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HOMOLOGY MODELING OF SUBCUTANEOUS FILARIASIS DHFR PROTEINS

Dihydro-folate-reductase (DHFR) is a key enzyme in folate metabolism; it plays a major role in the biosynthesis of nucleic acid throught the catalysis of the NADPH reduction of 7, 8-dihydrofolate to 5, 6, 7, 8-tetrahydrofolate. DHFR protein is main target in subcutaneous filariasis. This inhibitor also have anticancer, antibacterial and even antimalarial activity, which is collectively known as anti-folates. Loa loa is a thread like warm that lives under the skin in subcutaneous fat causing loiasis [1]. It is one of three parasitic filarial nematodes that cause loiasis in human. The other two are monsonellastreptocerca and onchocercavolvulus [2]. Homology modeling also known as comparative modeling of protein is the technique which allows to construct an unknown atomicresolution model of target protein from its amino acid sequence and an 3D structure of a related homologous protein (the template) [3]. Therefore the present study focused on to build a high quality of protein 3D structure for subcutaneous filariasis DHFR protein.


INTRODUCTION
Dihydro-folate-reductase (DHFR) is a key enzyme in folate metabolism; it plays a major role in the biosynthesis of nucleic acid throught the catalysis of the NADPH reduction of 7, 8-dihydrofolate to 5, 6, 7, 8-tetrahydrofolate. DHFR protein is main target in subcutaneous filariasis. This inhibitor also have anticancer, antibacterial and even antimalarial activity, which is collectively known as anti-folates. Loa loa is a thread like warm that lives under the skin in subcutaneous fat causing loiasis [1]. It is one of three parasitic filarial nematodes that cause loiasis in human. The other two are monsonellastreptocerca and onchocercavolvulus [2]. Homology modeling also known as comparative modeling of protein is the technique which allows to construct an unknown atomicresolution model of target protein from its amino acid sequence and an 3D structure of a related homologous protein (the template) [3]. Therefore the present study focused on to build a high quality of protein 3D structure for subcutaneous filariasis DHFR protein.

Homology modeling
The protocol to the model building by homology modeling (scheme 1) involves template identification, alignment of the sequences, generation of the model coordinates, optimization, and validation of the model [4]. The process start with getting the amino acid sequence then identify related homologous sequence as template. In next step download the template sequence, the sequence alignment between target and selected template, then build up the model and optimize and evaluate ( fig. 1) [5][6].

MATERIALS AND METHODS
1) Amino acid sequence and identify related homologous sequence: Amino acid sequence of DHFR protein for subcutaneous filariasis was downloaded from www.unipro.org. And DHFR malaria select as template which is homologous to target protein. Template sequence also download from same website.
2) Sequence alignment: A search with BLAST against this database will give a list of known protein structures that match the sequence. BLAST will provide a list of possible templates for the unknown structure. In sequence alignmentalign the target sequence and template sequence.
3) Building the model: In this step factor involve to build the model is: 1) backbone generation 2) loop modeling 3) side chain modeling. There are two type of model: knowledge base model and energy based model. By using knowledge based model subcutaneousfilariais protein was build up. Further efficiency was improve by using refining loops. There are three refinement task available in refine structure stepi. e refine loops, predict side chain and minimization.

4) Evaluation:
The model was further evaluated by using ramachandran plot.

RESULTS AND DISCUSSION
1) Basic local alignment search tool: One of the most widely used algorithms for comparing primary biological sequences, such as aminoacid sequences, is BLAST (Basic Local Alignment Search Tool). In this search the DHFRloaloa amino acid sequence compare with DHFR malaria sequence shown in fig. 2.

Fig. 2: Basic local alignment search tool
2) Template selection: After BLAST, result as a list of 3D protein structures ordered according to their "sequence identity percentage" with the target sequence. The quality of the homology model is dependent on the quality of the sequence alignment and template structure. Model quality declines with decreasing sequence identity. Highest percentage identity template was selected shown in fig. 3. The structure produced in the Build Structure step is likely to represent only a local energy minimum and not the global minimum.
Therefore, regions with gaps in the alignment are likely to require refinement in the Refine Structure step shown in fig. 5.  The present study was carried out to examine the probable role of DHFR protein in subcutaneous filariasis. No X-ray crystal structure of DHFR subcutaneous filariasis was available at PDB. Hence, homology modelling approach was used to obtain the 3D-structure of DHFR subcutaneous filariasis protein using the structure of DHFR malaria as a template. The modelled structure was subjected to energy minimization and refinement. Refine structure was validated by using ramachandran plot. In ramchandran plot majority of the amino acids are in the phi-psi distribution and the develop model is reliable and of good quality.

CONFLICT OF INTERESTS
Declared none 1. Spinello A, Luca Schifanella. Imported loa loafilariasis: three cases and a review of cases reported in non-endemic countries in the past 25 y. Int J Infect Dis 2012;16:e649-e662.