Process of Drug Discovery

1. Process of Drug Discovery • History …… Plant products pre-date the rise of modern medicine …… Use of opium, licorice, ephedra, marijuana, camellia, alcohol, digitalis, cocoa, quinine etc… …… First discovered was morphine (opium), which was found to be alkaline (1817) …… Isolation of strychinine, brucine, quinine …. …… Diacetyl morphine (1898) was introduced as a pain reliever … ‘heroic drug’ PCEU 607 Fall 2007

2. Process of Drug Discovery • Target Identification …… Unmet Medical Need …… Evolving Scientific Knowledge …… Serendipitous Leads …… Human Genome Project PCEU 607 Fall 2007

3. Process of Drug Discovery • Target Optimization …… Preparation of Target Native or Recombinant? Soluble or Membrane? Expressible? …… Physicochemical Properties of the Target Structure (NMR, X-ray, Molecular Modeling) PCEU 607 Fall 2007

4. Cell Viability Assay Drug Mouse Tail Flick Assay Heat! Drug Heat! Process of Drug Discovery • Lead Identification …… Biochemical or pharmacological assays …… Terms used to describe molecules that bind ligand, inhibitor, antagonist, agonist, activator, partial agonist …… Quantitative measures of binding KI, IC50, EC50, LD50, …… Units of binding affinity M, mM, mM, nM PCEU 607 Fall 2007

5. 96-well microtiter plate Process of Drug Discovery • High Throughput Screening Assays …… Simultaneous …. 96- or 384-well microtiter plates …. robotic analysis …… Chromogenic substrates …. develop color …… Yes or No answer ….. fixed concentration of drug …… Quantitative …. may also give you KI values!! PCEU 607 Fall 2007

6. Process of Drug Discovery • Sources of Compounds …… Natural Products …… Synthetic Library …… Combinatorial Synthesis …… Diversity Oriented Synthesis PCEU 607 Fall 2007

7. Y SPLIT Y Y Y X X X MIX Combinatorial Synthesis Split & Mix Strategy PCEU 607 Fall 2007

8. SPLIT AND MIX STRATEGY MIX and SPLIT PCEU 607 Fall 2007

9. Process of Drug Discovery • Lead Optimization …… Synthetic Difficulty …… Bioavailability …… ADME properties …… Toxicity PCEU 607 Fall 2007

10. Process of Drug Discovery • Investigational New Drug (IND) …… Clinical Development …… Phase I (health human volunteers … tolerance, safety, pharmacokinetics, ….) …… Phase II (initial efficacy … ‘a’ trial limited; ‘b’ trial more extensive) …… Phase III (pivotal clinical trials in several clinical centers with different groups of people) PCEU 607 Fall 2007

11. Process of Drug Discovery • Drug Discovery Timeline …… Yr 01 Identify suitable disease, assemble team, select approach, budget, start chemistry, devise assays, select ‘hits’ …… Yr 12 Establish utility of ‘hits’ in animals …… Yr 25 Identify promising ‘hits’; ascertain freedom to operate (patents, literature, potency, acute toxicity, ADME, synthesis, feasibility) …… Yr 45 Patent protection …… Yr 49 Phase I to III clinical trials …… Yr 811 Regulatory review …… Yr 1015 Marketing and phase IV …… Yr 1517 Patent protection expires! PCEU 607 Fall 2007

12. Process of Drug Discovery • Drug Discovery Costs …… 1991 $359 million …… 1995 $627 million …… 2000 $1360 million …… 2005 $1800 million (projected) …… Success rate of a new drug ….. 1 in 5 …… Cost for four failed molecules ….. $1 billion …… Total cost ….. $3 billion …… Recovery time ….. 5 – 7 years ….. Requires a market of $600 million annually!! PCEU 607 Fall 2007

13. An Example of Combinatorial Approach in the Discovery of a Potent Ligand Parallel Synthesis and Screening of a Solid Phase Carbohydrate Library Rui Liang, Lin Yan, Jennifer Loebach, Min Ge, Yasuhiro Uozumi, Klara Sekanina, Nina Horan, Jeff Gildersleeve, Chris Thompson, Andri Smith, Kaustav Biswas, W. Clark Still, Daniel Kahne* A solid phase carbohydrate library was synthesized and screened against Bauhinia purpurea lectin. The library, which containsapproximately 1300 di- and trisaccharides, was synthesized withchemical encoding on TentaGel resin so that each bead containeda single carbohydrate. Two ligands that bind more tightly to thelectin than Gal-b-1,3-GalNAc (the known ligand) have been identified.The strategy outlined can be used to identify carbohydrate-basedligands for any receptor; however, because the derivatized beadsmimic the polyvalent presentation of cell surface carbohydrates,the screen may prove especially valuable for discovering new compoundsthat bind to proteins participating in cell adhesion. Science (1996) 274; 1520-1522 PCEU 607 Fall 2007

14. Synthesis of the Library PCEU 607 Fall 2007

15. High Throughput Screening of the Library Derivatized TentaGel beads were washed three times with 1 ml of PBST buffer and then suspended in1 ml of PBST containing 3% bovine serum albumin. The beads wereincubated at room temperature for 3 hours on a rotary shaker in1 ml of a biotin-labeled lectin solution and then washed three times with 1 ml of TBST buffer containing 1% BSA. The beadswere incubated on a rotary shaker for 20 min at room temperaturein 1 ml of alkaline phosphatase-coupled streptavidin. The beads were washed three timeswith 1 ml of alkaline phosphatase buffer and kept in the alkaline phosphatasebuffer prior to staining. A portion of the beads was transferredto a petri dish and the alkaline phosphatase buffer was replacedwith 200 µl of a solution containing 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitro blue tetrazolium (NBT). Color developmentwas observed under a low-power microscope. The staining was terminatedby washing the beads twice with 200 µl of sodium EDTA solution. The colored beads were picked out manually underthe microscope for decoding. PCEU 607 Fall 2007

16. High Throughput Screening of the Library A portion of the beads in the library after 5 min of staining. The dark bead in the center was identified as a hit. This levelof contrast shown in the photograph was representative [60X]. PCEU 607 Fall 2007