Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Site Mutations and Drug ResistanceSlide 30Rational Drug Design : HIV IntegraseA process for drug design which bases the design of the drug upon the structure of its protein target.1. Structural mapping of the receptor (protein, P) active site2. Identification of ligands (L) of complementary shape and appropriate functionality3. Docking of the ligand to the receptor site - predicting a range of PL complexes with different GPL values4. Scoring i.e. ranking GPL and correlating with experimentally determined properties such as IC50 valuesIn the frst step of the integration process, two nucleotides are removed fromeach 3’-end of the viral DNA. This reaction exposes the terminal 3’-hydroxylgroup that is to be joined to target DNA (Fig. 1B). In the second step, DNAstrand transfer, a pair of processed viral DNA ends is inserted into thetarget DNA (Fig. 1C). Integrase is responsible for 3’-processing and DNAstrand transfer, but the latter repair steps are likely to be carried out bycellular enzymes.The catalytic domainhas an RNaseH-type fold and belongs to the superfamily of polynucleotidyl transferases. The active site is comprised of two Asp residues and one Glu, in the typicalD,D(35)E motif, each of which is required for catalysis.de novo Ligand DesignDCQ acids; DCT acidsDKAsQuinolone derivedPDP SQL four criteria to conclude that integrase is theinhibitor target:1. found to be active against recombinant integrase.2. infected cells treated with the drug must show an accumulation of 2-LTR circles, resulting from the accumulation of viral cDNA and decreased HIV integration into host3. integrase mutations must be found in drug-resistant viruses4, the drug should be inactive in biochemical assays against recombinantintegrases bearing the mutations identifed in the drug-resistant virusesIssues in Protein Setup Crystal structure available for Integrase but :I. Limitations of crystal structure:only catalytic domainDNA binding not revealedcystal structure vs. physiologically active structureII. Position of hydrogens undetermined III. Residues missing or ill-definedIV. Protonation of His undeterminedV. SolvationIssues in Protein Setup Crystal structure available for Integrase Catalytic Domain but :I. Crystal reveals trimeric structureII. Position of hydrogens undetermined III. Residues missing or ill-definedIV. Protonation of His undeterminedV. SolvationIssues in Protein Setup Crystal structure available for Integrase Catalytic Domain but :I. Crystal reveals trimeric structureII. Position of hydrogens undetermined III. Residues missing or ill-definedIV. Protonation of His undeterminedV. SolvationIssues in Protein Setup Crystal structure available for Integrase Catalytic Domain but :I. Crystal reveals trimeric structureII. Position of hydrogens undetermined III. Residues missing or ill-definedIV. Protonation of His undeterminedV. SolvationIssues in Protein Setup Crystal structure available for Integrase Catalytic Domain but :I. Crystal reveals trimeric structureII. Position of hydrogens undetermined III. Residues missing or ill-definedIV. Protonation of His undeterminedV. SolvationIssues in Protein Setup Crystal structure available for Integrase Catalytic Domain but :I. Crystal reveals trimeric structureII. Position of hydrogens undetermined III. Residues missing or ill-definedIV. Protonation of His undeterminedV. SolvationIssues in Ligand Design Crystal structure available for CITEP bound to catalytic core but :I. Position of hydrogens undeterminedII. Tautomeric structures possibleIII. Influence of pHIV. Need to limit conformational flexibility based on experimental and theoretical crteriaIssues in Ligand Design Crystal structure available for CITEP bound to catalytic core but :I. Position of hydrogens undeterminedII. Tautomeric structures possibleIII. Influence of pHIV. Need to limit conformational flexibility based on experimental and theoretical crteriaIssues in Ligand Design Crystal structure available for CITEP bound to catalytic core but :I. Position of hydrogens undeterminedII. Tautomeric structures possibleIII. Influence of pHIV. Need to limit conformational flexibility based on experimental and theoretical crteriaIssues in Ligand Design Crystal structure available for CITEP bound to catalytic core but :I. Position of hydrogens undeterminedII. Tautomeric structures possibleIII. Influence of pHIV. Need to limit conformational flexibility based on experimental and theoretical crteriaTetrazole pKa=5Issues in Ligand Design Crystal structure available for CITEP bound to catalytic core but :I. Position of hydrogens undeterminedII. Tautomeric structures possibleIII. Influence of pHIV. Need to limit conformational flexibility based on experimental and theoretical crteriaFixed and planarBased on HF/6-31G* calculationsLimited to +/- 45 degreesIssues in DockingThe prediction of the ligand conformation and orientation within a targeted binding site involves:I. Positioning ligand and evaluating quality of bindingII. Manually refining ligand positionIII. Energy minimization (electrostatic, steric, strain and h-bond)Issues in DockingThe prediction of the ligand conformation and orientation within a targeted binding site involves:I. Positioning ligand and evaluating quality of bindingII. Manually refining ligand positionIII. Energy minimization (electrostatic, steric, strain and h-bond)Issues in DockingThe prediction of the ligand conformation and orientation within a targeted binding site involves:I. Positioning ligand and evaluating quality of bindingII. Manually refining ligand positionIII. Energy minimization (electrostatic, steric, strain and h-bond)Issues in ScoringThe prediction of the optimum ligand conformation and orientation within a targeted binding site involves:I. Posing : Determining the fit of the ligandII. Conformational SearchingIII. Scoring and RankingResultsResultsCriterion for Ligand Selection:I. Theoretical and experimental structuresII. Fill active siteIII. Conformational structuresLigand DesignCriterion for Ligand Selection:I. Theoretical and experimental structuresII. Fill active siteIII. Conformational structuresLigand DesignCriterion for Ligand Selection:I. Theoretical and experimental structuresII. Fill active