Section V. Summary and conclusions

Transcription

1 122 Section V Summary and conclusions

2 123 Modern approach to drug design employs a diverse range of computer applications which analyze a proposed candidate drug molecule for its efficacy and potency, model it to carry out simulation of its suggested activity, modifies it if necessary, find out the better option by substituting groups, remodel the modified molecule by docking it to the target site where it is supposed to work either by enhancing the tissue function or by inhibiting the undesirable function, analyze the results systematically and finally suggest an ideal pharmacophore that needs to be synthesized. It is CAAD (Computer Assisted Drug Design), which is fast and delivers a drug quickly in contrast to the conventional method of drug design which employed synthesis of a large array of compounds followed by their clinical trial one by one and delivering a potent drug after years. The interactions between the receptor and a ligand molecule (drug candidate) are fundamental to drug discovery. The process of computational modeling represents molecular structures numerically and simulates their behavior with the equations of quantum and classical physics. Computational programs allow to generate and present molecular data including geometries such as bond lengths, bond angles, torsion angles, energies such as heat of formation, activation energy, etc., electronic properties such as moments, charges, ionization potential, electron affinity, spectroscopic properties like vibrational modes, chemical shifts and bulk properties like volumes, surface areas, diffusion, viscosity, etc. (Richon 1994). Electrostatic interaction The modeling applications calculate electrostatics interactions between pairs of point charges of molecules by applying Coulomb s law -

3 124 Where and N a and N b are the numbers of point charges in two molecules. The charge distribution is represented as an arrangement of fractional point charges in the molecule. If charges are confined to nuclear centers they are called potential atomic charges or net atomic charges. Vanderwaals interaction Vanderwaals chemical interaction occurs between atoms at short distance, and so, it would get off as the interacting atoms move away from each other even by a few Angstroms. This effect is modeled using equations The parameters A and B control the depth and position as inter-atomic distance of the potential energy for a given pair of non bonded interacting atoms such as C:C, O:H etc. Hydrophobic interaction When a hydrophobic molecule is made to come in contact with water, water molecules get attracted to each other to maximize hydrogen bonding among them and build a quasi crystalline surface around the hydrophobic molecules. If hydrophobic molecules are near by, they would get held up by this quasi crystalline film of water while water molecules would get distributed back into the bulk solvent. This generates favorable entropy. This gain in entropic is responsible for all associations in water medium such as formation of membrane, micelles and even protein folding.

4 125 Hydrogen bonding Though hydrogen bonds energies may seem small in the order of 4 to 25 kj/mole, it is to be noted that they exist as a system of interconnections which are additive and make the system stable. When a ligand protein reaches within the desired distance from the targeted protein to which the former has been attempted to dock with minimum free energy involved, H bonds would form to make a complex of the molecules that would affect the overall structure and behaviour of the system (functional protein or enzyme in the present case) Energy Minimization In molecular modeling, one is concerned with the minimum points on the energy surface. Minimum energy arrangements of the atoms correspond to stable states of the system. Any movement away from a minimum gives a configuration with a higher energy. There may be a very large number of minima on the energy surface. The minimum with the very lowest energy is known as the global energy minimum. To identify those geometries of the system that correspond to minimum points on the energy surface, the modeling applications use minimization algorithm. Protein-protein docking procedures normally consist of two successive steps. First, a search algorithm generates a large number of candidate conformations a high CPU-intensive step, and then, a scoring function is used to rank them in order to extract a near-native conformation. Hex uses spherical polar Fourier correlations to accelerate docking calculations. Basic approach to the docking in Hex docking application is to represent the steric shape, electrostatic potential, and charge density of each protein as expansions of spherical polar Fourier basis functions (Ritchie and Kemp 2000). Unlike conventional three-dimensional (3D) fast Fourier transform (FFT) docking approaches (Katchalski et al. 1992; Gabb et al. 1997; Chen and Weng

5 ) which accelerate translational correlations, its approach uniquely favours rotational searches. However, translational correlations may also be calculated. Synthetic peptides have been proposed to be the ideal inhibitors of enzyme activity either alone or in combination with small-molecule drugs (Singh et al. 2001). High synthesis cost of peptide synthesis has been hampering the development of peptide-based drugs during the last decade. Recent techniques involving automated peptide synthesizer have led to a renewed investment in therapeutic peptides. Compared to small-molecule drugs, which are currently dominating the pharmaceutical market, peptide-based therapeutics offer several advantages, such as high specificity, lower accumulation in tissues, lower toxicity, and biological diversity (Lien and Lowman 2003). Non-ribosomal synthesis of peptides Solid-phase peptide synthesis (SPPS), pioneered by the Nobel Laureate Robert Bruce Merrifield (1963), is now the accepted method for producing peptides and proteins synthetically. The peptide is synthesized from C terminal to N terminal in contrast to that by ribosomal synthesis. The SPPS involves repeated coupling-wash-deprotection-wash cycles. The free N-terminal amine of a solid-phase attached amino acid is coupled to the carboxy terminal of another N- terminal protected amino acid residue by an amide bond. N terminal of the latter is then deprotected to expose it for linking of a further amino acid residue. Washing schedule after each reaction removes excess reagent with the growing peptide remaining covalently attached to the insoluble resin. The present Ph.D. project had mainly three study targets 1. To analyze some of the well known AMP sequences from different sources (organisms) to understand the parameters on the basis of which some new sequences could be designed with a view to obtain potentially highly efficacious antibiotic peptides,

6 To design new sequences de novo and analyze their required characteristics in terms of their antibiotic potentiality, 3. To model the designed sequences to predict their secondary 3D structure on the basis of some sequence that has maximum residual similarity to the former, and 4. To simulate the antibiotic activity of the designed sequences in the form of their functional secondary structure by subjecting them to docking into the reliable bacterial protein targets that might be a component of the cell membrane, or of the cell wall synthesizing enzyme system, or that of the transcriptional machinery. All the four above points of study outlined in the synopsis of the research project were successfully tackled and all the work related to them was accomplished within the specified time. The whole work may be summarized as follows 1. Known peptides, that were analyzed, belonged to bacteria, animal and plants. These were nisin, amoebapore A, mytilin B, mussel defensin-1, Cecropin, thanatin, melitin, gomesin, moricin, tachyplesins, polyphemusins, ARD1, sapecin, aurein, magainin, LL-37, indolicidins, lactoferricin, human alpha defensin, human beta defensin and protegrin-i, thionin and a plat defensin. 2. UCSF Chimera, VegaZZ, ArgusLab and Swiss-PdbViewer (all in their latest version) were the chief molecular modeling software that were made use of for analysis and visualization of molecules. 3. A hydrophobicity calculator program HYb V 1.0 was written by the author in Perl Version It was used to calculate hydrophobicity % of a peptide.

7 Two peptide sequences were hypothesized and designed in silico as potential antibiotic peptides. These were ArtAmp of 24 residues and AdAmp of 45 residues. 5. The two peptides were re-assessed online at Antimicrobial Peptide Database (APD) for their assumed features and intended efficacy. 6. ArtAmp and AdAmp were modeled for their predicted 3D functional structure by the application Modeller9V3 on the basis of somewhat similar AMPs whose 3D structure was already determined previously. These were lactoferricin and ARD1 respectively. 7. Modeled molecules of ArtAmp and AdAmp were subjected to the docking process into some pre-selected target proteins of bacteria employing the latest version of a well appreciated docking application Hex. 8. The target protein molecules were the outer membrane protein X from E. coli, a bacterial heteromeric transcription regulator protein, prokaryotic transcription elongation factor GreA/GreB from Nitrosomonas europaea, transglycosylase PBP1b from E. coli, an activator dependent bacterial transcription initiation complex and the cell wall forming bacterial enzyme D-alanyl-D-alanine peptidase from Sterptomyces. 9. The docking process was performed for the best poses of the ligand docking involving minimized free energy level that gave the best scores. 10. Ligand peptide- target protein docking successfully resulted into almost all types of chemical interactions hydrogen bonding, Vanderwaals interaction, electrostatic interaction and hydrophobic interaction.

8 129 The following figure presents the snapshot of the screen of Hex 6.3 with a popup showing the docking parameters along with docked complex of 3PTE and ArtAmp The docked results confirmed in silico that both the peptides must be highly potent antibiotic peptides. But, before a molecule is recommended as a clinical drug, its synthesis followed by its clinical trial is must. According to Lipinski (2001), those compounds are likely to have good absorption and permeation in biological systems and be successful drug candidates if they have five or fewer hydrogen-bond donors, ten or fewer hydrogen-bond acceptors, molecular weight less than or equal to 500 and calculated logp less than or equal to 5. Beyond the scope of this thesis, the project work is being followed further. As a first step, request for the synthesis of the ArtAmp peptide was put online to a company Bachem, Bubendorf, Switzerland well known for custom peptide

9 130 synthesis (Bachem AG, Hauptstrasse 144, CH-4416, Bubendorf) at (Quotation : Customer no. T ). PS: 3D structural modeling of AdAmp and ArtAmp was also done online on ESyPred3D Web Server 1.0 (Lambert et al. 2002). The results obtained were similar to those carried out with the help of script driven standalone Modeller application as presented in section IV. Following renderings of the two models have been achieved by Swiss-PdbViewer (DeepView) v AdAmp ArtAmp

10 131 In ArtAmp sequence, the models generated showed two short helix parts (colored red here), a two-residue coil and rest as extended segments which can associate as β sheet - FRCRRWNWRMHKLACSMTCVRRAFX. Secondary structure prediction and domain assignment at Swiss-Model application platform ( (Arnold et al. 2006; Kiefer et al. 2009; Peitsch 1995) involving InterPro Domain scan ( Zdobonov and Apweiler 2001) with the help of HMMPfam, HMMTigr, ProfileScan, Superfamily and BlastProDom, PsiPred Secondary structure prediction (Jones 1999) and MEMSAT Transmembrane segment Prediction (Jones et al. 1994) returned the following projection The models generated for AdAmp showed secondary structure as LLICVVGAAFWYYPPLVCCAFFYIIGGLWPAAAPPYVIICAFFWWX with only two short helices (colored red). Secondary structure prediction and domain assignment at Swiss-Model application platform (references cited above) projected the structure as