
Catalytic domain
Allosteric binding site
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Details of Protein
Web Source: RCSB PDB
Resolution: 2.3 Å
Crystal Code: 1V4S
Chain Length: 455 AA
Originally Bound Molecules
2-amino-4-fluoro-5-[(1-methyl-1H-imidazol-
2-yl)sulfanyl]-N-(1, 3-thiazol-2-yl)benzamide
α-D-glucose
Kamata, K., et al., Structure, 12, 2004, 429-438
Serendipitous discovery of small molecule GK activator: RO-28-1675
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
RO-28-1675
NH
O
S
O
OCH3
S
N
First GKA patented in year 2000, was used to validate the clinical relevance of
targeting GK to treat T2D , had potential cardiovascular risk, caused reversible
hepatic lipidosis in repeat dose toxicity studies due to oxidative metabolism of
thiazole group. Thus further development was abandoned.
NH
O
NH2
S
N
CH3
SN
N
N
CH3
Tyr215
Arg63
NH
O
N
N
Cl
S
O
OCH3
O
RO-4389620
Banyu•A 2-Aminobenzamide derivative
•Good binding abilities
•Good glucose lowering activity in diabetic models of rodents
T. O. Johnson, et al., Annl. Rep. Med. Chem., 41, 2006, 141-154
Piraglatin GK2 or R1440 and patented in 2007, reached phase II trial stage
Improves the OGTT in patients with T2DM by increasing insulin secretion
and glucose uptake and lowering hepatic glucose output
9
T. Nishimura, et al., Bioorg. Med. Chem. Lett., 19, 2009, 1357–1360
overall structure of GK protein with a ligand, having 2-amino group, bound to it and glucose molecule trapped 20 Ǻ away
Ligand shown in orange mesh, nitrogen in thiazole ring and amidine -NH interacting with ARG63 backbone
Amino –NH forming hydrogen bond (purple colour) with TYR215
3FRO
N
N
F
NH
O S
N
NH2
Importance of amino group
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Kumari, V., et al., Curr. Chem. Genomics, 2, 2008, 76-89
Pocket 1: Val62, Arg63, Ile159, Val255, Val452
Pocket 2: Pro66, Tyr 215
Pocket 3: Met210, Met235, Tyr 214
Amino Acid Residues in the Allosteric site
N
N
F
NH
O S
N
NH2
N
NNH
O S
N
NH2
N
NNH
O
NH2
FF
F
N
NNH
O
NH2
NH2
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
GKA showing good glycemic control in rodents
TASK
Mutagenic liabilitiesOGTT in Diabetic rodents
List of Crystal structures of Glucokinase enzymes
Sr. No. Protein PDB ID Resolution (Å) Residue Count
1 1V4S 2.30 455
2 1V4T 3.40 451
3 3FGU 2.15 470
4 3IDH 2.14 470
5 3QIC 2.20 470
6 3ID8 2.40 470
7 4IWV 2.10 456
8 4IXC 2.00 456
9 4DHY 2.38 469
10 3F9M 1.50 470
11 4ISE 1.78 458
12 3FRO 2.70 455
13 4ISF 2.09 458
14 3VEY 2.25 455
15 4ISG 2.64 458
16 3AOI 2.20 455
17 3H1V 2.11 451
18 3IMX 2.00 455
19 3GOI 2.52 455
20 4BB9 1.47 633
21 4BBA 1.92 633
22 3S41 2.18 469
23 3VEV 1.80 470
24 3VF6 1.86 470
25 4DCH 1.79 473
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Computational Details
CADD software Schrodinger’s
Molecular Modeling Suit
After initial cleaning and correction, the
allosteric site was maintained in flexible
mode.
Structures were drawn using Build panel in
Maestro GUI & optimized using LigPrep
(v2.2) module
where low energy conformations of all
ligands were prepared
using OPLS force field.
Ligands were docked into the active site of
crystal structures using Glide. The best
suited conformations of few ligands, which
were successful in reversing the protein in
its original conformation and produced
maximum dock score were studied
precisely.
N
N
F
NH
O S
N
NH2
Crystal Structure: 3FGU
Banyu
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Trials of docking in different crystal structures with Banyu
Dock score: -3.593
N
N
F
NH
O S
N
NH2
Crystal Structure: 1V4T
Banyu
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Trials of docking in different crystal structures with Banyu
Dock score: -8.048
Trials of docking in different crystal structures with Banyu
N
N
F
NH
O S
N
NH2
Crystal Structure: 1V4S
Banyu
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Dock score: -11.534
Hydrophobic group
NH
H
ANH
O N
S
CH3
Design on benzamide template
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Modification Dock Score Replacement Dock Score
-9.3462 -2.2765
-8.3462 -1.363
-8.3462 -2.7439
-10.021 -3.1282
Cyclization
in an
heterocycle
-8.578 Cyclization
in a fused
aromatic
nucleus
-3.845
R
O
Ar-
O
Ar-
OR
S O
O
R
Ar-
O
Ar-
Protein: 1V4S
Cut-off: -7.000
Ligands: 1400
S O
O
R
Design and Docking
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Crystal Structure: 1V4S
GK2f
Dock score: -9.121
NH
O N
S
CH3
N
H
NH
H
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Design and Docking
Crystal Structure: 1V4S
GK3l
Dock score: - -8.578
NH
N
S
CH3
N
H
NH
H
Synthesis
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
*Spectral and Elemental data obtained from SAIF, Chandigarh, well supported the proposed structures for the compounds
*Problematic compounds, very few, were re-purified using chromatography, and re-submitted for the analysis before in vitro tests
Total 23 benzamide derivatives, either with secondary amino function, amide function, or sulphonamido function
were selected for synthesis, based on high dock score.
B. S. Furniss, B. S. ,et al., Vogel’s Textbook of Practical Organic Chemistry, V (ed.), 1989, 413-69
Khadse S. C. et al., Arabian Journal of Chemistry, 2012, http://dx.doi.org/10.1016/j.arabjc.2012.12.020
N+
O-
O
N+
O-
O
O
O 2-Amino-4-methylthiazole
Et3N, reflux, 90° C, 4hN
+
O-
O
N+
O-
O
O
NH
N
S
O
NH
NH2
NH2
N
S
(NH4)2S, NH3
6 h
Schotten-Bowman’s Reaction
GK2 series
NH
O N
S
CH3
N
H
NH
H
N+
O-
O
NH
NH2
Ethyl acetoacetate
Glacial acetic acid
Synthesis
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
*Spectral and Elemental data obtained from SAIF, Chandigarh, well supported the proposed structures for the compounds
*Problematic compounds, very few, were re-purified using chromatography, and re-submitted for the analysis before in vitro tests
B. S. Furniss, B. S. ,et al., Vogel’s Textbook of Practical Organic Chemistry, V (ed.), 1989, 413-69
Khadse S. C. et al., Arabian Journal of Chemistry, 2012, http://dx.doi.org/10.1016/j.arabjc.2012.12.020
reflux, 4 hNH
O
N+
O-
O
2-Amino-4-methylthiazole
Et3N, reflux, 90° C, 4h
NH
ON
N+
O-
O
S
(NH4)2S, NH3 reflux, 4 h
NH
ON
SNH2
N-alkylation
NH
N
S
CH3
N
H
NH
H
CH3GK3
in vitro glucokinase assay
GK activity
Spectrophotometrically 340 nm
Room temperature (22 – 25 °C)
Contents of assay mixture
KCl 25 mmol
Hepes pH 7.1 25 mmol
β-mercaptoethanol 5 mmol
NADP 1 mmol
ATP 2.5 mmol
MgCl2 2.5 mmol
Glucose 6-phosphate
dehydrogenase 2 units/mL
GK 60 nmol
Glucose 10 mM
(simulating fed condition)
Brocklehurst, K. J., et al., Diabetes, 53 (2004), 535-541
Ligand EC 50 (µM)* E max† Ligand EC 50 (µM)* E max†
RO-28-1675 0.387------ GK3f 4.53 0.36
PSN-GK1 0.04 0.93 GK3h 4.82 0.37
GK1d 13.367 0.16 GK3j 4.98 0.36
GK1g 13.20 0.04 GK3k 5.38 0.23
GK1h 13.45 0.07 GK3l 0.89 0.68
GK1h 18.75 0.03 GK3m 4.86 0.02
GK2e 4.56 0.14 GK3p 5.65 0.02
GK2f 1.02 0.76 GK3r 3.41 0.02
GK2g 2.69 0.13 GK3v 6.45 0.03
GK2h 2.87 0.45 GK4b > 10 -
GK2m 2.93 0.45 G4g > 10 -
GK2p 2.75 0.82 GK4s > 10 -
* Values are mean of three readings
† Ratio of maximal activating response of ligand to standard at each concentration point
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
Progress so far
Docking studies assured replacing toxic primary amino function from Benzamide scaffold
could yield glucokinase activators.
in vitro Glucokinase assay differentiated the potent ligands that can be developed further
newer binding modes in GK enzyme was indicated
Ligands GK2f and GK3l have been subjected to glucose lowering
assays in rats with induced diabetes.
A report has been submitted to the Sponsorers, B. C. U. D., S. P. P. U.
Pune
Status
3rd Annual International Conference on Pharmaceutical Sciences, ATINER
O
NH
NH
N
N
S
R
H
R
GK2f
NH
N
S
CH3
N
H
NH
H
GK3l
Board of Colleges and University Development, Savitribai Phule Pune University
Athens Institute of Education and Research, Athens, Greece