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’ 11/24/2018 PCEU 607 Fall 2007

Process of Drug Discovery Target Identification …… Unmet Medical Need …… Evolving Scientific Knowledge …… Serendipitous Leads …… Human Genome Project 11/24/2018 PCEU 607 Fall 2007

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) 11/24/2018 PCEU 607 Fall 2007

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 Cell Viability Assay Drug Mouse Tail Flick Assay Drug Heat! 11/24/2018 PCEU 607 Fall 2007

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!! 96-well microtiter plate 11/24/2018 PCEU 607 Fall 2007

Process of Drug Discovery Sources of Compounds …… Natural Products …… Synthetic Library …… Combinatorial Synthesis …… Diversity Oriented Synthesis 11/24/2018 PCEU 607 Fall 2007

 Combinatorial Synthesis Split & Mix Strategy 11/24/2018 PCEU 607 Fall 2007

 SPLIT AND MIX STRATEGY MIX and SPLIT 11/24/2018 PCEU 607 Fall 2007

Process of Drug Discovery Lead Optimization …… Synthetic Difficulty …… Bioavailability …… ADME properties …… Toxicity 11/24/2018 PCEU 607 Fall 2007

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) 11/24/2018 PCEU 607 Fall 2007

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! 11/24/2018 PCEU 607 Fall 2007

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!! 11/24/2018 PCEU 607 Fall 2007

Parallel Synthesis and Screening of a Solid Phase Carbohydrate Library  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 contains approximately 1300 di- and trisaccharides, was synthesized with chemical encoding on TentaGel resin so that each bead contained a single carbohydrate. Two ligands that bind more tightly to the lectin than Gal-b-1,3-GalNAc (the known ligand) have been identified. The strategy outlined can be used to identify carbohydrate-based ligands for any receptor; however, because the derivatized beads mimic the polyvalent presentation of cell surface carbohydrates, the screen may prove especially valuable for discovering new compounds that bind to proteins participating in cell adhesion. Science (1996) 274; 1520-1522 11/24/2018 PCEU 607 Fall 2007

 Synthesis of the Library 11/24/2018 PCEU 607 Fall 2007

 High Throughput Screening of the Library Derivatized TentaGel beads were washed three times with 1 ml of PBST buffer and then suspended in 1 ml of PBST containing 3% bovine serum albumin. The beads were incubated at room temperature for 3 hours on a rotary shaker in 1 ml of a biotin-labeled lectin solution and then washed three times with 1 ml of TBST buffer containing 1% BSA. The beads were incubated on a rotary shaker for 20 min at room temperature in 1 ml of alkaline phosphatase-coupled streptavidin. The beads were washed three times with 1 ml of alkaline phosphatase buffer and kept in the alkaline phosphatase buffer prior to staining. A portion of the beads was transferred to a petri dish and the alkaline phosphatase buffer was replaced with 200 µl of a solution containing 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT). Color development was observed under a low-power microscope. The staining was terminated by washing the beads twice with 200 µl of sodium EDTA solution. The colored beads were picked out manually under the microscope for decoding. 11/24/2018 PCEU 607 Fall 2007

 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 level of contrast shown in the photograph was representative [60X]. 11/24/2018 PCEU 607 Fall 2007