Homology modeling, also known as comparative modeling of protein refers to constructing an atomic-resolution model of the "target" protein from its amino acid sequence and an experimental three-dimensional structure of a related homologous protein (the "template").
The weaker non nucleophilic base potassium tert-butoxide improves the yield.
RO-151788 was fluro analog of ry-80 ( ANTAGONIST)xlI356 A1 1851, A24202.5, A3 8545, A4 ND A5 100.5, A6 5000
The N-Boc-L-alanine,1 was employed for the synthesis of the S enantiomers, while N-Boc-Dalanine9 was employed for the synthesis of the R enantiomers with a method developed by Sheaco2
There are two potential reasons why it binds better which will be addressed later.
Smit and colleagues crystallized and collected xray structure for achbp
A1b2g2 homology model in an agonist bound open pore conformation.
Duncalf paper : The Major Site of Photoaffinity Labeling of the g-AminobutyricAcid Type A Receptor by [3H]Flunitrazepam Is Histidine 102of the a Subunit, 1996.
Agonist bound, open channel conformation
Possible docking pose of diazepam in the binding site
His102mckernanphotolabelingTyr210 site directed mutagenesis decreased binding of all ligandsThr142 site directed mutagen to serine caused antag - agonists.
Surface witin 5 angstoms of the ligand was shown with lipophilic potential
PART I. UNIFIED PHARMACOPHORIC PROTEIN MODELS OF THEBENZODIAZEPINE RECEPTOR SUBTYPES PART II. SUBTYPE SELECTIVE LIGANDS FOR 5 GABAA/Bz RECEPTORS Terry S. Clayton 1
Outline• Introduction• Part I • Chemistry• Part 2 • Pharmacophore Modeling• Part 3 • Homology Modeling• Part 4 • Protein Ligand Docking• Part 5 What is next?• Conclusion 2
GABAA/ BzR/ Chloride Channel Complex The major inhibitory neuro transmitter system in the CNS, which modulates many of the neurological functions in the CNS Complicated molecular composition:pentamaric protein ploymer comprised , and subunits which formed transmemberane ion channel. When GABAA binds to the receptor, it opened the Cl- ion channel resulting hyperpolarization in neuronal transmission. 21 subunits, no x- ray crystal structure Benzodiazepine receptor ligands, a class of the most popularly prescribed drugs, of which most are used as anxiolytic and anticonvulsant agents, offer no selectivity, broad pharmacological effect 3
Cross-section of GABAA Receptor Absolute subunit arrangement of the a1b2g2 GABAA receptor when viewed from the synaptic cleft. The GABA binding sites are located at the b+a- subunit interfaces and the modulatory Bz BS (Bz) is located at the a+g- subunit interface.18,[ The GABAA binding sites are located at the subunit interfaces and the modulatory benzodiazepine binding site is 4 located at the subunit interface. The part of the schematically drawn subunits marked by the + indicates loop C of the respective subunits .
• Figure I. GABA function in C. elegans. GABA released from neurons activates the inhibitory GABAA receptor; the influx of chloride ions causes the relaxation of the body muscles. GABA is cleared from the cleft by the plasma membrane transporter, SNF-11. (Adapted from Trends Neurosci 27, Schuske, K., Beg, A. A., and Jorgensen, E. M. The GABA nervous system in C. elegans. 407-414, 5
Action of benzodiazepines at GABAA receptor subtypes Subtype Associated Effect Sedation, anterograde amnesia, Some anticonvulsant action, ataxia, addiction at higher dose Anxiolytic, hypnotic (EEG), some muscle relaxation Some anxiolytic action, anticonvulsant action at higher dose Maybe some muscle relaxation Diazepam-insensitive site Cognition, temporal and spatial memory (Maybe memory component of anxiety) Diazepam-insensitive site 7
Synthesis of 8-substituted imidazobenzodiazepines NH2 NHCOCH2Br O NH3 / CH3OH O Br Br O reflux 5L NaHCO3 / CHCl3 5L* 83% 24 hours, r.t. 2 days* 500g H O H O N N 2eq Br2 / H2SO4 / HOAc N 66% Br N2L ~7 days solid 100g 8
Synthesis of 8-substituted imidazobenzodiazepines O N O H N 1, NaH, ClPO(C2H5)2 / THF N O C2H5 2, NaH, CNCH2COOC2H5 / THFBr N 45% Br N 3 4 O N N O C2H5 SnBu3 N reflux 12 hours Pd(PPh3)4 tol 64% 5 DM-I-81 9
Synthesis of 8-substituted Imidazobenzodiazepine Dimers H H O N O H COOH N Br2, NaOAc DMSO + N H O H 3C H reflux AcOH, rt. 90 % N O CH3 80 % O 6 7 N H O CO2C2H5 N 1). LDA, THF N ClPO(OEt)2 0 o CBr N 2). LDA, THF Br N CH3 CNCH2CO2Et CH3 O O 8 45% 9 10
Synthesis of 8-substituted Imidazobenzodiazepine Dimers N N N CO2C2H5 CO2C2H5 CO2C2H5 TMS H N N N TBAF, THF/H2O, rt Pd(OAc)2(PPh3)2 N Et3N/CH3CN, reflux N 88 % NBr CH3 CH3 80 % TMS CH3 H O O O O RY80 N O O N N 2 N NaOH N OH CDI, DMF N O O N C2H5OH, 70 o C HOCH2CH2CH2OH, DBU N 90 % CH3 60 % N N H O CH3 H3C H O O H XLi093 N O O N N O O Pd/C, H2 N N XLI-356C2H5OH, CH2Cl290% N O O 13
Synthesis of PWZ-029 N H THF/DMF, NaH H O COOEt N O N ClP(O)(OC2H5)2, 0 oC; N CH3NHCH2COOH O THF/DMF, NaHCl DMSO, 150oC Cl N Cl N o O 84% CNCH2COOC2H5, 0 C O O 1 2 45% 3 LiBH4-CH3OH, 67% THF/ethyl ether reflux N N CH2OCH3 CH2OH N N DMSO, KOH, CH3I, rt Cl N 95% Cl N O O 5 PWZ-029 4 16
Oocyte and Selectivity of PWZ-029Modulation of EC3 in oocytes currentsBy PWZ-029 N N O Cl N O Affinity of PWZ-029 for axb3g2 (x = 1-6) benzodiazepine receptor isoforms Alpha 1 Alpha 2 Alpha 3 Alpha 4 Alpha 5 Alpha 6 Merck >300 >300 >300 ND 38.5 >300 Moltech 920 ND ND ND 30 ND UNC-Roth 17 362.4 180.330 328.2 ND 6.185 ND
Fear conditioned contextual memory 10 mg/kg PWZ-029 in mouse 18
Visual/Audio Cue Data for Xli356 Scopolamine (1 mg/kg) reduces freezing (ie impairs memory) typically caused by pairing the context (the cage) with a shock. The drug Xli356 when given at 10mg/kg attenuates the impairment of memory returning the freezing to the level that one typically sees the mouse freeze at (ie veh). In audio cued, memory is triggered by sound not the context. Xli356 is not able to reverse this type of memory. 19
Full Panel Receptor Binding Results from Case Western Reserve 20
Synthesis of Chiral Benzodiazepines O O NH2 N O O H DCC H NH O + N O HO CH2Cl2 O O (81%) 1. HCl(g), 20 min 2. MeOH/H2O(pH 8.5) r.t. 24 hrs (83%) O N H O O 1. NaH, THF/DMF H O N N ClPO(OEt)2, -40°C N Br2 con. H2SO4 glacial AcOH N Br N Br N 2. NaH, DMF 81% CNCH2CO2Et, -40°C (40%) Pd(OAc)2(PPh3)2 84% TEA, CH3CN reflux TMS O O N N O O TBAF, THF N N 85% N NTMS TMS 22 Sunjie, v.; Kajfez, F.; Stromar, I.; Blazevic, N.; Kolbah, D. J. Heterocycl. Chem. 1973, 10, 591.
Chiral HPLC • Regis Whelk 0,1 SS • 45% CH2Cl2, 42% Hex, 3% IPA, 10% EtOAc 23
Chromatograms of SH-053-S-CH3 (8) and SH-053-R-CH3 (15) by chiral HPLC 24
Chromatograms of SH-I-S66 and SH-I-R66 by chiral HPLC. 25
Benzodiazepine ConformationScheme 1. Dynamic Chirality of 1a-c and Stereochemical Cooperativity in 2b ( G Values Were Determined by 1H NMRSpectroscopy (Coalescense)) Carlier, P., Zhao, H., Deguzman, J., Lam, P., Enantioselective Synthesis of “Quaternary” 26 1,4-Benzodiazepin-2-one Scaffolds via Memory of Chirality, J. Am. Chem. Soc. 2003, 125, 11482-11483.
Developed Vehicle Options*1. 10% cyclodextrin in DI water2. 85% DI water: 14% propylene glycol: 1% Tween 803. a) dissolve in propylene glycol, dilute 50% in DI water4. 60% propylene glycol: 20% ethanol: 20% water* •Mortar and Pestle and sonication in all vehicles •when traditional saline is not working 28
Chemistry Summary• Scale up to 200 grams and faster work up using 10 Liter rotovap• Improved yield 30% with imidazo process improvements• Easier work up of dimers• R/S chiral benzo and natural products intermediates resolution using chiral HPLC• Developed some recipes for vehicles for our collaborators 29
Classes of Ligands Employed for the Study of Pharmacophore/ Receptor Models of BzR Subtypes N CO2Et CO2tBu N H N N N N N N N H CH3 N O N Ro15-4513 BCCT H pyridodiindole CH3 O N N CH3 N N N H3 C CH3 Cl N N O N N(CH3)2 diazepam zolpidem CF3 CL218,872 H3 C N N iPr N N N N N O N N O Cl N N N CH3 H Cl O CGS-8216 triazolam FG 8205 31
Pharmacophore Modeling LDiThe pyrazolo [3,4-c]quinolin-3-one ligand CGS-9896 (dotted line), diazepam (thick line), and planar diindoles (thin line)fitted to a schematic representation of the inclusive pharmacophore model for the BzR. The descriptors H1 and H2designate hydrogen bond donor sites on the receptor protein while A2 represents a hydrogen bond acceptor site necessary 32for potent inverse agonist activity in vivo. L1, L2, L3 and LDi are four lipophilic regions in the binding
Interactive Ligand Library• Single largest BzR database at present.• Features include: – Selectivity Filtering Ki value (nm) – Log P – Volume – MW – Query based on mathematical expressions • (Ex. = min (A1:A5)/A1) – Substructure search – Similarity search and R-group analysis – Import/ export• Today over 600 compounds in an interactive table 33
Molecular DatabasesChemDraw for Excel ChemDBsoft 34
Hardware for ModelingCurrent SYBYL Version: SYBYL X 1.3• OS: Linux RedHat 4• 2 Intel(R) Pentium(R) 4 CPU 3.20GHz, 3200.763, 1024 KB• Video Card NVIDIA Corporation 35
Synthesis of 5 selective BzR bivalent ligands (XLi093) N CO2C2H5 N O N CO2C2H5 N TBAF, THF/H2O, rt N OH 2N NaOH N N 88% C2H5OH, 70 oC TMS O CH3 N CH3 90% N H O CH3 RY79 RY80 H O O O N N CDI, DMF O O N N HOCH2CH2CH2OH, DBU 60 % N N CH3 XLi093 H3C H O O H O N 5g RY24 OtBu N (nM) N >1000 >1000 858 1550 15 >2000 CH3 H O (nM) RY24 26.9 26.3 18.7 N/A 0.4 5.1 (10g) RY79 121 142 198 159 5.0 114 (10g) RY80 28.4 21.4 25.8 53 0.49 28.8 (10g) 36
X-ray structure Xli093 Jeffrey R. Deschamps, Ph.D. Research Chemist Naval Research Laboratory 37
Receptor binding data of methyl ester analogs related to RY 80 and PWZ-029 in nM. 32.74 13.22 24.1 ND 3.548 ND Binding affinity at αxβ3γ2 GABA A/BzR subtypes (Values are reported in nM). 2.531 5.786 5.691 ND 0.095 ND Binding affinity at αxβ3γ2 GABA A/BzR subtypes (Values are reported in nM). 41
Table 3. Receptor binding data of methyl ester analogs related to RY 80 and PWZ-029 in nM. 15.31 87.8 60.49 ND 1.039 ND Binding affinity at αxβ3γ2 GABA A/BzR subtypes (Values are reported in nM). 945.9 326.8 245.9 ND 4.07 ND Binding affinity at αxβ3γ2 GABA A/BzR subtypes (Values are reported in nM). 42
PWZ-029 and Roche Cmpd 7 N CF2H NBr N N N Affinity for x (x = 1-6) benzodiazepine receptor isoforms Alpha 1 Alpha 2 Alpha 3 Alpha 4 Alpha 5 Alpha 6 Roche 174.3 185.4 79.6 ND 4.6 ND N N O Cl N O Affinity of PWZ-029 for x (x = 1-6) benzodiazepine receptor isoforms Alpha 1 Alpha 2 Alpha 3 Alpha 4 Alpha 5 Alpha 6 Merck >300 >300 >300 ND 38.5 >300 Moltech 920 ND ND ND 30 ND UNC-Roth 43 362.4 180.330 328.2 ND 6.185 ND
Strategies for improving selectivity• By reducing the electrostatic potential of ester terminal group some potency is lost but selectivity is improved for 5 over 1• Utilize bulkier groups extending into L2 pocket at the 8 position• Certain R isomers may bind to 5 selectivity 44
&Alpha 1 is represented by the red volume. Alpha 2 is represented by the yellow, where orange are regions of overlap. 46
Alpha 2 & Alpha3Alpha 2 is in red. Alpha 3 is represented in yellow. Regions of overlap are represented by orange. 47
Oocyte Data on SH-053-2F-R-CH3 CompoundSH-053-2’F-R-CH3 759.1 948.2 768.8 >5000 95.17 >5000 48
R and S ConformationS Conformation R Conformation 49
&Overlay of the a5b3g2 receptor (yellow) subtype with the a1b3g2 receptor(magenta) subtype. Orange surfaces indicate overlapping regions. 50
Subtype (solid) overlayed with the previous model (line). 51
Alpha 2 & Alpha 5The alpha 5 included volume is illustrated in red. The alpha 2 is in yellow. Regions of overlap appear orange. 52
Alpha 5 & Alpha 6Alpha 5 is represented in red. Alpha 6 is in yellow. Regions of overlap are orange. 53
New Conclusions Modeling 1. Discovery of an additional lipophilic pocket (L4) 2. L2 pocket has been consistency manipulated to enhance a5 selectivity on the oxo scaffold 3. Elimination of the ester moiety from the 1,4- benzodiazpine template offers discrimination between x and x receptor compositions a. Supported by Roche Cmpd 7 binding data 4. Evidence of conserved site entrance supported by of dimer protrusion 54
GABAA Receptor Homology Modeling ] Pritchett, D., Sontheimer, H., Shivers, B., Ymer, S., Kettenmann, H., Schofield, P., and Seeburg, P., Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology, Nature, 1989, 338, 582-585. J Neurochem 62(2):815-818, 1994 55
Figure 2. Longitudinal (A) and cross-sectional (B) schematic representations of a ligand-gated ion channel. The numbers 1-4 referto the M1-M4 segments. The M2 segment contributes to the majority of the pore lining within the membrane lipid bilayer. 56
Figure 1. Proposed topology of a GABAA receptor subunit. The extracellular domain begins with the N-terminus and M1-M4 represent the four transmembrane domains. 57
Cross-section of GABAA Receptor Absolute subunit arrangement of the a1b2g2 GABAA receptor when viewed from the synaptic cleft. The GABA binding sites are located at the b+a- subunit interfaces and the modulatory Bz BS (Bz) is located at the a+g- subunit interface.18,[i] [i 60
Pharmacophore ModelingThe pyrazolo [3,4-c]quinolin-3-one ligand CGS-9896 (dotted line), diazepam (thick line), and planar diindoles (thin line)fitted to a schematic representation of the inclusive pharmacophore model for the BzR. The descriptors H1 and H2designate hydrogen bond donor sites on the receptor protein while A2 represents a hydrogen bond acceptor site necessary 67for potent inverse agonist activity in vivo. L1, L2, L3 and LDi are four lipophilic regions in the binding
Homology Modeling Conclusions• H1 is Tyrosine 210• H2 is istidine 102• A2 is Threonine 142• Current Model assumes agonist bound open channel conformation• Further refinements necessary 68
AutoDock 4.2• Prepare PDB files of Protein and Ligand• Gasteiger (partial) charges are added• non-polar hydrogens were merged• aromatic carbons were identified• rotatable bonds detected, and TORSDOF set 69
Protein• Identify Key residues• Remove water molecules• Add gasteiger charges• Create a flexible residue file• Choose an algorithm (unique) 70
Homology &Docking Conclusions• Agreement with site directed mutagenesis• Agreement with Photoaffinity labeling• Entrance Theory supported• Dimer extending to extracellular space• Next steps• Continued refinement• Theoretical site directed mutagenesis• Site directed mutagenesis 74
Overall Conclusions of Research• Synthesized a number of benzodiazepine ligands and dimers and made improvements to synthetic procedures (pendant and oxo series)• Constructed a powerful Bz database and further refined the Included volume pharmacophores• Correlated the unified pharmacophore to a homology model 75
Thank You Committee Members Dr. Chen Dr. Peng Dr. Schwabacher Funding Dr. Cook o NIH Dr. Arnold o NIMH Dr. Silvaggi o UWMAnd to our collaborators:Dr. Cromer RMIT UniversityDr. Ramerstorfer Medical University ViennaProfessor Miroslav Savic University of BelgradeProfessor Werner Sieghart Medical University ViennaDr. Angela Duke Harvard Medical SchoolProfessor James Rowlett Harvard Medical SchoolDr. Cook’s Research Group University Wisconsin Milwaukee 76
Previous Collaborators 5• Subramaniam Sanker Case Western Reserve – Binding affinity at GABA subtypes• Mike Weed John Hopkins University – Delayed matching to position in rhesus monkeys• Klaus Miczek Tufts University – Modulating alcohol heightened aggression• Tim Delorey Molecular Research Institute – Audio and Contextual Memory• Jim Rowlett Harvard University – Alcohol self-administration (primates)• Harry June University of Maryland – Alcohol self-administration (rats)• Mark Galizio University of N.C.-Wilmington – Anxiety and cognitive enhancement in rats• Roman Furtmueller Brain Research Institute, Vienna Austria – Electrophysiology traces of compounds in oocytes• Galen Wenger University of Arkansas College of Medicine – Titrated matching-to-position in rats and pigeons 78