1Dr. Kalju KahnDepartment of Chemistry and BiochemistryUniversity of California, Santa BarbaraCurrent Approaches to Drug DiscoveryBiological ConceptValidated TargetLead CompoundModificationsTestsDRUGDISEASEUCSB Chem 162Dr. Kalju KahnWhat is a Target?• Biological macromolecule or molecular complex that is critical for the disease– e.g. an enzyme that is required for the growth of the infecting bacterium transpeptidasetransglycosylaseMore: The alternative to penicillinhttp://www.nature.com/cgi-taf/DynaPage.taf?file=/nm/journal/v7/n10/full/nm1001-1100.htmlUCSB Chem 162Dr. Kalju Kahn2Drug Targets andMechanism of Drug ActionTARGET MECHANISMEnzymes Reversible & Irreversible InhibitorsReceptors Agonist and AntagonistsViral Surface Proteins Blocking the Entry to CellIon Channels Blockers and OpenersTransporters Uptake Inhibitors or EnhancersDNA, RNA Alkylating Agents, Binders, Wrong Substrates (trojan horses)Source: Hugo Kubinyi lecture slides. University of Heidelberg and BASF AGUCSB Chem 162Dr. Kalju KahnTargets of Marketed DrugsHopkins and Groom: Nature Rev. Drug Discov. 1, 727-730 (2002)UCSB Chem 162Dr. Kalju KahnExample Target: HIV Surface ProteinsImage: http://www.healthinitiative.org/html/hiv/firstcontact/hivbig.htm Image: http://www.accessexcellence.org/AB/GG/hiv.htmlUCSB Chem 162Dr. Kalju Kahn3HIV: Virus-Cell InteractionA: gp120 binds to CD4+receptor on the surface of T-cells B: gp120 undergoes conformational change and binds to chemokine co-receptor C: gp41 becomes exposed and forms a three-helix bundleImage: www.medscape.comCD4+receptorUCSB Chem 162Dr. Kalju KahnHIV gp41 and gp120¾ gp41: viral fusion machinery¾ gp41 is highly conserved¾ gp120 is highly variableStructure before fusionUCSB Chem 162Dr. Kalju KahnHIV gp41 Hairpin Formation Triggers FusionFUSIONNHRX-ray structureUCSB Chem 162Dr. Kalju Kahn4HIV gp41: ValidationPNAS (1997) vol. 97 343–348UCSB Chem 162Dr. Kalju KahnTarget Validation"Target validation is one of the most pressing problems in pharmaceutical R&D. Many industry experts believe that without additional well-validated targets, pharmaceutical companies are unlikely to be able to maintain current levels of profitability." From the executive summary of Post-Genomic Target Validation: Next Generation Approaches and Tools for Optimizing Target Selection.UCSB Chem 162Dr. Kalju KahnValidated Target¾ Identification of a pathophysiologically relevantmolecular target, e.g. an enzyme, receptor, ion channel, or transporter¾ Determination of the DNA and protein sequence¾ Elucidation of the function and mechanism of the protein¾ Proof of the therapeutic concept in animalsSource: Hugo Kubinyi lecture slides. University of Heidelberg and BASF AGUCSB Chem 162Dr. Kalju Kahn5Any Targets Left?• Human genome: ca. 30,000 predicted genes• Currently known targets: ca. 500Human genome (30,000 genes)Disease-modifying genes ~3,000Druggable genome~ 3,000Drug targets600-1,500UCSB Chem 162Dr. Kalju KahnTarget Validation Methods: Pre-genomic•Goals:– Identify protein function•Strategies:– Find out what does it interact with– Find out where in the cell it is• Methods:– Systematic alteration of a gene– Phage display– Yeast-two-hybrid system– Expression cloningUCSB Chem 162Dr. Kalju KahnSystematic Alteration of a Gene (Knock-out)• Leishmaniasis (kala azar): • Parasite infection• 12 M infected worldwide• Dihydrofolate reductase-thymidylate synthase deficient Leishmania major parasite does not cause diseasedUMP + 5,10-Methylene-THF dTMP + DihydrofolateTS6Phage Display: Principle• Screens for protein-protein interactions in vitro• Living library in bacteriophages• Unraveling signal-transduction pathways• Hunt for viral surface proteinsMore: http://www.biotech.missouri.edu/mbp/exchange/tips/3-99tips.htmlUCSB Chem 162Dr. Kalju KahnPhage Display: PracticeImage: http://www.bio.anl.gov/research/brian_kay.htmlUCSB Chem 162Dr. Kalju KahnYeast Two-hybrid System: Principle• Screens for protein-protein interactions in vivo• Living library in yeast• Bait: Known protein fused to a DNA binding domain• Prey: Protein from library fusedto transcription activation domain• Binding Reporter gene ONMore: http://www.fccc.edu/research/labs/golemis/YTH_overview.htmlUCSB Chem 162Dr. Kalju Kahn7Yeast Two-hybrid System: PracticeImage & More: Pierre Legrain*, Luc Selig “Genome-wide protein interaction maps using two-hybrid systems” FEBS Letters 480 (2000) 32-36UCSB Chem 162Dr. Kalju KahnExpression cloning• Gene library in mammalian cells• Identification of protein function byobserving alterations in phenotype• Identification of protein localization by monitoring protein-GFP fusions• Function in normal environment• Post-translational modificationsMore: http://www.dkfz-heidelberg.de/LIFEdb/Cloning/strategy.htmlUCSB Chem 162Dr. Kalju KahnPost-genomics methods:Identifying genes• Goals:– Identify gene/protein function– Identify disease-modifying genes• Strategies– Take advantage of human genome sequence– High-throughput screening methods– Automation, robotics, microfluidics, bioinformatics• Methods:– DNA Microarrays– Single Nucleotide Polymorphisms– Proteomics with MS sequencing– RNA knockdown: antisense, ribozymes, RNAiUCSB Chem 162Dr. Kalju Kahn8Is it Important?Published in September 2003 by Cambridge Healthtech, 128 pages.•Print $2,500.00•Single-site license $5,000.00•Enterprisewide license $7,500.00UCSB Chem 162Dr. Kalju KahnDNA MicroarraysTwo fundamental approaches1) One-color array (Affymetrix)2) Two-color array (Stanford, 1996)Images and more: http://www.technologiesnews.net/news.html?view=204http://www.dhgp.de/ethics/ethics02.htmlUCSB Chem 162Dr. Kalju KahnOne-color Quantitative Microarray technology¾ Affymetrix GeneChip®¾ Target is a biotin-labeledcRNA¾ Probe is a single-stranded DNA oligo attached to the wafer¾ Complementary target and probe hybridize¾ Duplex is stained with the streptavidin-bound fluorescent marker http://www.affymetrix.com/technology/ge_analysis/index.affxUCSB Chem 162Dr. Kalju Kahn9GeneChip® Technology• In situ oligonucleotide synthesis (Affymetrix U.S. patent 5,861,242 )– 5-inch square quartz wafer with covalently bound layer of silane– Parallel synthesis with photolithographic masks– Makes oligos 20-25 nucleotides long; 400,000 per chipMore: http://www.affymetrix.com/technology/manufacturing/index.affxhttp://www.devicelink.com/ivdt/archive/98/09/009.htmlUCSB