Hedgehog signaling pathway is one of the important signal transduction pathways which leads to cell differentiation in an organism. Mutation in any one of the components of this pathway leads to various malformation. One of the rare syndrome is a consequence of mutation in patched (PTCH gene) Known as Gorlin syndrome. Gorlin syndrome is a rare autosomal dominant disorder with a birth incidence of 1 in 19,000. This mutated patched which is a tumor suppressor protein gives rise to tumorgenesis. The major clinical manifestation of gorlin syndrome is Basal Cell Carcinoma (BCC) and medulloblastoma and is associated with various other developmental deformities. The patched family of transmembrane protein is responsible for inhibition of smoothened(SMO) when hedgehog ligand is not bound to it. This inhibition by patched is due to the presence of sterol sensing domain which pumps out the sterols, mainly oxysterol. But when hedgehog is bound to patched this inhibition is withdrawn. There are certain transcription factor which gets activated by SMO . This factors are responsible for transcription of hedgehog responsive genes. In gorlin syndrome and other types of cancer this factors are hyperactivated.
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We used bioinformatics tool to analyse the GLI mutant obtained from uniport .And we compared the two inhibitory molecule that is physalin F and gant 61 about their inhibitory action upon GLI.
Keywords: gorlin syndrome , hedgehog pathway, basal cell carcinoma, gli.
Gorlin syndrome is one of the rare genetic dominant syndrome characterized by a variety of development deformities and increased probability of developing cancer that is multiple basal cell carcinoma , medulloblastoma and ovarian cancer being the most prevalent type due to mutation in patched protein which is the transmembrane receptor for binding to hedgehog. Germline mutation being the prime cause as the first hit and the second hit being exposure to mutagen such as UV and IR. Most of the mutation type are truncating mutation, missense mutation , large scale insertion or multiexon deletion.
PTCH is a transmembrane protein which inhibits smoothened(SMO) protein by pumping sterol through the sterol sensing domain(SSD) . Thus the transcription factors GLI remains inactivated as such hedgehog responsive genes are not transcribed. But when hedgehog ligands is bound to patched transmembrane protein, inhibition on SMO is inhibited which activates GLI transcription factors causing transcription of hedgehog responsive gene. So when patched protein is mutated constitutive expression of GLI transcription factor occurs giving rise to tumorigenesis. There is also a possibility that apart from pathched mutation there is a presence of mutated Gli protein. Thus GLI inhibition can be an effective measure against the development of tumors and cancer.
There are few compounds which show inhibition on gli transcription factors such as physalin F and gant 61. GANT 61 is a hexahydropyrimidine compound inhibiting GLI mediated transcription.
GANT 61 cannot be used in therapeutic purpose, it is only for research use. But physalin F is natural plant product that is isolated from physalis. 
Materials and methods
The GLI protein and the variant (single nucleotide polymorphism,SNP) was obtained from uniport database . Gli have some natural variants which have a probability of being associated with cancer and other type of hyperactive transcription factor disorder. Generally there were 7 natural variants of GLI protein.
Stability prediction due to single chain amino acid variation
I-mutant was used to check the stability for single chain amino acid variation. The variant protein sequence was submitted to I-mutant 2.0.I-mutant2.0 is a automatic prediction tool which has a support vector machine. It calculates the free energy change in a protein upon alteration. It is the most reliable database for thermodynamic based attributes. It mainly calculates the gibbs free energy (âˆ†G) change for the native and the variant protein.âˆ†âˆ†G is obtained by subtracting the unfolding free energy of variant from that of the native. Gibbs free energy is that potential of the compound or biomolecule to perform work upon stability. Positive value of âˆ†âˆ†G indicates high stability of the protein and vice-versa.
Change of function of the variant by point mutation
SIFT was used for detection of point mutation. It also predicts the functional change of the protein upon substitution. It uses closely related amino acid sequence from PSI-BLAST. It uses homology based tools which assumes that the amino acid will be conserved in the protein family. So there is a change in the amino acid sequence it is considered as destructive. Submission of the protein sequence was done in SIFT. It uses the similar sequence and compares the changes in the mutant protein. The value used in SIFT is tolerance index. Tolerance index should be greater than equal to 0.05. This higher value suggest that the mutation is causing lesser effect on the functionality of protein due to amino acid substitution.
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Another structural homology based database was used for prediction of functional change due to mutation that is polyphen .Here also the protein sequence was submitted and the substitution type , homologous sequence profiling were used as profiling. It calculates the PSIC that is position specific independent counts. The higher value of PSIC indicates greater variation of function in variant protein than in native.
Energy minimization of the protein
For energy minimization of both the protein that is native and the mutated one nomad ref server was used. [8 ]This server used gromacs for energy minimization The pdb file was submitted to the server. Here the calculation of the normal modes was done.
Identification of intramolecular interaction in the protein
Intramolecular interaction were calculated using picweb server. It stands for protein interaction server. It calculates the hydrophobic interaction, hydrogen interaction, aromatic-aromatic interaction, aromatic sulfur interaction, cationic interaction etc. for this we submitted the pdb format of the protein molecule. But the hetero atoms should not be included in it.
Ligand physalin F and gant61 structure were obtained pubchem. And then the 3D structure of the ligands were obtained from corina  by submitting the canonical smiles. Then the pdb file were download for further analysis of the interaction.
Identification of binding sites along with the atomic contact energy between the protein and the ligand
For this purpose we used patchdock which is an online based docking tool that predicts the interaction between the protein and the ligand which is the most stable one.
We did the docking for both the native and the variant protein with the two ligands before as well as after the energy minimization. The ACE result was computed from it that is the atomic contact energy. The principle used by this server is based on the molecular shape, surface patch and the scoring. It aims for finding the docking transformation that produces good molecular shape complementarity. The complementarity is an important factor for docking.
The PatchDock algorithm divides the Connolly dot surface representation of the molecules into concave, convex and flat patches. Then, complementary patches were matched in order to generate
candidate transformations. Each candidate transformation was further evaluated by a
scoring function that considers both geometric fit and atomic desolvation energy.
The advantage of high efficiency of Patch Dock was the fast transformation search which by local feature matching rather than searching forcefully of the six dimensional transformation spaces.
The speed is also high because it uses advanced data structures and spatial pattern detection.
Results and discussion
In the stability prediction study using I-mutant , SIFT and Polyphen only one of the natural variant were in the satisfactory region of âˆ†âˆ†G, Tolerance index and PSIC SD. And rest were not in the satisfactory region of three parameters at the same time.so only one variant that is P210A was stable with the single amino acid change. So we took this variant for our further study. In table 1 the âˆ†âˆ†G, Tolerance index and PSIC SD were listed out for all the variants obtained from uniport.
Table 1 List of variants that were predicted to be functionally significant
by I-Mutant 2.0, SIFT and PolyPhen
AA change amino acid change
Letters in bold indicate less stable, deleterious and damaging by I-Mutant, SIFT and PolyPhen, respectively.
Then the energy minimization step was done by using nomadref. Both the mutant and the variant we energy minimized.For further use in calculation of intramolecular interaction, the energy minimized mutant form was always used.
Then to check out further the stability of the mutant compared to native intramolecular forces of the two are compared using PIC server. It was found that all the intramolecular forces parameter of the mutant were high as compared to the native which again indicates that the change in the amino acid is a mutated one and is unstable. The parameters are given in table 2.
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Table 2 PIC Server (intramolecular interaction)
HI- Hydrophobic Interactions MC-MC- Main chain- main chain hydrogen bonds, MC-SC- Main chain- Side chain hydrogen bonds, SC-SC- Intraprotein side chain - side chain hydrogen bonds, II- intraprotein ionic interactions, AA- Aromatic- Aromatic interactions, AS- aromatic sulphur interactions, C-PI- Cation- Pi
Finally coming to the parameter of docking of the protein that with the ligand. The measured parameter is the ACE that is the atomic contact energy. The atomic contact energy was lower for the physalin F compared to the gant 61. Physalin F is a natural component showing more inhibition on the GLI protein than gant 61. A part from this ,the literature survey data provides the information that Gant 61 cannot be used for the therapeutic purpose.
Table 4 Comparison of ACE of the GLI1 protein with the inhibitory ligands Physalin f and Gant61
Fig 1: GLI and gant 61 and physalin F
The structural level analysis is yet to be done for the inhibitory molecule with the the GLI protein.
Thus from the above study we can propose that the Gli protein's natural variant P210A is a mutated form and have a possibility of association with gorlin syndrome and cancer as it is hyper activated in this disease. And the inhibitor physalin F is more promising than Gant 61 suggested from the docking studies. The future structural study of the inhibitor and the Gli protein interaction can be important for the therapeutic purpose of this disorder.