Oral contributed paper “Insilico methods for design of novel inhibitors of Human leukocyte elastase” in the International conference on Systemics, Cybernetics and Informatics-2006

1. Insilico methods for design of novel inhibitors of Human leukocyte elastase by L. Jayashankar, M.Tech Pharma., GVK Biosciences, Hyderabad. (Contributed oral paper in ICSCI-2006)

3. Stages in Drug Discovery

4. Identify disease state Relevant biomolecular target Assay Development e.g. Receptor cloning and expression Compound Collections Primary Assay (high through-put, usually in vitro ) Secondary Assays (counter screens, bioavailability, toxicity, metabolism, etc.., usually in vivo ) Bioinformatics Protein Modeling Drug Discovery and Design Clinical Candidate Lead compounds and SAR Chemical Synthesis Design Mapping Fitting In Silico

6. Raw data X-ray NMR Homology Target definition via Structure determination and prediction

7. Get the diffraction pattern Phasing : MIR and molecular replacement Electron density map Refinement Fit sequence to density X-ray based Target Definition

8. Ligand design When the structure of an enzyme is known, It is possible to display in a modeling environment ( Insight II ) to select potential binding sites by inspection and to design an inhibitor that targets those sites.

9. De Novo (New) Ligand Design They analyze the properties of the active site Determine favorable-binding locations for individual atoms or small fragments. Although conceptually simple, these approaches are quite useful for successful ligand design.

18. Ramchandran plot for 1 EAS

20. Site Search Aim: To find all cavities inside a protein Protein is mapped on a grid. One must visually inspect, select, adjust and define the binding site.

22. . . . . . . Log P Log 1/C Y X Draw best possible lines through the data points on the graph

23. Best line will be the one close to the data points To measure how close the data points are vertical lines are drawn from each point These verticals are measured and then scored in order to eliminate the negative values Squares are then added up to give a total Best line through the points will be the line where the total is a minimum

24. The significance of the equation is know as regression coefficient(r) For a perfect fit r2=1 Good fit generally have r2 values of 0.95 or above Physiochemical properties Most commonly studied are Hydrophobic,Electrostatic and steric interaction Hydrophobic properties can easily quantified for complete molecules or for individual substituents

25. Electronic and steric properties are more difficult to quantify,and quantifications are only feasible for individual substituents QSAR studies are being carried out on compounds of the same general structure where substituents on aromatic rings or accessible functional groups are varied QSAR studies then considers how the hydrophobic,electronic,and steric properties of the substituents affect the biological activity

26. Hydrophobicity How easily it crosses the cell membranes and may well also be important in receptor interactions Changing substituents on a drug may well have significant effects on its hydrophobic character and hence its biological activity P= Concentration of the drug in octonol Concentration of drug in aqueous solution

27. Hydrophobic compounds - P-values hydrophilic compounds - low P values Biological activity = 1/C C=Concentration of the drug required to achieve a defined level of biological activity If Log P values is resticted to a small range (1-4) a straight line graph is obtained showing that there is a relation ship between hydrophobicity and biological activity Log(1/C)=K1 log P+k2

28. Increasing the hydrophobicity of a lead compound results in a increase in biological activity Compounds having a log P value close to 2 should be capable of entering the central nervous system effectively Drugs which are designed to act elsewhere in the body should have lop values significantly different from 2 in order to avoid possible central nervous system

29. Substituent Hydrophobicity constant Measure of how hydrophobic a substituent is relative to hydrogen πx = log Px - log PH PH-partition coefficient of a standard compound Px-partition coefficient of a standard compound with substituent +value –Substituent is more hydrophobic -value - Substituent is less hydrophobic

30. The electronic effects of various substituents will clearly have an effect on drugs ionisation or polarity Measured in terms of Hammet substituent constant σ σ-measure of electronic with drawing or electron donating ability of a substituent Determined by measuring the ability of a series of substituted benzoic acids compared to the dissociation of benzoic acid itself Cl, CN, CF3 –σ values positive (Electron withdrawing) Me ethyl and butyl – σ –values (electron donating) Values also depend whether the subsituent is meta or or para

31. σ values cant be measured for ortho substituents since such substituents have an important steric as well as electronic effects Electron withdrawing groups increase the rate of hydrolysis and have positive values Steric properties Bulk ,size,and shape of the drug may have influence on this process Tafts steric factor Molar refractivity-measure of volume occupied by an atom or group of atoms

35. Grid search

39. Typical QSAR study table

46. Comparision of different statistical analysis r^2=correlation coefficient .856 .739 2 .798 .013 9.26 .910 .839 2 .795 .0168 6.26 .839 .793 2 .845 .002 10.56 R^2 XVR^2 Outliers BSR^2 BSR^2ERROR PRESS 2D QSAR RSA MFA Statistical parameters

51. Pretein scffold with ligand docked at active site

52. The docked molecule in space filled model Ligand

53. Docking scores of the ligand in the active site of human leukocyte elastase

55. LIGAND INTERACTING WITH THE STATED AMINOACIDS IN THE ACTIVE SITE