Medicinal Chemistry & Drug Discovery Dr. Peter Wipf

Medicinal Chemistry & Drug Discovery Dr. Peter Wipf

Medicinal Chemistry & Drug Discovery Dr. Peter Wipf Department of Chemistry University of Pittsburgh http://ccc.chem.pitt.edu/wipf/index.html University of Pittsburgh Center for Chemical Methodologies & Library Development http://ccc.chem.pitt.edu/wipf/Courses.html Survey of current drugs Drug discovery challenges Methods to identify new leads Case studies from industry Forward chemical genetics reverse pharmacology Medicinal Chemistry The science that deals with the discovery or design of new therapeutic agents and their development into useful medicines. It involves: Synthesis Structure-Activity Relationships (SAR) Receptor interactions

Absorption, distribution, metabolism, and excretion (ADME) 2003 Blockbusters at the Drugstore (US/Worldwide; out of ~$500b) Lipitor (Pfizer) cholesterol $6.8/10.3 billion (66%) Zocor (Merck) cholesterol $4.4/6.1 billion (72%) Zyprexa (Eli Lilly) antipsychotic $3.2/4.8 billion (72%) Norvasc (Pfizer) blood pressure $2.2/4.5 billion (40%) Procrit (J&J) anemia $3.3/4.0 billion (83%) Prevacid (TAP) ulcers $4.0/4.0 billion (100%) Nexium (AstraZeneca) ulcers $3.1/3.8 billion (82%) Plavix (BMS-Sanofi) blood thinner $2.2/3.7 billion (59%) Seretide (GSK) asthma $2.3/3.7 billion (62%) Zoloft (Pfizer) depression $2.9/3.4 billion (85%) Epogen (Amgen) anemia $3.1/? billion (?%) Celebrex (Pfizer) arthritis $2.6/? billion (?%) Source: IMS Health, March 2004 HO O N N H N N O OH

Lipitor N H F O S H2N CH3 O H O OH OH N O Zyprexa S O H

H H3C CH3 CH3 Zocor OH O N N O S H N CF3 N O CF3 CH3 N HO Seretide

Prevacid H 3C Ph (H2 C)4 HN O (CH2 )6 OH HO CO 2 H Celebrex H N O NH2 NHCH3 HCl Me O 2 C CO 2 Et Cl Cl CO 2 Me

HSO 4 Norvasc Me O N Zoloft - Plavix N N S O S Cl Cl Ne xium N )2 Mg2+ Big Pharma Drug Discovery in the 21st Century The Problem: The pharmaceutical industry is short of new drugs. In the 2nd part of

the 20th century, about 50-60 new drugs (NCEs) were approved by the FDA every year. In contrast, in 2002, a historical low of 18 NCEs were approved (in 2001, 24 NCEs, in 2000, 27 NCEs, in 2003, 21 NCEs). Conversely, research costs for a new drug are estimated to be in the $11.5 Bi. range. Considering all high-profile failures in recent drug discovery, this figure is unlikely to drop substantially. gloom around the pharmaceuticals company.. Source: Wall Street J. 11/21/2003 Wallstreet (and companies) are looking for a scapegoat.. Drug Industry's Big Push Into Technology Falls Short. Testing Machines Were Built To Streamline Research -- But May Be Stifling It A decade ago, pharmaceutical companies announced a revolutionary new way of finding drugs. Instead of relying on scientists' hunches about what chemicals to experiment with, they brought in machines to create thousands of chemical combinations at once and tested them out with robots. The new technology was supposed to bring a flood of medicines to patients and profits to investors. Today, some leading chemists are calling the effort an expensive fiasco. Machines churned out chemical after chemical that didn't produce useful results. And chemicals that seemed promising often turned out to have big flaws that traditional testing might have caught earlier on. Some drugs couldn't dissolve in water or be turned into pills, for example. Critics believe these problems help explain the pharmaceutical industry's drought of new products. "That's the secret of why they're spending billions of dollars and getting nothing," says James Hussey, a former Bristol-Myers Squibb Co. manager who now leads biotech company NeoPharm Inc. ..

Source: Wall Street J. 2/24/2004 In Search of New Leads.. The decline in the number of new drugs is based, among other reasons, on the current high therapeutic standard in many indications, focusing research on chronic diseases such as coronary heart, Alzheimers, arthritis, cancer, and AIDS, as well as the enhanced regulatory requirements for efficacy and safety of new drugs. A lead can be characterized as a compound that has some desirable biological activity, not extremely polar or lipophilic, and not contain toxic or reactive functional groups. Often, molecular weight (<350) and lipophilicity (log P < 3) are considered the most obvious characteristics of a drug-like lead. The lead should also have a series of congeners that modulate biological activity, indicating that further structural modification will improve selectivity and potency. Why Is It So Difficult to Make Drugs? Estimates of the number of possible drug molecules average 1040. In contrast, the number of seconds since the Big Bang is only 1017. If 10,000 chemists were to prepare 1 compound each per second, it would take 10,000,000,000,000,000,000,000,000,000 years to finish the job. Drug Discovery Paradigm Currently: Biology-driven drug discovery Chemistry-driven drug discovery back to the future?

Where Did (Do) Our Drugs Come From? New drugs from old poisons CH3 O HO CH3O O O O H H N CH3 N CH3 O O H HO N CH3

HO morphine O CH3 codeine heroin The reductionist approach to medicine began with the isolation of opium alkaloids Medicinal Chemistry Folklore Earliest medicines ~ 5100 years ago Chinese emperor Shen Nung - book of herbs, Pen Tsao Chang Shan - contains alkaloids; used today in the treatment of malaria and for fevers Ma Huang - contains ephedrine; used as a heart stimulant and for asthma. Now used by body builders and endurance athletes because it quickly converts fat into energy and increases strength of muscle fibers. Modern therapeutics: Extract of foxglove plant, cited by Welsh physicians in 1250. Used to treat dropsy (congestive heart failure) in 1785 Contains digitoxin and digoxin; today called digitalis At least a quarter of all prescriptions dispensed in the US and UK contain, as active compounds, molecules derived from flowering plants. Other data show that 12 out of the top 25 highest earning drugs in 1995 were derived from natural products. The importance of plants as medicines in the developing world should also be acknowledged. Here, they are estimated to comprise 80% of the medication used in primary healthcare (Source: Houghton, P. J., "Roots of

remedies: Plants, people and pharmaceuticals." Chem. Ind. 1999, 15). Ethnographies show that humans are great botanical experimentors Perhaps human brains, drugs and spices evolved together! Examples of Natural Products as Leads & Drugs Cardiac glycosides, morphine, quinine, salicylic acid, taxol, camptothecin, penicillin, cyclosporin A, warfarin, artemisine. HO N H H O H OH H O norethindrone N HO O O HO

17-ethynylestradiol R = H: Morphine R = Me: Codeine (pain killer) OMe HO H H H HO RO O OH OH O O OMe O

(the "Pill"; contraceptive) OH Clarithromycin (antibacterial) HO O NH2 CO2H O O N H H S CO2H N O OH Clavulanic acid (-lactam ase inhiitor)

Ampicillin (antibiotic) OH O O N O 10 N 11 O 9 8 N H N N 4 O O 7

O Augm e ntin (antiiotic) N 2 H 1 O 3 N N 6 N H N 5 O HN O O Cyclosporine A

Current use of natural product extracts QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. High-Pressure Accelerated Solvent Extractor System QuickTime and aGraphics decompressorare needed to see this picture. DCM & MeOH Extracts QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and aGraphics decompressorare needed to see this picture. Multiwell Plates For HTS QuickTime and aGraphics decompressorare needed to see this picture. QuickTime and aGraphics decompressorare needed to see this picture. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. Parallel HPLC

HT Solvent Concentrator Drug Discovery One way to discover drugs Serendipitous Drug Discovery The use of nitrous oxide and ether as narcotic gases in surgery resulted from the observation that people who inhaled these chemicals [in parties] did not experience any pain after injury. The vasodilatory activity of amyl nitrite and nitroglycerin was discovered by chemists who developed strong headaches after inhaling or ingesting minor amounts. A wrong working hypothesis on chloral hydrate, which was supposed to degrade metabolically to narcotic chloroform, led to its application as a strong sedative (in reality, the metabolite trichloroethanol is the active form). Similarly, urethane was supposed to release ethanol but is a hypnotic by itself. Acetylsalicylic acid was thought to be just a better tolerable prodrug of salicylic acid, but turned out to have a unique mechanism. Phenolphthalein was considered as a useful dye for cheap wines; after a heroic self-experiment, a pharmacologist experienced its drastic diarrhoic activity. Warfarin was used a rat poison. Serendipitous Discovery of Librium without a Lead In 1955 Roche set out to prepare a series of benzheptoxdiazines as potential new tranquilizer drugs, but the actual structure was found to be that of a quinazoline 3-oxide. N R1

O N X Y R2 2.4 N R1 N+ O X R2 Y 2.5 No active compounds were found, so the project was abandoned In 1957, during a lab cleanup, a vial containing what was thought to be the latter compound (X = 7-Cl, R1 = CH2NHCH3, R2 = C6H5) was sent for testing, and it was highly active. Further analysis showed that the actual structure of the compound was the benzodiazepine 4-oxide, Librium, presumably produced in an unexpected reaction of the corresponding chloromethyl quinazoline 3-oxide with methylamine. N CH 2Cl N+ O

Cl H N .. CH3NH2 Cl NHCH 3 CH 2Cl N + O 2.6 CH 3NH2 N Cl CH 2NHCH 3 N+ O N Cl N NHCH 3 .. CH 2 Cl N OH Cl

NHCH 3. HCl N+ O- chlordiazepoxide HCl 2.3 Librium Rational Drug Discovery Nearly every modification of neurotransmitters dopamine, serotonin, histamine, or acetylcholine by classical medicinal chemistry led to a compound with modified activity and selectivity. Steroid hormone modifications led to similar success stories. Many enzyme inhibitors were developed from leads that mimic the transition state of the corresponding enzyme. Protease inhibitors started from cleavage-site peptides by converting the critical amide bond into another functionality. For example, aspartyl protease inhibitors should contain the amino acids at both sides of the cleavable peptide bond, and the latter bond needs to be replaced by a stable isostere that resembles the transition state. In the 1980s and 1990s, computer modeling of enzyme-substrate complexes became a major driving force for rational drug discovery and the interpretation of SAR results. Structure-Activity Relationships (SARs) 1868 - Crum-Brown and Fraser Examined neuromuscular blocking effects of a variety of simple quaternary ammonium salts to determine if the quaternary amine in curare was the cause for its muscle paralytic properties.

Conclusion: the physiological action is a function of chemical constitution Structurally specific drugs (most drugs): Act at specific sites (receptor or enzyme) Activity/potency susceptible to small changes in structure Structurally nonspecific drugs: No specific site of action Similar activities with varied structures (various gaseous anesthetics, sedatives, antiseptics) Example of SAR H 2N SO 2NHR sulfa drugs 2.1 Lead: sulfanilamide (R = H) Thousands of analogs synthesized From clinical trials, various analogs shown to possess three different activities: Antimicrobial Diuretic Antidiabetic SAR General Structure of Antimicrobial Agents NH 2 R

2.32 R = SO2NHR, SO3H Groups must be para Must be NH2 (or converted to NH2 in vivo) Replacement of benzene ring or added substituents decreases or abolishes activity O R can be SO 2 NH 2 , SO NH 2 , O CNH2 (but potency is reduced) R = SO2NR2 gives inactive compounds ,

C R SAR Antidiabetic Agents R H SO 2NHC N X 2.33 X = O, S, or N R' SAR Diuretics (2 types) R2 NH2 SO 2 N R1 NH S O O X

R2 R 1NHSO 2 CO 2H 2.34 2.35 hydrochlorothiazides high ceiling type R2 is an electrophilic group Rational Drug Discovery - Piroxicam It took Pfizer about 18 years to develop the anti-inflammatory drug piroxicam, which was launched in 1980 during the golden age of rational drug discovery. The starting point for the development was chemistry-driven, ie. to identify acidic, but not carboxylic acid-containing (salicylic acid) structurally novel compounds. Measurement of a physical property (pKa) as well as serum half-life in dogs was the guide for the synthesis program. Several generations of leads were refined and ultimately led to a successful structure with an acceptable safety and activity profile: O O O Ar

NHAr O Ar S O O O OH N O O O NHAr O NHAr S O N O R R S

O N O R Bioisosterism Bioisosteres - substituents or groups with chemical or physical similarities that produce similar biological properties. Can attenuate toxicity, modify activity of lead, and/or alter pharmacokinetics of lead. Classical Isosteres Table 2.2 1. Univalent atoms and groups a. b. c. d. CH3 NH2 OH F Cl Cl PH2 SH Br i-Pr I t-Bu 2. Bivalent atoms and groups a.

b. CH2 NH COCH2 R O CONHR S CO 2R Se COSR 3. Trivalent atoms and groups CH a. b. N P As 4. Tetravalent atoms

a. b. C Si C N P 5. Ring equivalents a. b. c. CH CH O CH S N S (e.g., benzene, thiophene) (e.g., benzene, pyridine) CH 2 NH

(e.g., tetrahydrofuran, tetrahydrothiophene, cyclopentane, pyrrolidine) Non-Classical Isosteres OO 1. O C CC OH NH 2 O Do not have the same number of atoms and do not fit steric and electronic rules of classical isosteres, but have similar biological activity. 2. C S N OH

N OH N O Amide group O O NOCH O O O CH H2 C N S 3 N S S C N Ar N Ar H O O O H NH NH 2 2 CH 3 O

N OH Carboxylic acid group N OH N 3. OO O O O NOH CN CH N S SArO -C OR N HHNO H O O CN C O HN O O O

O O OHS O O S S OH C P OH P OH C NH C N S C NH2 CH 2 CH 2 NH2 HN O NH2 OEt CN O R NC OO CN SC N H C HN O R Carbonyl group S

N OH N N O X N OH O O NH O OH N N OH N N N N N H OH F OH N

F OO group R' O NO 2 R' O SO2NH 2 NC CN O CNCN Ester CNN(CN) C(CN) S O CFS 3 2 N 3N NN O H N S N N HOOH O OH R CH NHCR S N NHSO2RC NHCNHO HN R' C 2 N 2

N N NH OR NH2 NRR' NH NH 2 NH NH 2 NH2 O SX O O- N N F HO HO HO HO NHCN CH(CN) 2 R' X = O, NR R' N N O N S O N N N N N N N N

OR N OR OR OR 4. X 5. Hydroxyl group 6. Catechol 7. Halogen 8. Thioether 9. Thiourea 10.Azomethine CN N

C 11.Pyridine N+ R N NO2 + NR3 12.Benzene N S S N O O O H N N

N N N N 13.Ring equivalents R N R 14.Spacer group (CH2) 3 15.Hydrogen H F R O R' R N

O H3 C R' H O H O N R O R N N H NH2 Examples of Bioisosteric Analogues Neuroleptics (antipsychotics) N (CH2) 3X R X=

O C or CHCN NH Anti-inflammatory agents X CH3O O CH3 N O X = OH (indomethacin) = NHOH N N N N H = Cl OH Y O CH3

Y = CH3 O Z = Cl Y = F Z = SCH 3 (sulindac) Z Ph Antihistamines R X (CH2) n Y Ph CO2H O X = NH, O, CH2 Y = N (CH 3) 2 (n = 2) N (n = 1) N H H N N (n = 1, 2) N

Diphenhydramine (Benadryl) Ph HO Ph N OH Fexofenadine (Allegra) Changes resulting from bioisosteric replacements: Size, shape, electronic distribution, lipid solubility, water solubility, pKa, chemical reactivity, hydrogen bonding Effects of bioisosteric replacement: 1. Structural (size, shape, H-bonding are important) 2. Receptor interactions (all but lipid/H2O solubility are important) 3. Pharmacokinetics (lipophilicity, hydrophilicity, pKa, H-bonding are important) 4. Metabolism (chemical reactivity is important) Bioisosteric replacements allow you to tinker with whichever parameters are necessary to increase potency or reduce toxicity. Bioisosterism allows modification of physicochemical parameters Multiple alterations may be necessary: If a bioisosteric modification for receptor binding decreases lipophilicity, you may have to modify a different part of the molecule with

a lipophilic group. Where on the molecule do you go to make the modification? The auxophoric groups that do not interfere with binding. Rational Drug Discovery - From Hit to Lead - Li, X.; Chu, S.; Feher, V. A.; Khalili, M.; Nie, Z.; Margosiak, S.; Nikulin, V.; Levin, J.; Sprankle, K. G.; Tedder, M. E.; Almassy, R.; Appelt, K.; Yager, K. M., "Structurebased design, synthesis, and antimicrobial activity of indazole-derived SAH/MTA nucleosidase inhibitors." J. Med. Chem. 2003, 46, 5663-5673. -Background: S-adenosyl homocysteine/methylthioadenosine (SAH/ MTA) nucleosidase presents a potentially useful target: -First, its active site is similarly highly conserved across bacterial species, while differing from that of the related mammalian proteins. -Second, SAH/MTA nucleosidase participates in the synthesis of the quorum sensing autoinducer AI-2, which in turn stimulates expression of virulence factors. Consequently, inhibiting SAH/MTA nucleosidase should attenuate bacterial virulence. -Third, and most importantly, such inhibition should kill bacteria because accumulation of SAH and MTA inhibits certain essential methyltransferase reactions and thereby impedes recycling of adenine and methionine, which are necessary for DNA and protein synthesis, respectively. Rational Drug Discovery - From Hit to Lead NH2 N N O R N MTA/SAH

Nucleosidase (MTAN) O R OH S OH OH S R= N OH NH2 N N H N N Adenine OH O2C NH3+

S-Adenosylhomocysteine (SAH) R = H3C Methylthioadenosine (MTA) R= O2C NH3+ S-Ribosylhomocysteine (SRH) R = H3C Methylthioribose (MTR) Rational Drug Discovery - From Hit to Lead -SAR: In Silico Lead Identification. Selection of compounds for screening in the SAH/MTA nucleosidase activity assay began by filtering a virtual library of 390 000 commercially available compounds to approximately 2000 satisfying the following criteria: (i) possessing three pharmacophoric features identified from inspection of homology modelsubstrate complexes, (ii) having molecular volumes less than that of the homology model binding pocket, and (iii) conforming to Lipinski guidelines (i.e., molecular weight < 500, -2.0 < ClogP < 5.0). The resulting data set was clustered for 2D fingerprint diversity and a representative selection of 1288 compounds identified using SELECTOR (Tripos, Inc.). X-ray structural determination of lead compounds cocrystallized with SAH/MTA nucleosidase derived from Escherichia coli and other pathogenic species revealed the mode of inhibitor binding within the active site. These co-structures provided the structural information for design of individual compounds and focused libraries. Use of 5-aminoindazole as the core scaffold provided a structure-guided series of low nanomolar inhibitors with broad-spectrum antimicrobial activity. The implementation of structure-based methodologies provided a 6000-fold increase in potency over a short timeline (several months) and an economy of synthesized compounds. The lead indazole (Ki=2.8

uM) bound in the active site of SAH/MTA nucleosidase as determined by X-ray crystallography. The solventaccessible surface of the active site (A) illustrates the general shape of the pocket. The hydrogen bonding pattern comprised of protein backbone and side-chain interactions are illustrated in (B, C). Inhibitor hydrophobic interactions with a cluster of phenylalanines and V102 are also illustrated; residues from the associated monomer are indicated by an asterisk (C). Bound water molecules are indicated as yellow spheres (A, B). -Synthetic work: H N N O2N Cl2 H N AcOH

O2N Cl H2NNH2 O O2N R = Cl R = Me R1 O2N SnCl2 N R NO2 HNO3 N H2SO4 R NH

O2N H N N R SnCl2 EtOH H2N acylations N S(NH4)2 EtOH NH R2 sulfonylations NH2 R R1 H

N N H2N EtOH R or: H2, Pd/C R = H, Cl, Me MeOH DMF H N O2N R = Cl R = Me H N H N O2N R1CHO N NaBH(OAc)3

R R1 H N N R R2SO2Cl O R1 5 O S R2 N H NH H N N R H N

N R -Synthetic work: NH R = Cl R = Me H2N H N 3-Ar-C6H4SO2Cl N pyridine R NH Ar NH Br O S

O N H H N ArB(OR3)2 N R Pd(PPh3)4 Na2CO3, DME O S O N H H N N R Rational Drug Discovery - From Hit to Lead -Conclusions: Structure-based design of a series of indazole-based S-adenosyl homocysteine/methylthioadenosine (SAH/ MTA) nucleosidase inhibitors led to the

discovery of a chlorinated indazole, a potent, low molecular weight inhibitor with nanomolar potency in an enzyme inhibition assay. In antimicrobial assays, this compound inhibits the growth of three important pathogenic species, showing MIC values of less than 10 uM. Cl Cl NH O S O N H H N N Cl Me Too Compounds Copying existing drugs with only minor chemical variations is usually referred to as me too research. Interestingly, sometimes these close analogs demonstrate major (usually unexpected) advantages, like the bioavailable, broad-spectrum lactamase-resistant penicillins, polar H1 antihistamins without sedative side effects, statins, or PDE5 inhibitors. Case Study: Use of a combined rational design - combinatorial chemistry strategy Gadek, T. R.; Burdick, D. J.; McDowell, R. S.; Stanley, M. S.;

Marsters Jr., J. C.; Paris, K. J.; Oare, D. A.; Reynolds, M. E.; Ladner, C.; Zioncheck, K. A.; Lee, W. P.; Gribling, P.; Dennis, M. S.; Skelton, N. J.; Tumas, D. B.; Clark, K. R.; Keating, S. M.; Beresini, M. H.; Tilley, J. W.; Presta, L. G.; Bodary, S. C., "Generation of an LFA-1 (leukocyte functional antigen1) antagonist by the transfer of the ICAM-1 (intercellular adhesion molecule1) immunoregulatory epitope to a small molecule." Science 2002, 295, 1086-1089. The interaction of LFA-1 with the ICAM proteins 1, 2, and 3 is critical to the adhesion, migration, and proliferation of lymphocytes. A disruption of these protein-protein interactions could lead to agents for the treatment of psoriasis and transplant rejection. An epitope comprising residues E34, K39, M64, Y66, N68, and Q73 within ICAM-1s first domain has been identified as essential for its interaction with LFA-1. The function of this epitope is embedded in the carboxylic acid, amine, sulfide, phenol, and carboxamide chemical functionalities of the amino acid side chains of these six residues and their display in three dimensions along one face of the protein. Molecules which mimic this epitope could capture the LFA-1 binding specificity and safety inherent in ICAM-1s function as a regulator of the immune system. More or less serendipitously, compound 1 was found to be an inhibitor of LFA-1. Comparison of the inhibition of ICAM-1/LFA-1 binding and the inhibition of mixed lymphocyte reaction (MLR). IC50 values were determined from a 4P fit of data from titrations over concentrations of 10 -3 to 10-10 M. Values reported are the meanstandard deviation for n>2 of experiments run in triplicate. ND, not

determined. NA, not applicable. Two orthogonal views of the superimposition of compound 4 on the crystal structure of the first domain of ICAM-1 indicating that compound 4 mimics the ICAM-1 epitope. Residues highlighted in blue contribute significantly to LFA-1 binding. The E34 side chain of ICAM-1 has been rotated to a low-energy conformation to enhance the overlay with compound 4. Conclusions: Compounds 2 through 4 appear to be mimics of ICAM-1 resulting from the transfer of the ICAM epitope to a small molecule. Compound 4 is a potent LFA-1 antagonist, which binds LFA-1, blocks the binding of ICAM-1, and inhibits LFA-1 mediated lymphocyte proliferation and adhesion in vitro. This work represents the first reduction of a nonlinear, discontinuous but contiguous protein epitope (encompassing five residues spanning three different -strands across the face of a protein surface) from a protein to a small molecule. Peptides to Peptidomimetics H N R O R NH O O

H O H N O N H H N Tyr82 O H N N H O to a he lix O Me O O Me O

H O H H N Me O O O H H N O H O O N H OH O H N O O

Tyr82 to a he lix O Evelyn Paul, "It Is Indeed the Cup" from: Claire de Lune and Other Troubadour Romances. London: G. G. Harrap & Co., Ltd., 1913 Peptides to Peptidomimetics Many substrates of enzymes, ie. angiotensinogen, fibrinogen, HIV protease, and receptors are either peptides or proteins, and the interaction of these ligands with their target is often mediated by only a few amino acid side chains; the rest of the protein stabilizes a certain 3D conformation. The RGD motif which interacts with different integrins in different conformations is a striking example. Peptides can be easily synthesized in large numbers, but the next step, the chemical conversion of such a peptide lead into a non-peptide mimetic is far from trivial. Achieving oral activity and bioavailability is even more challenging. The promiscuous benzodiazepines are examples of scaffold mimetics of peptide loops, but most other scaffolds described in the literature have not yet yielded similar successes. The comparison between enkephalins and morphine is often used as an example for peptide mimicry. However, this is obviously flawed because morphine was discovered first. Metabolic Stability of Biologically Active Peptides Enkephalin Analogs Sequence Se rum (Plasm a) Half-live [m in]

Tyr-Gly-Gly-Phe-Leu 7 (1.3) Tyr-Gly-Gly-Phe-Met (0.4, rat) Tyr-D-Ala-Gly-Phe-Leu 63 Tyr-y[CH2 S]-G ly-G ly-Phe -Le u 47 Tyr-G ly-y[CH2 S]-G ly-Phe -Le u 35 Tyr-G ly-G ly-y[CH2 S]-Phe -Le u 67 Tyr-G ly-G ly-Phe -y[CH2 S]-Le u 157 Tyr-D -Ala-G ly-y[CH2 S]-Phe -Le u >500

The prim ary de gradation route of sm allpe ptide s in se rum is y e xope ptidase activity, m ostly am inope ptidase (AP) and dipe ptidylam inope ptidase (D PAP) Source : Powe ll, M. F. Ann. R e p. Me d. Che m . 1993, 28 , 285. Preparation of a 27 nM Farnesyl Transferase Inhibitor based on Peptide Mimicry C - A - A - X HS H N H2N O HS O N H Ph H N O CO2H SMe Ph H N

H2N O CO2H SMe Yang, H.; Sheng, X. C.; Harrington, E. M.; Ackermann, K.; Garcia, A. M.; Lewis, M. D. J. Org. Chem. 1999, 64, 242. Amide Bond Replacements in Cholecystokinin Dipeptoids CCK-B R e ce por Binding Affinity: IC50 [nM] -CONH- 32 -CH2 NH2 - 1080 -CSNH- 808 -COO- 547 -COS- 932

HN 2-AdocHN -CON(Me )- 123 -HC=CH- 6340 -CH2 CH2 - 8690 -CH(OH)CH2 - 595 Ph Source: Fincham, C. I.; Higginbottom, M.; Hill, D. R.; Horwell, D. C.; O'Toole, J. C.; Ratcliffe, G. S.; Rees, D. C.; Roberts, E. J. Med. Chem. 1992, 35, 1472-84. angiotensinogen renin angiotensin II

angiotensin I IC50 5.7 nM Boc-Phe-His-Sta-Leu-NHCH2Ph raises blood pressure ..We hoped that [replacing the P3-P2 amide bond with 13 different isosteres] would lead to enhanced stability to enzymatic hydrolysis and longer acting, possibly orally active, renin inhibitors. Kaltenbronn, J. S. et al. J. Med. Chem. 1990, 33, 838-845. The two most potent isosteres had IC50 values of 61 and 22 nM aldosterone salt and water retention potassium loss Complete lack of response upon oral administration to high-renin monkeys! ..a rational mechanism for transforming [peptides] into non-peptide peptidomimetics has not been evident. Bursavich, M. G.; Rich, D. H. J. Med. Chem. 2002, 45, 541-558. Peptide mimicry by design used to be touted as a solution to [the limited bioavailability and oral activity of peptides] and was focused on impersonating secondary structure motifs, particularly turns, but this approach has yielded few pharmaceutical products. Beeley, N. R. A. Drug Disc. Today 2000, 5, 354-363. What About Drug Discovery in Academia? Forward Chemical Genetics = screening small molecules for their ability to perturb cellular

pathways, followed by identifying the specific targets of the active compounds. In general, whole cell assays are used. This approach may lead to the identification of new regulatory mediators of biochemical pathways and the validation of molecular targets for therapeutic intervention. It is used to systematize the discovery and use of small molecules as tools for biological research. Reverse Pharmacology enables identification of the molecular targets of efficacious agents with unknown mechanisms of action. Ref: Lokey, R. S., "Forward chemical genetics: Progress and obstacles on the path to a new pharmacopoeia." Current Opinion in Chemical Biology 2003, 7, 91-96. Forward chemical genetics involves 3 basic stages: a library of compounds an assay, usually a phenotypic assay a strategy to trace an active compound to its biological target Natural Products Diversity-Oriented Synthesis (DOS) Commercial Collections QuickTime and aGraphics decompressorare needed to see this picture. HITS A Phenotypic Screen Protein HITS Success Stories of Forward Chemical Genetics and Phenotypic Screens:

The study of the cellular effects of Colchicine was instrumental in the discovery of the cytoskeletal protein tubulin. O MeO NH MeO MeO O Colchicine OMe Similarly, The Hsp90 inhibitor geldanamycin, the Golgi disruptor brefeldin A and the the G2 checkpoint inhibitor isogranulatimide were initially identified by their phenotypic effects, prior to the identification of their molecular targets. In some respects, Chemical Genetics is equivalent to classical pharmacology on (technological) steroids. Success Stories of Forward Chemical Genetics and Phenotypic Assays from Chemical Library Screens: A 16,600-member library from Chembridge was used in a phenotypic assay for mitotic spindle disruptors, leading to the identification of the kinesin inhibitor monastrol ( Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R. W.; Schreiber, S. L.; Mitchison, T. J., "Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen." Science 1999, 286, 971-974). Phenotypic screens of commercial collections led to compounds that inhibit the Ras pathway, Sir2-mediated transcriptional silencing in yeast, alter the developmental program of zebrafish, and modulate TGF- signaling in mammalian cells.

Jiang, X., H.-E. Kim, et al. (2003). "Distinctive roles of PHAP proteins and prothymosin-a in a death regulatory pathway." Science 299(5604): 223-226. An example of an empirical forward chemical genetics search for pharmacological agents that induce apoptosis only in transformed cells. HTS led to the identification of a small molecule, PETCM, an Abbot compound, that activates caspase-3 in cell extracts. In elucidating the mechanism of action of PETCM, the oncoprotein prothymosin-a (Pro-T) and the tumor suppressor putative HLA-DRassociated proteins (PHAP) as important regulators of caspase-3 activation. These proteins appear to mediate distinct steps in the mitochondrial pathway: ProT blocks formation of the apoptosome, an event inhibited by PETCM. In contrast, PHAP appears to facilitate apoptosome-mediated caspase-9 activation. The apoptosome is a macromolecular complex that is formed in response to the cellular commitment to apoptodic death. The apoptosome is crucial to activation of caspase-9. Controlling the caspase cascade. Formation of the heptameric "apoptosome" is a crucial step in the initiation of apoptosis. Selective mitochondrial proteins such as cytochrome c (cyt c) (red) are released from the mitochondrial intermembrane space in response to apoptotic stimuli. Cytochrome c induces the assembly of Apaf-1 monomers (blue) into the apoptosome, which recruits and activates caspase-9 (C9), which in turn activates caspase-3 (C3) and -7 (C7). IAP proteins inhibit rogue caspases, but their inhibitory actions are blocked by antagonists (such as Smac/Diablo and Omi/HtrA2) that

are co-released with cytochrome c. Two additional tiers of apoptosome regulation have been identified: the oncoprotein proT, which retards assembly of the apoptosome complex, and tumor suppressor PHAP proteins, which stimulate the apoptosome's deadly activities This work nicely illustrates the opportunity of forward chemical genetics to identify novel players in a complex biochemical pathway. It also illustrates some potential pitfalls: more work is needed to definitively establish the in vivo significance of the PHAP finding. High (0.2 mM) concentrations of PETCM are necessary to stimulate caspase-3 activity and drive apoptosome formation. Under these high, physiologially not relevant concentrations, many other signaling processes might be partially influenced. PETCM, identified by HTS, is not a drug-like molecule, and therefore is of questionable value as a lead structure for drug discovery. Our Strategy for Integrating Organic Synthesis with Biology, Pharmacology and Drug Discovery Diversity-Oriented Synthesis Represents the synthesis of relatively small libraries of organic molecules that are structurally more complex, have a greater variety of core structures, and possess richer stereochemical variations than those produced by traditional combichem. DOS Strategies include: Tandem or Multi-Component Reactions in which complex polycyclic products are synthesized in a minimum of steps

Bi- and polyfurcating reaction pathways, giving access to different scaffolds depending on reaction conditions Biomimetic synthetic approaches for the preparation of complex polycyclic molecules C&E News, October 4, 2004, Volume 82, Number 40 pp. 32-40 Further reading: Lombardino, J. G.; Lowe, J. A., "A guide to drug discovery: The role of the medicinal chemist in drug discovery - then and now." Nat. Rev. Drug Disc. 2004, 3, 853-862. Jorgensen, W. L., "The many roles of computation in drug discovery." Science 2004, 303, 1813-1818. Gustafsson, D.; Bylund, R.; Antonsson, T.; Nilsson, I.; Nystroem, J.-E.; Eriksson, U.; Bredberg, U.; Teger-Nilsson, A.-C., "Case history: A new oral anticoagulant: The 50year challenge." Nat. Rev. Drug Disc. 2004, 3, 649-659.

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