Supporting Information

Synthesis and Biology of Ring-Modified L-Histidine Containing Thyrotropin-Releasing Hormone (TRH) Analogues

Chhuttan L. Meena, Avinash Thakur, Prajwal P. Nandeker, Shyam S. Sharma, Abhay T. Sangamwar, and Rahul Jain

CONTENTS1H-NMR Spectrum of 7d13C-NMR Spectrum of 7d HPLC chromatogram of 7d1H-NMR Spectrum of 7i13C-NMR Spectrum of 7iHPLC chromatogram of 7i1H-NMR Spectrum of 7k13C-NMR Spectrum of 7kHPLC chromatogram of 7kHomology modeling of TRH receptors

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Experimental

The reaction monitoring and compounds purity was checked on pre-coated silica gel G254 TLC plates (Merck) through the spotting and visualization under UV spectrophotometer or by exposing them to iodine vapors. Column chromatographic purification was carried out on Merck silica gel (230-400 mesh) or neutral alumina. IR spectra (max in cm-1) were recorded on Nicolet FT-IR Impact 410 instrument either as neat or with KBr pellets. 1H NMR spectra were recorded on 400 MHz Bruker FT-NMR (Advance DP X 400) spectrometer-using tetramethylsilane as the internal standard and the chemical shifts are reported in (ppm) units. Coupling constants are given in Hz. The following abbreviations are used to indicate the multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; bs, broad singlet. The sample concentration in each case was approximately 7 mg in 0.5 mL of the solvent. Mass spectra were recorded on either GCMS (Shimazdu QP 5000 spectrometer) auto sampler/direct injection (EI/CI) or LC (Finnigan Mat LCQ spectrometer) (APCI/ESI). Optical rotations were recorded on a Perkin-Elmer 241MC polarimeter. The HRMS spectra were recorded on Bruker Maxis mass spectrometer. The melting points were recorded on capillary melting point apparatus or on the PerkinElmer DSC instrument and are uncorrected. All final peptides were checked on TLC using a solvent system of CH3OH:10%NH4OH:CH2Cl2 [10:.2.0:88]. All final peptides were checked for their homogeneity on a Shimadzu SPD-M20A HPLC system using a SupelcosilTM LC-8, 5 min (25 cm x 4.6 mm ID) column. The peptides were analyzed by using a solvent system of CH3CN-H2O-TFA (70:30:0.8%) using a SUPELCOSIL™ C-18 column. The peptides were ≥ 95% pure on HPLC analysis. Amino acids, and coupling reagents, DMF, and TFA were purchased from either Chem-Impex International or NovaBiochem (Merck Ltd.).

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1H-NMR Spectrum of 7d

13C-NMR Spectrum of 7d

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HPLC chrmoatogram of 7d

1H-NMR Spectrum of 7i

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13C-NMR Spectrum of 7i

HPLC chromatogram of 7i

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1H-NMR Spectrum of 7k

13C-NMR Spectrum of 7k

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HPLC chromatogram of 7k

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Homology modeling of TRH receptors

The homology modeling of TRH-R1 and TRH-R2 involved retrieval of amino acid sequences

from the UniProtKB/TrEMBL database, secondary structure prediction using online servers,

selection of suitable template for homology modeling, target-template sequence alignment,

model building, model refinement and model validation [1]. The amino acid sequence of human

TRH-R1 (Uniprot id P34981) was retrieved from the UniProtKB/TrEMBL database and was

subjected to NCBI BlastP search against Protein Data Bank (PDB) for finding crystal structure

templates with suitable sequence homology. The Basic Local Alignment Search Tool (BLAST)

finds regions of global/local similarity between amino acid sequences. The program compares

protein sequences to the sequence databases and calculates the statistical significance of matches.

BLAST can also be useful to understand functional relationships between sequences and help to

identify close congeners. The protein topologies were predicted using MEMSAT-SVM

Prediction, TMHMM prediction and TMpred server. The MEMSAT algorithm employs

empirically derived topological data, from available membrane proteins [2]. All residues were

assigned to one of five states (inner helix end/middle helix/outer helix end and within cell/out-of-

cell). Statistical propensities were normalized and occurrence of amino acids in each state was

incorporated in five frequency tables [3]. Each residue in a query protein was scored according to

its state of occurring in a series of dynamically generated models and highest scoring topologies

represent the global optimum models. The primary sequence alignment was taken from BlastP

sequence alignment of TRH-R1 with amino acid sequence of the 2VT4 (turkey beta1 adrenergic

receptor) crystal structure using the Blosum62 substitution matrix. The manual sequence

alignment was done to maintain the topology of TRH-R1 with respect to the 2VT4 crystal

structure. Previously reported and validated interacting amino acids such as Cys98, Tyr106,

Asn110, Cys179, Tyr181, Lys182, Tyr282, Asn289, Phe296, Glu298 and Arg306 were defined

as anchor groups [4]. The gaps between amino acid sequences within helical regions were moved

to loop regions by shifting the sequence towards the next anchor group. No gaps were allowed in

the helical regions to maintain the alpha-helical secondary structure for these domains.

Furthermore, intra-protein hydrogen bonds and the positioning of amino acids within the protein

structure were taken into consideration as structural restraints. The hydrophilic residues within

TM domain were placed at positions in which they were oriented towards the protein core as per

previous reports. The sequence alignment of TRH-R1 with 2VT4 (Fig. 1A) was used to build a

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homology model of TRH-R1. The homology model of TRH-R1 was built using Modeller9v8

software. The structural quality of the homology models was evaluated using the Ramachandran

plot and PROCHECK program in SAVES server developed by NIH MBI Laboratory for

Structural Genomics and Proteomics (http://nihserver.mbi.ucla.edu/SAVES). The iterative loop

refinement and energy minimization were performed to refine the obtained homology models.

The amino acid sequence of human TRH-R2 is not available till date; indeed, there does not

appear to be a TRH-R2 in the human genome. The mouse TRH-R2 amino acid sequence was

used to model 3D structure of TRH-R2 receptor. The methodology applied was similar as

described for TRH-R1 3D structure. The sequence alignment of TRH-R2 with TRH-R1 model

(Fig. 1B) was used to build a homology model of TRH-R2. The active site analysis of homology

models was performed in Pocket-finder server provided by the University of Leads. Pocket-

Finder is based on the Ligsite algorithm written by Hendlich et al [3]. Pocket-Finder was written

to compare pocket detection with ligand binding site detection algorithm Q-Site Finder.

Figure 1. Sequence alignment between: A) TRH-R1 and turkey beta1 adrenergic receptor crystal structure (PDB id: 2VT4) and B) TRH-R2 and TRH-R1 homology model.

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The construction of the homology model of TRH-R1 was performed using Modeller9v8

software. The BlastP search was performed to identify the suitable template from PDB. The top

hit from BlastP search was the crystal structure of turkey beta1 adrenergic receptor (PDB ID

2VT4) and it was selected as the template for TRH-R1 model building. 2VT4 is a nearest

congener of TRH-R1 and has 7 transmembrane (TM) domains with resolution of 2.7Å and

composed of 313 amino acids residues. The protein topology prediction servers were used to

predict the topology of transmembrane protein TRH-R1. According to consensus topology

prediction servers, the protein is composed of 7 transmembrane helices, 4 cytoplasmic loops

including C-terminal end of protein and 4 extra cellular loops acts as connecting bridge between

7 TM helices as depicted in Fig. 2.

The target-template sequence alignment was done manually to obtain reported protein topology

and amino acid orientations. The final model obtained after loop modeling and structure

refinement has a backbone dihedral distribution of all amino acid residues calculated by

Ramachandran plot as shown in Fig. 3A. It demonstrated that 88.8% (83.2) residues are in the

most favored region, 9.8% (15.6) in allowed region, 1% (1.2) in generously allowed region and

0.3% (no) residue in disallowed region. This indicates that the built homology model is

reasonably accurate in terms of dihedral distribution and steric clashes.

Figure 2. Protein topology of TRH-R1 and TRH-R2 homology models showing seven transmembrane domains (TM), extracellular loops (ECL), intracellular loops (ICL) and highlighted anchor residues embedded in cell membrane lipid bilayer.

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The overall quality of both models was found to be 81.2% (90.675) in Errat plot, which further

increases the confidence of the homology model (Fig. 4A). The active site analysis of the

homology model was performed in Pocket-finder server provided by the University of Leeds.

Figure 3. Ramachandran plots of A) TRH-R1 and B) TRH-R2 receptors.

Figure 4. Errat plot of A) TRH-R1 and B) TRH-R2 receptors.

The resulting homology model has several cavities that were visually inspected to detect the

appropriate active site cavity where substrates bind to initiate the cascade of signaling events.

The volume of the active site cavity was found to be 1169 Å. The amino acid residues Cys98 is

present in ECL1, Tyr106 and Asn110 in TM3, Cys179 in ECL2, Tyr181 at protein surface,

Tyr282 in TM6, Asn289 in ECL3, and Arg306 in TM7 as reported in previous studies. Also, the

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amino acid residues Tyr106, Asn110, Tyr282, Arg306, Asn289, Phe296 and Glu298 surround its

active site cavity. The results obtained correlated well with the previously reported observations

about 3D structure of TRH-R1.

An amino acid sequence for human TRH-R2 is not available in UniProtKB/TrEMBL database

till date. Indeed, it is likely that humans express only a single TRH receptor as stated above. The

sequence similarity between TRH-R1 and TRH-R2 for various species was compared. The

sequence similarity analysis showed that the sequence identity between mouse TRH-R1 and

TRH-R2, rat TRH-R1 and TRH-R2 and frog TRH-R1 and TRH-R2 was ~55% (Table 1). For

example, the in-depth sequence similarity comparison between human TRH-R and mouse TRH-

R1, Rat TRH-R1, frog TRH-R1 showed that the maximum sequence identity of 94% and

similarity of 97% was found between human TRH-R and mouse TRH-R1 (Table 1). Hence the

amino acid sequence of mouse TRH-R2 (Uniprot id Q9ERT2) was used to build the homology

model of TRH-R2.

Table 1. Sequence identity and similarity analysis between TRH-R1 and TRH-R2 receptors of human, mouse, rat and frog.

S. No. Compared Amino acid sequences (%)Identity

(%) Similarity

1. Human TRH-R (P34981 – –2. Mouse TRH-R1 (P21761) and Mouse TRH-R2 (Q9ERT2) 55% 75%3. Rat TRH-R1 (Q01717) and Rat TRH-R2 (Q9R297) 54% 73%4. Frog TRH-R1 (Q9DDR1) and Frog TRH-R2 (Q9DDR0) 57% 76%5. Human TRH-R (P34981) and Mouse TRH-R1 (P21761) 94% 97%6. Human TRH-R (P34981) and Rat TRH-R1 (Q01717) 93% 96%7. Human TRH-R (P34981) and Frog TRH-R1 (Q9DDR1) 77% 88%

The modeled 3D structure of TRH-R1 was used as a suitable starting template to build the

homology model of TRH-R2. The target template sequence alignment was done by modeller9v8

using default parameter set (Fig. 1B). The amino acid residues Cys95, Tyr105, Asn107, Cys175,

Lys239, Tyr270 and Asn294 were aligned to respective amino acid residues of TRH-R1

homology model hence they were considered as anchor residues, while developing homology

model of TRH-R2. The similar protocol was followed to build and validate the homology model

of TRH-R2 using Modeller9v8 software as described for TRH-R1. The Ramachandran plot (Fig.

3B) demonstrated that 83.2% residues are in the most favored region, 15.6% in allowed region,

1.2% in generously allowed region and no residue in disallowed region. This indicated the

accuracy of built homology model in terms of dihedral distribution and steric clashes. The

overall quality of model was found to be 90.65% in Errat plot, which further increases the

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confidence of homology model (Fig. 4B). The active site analysis using Pocket-Finder server

show that amino acids Tyr103, Asn107, Tyr270, Arg294, Asn277, Phe279 and Glu283 were

contributing in active site cavity of TRH-R2. These findings increase the level of confidence for

the built homology model of TRH-R2 for further molecular docking studies of designed TRH

analogues.

5.1.3. Docking methodology

The energy minimized structures for both the proteins TRH-R1 and TRH-R2 were used for

molecular docking using the Glide docking program included in the Schrodinger Suite 9.0.02

software package. Prior to molecular docking, protein models were prepared, hydrogen-bonding

networks were optimized, protonated forms of amino acid residues were generated and atom

type’s assignment was performed in protein preparation wizard of Schrodinger software package.

The grid was defined as centroid of residues Tyr106, Tyr282 and Arg306 in the case of TRH-R1

model and Tyr103, Tyr270, Arg294 and Cys293 in the case of TRH-R2 model at 25 Å box so

that all reported active site residues should be included in grid box. The 3D structure of all

designed TRH analogues were drawn and optimized in ligprep module of Schrodinger software

package and used for molecular docking in prepared homology models of TRH-R1 and TRH-R2.

The molecular docking was performed with standard precision, OPLS 2001 force field, scaling

factor of 0.80 with partial cut-off of 0.25 and Coulomb-vdW cut-off of 50 kcal/mol. The best-

docked pose for each ligand was selected using a binding affinity predicted by Glide Score.

1. UniPort Consortium. The universal protein resource(UniProt). Nucleic Acids Res. 2008, 36,

D190-195.

2. Hooft, R. W. W.; Sander, C.; Vriend, G. Objectively judging the quality of a protein

structure from a ramachandran plot. Comput. Appl. Biosci. 1997, 13, 425-430.

3. Hendlich, M.; Rippmann, F.; Barnickel, G. LIGSITE: automatic and efficient detection of

potential small molecule-binding sites in proteins. J. Mol. Graphics 1997, 15, 359-363.

4. Deflorian, F.; Engel, S.; Colson, A. O.; Raaka, B. M.; Gershengorn, M. C.; Costanzi, S.

Understanding the structural and functional differences between mouse thyrotropin-

releasing hormone receptors 1 and 2. Proteins 2008, 71, 783-794.

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