2024 DTDD Final Study Guide PDF

Core Concept 1: History of pharmacology and pharmaceutics provides a basis for modern drug discovery and design ‘Drugs do not act unless they bind’-Ehrlich knowledge of pharmacophores provides a platform for drug design knowledge of SAR provides a platform for drug modification *pharmacophore is the...

Core Concept 1: History of pharmacology and pharmaceutics provides a basis for modern drug discovery and design ‘Drugs do not act unless they bind’-Ehrlich knowledge of pharmacophores provides a platform for drug design knowledge of SAR provides a platform for drug modification *pharmacophore is the portion of the drug that binds to the receptor and causes drug action Economics of Drug Development National health expenditure* (NHE), U.S., 2022: ~$4.5 trillion (~17.3 % GDP; ~$13,554 per each of the ~332 million people in U.S.) This means 1 in 5 dollars spent in U.S. is spent on health care. Note that this is holding fairly steady as a percentage of GDP (but 2020-2022 aberrant due to COVID -although capacity can cap expenditures!) U.S. retail drug sales ($535 billion in 2020) constitute about 50% of the world’s retail drug sale *total health expenditure is the consumption of health goods and services plus capital investment in health care infrastructure Ask each other questions, and if you can’t answer them-let me know! Global Drug Expenditures Numbers somewhat difficult to obtain on yearly basis as record keeping is not uniform across the planet. A reasonable guess for 2020 would be just over $1 trillion, suggesting that U.S. retail drug sales ($535 billion in 2020) constitute about 50% of the world’s retail drug sales. Against that background, total global pharmaceutical research investment would be around 14% of the retail gross for international sales. -CMS.gov 2020 Planetary Drug Sales, Top Ten statista.com Retail, 2017; $397 Billion Abilify (depression) (approx. 1.8%) Humira (arthritis) (approx. 1.6%) Nexium (acid reflux) (approx. 1.6%) Crestor (anti-cholesterol) (approx. 1.4%) Enbrel (arthritis & plaque psoriasis) (approx. 1.2%) Advair Diskus (GSK; indication asthma) (approx. 1.2%) kff.org Top Six Drugs, Non-retail (2017; $Billion) Humira (adalimumab; AbbVie; indication arthritis) (18—#1; as last year) Rituxan (rituxamab; MabThera; lymphoma and RA) (9.2--#2; was #4 last year) Revlimid (lenalidomide; Celgene; indication multiple myeloma) (8.2--#3, was #6 last year) Enbrel (etanercept; Amgen and Pfizer; rheumatoid arthritis) (7.9--#4; was #3 last year) Herceptin (trastuzumab; HER2-positive metastatic breast cancer) (7.4--#5; new to top six) Eliquis (apixaban; anticoagulant) (7.4--#6; new to top six) statista.com What makes a blockbuster drug? Broad base of patients Treatment is chronic Patent protection is robust http://www.drugs.com/top200.html Cardiac Glycosides Foxglove was introduced for therapy of dropsy (CHF) in a treatise by William Withering in 1785. Stophanthus was introduced into therapy in 1890 by Sir Thomas Fraser -discovered its digitalis-like action while studying arrow tip poisons. The sea onion, or squill, has been used in medicine since 1500 B.C. For centuries the Chinese have employed a Galenical preparation from toads (Ch’an-su) for dropsy. The related glycosides (cardenolides) are also potent inhibitors of (Na+/K+)ATPase. Cardiac glycosides consist of a steroid nucleus containing the drug pharmacophore connected to unique sugars (hence the name glycoside). The glycoside moiety is not part of the pharmacophore, but is important in drug pharmacokinetics and pharmacodynamics. The cardiac glycosides act to increase intracellular calcium in cardiac myocytes. Increased force of contraction of the heart results. Compounds active at voltage-sensitive calcium channels are useful for arrhythmia. History of Pharmacology--Antiquity 1552 B. C. The Ebers Papyrus--700 prescriptions/anthelmintics History of Pharmacology--Greco-Roman 400 B.C. Hippocrates; Father of medicine; ~400 ‘simples’; diagnosis, prognosis and animal dissection 130 A.D. Galen--galenical, descriptions of herbal mixtures Last of the great Alexandrians Strong believer in the therapeutic value of wine History of Pharmacology--Arabic Period Geber (b. 721, Abu Musa Jabir ibn Hayyan; ‘father of Arab chemistry’ -cannabis. Systematic experimentation in alchemy; crystallization. Rhazes (aka Ibn Zakariya, al-Razi; 841 A.D.; ‘the Islamic Hippocrates’). Discoverer of ethanol; recommended testing of drugs on animals; opium for sedation History of Pharmacology--Medieval Medieval therapeutics accompanied by recitation of prayers, often to regulate duration of treatments with poultices, etc. Nicholas of Salerno--grain, scruple, drachm system of weights; Antidotes History of Pharmacology--Renaissance Printing press assures wide dissemination and high-fidelity replication of formularies of Valerius Cordus and others. History of Pharmacology--Enlightenment 17th was the century of famous pharmacopoeias (e.g. London Pharmacopoeia of de Mayherne incorporated Paracelsan Reforms) Lemonade as anti-scurvy medication pass to Lemonadiers in 1676. History of Pharmacology--19th & 20th Centuries Isolation of natural products, 1800’s (morphine, quinine, strychine, emetine, atropine, codeine, nicotine)—the alkaloids that could be purified in a similar way, came from plants, formed the backbone of the modern drug armamentarium Paul Ehrlich (1909) “Corpora non agunt nisi fixata” (Drugs do not act unless they bind) Structures of norepinephrine, acetylcholine, prostaglandins Molecular Pharmacology “A drug is a substance of well-defined chemical structure used for therapeutical purposes”—modern definition of a drug --Koralkovas, Essentials of Molecular Pharmacology, 1970 Molecular pharmacology creates a common platform for integrating chemistry, physiology, molecular biology, biotechnology and biocomputation into drug discovery and design Mechanism of action of warfarin as an anti-coagulant: *By inhibiting Vitamin-K epoxide reductase, warfarin limits the amount of reduced Vitamin K available to help catalyze the gammacarboxylation of clotting factors. *Gamma-carboxylation of clotting factors is required for their activation. *Activation of clotting factors is required for their ability to convert prothrombin to thrombin. *Activated thrombin is responsible for conversion of fibrinogen to fibrin. *Conversion of fibrinogen to fibrin causes the blot clot to form. **So warfarin ultimately inhibits conversion of fibrinogen to fibrin, and acts as an anticoagulant (anti-clotting agent). Eliquis works via inhibition of Xa: same target as heparin, but different mechanism Prothrombin (II) Complex of Xa Thrombin (IIa) M 72,000 MW 36,000 Va, Ca ++, Platelet surface (inactive for clot formation) (catalyzes clot formation) Why is Eliquis a good substitute for warfarin? Because it has a higher TI. Drug Discovery by Serendipity (Luck) 1928-Alexander Fleming, St.Mary’s Hospital in London, noticed lysis of staph colonies by mold. Named factor penicillin for Penicillium notatum Observation unnoticed until 1939-by H.W. Florey at Oxford University. 1941- Therapeutic trials using crude culture broth. Bedpans used as culture vessels. Penicillin recovered from the urine of treated patients to treat other patients. First patient treated was an Oxford police officer. Large-scale production fueled in US as war effort. 1942-122 million units produced. By 1949 production at 800 billion units/month. By 1950, 222 trillion units (148 tons). Drug Discovery by Screening AMINOGLYCOSIDE ANTIOBIOTICS Selman Waksman of the New Jersey Agricultural Experimental Station discovered Streptomycin generated by Streptomyces griseus in 1943 following an extensive soil sample screening program. He received the Nobel Prize for that work. Readings #1a and #1b: ISRIB as an anti-Alzheimer’s drug: BACKGROUND Cell stressàeIF2a is phosphorylatedàmRNA translation to protein inhibited; brain eIF2a phosphorylation correlates with AD. What is ISRB? Where does it act? Experts estimate the cost of treating dementia could jump to $1 trillion in just three years and called for governments to adopt legislation to ensure better treatment for people with the disease. There is no known cure for dementia. Source: Associated Press Core Concept 2: Differential toxicity as a basis for drug design Toxicity and Side Effects Drugs can cause toxicity due directly to their mechanisms of action (intrinsic), or due to side effects unrelated to mechanisms of action (extrinsic). Toxicity is a ‘negative effect’, side effects are ‘neutral’ or ‘negative’ and sometimes even ‘positive’ Sildenafil DRUG EFFECTS DESIRABLE (therapeutic) Treat hypertension UNDESIRABLE Treat erectile dysfunction NON-DELETERIOUS (side effects) Treat erectile dysfunction Pharmacological DELETERIOUS (toxic effects) Pathological Genotoxic Therapeutic Index The ratio between the toxic dose (TD50) and the therapeutically effective dose (ED50) of a drug determines its safety. This ratio is called the therapeutic index (T.I.). The higher the T.I., the safer the drug. A drug with a low T.I. is said to have a narrow therapeutic window, and may require close monitoring of drug levels in the patient in order to be used at all. The T.I. for one therapeutic application of a drug (e.g. cough suppression by codeine) could be different than the TI for a different therapeutic application of a drug (e.g. pain relief by codeine) Cancer Chemotherapeutics Standard cancer chemotherapeutic compounds rely on differential toxicity* towards normal, even rapidly proliferating, and transformed cells. *Paul Ehrlich’s legacy to drug discovery! Antimicrobials *Penicillins/cephalosporins inhibit bacterial cell wall synthesis (differential bug toxicity) *Tetracyclines and erythrocin bind 30S or 50S ribosomal subunits and inhibit protein synthesis *Aminoglycosides bind 30S ribosomal subunit and alter protein synthesis *Rifamycins inhibit RNA polymerasae *Antimetabolites inhibit essential enzymes of folate metabolism. There is no currently effective antibiotic for vancomycin-resistant Staphylococcus Aureus—the Superbug. The phosphotransferase system (PTS) of bacteria is now a target for novel antibiotics because resistance is less likely to develop to antibiotics targeted to this system. Superbugs-our latest antibacterial challenge Bacteria that are resistant to multiple antibiotics are categorized as ‘multi-drug resistant’ or ‘superbugs’. VRSA (vancomycin-resistant Staph. Aureus) and MRSA (methacillin-resistant Staph. aureus) are two common Staph superbugs. Most are hospital acquired. Superdrugs for superbugs are a vast new market. Future of antibiotics: AMPs Antimicrobial peptides (AMPs) are both new and old antibiotics. Old because they are derived from organisms as their endogenous defense against infection. New, because they are just now being mobilized for clinical use. Magainin-developed by Georgetown University Professor Michael Zasloff while at NIH. Derived from frog skin. Amphipathic helical, acts at cell membrane. GIGKFLHSAKKFGKAFVGEIMNS Failed FDA approval-perhaps because antibiotic resistance is not yet an existential issue... Future of antibiotics: PTS PTS-phosphoenolpyruvate-dependent phosphotransferase system. Used at bacterial cell membrane to transport sugars. Xanthone inhibit it, and therefore may be future antibiotics. Peptic Ulcer: Two Treatment Approaches 1. Peptide ulcer due to excessive proton secretion from parietal cells in stomach. Proton pump inhibitors (Nexium, Prilosec) block acid secretion; provide symptomatic relief. 2. Discovery of Helicobacter pylori by Barry Marshall (Nobel Prize in Physiology or Medicine, 2005). These bacteria infect the stomach and cause acid hypersecretion. Peptic ulcer could thus be treated with antibiotics (tetracycline). H. pylori, courtesy of Marshall website, www.helico.com A Brief Word on How Drugs are Named… Generic-refers to the structure of the drug-one name, one chemical entity. Trade or brand name-refers to the particular formulation by a particular company. Note-generic drugs with closely-related structures can have different trade/brand names but are essentially identical pharmacologically. ‘informative suffixes’ include -mab (monoclonal antibody), -statin (inhibitor), -an (antagonist), etc. Trade/Generic examples (cont.) Zoloft/sertraline Prozac/fluoxetine Celexa/citalopram Luvox/fluvoxamine Serotonin (5-HT) Trade name Capitalized; generic name lower case Currently used antidepressants are biogenic amine re-uptake inhibitors Trade/Generic examples (cont.) Zoloft/sertraline Prozac/fluoxetine Celexa/citalopram Luvox/fluvoxamine Serotonin (5-HT) What happened to Cymbalta? Was #5 retail drug in 2013; used for treatment of anxiety and depression Duloxetine, generic equivalent, is now available Trade name Generic name duloxetine duloxetine duloxetine Cymbalta Ariclaim Xeristar Structure of duloxetine, an SNRI A note on drug re-purposing: Thalidomide Thalidomide: a tragic example of teratological differential toxicity to mother and infant. Thalidomide (Thalomid) was first introduced as a drug for treatment of nausea during pregnancy. Drug targets in senescing cells Core Concept 3: Drug Receptors With few exceptions (such as antacids, purgatives and colligatives) drugs bind at specific receptors, with a reasonable affinity, to exert a pharmacological effect. Drug receptors are often receptors for endogenous compounds whose actions are mimicked or blocked by the drug. However the term ‘drug receptor’ is more general than the term ‘biological receptor’. All biological receptors are potentially drug receptors; not all drug receptors are biological receptors (e.g., some are enzymes). Five major classes of biological receptors Receptor tyrosine kinases (RTKs) are localized at the plasma membrane Nuclear/Lipophilic hormone receptors (NRs) are localized in the nucleus Ionotropic receptors (inc. Ca2+, Na+, nicotinic receptors-see ‘channelopathies’) are localized at the plasma membrane Guanylate cyclase (NO receptor) is localized in the cytoplasms G-protein coupled receptor (GPCRs) are localized at the plasma membrane Drugs can be agonists or antagonists Now consider effects when endogenous ligand is present at receptor… General Feature of Receptors **Most are proteins (nucleic acids can also be receptors, particularly for anti-cancer drugs) only gunalyate cyclase is an enzyme that is abiological receptor **Many are enzymes (NONE OF WHICH ARE BIOLOGICAL RECEPTORS) **Drug can block the action of receptor ligands (antagonists), mimick the action of receptor ligands (agonists), weakly activate receptors (partial agonists), or stabilize receptors in an inactive conformation (inverse agonists) A Few Well-known Drugs & Their Receptors (which are also biological receptors) LSD--5-HT2 receptor Morphine--µ opioid receptor (first, and only widely known, ‘druglike’ neuropeptide receptor ligand) THC—CB1 (CNS) cannabinoid receptor (several emerging medical uses, e.g. SR141716A, aka rimonobant) A Few Well-known Drugs & Their Receptors (which are not biological receptors) Aspirin—receptors are cyclooxygenases 1 and 2 (COX-1, COX-2) Reserpine/tetrabenazine—receptors are VMAT1 and VMAT2/VMAT2 Note: deutetrabenazine (Austedo) now used to treat involuntary movements in Huntington’s disease!) Immunosuppressants: Cyclosporin A—cyclophilin Rapamycin—TOR (target of rapamycin) FK506--FKBP12 Mechanisms of action--initially thought to be related to proline isomerase activity, now linked to inhibition of the phosphatase calcineurin (CsA, FK506) or various intracellular kinases (TOR) There are no known endogenous ligands for cyclophilin, TOR or FKBP12! ! Drug Receptors Biological Receptors GPCRs as Drug Targets *35-45% of marketed drugs target GPCRs *Human genome has 750 putative GPCRs *Ligands: 50% visual and olfactory; 7% proteins; 7% biogenic amines; 8% peptides; 7% viral, lipids, nucleotides, calcium, viral; 20% orphans G-protein activation is receptor- and ligand-specific Receptor Tyrosine Kinases (RTKs) Most growth factors use RTKs as their receptors Most if not all RTK ligands are dimers, and act to dimerize the RTK Protein Tyrosine Kinase Receptor Action Is Terminated by PTPs, or Protein Tyrosine Phosphatases Phospho-tyrosines Lipophilic hormone receptors PPARs: Lipid Receptors and New Drug Targets PPARs activate transcription of specific target genes What are peroxisome proliferators? Peroxisomes are intracellular organelles found in large numbers in hepatocytes (liver cells). They contain enzymes, like catalase, that help metabolism hydrogen peroxide, as well as H2O2generating oxidases, and a complete fatty acid beta-oxidation cycle. So they are important for cholesterol metabolism, drug and xenobiotic metabolism, lipid metabolism. Peroxisome proliferators increase peroxisome number in liver cells. They can be carcinogenic. The most potent are hypolipidemic agents like clofibrate, ciprofibrate, Wy-14,643. Peroxisome proliferator-activated receptors bind peroxisome proliferators, which control the transcription of cohorts of genes involved in lipid metabolism, etc. Glitazone—a thiazolidinedione antidiabetic drug and PPARgamma ligand Clofibrate—a fibrate triglyeride and LDL-lowering drug and PPARalpha ligand *Both ‘classic’ drugs in wide use prior to identification of their receptors *More on the ‘offspring’ of these prototype drugs when we discuss Target Validation Guanylate cyclase—the NO receptor Unlike the other four types of biological receptors—RTKs, LHRs, Channels, and GPCRs, which all constitute fairly large gene families, guanylate cyclase is a receptor for just one biological ligand. GC has two isoforms, and is heterodimeric. There are other guanylyl cyclases, but only one that is NO-sensitive. NO activates GC by binding to its heme prosthetic group, which increases enzyme activity some 200-fold. (or do you say guanylyl cyclase—you should!) Nitric Oxide and Vasodilatation GC = Guanylate cyclase cGMPàMLCKàinhibition of calcium uptakeàdecreased contractilityàvasodilatation Exploiting PDEs to Develop Drugs as Cognitive Enhancers PDE5 inhibitors improve object recognition memory in rats: *Is PDE5 the only PDE in the hippocampus? *Do other brain areas require cGMP for other types of cognition? *Is cGMP requirement for memory function conserved in other species (e.g. primates?) *Is PDE5 the major enzyme for degradation of cGMP in other species (e.g. primates?) Steinbusch et al.: Animal Model for Cognition The Ionotropic Receptors Ionotropic receptors include calcium and sodium channels, and ionotropic (versus metabotropic) receptors that are operated by the neurotransmitters acetylcholine, glutamate and GABA Ionotropic receptors are well-established drug targets (they are BOTH ’biological’ and ‘drug’ receptors) Actions of drugs at neurotransmitter-operated channels (ionotropic receptors) are often ALLOSTERIC (i.e. the pharmacophoric site is not the same as the site at which the neurotransmitter binds to the receptor) Ionotropic and Metabotropic Acetylcholine Receptors (“own the surrounding biology”) O Na+ Ca++ Nicotinic Receptor O N + ACETYLCHOLINE G Muscarinic Receptor Anti-arrythmic drugs act at ion channels GPCRs as Drug Targets *About 35% of marketed drugs target GPCRs *Human genome has 750 putative GPCRs *Ligands: 50% visual and olfactory; 7% proteins; 7% biogenic amines; 8% peptides; 7% viral, lipids, nucleotides, calcium, viral; 20% orphans G-proteins are ‘clocks’ for first-messenger signaling G-protein activation is receptor- and ligand-specific G protein coupled receptors and RAMPs (Receptor Activity Modifying Proteins) G protein coupled receptors and RAMPs CTR-calcitonin receptor (continued) CLR-calcitonin-like receptor AM-adrenomedullin CT-calcitonin ! D. Hay, Br. J. Pharmacol. 175: 3-17, 2018 Rule of Five In the discovery setting ‘the rule of 5’ predicts that poor absorption or permeation is more likely when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight (MWT) is greater than 500 and the calculated Log P (CLogP) is greater than 5. Lipinski et al. Adv. Drug Delivery Reviews 23: 3-25, 1997 ! Kaspar, Drug Discovery, 2013 Reading #4: Alternative drug discovery approaches for orphan GPCRs Reading #5: Ion Channels as Drug Targets: The Next GPCRs Reading #6 ! Bruzzoni-Giovanelli et al., Interfering peptides targeting protein-protein interactions: the next generation of drugs? Drug Discovery today 23: Feb. 2018 Bruzzoni-Giovanelli et al., Interfering peptides targeting protein-protein interactions: the next generation of drugs? Drug Discovery today 23: Feb. 2018 Core Concept 4: SAR Definition— Structure-activity relationships (SAR) are obtained by first choosing a prototype drug that causes a biological effect that can be easily measured and is a surrogate for drug action. The molecular structure of the prototype is then modified systematically with addition and subtraction of substituents (“Rgroups”) at various positions, and steric alterations, in order to draw conclusions about drug-receptor interactions and define the drug pharmacophore. SAR—Cholinergics (nicotinic) Nicotinic Cholinergic Receptor O (CH3)3N-CH2-CH2-O-C-CH3 = + 5Å + + SAR—Cholinergics (muscarinic) muscarine Cardiac Glycosides The following represents the minimal structural requirements for inotropic activity of a cardiac glycoside. O O H 17b-butenolide CH3 cis-A/B ring juncture H HO H cis C/D ring juncture OH 14b-OH H Digitoxigenin 3b-OH MORPHINE 17N 17 14-OH 3,6-diacetyl=heroin 3-methoxy=codeine 6-oxy, N17-allyl= naloxone 3-methoxy, 6-oxy, 14-hydroxy=oxycodone 10 1 2 3 HO 3-OH 9 CH3 N D-7,8 16 11 8 14 12 13 4 O 15 5 6 7 OH 6-OH if you sub the 17-n for a larger group, you make a n antagonist of e mu opiae receptor )e.g., naloxone) ! N-terminus ICL1 ECL1 ECL2 ICL2 ECL3 ICL3 C-terminal tail Swapping domains among three types of opiate receptors allows the region of the receptor that selectively binds opiate ligands to be identified. In the opiate receptor family, this domain is within the third extracellular loop (EL-III) of the receptor. the EC parts are for receptor action, while ICL domains are for signalling Opiate receptors as drug targets (cont.) Selectivity of opioid ligands viewed in terms of ‘message/address’. Several selective antagonists derived from common pharmacophore: d-selective antagonist naltrindole (NTI) k-selective antagonists norbinaltorphimine (norBNI) and 5’-guanidinonaltrindole (GNTI) derived from ‘universal antagonist’ naltrexone. Next step: biased ligands-activate only one of the several downstream signaling pathways normally activated by ligand occupancy of GPCRs (e.g. mu receptor via Gproteinsèanalgesia and euphoria, via arrestinsèconstipation, tolerance and respiratory depression. SAR Within the GPCR Third intracellular loop and C-terminal tail a locus for second-messenger coupling and signal propagation Drugs can be targeted at this region of the receptor Drug action can be highly specific GPCR-based pepducins as potential drugs Covic, Lidija et al. (2002) Proc. Natl. Acad. Sci. USA 99, 643-648 Copyright ©2002 by the National Academy of Sciences http://www.trevenainc.com/technology.php ! Cast of characters: Receptor—AT1R Endogenous ligand-angiotensin II Unbiased antagonist-Valsartan (an ARB) Unbiased antagonist-Losartan (an ARB) Biased ligand-TRV120027 IP-One assay Beta-arrestin assay AIIàATR1àGqàMAP TRV blocks AII Gq effects AIIàATR1àbArràdVmax TRV mimics AII bArr effects Violin et al., JPET 335: 572, 2010 ! analgesia cant be occuring thru the b arrestin apathway, bc if you take that pathway you get even more analgesia bc you're not desensitzing the receptor, the analgesisa must be mediated thru Gi Bohn et al., Science 286: 2495, 1999 Chemical basis of pharmacology— ‘Moving on’ from molecular biology-driven drug discovery Reading #7 Chemical basis of pharmacology Reading #7 Specific receptor-classifying molecules can lead to new drugs Core Concept 5: Drug Screening Drug screening is carried out at various levels of specificity, and is scaled appropriately to each level. Drug screening is downstream of target validation. Drug screening generally requires a surrogate read-out for ultimate therapeutic action. Examples: *soil screens for antibiotics *receptor binding screens for antipsychotics High-Throughput Screening (HTS) ! made possible by advances in engineering (robotics and microfluidics), physics (fluorescence), chemistry (combinatorial library fabrication), molecular biology (target cloning) contributing to both structure-based rational drug design and to traditional random screening approaches t Drug screening with combinatorial libraries & high-throughput assays Encoded Combinatorial Libraries on Polymeric Support O N N O O Compound linker Bead Tag linker N ® Douglas Auld Pharmacopeia 2002 Displacement of the binding of a fluorescent ligand by novel compounds being tested as drugs Note that this assay requires single labeled ligand, and both agonists and antagonists can be identified in assay. Principles of Scanning Image Analysis Zuck et al., PNAS 96:11122, 1999 The Corning 1536 well wafer plate 1,536 samples in 96-well plates S = 154 mL 1,536 samples in 1536-well plates S = 1.5 mL Retroviral"cAMP"sensor"genome" LTR" Ψ N"terminal" luciferase"3592544" LETTER RIIβB" C"terminal" luciferase"42355" Hygror& LTR" HEK"cAMP"Sensor""Cells" uciferase a b !Luc) was created by Open Closed ing the coding reN Ψ Open Covalent C N LTR LTR PROMOTER GPCR V5 blasticidin C amino acids 4−233 C N 234−544 44 ahead of the recoding 4!233, mino acids 4 and C connected by a Noncovalent HEK"cAMP"Sensor""Cells" N tide linker (Figure 1, +"single"copy"GPCR" 4−547 FKBP C N FRB. Several protease Closed Analyte&binding& re separately incorC C N N into this polypeptide C N igure 2, panel a), Allosteric C N C genes were exRIIβB 4−355 C N 359−544 in cell-free translactions. The translaN ducts yielded very minescence, several Figure 1. Design strategies for biosensors using firefly luciferase. a) Molecular models of firefly luciferase in the d-fold below the pa- absence of substrates (open conformation) or bound to an analog of the luciferyl-adenylate reaction intermediate (closed conformation, analog colored yellow) generated using PDB files 1LCI and 2D1S, respectively. The ciferase expressed quivalent conditions smaller C-terminal domain of luciferase (residues 441!544) is predicted to rotate and translocate toward the 2, panel b). However, larger N-terminal domain during the catalytic cycle. Residues 4!233 and 234!544 are colored blue and gray, respectively, for the “open” conformation. Residues 4!355 and 359!544 are colored blue and gray, respeccence activity intively, for the “closed” conformation. Nonvisible residues at the N- and C-termini of PDB file 1LCI were typically between 185- and excluded from the various biosensor design strategies. b) Schematic representation of the three design strateld following protease gies used to generate luciferase biosensors. Covalent. Fusion of the wild-type N- and C-termini with a polypeptide containing a protease cleavage site inhibits formation of the closed conformation. Cleavage by protease rent, raising the light lieves this constraint, allowing increased luminescence. Noncovalent. Association of polypeptides FRB and y to 3!16% of the FKBP12 in the presence of rapamycin inhibits formation of the closed conformation, causing decreased luminesluciferase. cence. Allosteric. The conformational change of an analyte binding domain modulates luminescence, e.g., cAMP rther characterized binding to RII"B promotes increased luminescence. y of these protease ors by evaluating a CP234!Luc con- sensitive than was detection by SDS-PAGE present on both sides of the scissile peptide bond; and they provide the sensitivity ontaining a caspase 3 cleavage site (Supplementary Figure 1). To optimize the and large dynamic range characteristic of lu!Luc/DEVDG). After cell-free expres- response of this sensor, the length of the polypeptide linker containing the DEVDG minescent assays. Assays using these send fluorescent labeling, the time decleavage sequence was systematically varsors can be readily employed to quantify ce of protease cleavage was moniied (Supplementary Figure 2). Longer linkers protease activity, analyze substrate specificboth luminescence activity and Construction of cAMP indicator clonal cell lines for high-throughput GPCR screening. A retroviral vector containing a cAMP sensor genome comprising a permuted circular luciferase gene and a cAMP binding domain derived from the regulatory subunit of PKA was used to transduce HEK cells. Hygromycin resistant single copy clones derived from the transduced target cells aretransduced by retroviral vectors expressing any GPCR of interest with a C-terminal epitope tag (V5). GPCR-expressing cAMP sensor are analyzed for the ability of test compounds (including the full spectrum of orthosteric and allosteric agonists, antagonists, and modulators) to mediate luciferase activity in living cells. Criteria for selection of CNS-relevant putatively Gi/Gs-coupled orphan GPCRs for translational focusing by ligand screening. Receptor CNS/non-CNS EV Ratio NS-1/non-CNS EV Ratio Disease Involvement Pred-couple scores Gs GPR3 GPR12 GPR50 GPR63 GPR83 0.95 1.83 -0.08 1.11 0.93 -0.12 Alzheimer’s Disease -0.03 Cognitive disorders 1.32 Mood disorders 0.98 Addiction -0.39 MS 0.90 0.97 0.92 0.99 0.02 Gi/o 0.79 0.52 0.83 0.51 0.99 Gq/11 G12/13 0.15 0.00 0.10 0.13 0.02 0.05 0.00 0.04 0.05 0.00 ! The Pipeline Substances emerging from initial screening are rarely powerful enough to be effective as they stand. The next step…is for chemists to fiddle with the exact arrangement of a promising compound’s atoms…to increase its potency [or decrease its toxicity, or both]. The “lead compound” which results from this tinkering is then subjected to further tests [to] show how well it is absorbed by the body, how it stand up to the biochemical rigours it meets there, how poisonous it is, and what sort of side effects it might… produce. Only then is it allowed to go into clinical trails in people--first...to test its safety, and then…to prove its effectiveness for its intended job. If, after going through all this, the company thinks that it has a winner, it [must] persuade the regulatory authorities to agree. Only when a molecule has passed this final test does it pop out of the other end of the pipeline and on to the revenue line of the company’s accounts. -- The Economist, Feb 21, 1998, pp.1-8, cited in M. J. Plotkin, Medicine Quest, 2000. Drug targeting/design within the drug development pipeline Typically, drugs are designed based on target validation and proof of concept, high-throughput screening identifies multiple candidates (hits), these are further winnowed (leads) and ADME/T.I. considerations are addressed (development). Proposals for future research include integration of pharmacokinetic/dynamic/toxicological considerations directly into drug design. Drug pipelines have bottlenecks In drug design, target identification, production. Bottlenecks can change depending on: -->changes in rate-limiting steps -->re-design of the pipeline Main impact on pipeline last 10 years: HTS Rate of drug production can be increased by identifying local/global bottlenecks and/or attempting to alter the overall shape of the pipeline Summary: Cheminformatics The impact of computing on drug discovery is limited by cost of software development – Opportunities for new approaches and pooling of resources Major increases in R&D throughput achieved for lead identification (HTS) – Informatics challenge is mining the wealth of data: 200 MM datapoints per year Diversity is major success factor for both lead identification and lead optimization – Redesign of compound collections is a necessary strategy but not a complete solution – Recent computational methods are breakthrough technologies but dependent on crystallization of target protein – Structure clustering of compounds allows identification of hit clusters’ vs compounds First time in vivo principle designed to extract maximum value from most expensive experiments – 10x faster gene expression profiling would revolutionize biology – Description of biochemical pathways is most important problem in modern genomics Ultimate goal for cheminformatics is genome-wide pharmacological selectivity predictions – Activity profiling still expensive and low resolution – Computational binding site maps depends on protein crystallography Virtual Drug Screening: An Example Virtual Drug Screening: Filtering Virtual Drug Screening: Structural Rationale Reading #8: Takasaki et al., 2018 Virtual Drug Screening: The Compounds Reading #8: Takasaki et al., 2018 Virtual Drug Screening: The Results Reading #8: Takasaki et al., 2018 Core Concept 6: Systems-based targets, pharmacophores, and drug design Target discovery and drug design within large target classes--the kinome as a systems target. Systems-Based Research/Development Start with a target family, not disease Allows knowledge transfer from one member of the class to other targets of interest Therefore more efficient - not a fresh start with each new target Build and use a knowledge base Gene Families That Form Major Drug Target Classes Integrins 7TM Receptors Informatics Biology Chemistry Nuclear Receptors Proteases MEDICINES Ion Channels Kinases Homologs of Yeast STE7,11,20 Casein kinase 1 PKA, PKG,PKC families Tyrosine Kinase-like Calcium/Calmodulindependent kinases Tyrosine Kinase Receptor Guanylate Cyclase (inactive ePKs) Contains CDK, MAPK, GSK3, CLK The Kinome & High-throughput Drug Discovery and SAR of TKIs Protein Kinases as Targets Most common post-translational modification. Regulates: activity, location, degradation, conformation………. Many implicated in disease - especially cancer, inflammation….. Kinase Targets: Linking Structure and Chemistry all ser/thr/tyr kinases share the same 3 dimensional fold ATP binding site is conserved i.e.. the catalytic residues and their positions within the active site are conserved How it Works A number of kinases are available whose structure can be readily solved with different ligands solve the same template with many substitutions - lead optimization solve the same template in many kinases selectivity, binding modes Why it Works ATP site - conserved but not optimized for ATP large database of structures allows for: greater understanding of key pharmacophores and SAR improved homology models novel template design Exploit unique features of ATP site to achieve potency and selectivity New scaffold Development Scaffold- the moiety GW0665 H H N N within the core structure N C-FMS 6.23 VEGFR2 6.10 of a drug from which F TrJNK3 5.82 additional functionality Br GSK3 5.87 Core Structure can be built out by N adding pharmacophore features to enhance potency or selectivity. Gate Keeper induced fit O N H H N inner hydrophobic N Scaffold H O N Outer Hydrophobic Lys N Glu H H O H Lys area O O O Asp Sugar Pocket Solvent Front ! Tang, et al. Bioorg. Med. Chem. Lett. 2003, 13(18), 2985-2988. Kinase Targets: Linking Structure and Chemistry How it Works A number of kinases are available whose structure can be readily solved with different ligands solve the same template with many substitutions - lead optimization solve the same template in many kinases - selectivity, binding modes Why it Works ATP site - conserved but not optimized for ATP Exploit unique features of ATP site to achieve potency and selectivity Large database of structures allows for: --greater understanding of key pharmacophores and SAR --improved homology models --novel template design Kinases: Ideal for Systems-Based Research One scaffold can yield multiple inhibition profiles by changing side chains and taking advantage of multiple binding modes Crystallography of multiple templates and multiple kinases fantastic for Compound design (via surrogate crystal structures) Kinome-based assay systems help us understand mechanism of action and effective inhibition profiles for drug-like molecules Protein Tyrosine Kinase Receptor Action Is Terminated by PTPs, or Protein Tyrosine Phosphatases O O N H CH C N H N H CH C N H CH2 CH2 PTPase -phosphate OH O -O P O -O much easier to find inhibitors for targets than activators (this is true for enzymes) *go with the inhibitor Phospho-tyrosines Introduction – Protein Tyrosine Phosphatases O O N H CH C N H N H CH C N H CH2 CH2 PTPase -phosphate OH O -O P O -O Protein Tyrosine Phosphatases (PTPs) are a diverse group of proteins that comprise over 300 independent genes in Homo sapiens The first PTPase (placental PTP1B) was isolated in 1988 by Tonks, Diltz, and Fischer. Discovery target Phosphatases HCPTPA Targets VEGFR2, relevant for wound healing & angiogenesis Small (~20 kDa), soluble, intracellular Structure of a very close analogue is known Some inhibitor information available PTP1B Targets insulin receptor, relevant for diabetes & obesity Medium size (~45 kDa), soluble, intracellular Multiple structures solved Multiple inhibitors developed PTPb Targets VEGFR, TGFR, etc. – relevant for angiogenesis & cancer Large (~230 kDa), transmembrane No structures at the time of project start Very little inhibitor information available Preparatory work HCPTPA Cloned & expressed full-length protein Purified >100 mg of protein per batch of cells Crystallized in ~2 weeks post production Solved by molecular replacement with bovine HCPTPA PTP1B Cloned/expressed catalytic domain (residues 1-298) of the protein Purified >150 mg of protein per batch of cells Crystallized 3-4 days post production Solved by molecular replacement with known structure PTPb Cloned/expressed several catalytic domain constructs Selected two constructs for crystallization Purified >100 mg of protein per batch of cells One of the constructs crystallized ~2-3 weeks post production Solved by molecular replacement with PTPµ-CD Core Concept 7. Target validation versus better drugs for validated targets Target Validation=Verification of the predicted molecular target for a small molecule (i.e. for a ‘drug’ as defined by Karolkovas) The First Step in Drug Design! Types of target validation: *Human gene mutation and disease (cancers; y-kinase mutations) *Receptor identification and knock-out in animal models *Identification of receptors for known drugs (PPARs) *Drug/treatment-based target validation (antihistaminics) Aspirin and NSAIDs *Salicylic acid an effective analgesic, but very unpleasant taste (derived from willow bark) *Acetylsalicylic acid-re-discovered by Felix Hofmann at Bayer as a favor to his father for rheumatism *Other aspirin-like drugs developed without toxic side effects due to understanding of aspirin metabolism *cyclooxygenase discovered as a target of aspirin, and of other nonsteroidal anti-inflammatory drugs (NSAIDs) NHCOCH3 Acetanilide NHCOCH3 OH N-acetyl-p-aminophenol NH2 Aniline Metabolism of acetanilide, from Axelrod JBC 2003: discovery of acetaminophen made in 1946. Example of extrinsic toxicity. ! Aspirin and Cyclooxygenase *Aspirin found to be inhibitor of rate-limiting enzyme in prostaglandin synthesis, cyclooxygenase *Two forms of COX, COX1 and COX2 *Differential distributions of COX 1/2 in gut, brain, muscle (more COX1 in gut; more COX2 in muscle) leads to COX2specific NSAIDs with decreased gut side effects (e.g. Celebrex/Celecoxib; Vioxx/Rofecoxib) Extensive use of COX2-specific NSAIDSàcardiotoxic side effects emerge, curtailing their wide usage. Further mechanistic studies will likely lead to new types of NSAIDs, post-Celebrex, since COX2 is a validated target Success will depend on whether cardiovascular side-effects are intrinsic or extrinsic (spoiler alert—they are intrinsic!) Types of Target Validation Human gene mutation and disease (cancers; y-kinase mutations) Receptor identification and knock-out in animal models Identification of receptors for known drugs (PPARs; ’target re-validation’) Drug/treatment-based target validation (e.g. antihistaminics) Why is target validation the first step in drug design? PPAR-based drug development: Targets validated by previous drug efficacy, and subsequent identification of receptors Fibrates (clofibrate)—lowers triglycerides and LDLs— PPARalpha ligand Thiazolidinediones (glitazone)—antidiabetic; increases insulin sensitivity-PPARgamma ligand Two validated targets—PPARalpha and PPARgamma Tissue-specific actions of PPARs Reading #9 Gervois Reading #9—Selective modulation of PPAR activity Challenges and Opportunities in Development of PPAR Agonists Wright, Bortolini, Tadayyon and Bopst, Mol. Endocrinol. 28: 1756, 2014 Selective PPAR agonists are hard to make because we still don’t have a good handle on what happens when each of the many PPARalpha and PPARgamma ‘complexes’ get activated by a PPAR ‘agonist’ Reading #9 Gleevec: a classic example of a search initiated by genetic TARGET VALIDATION https://www.cancer.gov/research/progress/discovery/gleevec Gleevec: a classic example of a search initiated by genetic TARGET VALIDATION https://www.cancer.gov/research/progress/discovery/gleevec Gleevec, the ATP-binding pocket, and other possibilities for kinase inhibition Reading #10: Quintas-Cardama, 2007 Resistance, resistance, resistance… Reading #10: Quintas-Cardama, 2007 Compare critical path for anti-stroke versus antidepressant drug development for PACAP agonist/antagonist. Are they mutually incompatible? Anti-depressant: PACAP antagonist Stroke medication: PACAP agonist In each case, what does/would constitute target validation? Core Concept 8: Combinatorics And Combination Therapies v Signaling pathways converge on multiple targets, and first messengers diverge to multiple targets. v There may be modulation of more than a single signaling pathway in treatment of cancer, mental illness, stroke, neuro-AIDS. v Combinatorics can help attack multiple resistance mechanisms, multiple phases of disease that can be attacked individually; and multiple targets within given disease pathways. v FDA discourages multiple-drug formulation. However, pharmacology and therapeutics of viral infection (HIV) now involves multiple target selection in a single therapeutic regimen (HAART), which caused a paradigm shift in combinatoric drug development for many diseases besides HIV/AIDS. HIV Virus Life Cycle AZT-First Drug Developed for Treatment of AIDS Major Problems with AZT: *resistance *resistance *resistance NRTIs-varied points of attack, requiring multiresistance Emtricitabine (Emtriva) Cytidine analog AZT (Zidovudine) Thymidine analog HAART—Highly active anti-retroviral therapy E.g., protease inhibitor, nucleoside RT inhibitor, and non-nucleoside RT inhibitor or fusion inhibitor COVID-19 life cycle—points of intervention Vaccination/ Passive immunization Virus entry Hydroxychloroquineendosome basification Remdesivir COVID treatment: more interfering peptides Dahal et al., PeptideScience 114:e24245, 2022 COVID infection and ACE2 down-regulation Zhang et al., J. Neuroendocrinol. 33e12935, 2021 Therapies for Stroke: Case Study For Combinatorial Drug Development Cerebral ischemia (stroke)-3rd leading cause of death, & leading cause of disability, in U.S. 600,000 strokes each year, 25% fatal. Cost of stroke to the United States (1997 dollars) $43 billion / year Direct medical + therapy: $28 billion / year Indirect lost productivity and other factors: $15 billion / year Average cost of care for a patient up to 90 days after a stroke: $15,000. Similar impact for traumatic brain injury, with similar ‘salvage profile’ Treatment of acute stroke with tissue plasminogen activator As reported in: NEJM 340: 1781, 1999 Treatment of acute stroke with tissue plasminogen activator Stages in tissue damage/targets for therapy in ischemic stroke Neuroprotection Dirnagl and Hallenbeck, TINS 26:248,2003 Neuroprotection—need to block multiple parallel pathways Leker RR, Shohami E: Cerebral ischemia and trauma-different etiologies yet similar mechanisms: neuroprotective opportunities. Brain Res Brain Res Rev 2002, 39:55-73. Beauchamp et al., Pharmacology of Brain Injury, 2008 Drugs for TBI and stroke NMDA antagonists-Traxoprodil (NMDA antagonist); D-CCP-ene (NMDA agonist); magnesium sulfate (NMDA blocker). All no effect, or harmful, in TBI patients. Corticosteroids- CRASH Trial Aminosteroids-e.g. Tirilazad; acts at vascular endothelium; inhibits lipid peroxidation; ischemia/hypoxia->mitochondrial damage->free radicals inc lipid peroxidation. No evidence for therapeutic effect. Progesterone-neuroprotective. An actual ‘golden bullet’ for TBI? Stroke? PROTECTIII (2014)-no clinical benefit of progesterone for acute TBI Cannabinoids-dexanabinol-no benefits despite neuroprotection in animal models. Sequenced Treatment Alternatives to Relieve Depression (STAR*D) Study *Level 1 Citalopram. 33% remission; 10-15% responsive. *Level 2 Switch, 25% remission; Add-on, 33% remission. *Level 3 Switch, 12-20% remission; Add-on, 20% remission. *Level 4 Switch, 7-10% remission, same across treatments, but compliance least good with tranylcypromine. In the end, 50% remission after two treatment levels, and 70% remission after four treatment levels (but note withdrawal rates21% after level 1, 30% after level 2, and 42% after level 3). Treatment of chronic versus acute (emergency-room) depression Treatments cited previously require up to 4 weeks of treatment before a therapeutic response occurs. Emergency-room depression requires sedation/supervision during this period, or a faster-acting anti-depressant. There are now several possibilities for the latter, including ketamine and other glutamate receptor-directed drugs. Combinatorics for metabolic disease Clemmensen et al. Nature Reviews/Endocrinology 2018 Combinatorics for metabolic disease Clemmensen et al. Nature Reviews/Endocrinology 2018 Combinatorics for metabolic disease Clemmensen et al. Nature Reviews/Endocrinology 2018 Clemmensen et al.: Combinatorics for metabolic disease Terzepatid: a chimeric GLP-1/GIP-based obesity treatment is a single drug with two different targets GPCR SAR Coda: Peptide-liganded GPCRs Reading 12: Advances in therapeutic peptides targeting GPCRs GPCR-targeted Peptide Drugs Reading 12: Advances in therapeutic peptides targeting GPCRs Endogenous peptides for GPCR potential drug targets Reading 12: Advances in therapeutic peptides targeting GPCRs A short history of peptide drug development/approval Reading 12: Advances in therapeutic peptides targeting GPCRs Peptide and peptide agonist pharmacokinetics and pharmacodynamics Reading 12: Advances in therapeutic peptides targeting GPCRs Core Concept 9: Genomics, proteomics and expression profiling in drug design *microarray—analysis of the transcriptome. Technology has changed radically over last several years. *proteomics and phosphoproteomics relies on highthroughput mass spec analysis of proteins/peptides enriched by affinity methods and/or purified by 2DPAGE or HPLC STRATEGY TARGET GENES Gene transfer Rat genome sequenced Rat models Physiology ***Bioinformatics: a converging science comprised of biology, computer science and information technology GENOME TRANSCRIPTOME PROTEOME ~20,000 genes in the genome. But only 2000 studied. “We find that biomedical research is primarily guided by a handful of generic chemical and biological characteristics of genes, which facilitated experimentation during the 1980s and 1990s, rather than the physiological importance of individual genes or their relevance to human disease.” Stoeger et al., (2018) Large-scale investigation of the reasons why potentially important genes are ignored. PLoS Biology https://doi.org/10.1371/journal.pbio.2006643 EXPERIMENTAL DESIGN MICROARRAYS EXPERIMENT 1 2 SAMPLES RNA 1 RNA 2 Target 1 Target 2 HYBRIDISE TO MICROARRAY PROBE Affymetrix GeneChips BIOINFORMATICS (BIOLOGICAL OUTCOMES) What will Sequencing tell me? ALL transcripts including non-coding transcripts (depending on amplification protocol) Splice variants Quantification of differentially regulated RNAs Single Nucleotide Polymorphisms/Indels Hundreds of potential targets for study How do we choose? LOGIC CHERRY PICKING BIOINFORMATICS BIOINFORMATICS Logistical logjam A single microarray/RNAseq run can produce between 100,000 and a million data points A typical experiment may requires tens or hundreds of runs *Analysis LOW-LEVEL ANALYSIS - background elimination, filtration and normalisation removal of systematic variation between chips, enabling group comparisons HIGH-LEVEL ANALYSIS ( "data mining”) - the uncovering of relevant patterns of interest in data from a particular problem domain * Statistics * Presentation - eg CLUSTERING * Archiving * Relational and functional databases - Integrate with other type of information - Search literature on genes and gene-gene relationships - Known regulatory circuits, chromosomal localisation, cellular localisation of gene product etc. VALIDATION Confirmatory studies “ Because of the statistical issues raised by microarray technology, it is very important that the findings be confirmed using an independent method, preferably with separate samples rather than retesting of the original mRNA. Because data resulting from a microarray are so extensive, it is impossible to retest all of the data. Nevertheless, it is incumbent upon investigators to evaluate a reasonable number of genes.” Gary S Firestein and David S. Pisetsky (2002) DNA microarrays: Boundless technology or bound by technology? Guidelines for studies using microarray technology Arthritis & Rheumatism 46: 859-861 *RNA Quantitative (real-time) reverse transcription (RT) PCR In situ hybridisation - RNAscope *Protein Western blotting Immunocytochemistry *Clarity Polymerase Chain Reaction PCR is a simple technique for amplifying specific segments of DNA sequence Coupled with reverse transcription (RT) of mRNA the technique of RT-PCR is used to amplify specific RNA sequences PCR is dependent upon some prior knowledge of the sequence because specific targets are defined by two flanking primers (15-25 nt synthetic oligonucleotides) Exquisitely sensitive ( Peptides DNA miRNA transcriptional control Primary mRNA transcript processing; transport miRNA Active mRNA Protein Primary mRNA transcript and miRNA transcripts translational control chaperoning; post-translational modification; processing; trafficking Functional protein Active mRNA Protein Functional protein Degradation; processing; modification Peptides PROTEOMICS, GENOMICS AND PEPTIDOMICS Transcription controls expression of functional proteins & proteins supporting function (genomics) Translation controls protein abundance (genomics/proteomics) Post-translational modifications and events control protein trafficking and activity (proteomics; phosphoproteomics; glycoproteomics, etc) Final protein/peptide products (peptidomics) 2-D Gel Electrophoresis Sample Protein pI: 4(+) 7(-) 1st Dimension (Isoelectric Focusing) MW 150K 2nd Dimension (Separation by MW) 10K Gel Analysis Normal Cells/Serum Diseased Cells/Serum Removal of the “unknown” protein for mass spec analysis. Courtesy of H. Pollard Instrumentation – Automatic in-gel digestion ProGest Sample preparation Sample (cell extract, IP) 1D and/or 2D gel Excised protein gel spots Gel imaging Gel Processing (PDQuest, Typhoon) Excision of protein spots Courtesy of Vivian Hook, UCSD Automatic in-gel digestion in 96 well plate Gel spots are reduced, alkylated and digested with protease (i.e. trypsin) Extraction with H2O/formic acid/ACN Instrumentation – Proteomics Solution 1 Symbiot Voyager DE STR automatic sample loading samples from 96 well plate are loaded onto MALDI target MALDI – TOF MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Courtesy of Vivian Hook, UCSD Mass Spectrometry Measurement of the mass of organic molecules such as protein fragments (peptides) collected from 2D gels or online after HPLC Various methods for MS determination: SELDI/MALDI-ToF-MS (sample adsorbed onto surfaceenhanced chip (SELDI) or matrix (MALDI), then laser desorbed and ionized with mass analysis by time-of-flight) 2D gel/MALDI-MS Capillary electrophoresis-MS (CE-MS) Liquid chromatography-MS (LC-MS) Trade-offs include bias, reproducibility, sensitivity, diagnosis vs. discovery MALDI-ToF Prism Sample (peptide fragment) is vaporized and ionized by the laser in the sample chamber. Sample plate is given a positive charge and the ion is repelled through the flight chamber to the ion detector. Laser Flight Chamber Ionized Sample Sample Ion Detector intensity Sample Plate m/z Green-strong responders Red-non-responders 16961.4 is a marker that predicts poor response to celecoxib in colon cancer clinical trial Fig. 1. The central 47 nucleotides of RNA B11 are sufficient for binding to D-ghrelin SELEX-selected RNA Aptamers (Spiegelmers) Bind Peptides An alternate technology for generating recognition reagents for high-throughput screening. SELEX (Systematic evolution of ligands by exponential enrichment) technology can be used to generate RNA-based aptamers for peptides and peptide fragments with N- and C-terminal specificity and recognizing a relatively limited epitope (e.g. Helmling et al., PNAS 101: 13174, 2004) Helmling, Steffen et al. (2004) Proc. Natl. Acad. Sci. USA 101, 13174-13179 Copyright ©2004 by the National Academy of Sciences Expanded bioinformatics definitions… *Bioinformatics: a converging science comprised of biology, computer science and information technology *Transcriptome-the collection of RNAs expressed by the genome of a cell, tissue or organism (in a specific state) *Proteome-the collection of proteins that are encoded by the transcriptome of a cell, tissue or organism (in a specific state) -->Transitional transcriptomes/proteomes and their role in drug discovery Core Concept 10: Pharmacogenetics, pharmacokinetics, and pharmacotoxicology are partners in drug design *ADME-absorption, distribution, metabolism and excretion *pharmacogenetics/pharmacogenomics-drug & dosage personalization to minimize side effects and increase drug efficacy *drug-like properties enhance predictability of drug development Pharmacokinetics/ADME LOCUS OF ACTION (RECEPTORS) BOUND FREE TISSUE RESERVOIRS FREE BOUND circulation ABSORPTION FREE DRUG BOUND DRUG Serum levels of drug=efficacy Not always the case… Target engagement-Fos and pharmacodynamics EXCRETION metabolites METABOLISM (phase I/phase II) Adapted from The Pharmacological Basis of Therapeutics, Hardman & Limbird, eds.,10th Ed,2001 Pharmacogenetics ‘started off’ with a focus on drug metabolism, but the discipline applies to absorption, distribution, excretion, toxicity and receptor efficacy as well!! Pharmacogenetics The study of the role of inheritance in individual variation in response to xenobiotics, including drugs. Slides courtesy of Richard Weinshilboum, Mayo Clinic Cancer Center Pharmacogenetics of Phase I Drug Metabolism CYP2D6 pharmacogenetics CYP2C9 pharmacogenetics CYP2D6 and CYP2C9-each associated with a few prototypes Pharmacogenetics-Pharmacogenomics Clinical Goals Avoid adverse drug reactions Maximize drug efficacy Select responsive patients Pharmacogenetics Scientific Goal Correlation of variation in DNA sequence and/or structure with variation in drug response phenotype. Genotype-Phenotype Correlation Evolution from phenotypeàgenotype, to genotypeàphenotype Pharmacogenetics-Pharmacogenomics Genomic Variation Single nucleotide polymorphisms (SNPs) Insertion-deletions (Indels) Variable number of tandem repeats (VNTRs) Gene deletion and/or duplication Large segmental duplications Gene sequence variation resulting in alternative splicing Epigenetic variation Epigenetic patterns can be inherited.. Pharmacogenetic “Targets” Drug absorption Drug distribution Drug-target interaction Drug metabolism Drug excretion Keep this in ‘back of mind’—look for development in other 4 areas in coming years Bradykinin, ACE inhibitors, and cough Drug Metabolism Phase I Oxidation Reduction Hydrolysis Phase II Glucuronide conjugation Sulfate conjugation Glutathione conjugation Glycine conjugation Acetylation Methylation FunctionalizeàConjugate Genetically Polymorphic Cytochrome P450s CYP2C9 - Warfarin CYP2C19 - Omeprazole CYP2D6 - Metoprolol (and > 40 other drugs) Remember 2C9 and 2D6! Genetic Polymorphism Frequency of least common allele 0.01 (1%) or greater Polymorphism versus mutation…the Philadelphia translocation (the granddaddy of all INDELs is a mutation not a polymorphism Codeine Biotransformation to Morphine HO CH3O CYP2D6 O O N — CH3 HO N — CH3 HO Codeine Morphine Codeine Toxicity Case Report NEJM December 30, 2004 62 year old man hospitalized for pneumonia Treated with “standard” doses of codeine as a cough suppressant Coma – morphine levels 20 times expected levels What CYP polymorphism involved? Patients was higher or lower than average? And this tells us what about TI and indication? CYP2D-not just for first-pass metabolism anymore! …relevance to pharmacokinetics and serum sampling CYP2D6 Polymorphism Selected Substrate Drugs Drug Metabolic Pathway à Alprenolol Aromatic hydroxylation Amitriptyline Benzylic hydroxylation Bufuralol Aliphatic and aromatic hydroxylation O-Demethylation à Codeine à Dextromethorphan O-Demethylation à Dihydrocodeine O-Demethylation Flecainide O-Dealkylation Imipramine Aromatic hydroxylation Metroprolol Aliphatic hydroxylation and O-dealkylation Perhexiline Aliphatic hydroxylation Aromatic hydroxylation à Propranolol Timolol O-Dealkylation Selected from a list of 41 drugs (Kroemer and Eichelbaum, Life Sci. 56:2285, 1995) CYP2D6 Population Frequency of Poor Metabolizers Asian 1-2% Caucasian 5-10% African 2-10% Clinically meaningful for pharmacist? Hospitalist? Physician? On the street? Vitamin K Cycle VKORC1 February 5, 2004 Warfarin Pharmacogenomics CYP2C9 Metabolites Metabolism (Pharmacokinetics) Warfarin Therapeutic Effect (Pharmacodynamics) o VKORC1 Inhibition New England Journal of Medicine June 2, 2005 Warfarin Pharmacogenetics CYP2?? VKORC1 gene resequenced 10 common SNPs and 5 common haplotypes Low-dose (A) and high-dose (B) haplotypes Mean maintenance dose 2.7 ± 0.2 mg/day for AA, 4.9 ± 0.2 for A/B and 6.2 ± 0.3 for BB (P < 0.001) CYP/VKORC1 combos of concern? Drug Metabolism Phase II Glucuronide conjugation Sulfate conjugation Glutathione conjugation Glycine conjugation Acetylation Methylation Put acetylation and methylation in ‘conjugation’ terms? Pharmacogenetics of Phase II Drug Metabolism NAT-2 pharmacogenetics TPMT pharmacogenetics N-Acetyltransferase Polymorphic HNNH2 O C H NNH2 Isoniazid treatment of tuberculosis NH2 N N N Hydralazine Isoniazid O C H NCH2CH2N(CH2CH3)2 Procainamide Monomorphic NH2 NH2 OH COOH COOH p-Aminosalicylic Acid (PAS) NAT1 same in everyone; NAT2 quite different Acetylation inactivates isoniazid— opposite of effect of demethylation on codeine! p-Aminobenzoic Acid (PABA) Number of Subjects Acetylation Pharmacogenetics 24 "Fast" Acetylators 267 Subjects "Slow" Acetylators 12 0 4 8 12 Plasma Isoniazid Concentration (µg/ml) Slow acetylators 5x more frequent in Caucasian than Japanese populations: relevance to personalized medicine Thiopurines and Natural Purines SH N N N H N H2N 6-Mercaptopurine SH N N N N H Reactivity, drug action, drug metabolism 6-Thioguanine Hypoxanthine OH N N N N H Guanine Metabolism of 6-Mercaptopurine SH N Thiopurine Methyltransferase (TPMT) Active drug, active toxin (intrinsic toxicity) N N H N Xanthine Oxidase (XO) AdoMet AdoHcy SH SCH 3 N N N N OH N N H HO AdoMet N N H AdoHcy XO TPMT SCH3 N N OH OH N N H Inactive as drug or toxin 2,8-Dihydroxy-6-Methylmercaptopurine TPMT Genetic Polymorphism Clinical Consequences Patient with low TPMT activity – – Increased thiopurine toxicity Increased risk for secondary neoplasm Patient with high TPMT activity – Decreased therapeutic effect Worthwhile to measure patients TPMT activity prior to chemotherapy? Molecular Pharmacogenetics Principles Allelic heterogeneity Ethnic variation in allele frequencies Pharmacogenomics: “Traditional” Paradigm Phenotype Genotype The Human Genome February 2001 Genotype-to-Phenotype Strategy Gene sequence Variation in gene sequence Functionally significant variation in gene sequence Clinical important functionally significant variation in gene sequence Pharmacogenomics The Future The Vision The right drug, at the right dose for every patient. Genetics of Drug Efficacy/Toxicity: Pharmacogenomics Johnson & Evans, Trends in Molec. Med. 8: 300, 2002 Treatment modifications and patient genotypes Johnson & Evans, Trends in Molec. Med. 8: 300, 2002 Think of this as ‘real-time personalized genomics’-– what would be good markers to predict developing resistance? Core Concept 11:Critical Path to Drug Development empirical HTS structural bioinformatics You are here DRUG observation Drug design/development pipeline Genomics, proteomics and bioinformatics have produced an increasing number of targets, HTS and combinatorial chemistry have increased the number of leads, and rational drug design strategies have optimized refinement of leads to new chemical entities (NCEs, a.k.a. NMEs). However, NCE-->drug development has a >90% attrition rate. May be due to failure to incorporate drug-like characteristics into the pipeline early enough. -- “Integrating Drug Discovery and Development”, R. Borchardt, The Scientist 15: 43, 2001 23&Me-Pharmacogenomic Snapshot Critical Path *Particular to each drug development process but always begins with target validation, which can arise from genetic information; previous clinical experience with known drugs; known mechanisms or etiologies of disease *Milestones from target identificationàvalidationàhit-toleadàmarketable/marketed drug. *So the critical path is a ‘critical algorithm’ for drug development *A validated target is “an effector of a therapeutic compound that, when modulated in humans, has desirable therapeutic use” T. Reiss, Trends in Biotech.19:496,2001. This means that a validated target is a bona fide receptor for a drug. DRUG DEVELOPMENT PIPELINE Discovery Research Clinical Trials Hypothesis Generation Commercialize Candidate Development Commercialization 200-400 100-200 250-400 3-5 1-3 4-8 Cost $M Years SOURCE: Tufts\McKinzie Analysis Target Identification and Validation Assay Development Lead Optimization FHD Phase IA Phase IB / II Phase III Submit Global launch Lead Generation Project sanction Screen sanction Program sanction Candidate(s) Phase IA selection Strategy (Clinical Trial Application DRUGS PHARMACOLOGICAL TOOLS Product decision Submit (Marketing Application) Launch Global optimization Lead Discovery Science Learning Probabilistic Knowledge Based Technology 1996-1999 Dominant Influences HTS Driven Hit Discovery Combi-Chem Maximum Diversity “Screen for Drug” Promise 2000-2008 Dominant Influences Target Validation Science Bioinformatics Target Platforms Target Drugability Quantitative Biology Biomarkers ADMET Profiling Structural Biology IP Strategies Testing Flowschemes Diversified Lead Generation Strategies Computational Science Library Sciences Compound Quality Targeted Diversity KOs/Transgenics Receptor Panel Profiling Chemogenomics Systems Biology Factored into Contemporary Lead Generation Partial List of Drug Development Team Participants The Critical Path and Drug Development Pipeline The critical path is the individual action plan for a specific drug candidate (lead compound) as it makes its way through the drug development pipeline. Critical Path ‘Case Study: Re-consideration of PACAP as a Neuroprotective v. Anti-depressive Agent Class discussion (as development team) of: --target validation --animal models --proof-of-concept --drug-like molecule development --high-throughput hit identification --ADME in hit-to-lead --single-drug or combinatorial niche --tentative development plan with milestones --when does a drug enter a critical path for development? Experimental Flowchart: 0 hr 1 hr Ischemia-induced specific regulation of gene expression was analyzed with a NIH Mouse 36K cDNA microarray. Equal amounts (2-5 µg/15.5 µl) of total RNA were separately labeled with two different fluorescent dyes (Cy3 or Cy5) and applied on the same chip. \ Pre-ischemic period 24 hr Post-ischemic period Neurological Function Tasks MCAO Infarct Volume Measurement PACAP administration PACAP knockout and wildtype mice subjected to left middle cerebral artery occlusion (MCAO). Motor function evaluated with modified neurological severity scoring system (NSS) and walking fault task (WF). 0 PACAP administered i.c.v. (160 pmol/ml/hour ) or i.v. (0.75 nmol/mouse) 1 hr after MCAO. Infarct volume measured with NIH Image analyzing system, in whole brain serial cryosections stained with cresyl violet. Neurological Severity Score at 1 hr A. No treatment PACAP treatment (i.v) PACAP treatment (i.c.v) B. delta-Neurological Severity Score No treatment PACAP treatment (i.v) PACAP treatment (i.c.v) 12 8 10 * * * * 6 dalta-NSS NSS 8 6 4 4 2 2 0 0 C. PACAP-deficient Mice delta-NWF (Number of Walking Faults) Number of Walking Faults No treatment PACAP treatment (i.v) PACAP treatment (i.c.v) 30 25 * * 20 * * # 15 10 5 0 Wild Type Mice PACAP-deficient Mice Infarct Volume D. # 35 Infarct Volume (mm3) Wild Type Mice 30 25 * * No treatment PACAP treatment (i.v.) PACAP treatment (i.c.v.) * * 20 15 10 5 0 Wild Type Mice PACAP-deficient Mice Wild Type Mice PACAP-deficient Mice HPA: PACAP regulates the PVN in a stress specific manner PACAP controls CORT elevation in response to acute psychogenic stress (Stroth and Eiden 2010, Tsukiyama et al., 2011 ). PACAP does not regulate the PVN in response to systemic stressors (Lehmann et al., in press, Psychoneuroendocrinology, 2012, Tsukiyama et al., 2011). * * HPA: PACAP is required for prolonged CORT elevation PACAP-KO mice subjected to daily stressful encounters experience less stress (CORT elevation) than wild-type mice Social defeat is stressful! Lehmann et al. Psychoneuroendocrinology 38: 702-715, 2013 HPA: PACAP-deficiency offers protection against depression Behavioral affects following social defeat – DEPRESSION Learned Helplessness Social Avoidance SD PACAP ( -/- ) = Reduced despair driven immobility SD PACAP ( -/- ) = Reduced social avoidance Lehmann et al. Psychoneuroendocrinology, in press, 2012 = ê Depression = ê Depression Compare critical path for anti-stroke (PACAP agonist) versus anti-depressant (PACAP antagonist) drug development In each case, what would constitute target validation? Critical Path: General Principles Decision branchpoints include: --choice of animal models for proof-of-concept, versus development, versus preclinical --drug-like molecule development: at which stage? --clinical targets: should drug purposing be broad or narrow? Critical path is different for each drug: --toxicology familiar or new? --compound ‘druggable’ or a wholly new entity? --validation is disease-based, receptor-based, toxicology-based? The development of GLYX-13 as an antidepressant drug Joseph R. Moskal, Ph.D. Director, Falk Center for Molecular Therapeutics McCormick School of Engineering and Applied Sciences Professor, Dept. of Biomedical Engineering Northwestern University Founder, President and CSO Naurex Inc. Rapastinel (GLYX-13): acute treatment for depression Core Concept 12: Drug Hunting, Trapping, and Development Two case histories and general principles of drug development 1. Chris Felder, Eli Lilly, Inc., Cholinergic anti-Alzheimer’s drug development 2. Michael Iadarola, NIDCR, TRPV-based cell deletion therapy for intractable pain Cholinergic Hypothesis & AD Cognitive changes in Alzheimer's disease (AD) are due to the loss of cholinergic innervation from the basal forebrain (Whitehouse et al. 1982 Science 215,1237). Cholinesterase inhibitors are the only currently approved Muscarinic-based treatment for the symptoms of AD Objective Identify a potent, selective, and orally active M1 receptor agonist appropriate for the treatment of cognitive deficits associated with psychiatric and neurological diseases including Alzheimer's disease. more acetylcholine --> can inhibit the breakdown of acetylcholine. or stimulate an activator Pathways of Muscarinic M1 Neuroprotection Xanomeline Milameline Sabcomeline Tilsacledine ACh M1 Agonist ACh Cholinesterase Inhibitors Cognex(tacrine) Aricept(donepezil) Exelon(rivastigmine) X ßAPPs APP Aß g/b SECRETASE a-SECRETASE PLC Gq PKC MAPK X GSK-3 β Hyper-P-Tau Tau ßAPPs Paired Helical Filaments Neurofibrillary Tangles Ionotropic and Metabotropic Acetylcholine Receptors (“own the surrounding biology”) O Na+ Ca++ Nicotinic Receptor O N + ACETYLCHOLINE G Muscarinic Receptor The Muscarinic Acetylcholine Receptor Family M1 M3 M5 M2 M4 PLC AC Gq Gi STIMULATORY Signal Stimulates PLC and Ca++ Transduction: through Gq INHIBITORY Blocks AC through Gi 5 well characterized muscarinic receptor subtypes All 5 receptor genes cloned from human, rat, mouse Widely distributed throughout brain and periphery Per cent Phosphoinositide Conversion In vivo hydrolysis of PI in hippocampus of M1-5 knockout mice Summary: Pilocarpine-stimulated PI hydrolysis is lost in hippocampus of M1 knockout mice but not in mice deficient in M2M5 receptors. Is the target available in the patient? M1 muscarinic receptor activity remains in hippocampus of Alzheimer's Disease patients Drug Development Example: Testing the Cholinergic Hypothesis that an M1 receptor partial agonist would enhance declining memory in the elderly and provide neuroprotection from Abeta M1 Muscarinic Receptor Agonist Development 500,000 Compounds hM1 Receptor Ca2+ Mobilization Assay 40 aa PHS Act under section 351: 351(a), 351(k) [see the definition of “protein” at URL] FD&C Act = Food Drug & Cosmetic Act PHS Act = Public Health Service Act User Fee Programs PDUFA GDUFA BsUFA Biological Products Reviewed by CDER vs. CBER Center for Drug Evaluation and Research (CDER) -- examples Center for Biologics Evaluation and Research (CBER) -- examples monoclonal antibodies (mAb) allergenic extracts (e.g., for allergy shots and tests) blood and blood components gene therapy products devices and test kits human tissue and cellular products used in transplantation vaccines targeted therapies in cancer and other diseases cytokines proteins involved in immune response growth factors proteins that affect the growth of a cell enzymes proteins that speed up biochemical reactions immunomodulators proteins that affect immune response (mostly produced by biotechnology methods) Information from legacy FDA website - Therapeutic Proteins Protein Therapeutic Agents Biotherapeutics Biologics Final Prescribing Information Summarizes Important Information for Patient Care Regulatory Submission Package (Common Technical Document, CTD) contain scientific data & evidence of safety and effectiveness 2022 UH-Regulatory Affairs Course (YM-Wang) Getting It Right in Drug Development Information Drug Dev Phases Studies (leveraging clinical pharmacology in stand-alone therapeutic proteins programs) BLA/NDA IND Pharmacology (PD) In vitro In vivo PK Discovery Research & Preclinical Development Tox & TK PK & PD + Clinical Response Phase 1 / FIH, MD Phase 2 / Proof of Concept Phase 2 / Dose ranging Phase 3 / Pivotal Trials / Extension safety Trials BLA/NDA Supplements Phase 4 / Post-marketing Trials Preclinical Development – safety studies Pre-IND Exposure (animal) Safety Response PK-PD Modeling Human exposure projection Impact Go/No-Go Learn and confirm IND FIH dose selection Exposure (short term) Safety Biomarker PD PK-PD Modeling (exposure response) NDA dose selections Clinical response Exposure (longer term) Dosing recommendation for labeling Goals of “Stand-alone” Development and Biosimilar Development are Different “Stand-alone” Development Program, 351(a) Goal: To establish safety and efficacy of a new product “Abbreviated” Development Program, 351(k) Goal: To demonstrate biosimilarity (or interchangeability) to a reference product Link to The Statute (PHS Act Section 351) https://www.fda.gov/drugs/therapeuticbiologics-applications-bla/biosimilars 2022 UH-Regulatory Affairs Course (YM-Wang) Multi-Disciplinary Review Team Sponsors Project manager Chemistry Clinical Pharmacology Medical Preclinical Pharm/Tox Statistics Decision: Safe & Efficacious? Some products may require additional disciplines such as microbiology and virology 290 Advisory Committees Panels of experts and patient advocates who provide outside perspectives to FDA Discordance between FDA decisions and AC recommendations was 22% between 2008 and 2015 (Zhang et al. 2019) Risk Benefit Unmet need Toxicity Convenience of administration Inappropriate use Reduced toxicity Drug-drug interactions Superior efficacy Product labeling Provides information for healthcare professionals and patients on safe an effective use including: Approved indication and use Dosage and administration Warnings an adverse reactions Drug interactions Use in specific populations Clinical studies Important implications for advertising and promotional claims subject to review by the Office of Prescription Drug Promotion (OPDP) Reasons Drugs Fail to Get Approved Sacks et al. JAMA 2014;311(4):378-384 Clinical Trials for Novel Drug Approval Investigation New Drug (IND) Process Required before beginning clinical research for a novel drug without past clinical experience (can submit data obtained in other countries) Sponsors must include: Submission of animal and toxicity data Clinical protocols Information about investigator(s) https://www.fda.gov/patients/drug-development-process/step-3-clinical-research Phase 1 No observable adverse event level (NOAEL) and MABEL (minimal anticipated biological effect level) approaches 20-100 healthy volunteers (HVs) or patients Nearly always done in HVs Duration is several months Goal: safety and dosage ~70% of drugs move on to the next phase Phase 2 Hundreds of patients with disease for which approved indication is sought Can requires years of follow-up Goal: demonstrate efficacy and identify side effects ~33% of drugs move on to the next phase Phase 3 300-3000 patients Duration of 1 to 4 years Goal: confirm efficacy at recommended dose and further monitoring of adverse effects ~25-30% of drugs move on to the next phase Phase 4 – post-marketing surveillance After drug approval Pharmacovigilance Satisfy any post-marketing requirements No Adverse Effect Level (NOAEL) Highest dose of a compound that, even when repeatedly administered, yields no measurable toxicologic effects or adverse effects. Toxicology-based know that these are two different approaches to find the safe level of drug to use for a phase 1 trial Minimum Anticipated Biological Effect Level (MABEL) Lowest dose of a compound that results in a detectable effect Receptor occupancy is one criterion used to help define the MABEL Pharmacology-based