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1、Medicinal Chemistry &Drug DiscoveryDr. Peter WipfDepartment of ChemistryUniversity of Pittsburghhttp:/ccc.chem.pitt.edu/wipf/index.htmlhttp:/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
2、 genetics - reverse pharmacologyMedicinal ChemistryThe 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 e
3、xcretion (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
4、&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 $
5、3.1/? billion (?%) Celebrex (Pfizer) arthritis $2.6/? billion (?%)Source: IMS Health, March 2004“Big Pharma” Drug Discovery in the 21st CenturyThe 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 ev
6、ery 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 $1-1.5 Bi. range. Considering all high-profile failures in recent drug discovery, this figure is unli
7、kely to drop substantially. Source: Wall Street J. 11/21/2003“gloom around the pharmaceuticals company.”Source: Wall Street J. 2/24/2004Wallstreet (and companies) are looking for a scapegoat.”Drug Industrys Big Push Into Technology Falls Short. Testing Machines Were Built To Streamline Research - Bu
8、t 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 rob
9、ots. 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 didnt produce useful results. And chemicals that seemed promising often tu
10、rned out to have big flaws that traditional testing might have caught earlier on. Some drugs couldnt dissolve in water or be turned into pills, for example.Critics believe these problems help explain the pharmaceutical industrys drought of new products. Thats the secret of why theyre spending billio
11、ns of dollars and getting nothing, says James Hussey, a former Bristol-Myers Squibb Co. manager who now leads biotech company NeoPharm Inc.”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, focus
12、ing 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 lipophi
13、lic, 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 structura
14、l modification will improve selectivity and potency.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. Why Is It So Difficult to Make Drugs?Drug Discovery ParadigmChemistry-drivendrug discovery -back to the fut
15、ure?Currently:Biology-drivendrug discoveryWhere Did (Do) Our Drugs Come From?New drugs from old poisonsThe reductionist approach to medicine began with the isolation of opium alkaloidsMedicinal Chemistry FolkloreEarliest medicines 5100 years agoChinese emperor Shen Nung - book of herbs, Pen TsaoChan
16、g Shan - contains alkaloids; used today in the treatment of malaria and for feversMa 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 thera
17、peutics: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 Ethnographies show that humans are great botanical experimentors Perhaps human brains, drugs and spices evolved togethe
18、r!Examples of Natural Products as Leads & DrugsCardiac glycosides, morphine, quinine, salicylic acid, taxol, camptothecin, penicillin, cyclosporin A, warfarin, artemisine.Current use of natural product extractsHigh-Pressure AcceleratedSolvent Extractor SystemDCM & MeOHExtractsParallel HPLCHT Solvent
19、 ConcentratorMultiwell PlatesFor HTSDrug DiscoveryOne way to “discover” drugsSerendipitous 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 vas
20、odilatory 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 str
21、ong 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. Phenolp
22、hthalein 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 LeadIn 1955 Roche set out to prepare a series of benzheptoxdiazines as pot
23、ential new tranquilizer drugs, but the actual structure was found to be that of a quinazoline 3-oxide.No active compounds were found, so the project was abandonedIn 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
24、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.LibriumRational Drug Discovery Nearly
25、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 mimi
26、c 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
27、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)186
28、8 - Crum-Brown and FraserExamined 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 constitutionStructurally s
29、pecific drugs (most drugs):Act at specific sites (receptor or enzyme)Activity/potency susceptible to small changes in structureStructurally nonspecific drugs:No specific site of actionSimilar activities with varied structures (various gaseous anesthetics, sedatives, antiseptics)Example of SARLead: s
30、ulfanilamide (R = H)Thousands of analogs synthesizedFrom clinical trials, various analogs shown to possess three different activities: Antimicrobial Diuretic AntidiabeticSARGeneral Structure of Antimicrobial AgentsR = SO2NHR, SO3H Groups must be para Must be NH2 (or converted to NH2 in vivo) Replace
31、ment of benzene ring or added substituents decreases or abolishes activity R can be , , , (but potency is reduced) R = SO2NR2 gives inactive compoundsSARAntidiabetic AgentsX = O, S, or NSARDiuretics (2 types)hydrochlorothiazidesR2 is an electrophilic grouphigh ceiling typeRational Drug Discovery - P
32、iroxicam 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 ac
33、id) 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:BioisosterismBi
34、oisosteres - 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 IsosteresDo not have the same number of atoms and do not fit steric and electronic r
35、ules of classical isosteres, but have similar biological activity.Non-Classical IsosteresExamples of Bioisosteric AnaloguesChanges resulting from bioisosteric replacements:Size, shape, electronic distribution, lipid solubility, water solubility, pKa, chemical reactivity, hydrogen bondingEffects of b
36、ioisosteric 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
37、allow you to tinker with whichever parameters are necessary to increase potency or reduce toxicity.Bioisosterism allows modification of physicochemical parametersMultiple alterations may be necessary:If a bioisosteric modification for receptor binding decreases lipophilicity, you may have to modify
38、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.; Lev
39、in, J.; Sprankle, K. G.; Tedder, M. E.; Almassy, R.; Appelt, K.; Yager, K. M., Structure-based 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) nuc
40、leosidase 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 tu
41、rn 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
42、 impedes recycling of adenine and methionine, which are necessary for DNA and protein synthesis, respectively.Rational Drug Discovery - From Hit to LeadRational Drug Discovery - From Hit to Lead-SAR: In Silico Lead Identification. “Selection of compounds for screening in the SAH/MTA nucleosidase act
43、ivity 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 model-substrate complexes, (ii) having molecular volumes less than
44、 that of the homology model binding pocket, and (iii) conforming to Lipinski guidelines (i.e., molecular weight 500, -2.0 ClogP 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 d
45、omain 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
46、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 n
47、onlinear, 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 Evelyn Paul, It Is Indeed the Cup from: Claire de Lune and Other Troubadour Romanc
48、es. London: G. G. Harrap & Co., Ltd., 1913Peptides 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 chain
49、s; 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-
50、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 b
51、etween 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 PeptidesYang, H.; Sheng, X. C.; Harrington, E. M.; Ackermann, K.; Garcia, A. M.; Lewis, M. D. J. Org. Ch
52、em. 1999, 64, 242.Preparation of a 27 nM Farnesyl Transferase Inhibitor based on Peptide MimicryAmide Bond Replacements in Cholecystokinin Dipeptoids“.a rational mechanism for transforming peptides into non-peptide peptidomimetics has not been evident.” Bursavich, M. G.; Rich, D. H. J. Med. Chem. 20
53、02, 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 b b-turns, but this approach has yielded few pharmaceutical products.” Beeley, N. R. A. Dr
54、ug Disc. Today 2000, 5, 354-363.“.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.reninangiotensin
55、 Iangiotensin IIraises blood pressurealdosteronesalt and water retentionpotassium lossangiotensinogenBoc-Phe-His-Sta-Leu-NHCH2PhIC50 5.7 nMThe two most potent isosteres had IC50 values of 61 and 22 nMComplete lack of response upon oral administration to high-renin monkeys!What About Drug Discovery i
56、n 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 bio
57、chemical 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 mechan
58、isms 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 trac
59、e an active compound to its biological targetNatural ProductsDiversity-OrientedSynthesis (DOS)Commercial CollectionsA Phenotypic ScreenHITSHITSProteinSuccess Stories of Forward Chemical Genetics and Phenotypic Screens: The study of the cellular effects of Colchicine was instrumental in the discovery
60、 of the cytoskeletal protein tubulin. 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, Che
61、mical 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
62、 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 commerc
63、ial 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
64、 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
65、extracts.In elucidating the mechanism of action of PETCM, the oncoprotein prothymosin-a (Pro-T) and the tumor suppressor putative HLA-DR-associated proteins (PHAP) as important regulators of caspase-3 activation. These proteins appear to mediate distinct steps in the mitochondrial pathway: ProT bloc
66、ks 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 o
67、f 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. Cytochr
68、ome 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)
69、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 apoptosomes deadly activitiesThis work nicely illustrates the op
70、portunity 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
71、 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
72、 discovery.Our Strategy for Integrating Organic Synthesis with Biology, Pharmacology and Drug DiscoveryDiversity-Oriented SynthesisRepresents the synthesis of relatively small libraries of organic molecules that are structurally more complex, have a greater variety of core structures, and possess ri
73、cher 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 o
74、n reaction conditions Biomimetic synthetic approaches for the preparation of complex polycyclic moleculesC&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
75、 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 50-year challenge. Nat. Rev. Drug Disc. 2004, 3, 649-659.
