NEPRILYSIN LIKE PEPTIDES AND SMALL MOLECULES IN THE COMBAT AGAINST AMYLOID BETA PLAQUES OF ALZHEIMER’S DISEASE THROUGH IN-SILICO BINDING STUDIES

* Uma Anusha Nukala 1 and Mrs. P. Sahithi 2 . 1. M.Tech (Biochemical Engineering & Biotechnology), University College of Technology, Osmania University, Hyderabad, India. 2. Former Assistant professor, University College of Technology, Osmania University, Hyderabad, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

1068 incrementation will be in developing countries, with the most rapid magnification in the elderly population taking place in China, India, and their south Asian and western Pacific neighbours. In 2010, Europe had an estimated 10 million disease cases and predicated on Amalgamated Nation"s demographic forecast this figure will elevate to 14 million in 2030. It is projected that by 2050, people aged 60 and over will account for 22% of the world"s population with four-fifths living in Asia, Latin America or Africa. Visually examining these data, it is ostensible that there is an imperative duress for action owing to the fact that the Alzheimer"s disease has become a major public health concern as the world"s population ages. For the same, many drug targets were identified among which one of the most potential one is the Amyloid Beta protein that is biosynthesized from the amyloid precursor protein (APP) by the operation of the β-and γsecretases.
As an additional novel approach to the Alzheimer"s disease therapy, the focus of the therapeutic strategies of Objective -2 has been aimed at fostering the Aβ degradation that aids in the maintenance of the steady-state level of amyloid β-peptide (Aβ) by promoting the catabolism of Aβ by a variety of proteolytic enzymes, thereby balancing the Aβ biosynthesis from APP. For the same, as a matter of paramount importance, this study has focussed on Neprilysin (NEP), family of zinc metalloproteinases, as it is known to play a major role in the degradation of both Aβ 1-40 and Aβ 1-42 in vitro and in vivo and also in terminating neuropeptide signalling aiding the brain functioning, while it"s decrease during ageing or after pathologies such as hypoxia or ischemia contribute significantly to the development of AD pathology. On the grounds of Structure Activity Relationships (SAR) which states that similar structures possess similar biological activities / functionalities, this task has been consummated by taking / constructing peptide molecules with the following 10 sequences similar to Neprilysin in order to perform proteinpeptide docking studies and identify the best peptide molecule: A4TA86; B1MB62; ELNFTPNWGT (pepbank); HFPGP (pepbank); MEHFPGP (pepbank); P24559; Q03R34; Q18G54; TKPRP (pepbank); TKPRPGP (pepbank) Although, there are a few drugs commercially available so far, these drugs are not devoid of their side effects. Hence, this in-silico study is an attempt to detect more physiologically suitable and compatible therapeutic molecules with least possible side effects.

Tools & Software:-
The online similarity search & sequence analysis tool BLAST (www.ncbi.nlm.nih.gov/blast) and the Accelrys Discovery Studio 2.5 software have been employed to carry out the objectives of the study to completion.

Methodology:-Target Protein Structure Preparation:-
With the target protein being Amyloid Beta (Aβ) for both Objective -1 & Objective -2, the Uniprot is searched for all the available Amyloid Beta human (Aβ) structures of which structures of the Aβ human protein with Accession number: P05067 (A4_HUMAN) have been submitted to RAMPAGE server for Ramachandran Plot Analysis that yielded the best protein structure bearing PDB ID: "1IYT" (Figure 1), which was downloaded from PDB for protein preparation. Thus, the obtained Aβ protein structure is loaded into the 3D-Window of Discovery Studio 2.5 and then submitted to protein modelling by enabling the parameters viz., Incomplete residue, Atom order, Add hydrogens, Valency, & Charge, which is then subjected to energy minimization to obtain a stable configuration by applying "Forcefield" and "Simulation" with the aid of "Steepest Descent" and "Conjugate Gradient" minimization algorithms which are repeated until the minimization criteria shows a "Gradient Tolerance Satisfied" message indicating that the protein has reached a stable configuration which is then saved in ".mol" format ( Figure 2).   Table 1, were identified and selected for further analysis. The fasta sequence of the Neprilsyin Human protein structure with PDB ID: "1DMT", that was selected by Ramachandran Plot Analysis through RAMPAGE server, was employed for running the Protein BLAST and the sequences with highest identity % were selected for further analysis. While the Pepbank Server, peptide database, was ransacked for Neprilysin like sequences and the sequences with high interaction score were selected for further analysis.

Construction & Minimization of peptide molecules:-
The selected peptide sequences were then employed in the construction of peptide molecules for further studies to be performed with the aid of Protein Modelling tool in Discovery Studio by selecting Build and Edit protein with the Conformation as "Left-hand alpha helix" as the protein structure originally seems to have a left-hand alpha helix conformation and at Choose Amino acid the peptide sequences that have been selected for study were pasted individually to generate the respective peptide molecules. Thus, constructed peptide molecules were then subjected to Dreiding minimize for approximately 20 times until there is no significant change in the energy of the molecule and then to Forcefield: CharmM followed by the Simulation through Minimization by employing the Smart minimizer algorithm with maximum steps of 500.

Docking by Z-Dock & R-Dock Algorithms:-
The minimized Aβ protein structure (PDB ID: "1IYT") and the minimized peptide molecules were then subjected to proteinpeptide docking process through Discovery studio with the help of two different algorithms namely Z-dock and R-dock.  Since the docking results of the active site 16 -21 amino acid region showed that there were no ligands docked, the complete docking scores of the all small molecule compounds docked along with the Interacting amino acids for the active site 32 -36 amino acid region were tabulated in the following Table 2 that yielded in the best Ligand molecule as Octadecylsulfate with highest libdock score as "84.927" (Figure 6).

Z-Dock & R-Dock Docking Results for Objective 2:-
The results of the proteinpeptide docking studies consummated with the aid of Z-Dock algorithm followed by the R-Dock algorithm for the selected & constructed 10 peptide molecules have been tabulated below in Table 3 with their respective poses having highest Z-Dock score and the cluster with the (-ve) highest R-Dock score.

Discussions:-
In this study, the main objective here was to target the major pathological lesions of Alzheimer"s disease i.e., the Amyloid beta plaques with the help of Bioinformatics tools and design therapeutic molecules that would combat against these plaques and reduce their content in neurons which might result in an improved communication and thereby enhancing the condition of the diseased person.
Albeit, the current structure "PDB ID -1IYT", culled as the query, is a resultant of NMR, it has been gleaned as the query as it was found to be the most paramount protein structure involved in the formation of Amyloid beta plaques, wherein the amyloidogenic regions were found to be around the regions in the length of 16 -21 amino acids and 32 -36 amino acids around which the active site spheres were constructed. With the 42 small molecules or analogues (ref Introduction) of small molecules taken for performing protein -ligand docking studies with Aβ minimized protein structure, the docking studies of the 42 small molecules with the amyloidogenic regions of the Aβ gave no docked poses for the first active site viz., 16 -21 amyloidogenic region and gave 381 poses for the second active site viz., 32 -36 amyloidogenic region among which the best ligand molecule was found to be Octadecylsulfate with the highest libdock score of 84.927.
Since the first objective had no significant effect on the first amyloidogenic region viz., 16 -21 amino acid region, the second objective was consummated which showed a significant effect on both the amyloidogenic regions. Wherein, in the second objective, on the basis of Structure Activity Relationships (SAR) peptide sequences, similar to a physiological metalloproteinase named Neprilysin, were taken and subjected for construction of peptides which were later submitted for proteinpeptide docking studies with the same amyloid beta protein structure which was used for the first objective by the aid of Z -Dock and R -Dock algorithms with the 16 -21 and 32 -36 amino acid regions as the active sites. By the whole of 10 best peptide sequences (ref Introduction) obtained by means of both protein blast and pepbank server the best peptide molecule was found to have the sequence ELNFTPNWGT which gave the highest z-dock score of 10.34 for the Pose168.
Although in silico studies represent a relatively new avenue of inquiry, it has begun to be used widely in studies which predict how drugs interact with the body and with pathogens. These in silico models exactly mimic the real models hence reducing lot of money and time. But as it stands that it still is an artificial environment quite different from that of living models, it is imperative that these molecules be tested under wet lab conditions to ensure the competence and efficacy of the conclusive data obtained from the bioinformatics work.