Reverse Pharmacology Flashcards
Classical Pharmacology
1804 Friedrich Sertürner, pharmacist, isolated morphine from raw opium.
1832 Pierre-Jean Robiquet isolated codeine.
1848 Georg Merck isolated papaverine.
1898 Production of heroin (diacetylmorphine) by Bayer
1952 First synthesis of morphine
1971-73 Receptor binding studies using radioligands
1975 Enkephalins (endogenous opioids = endorphins)
1992/93 Opioid-Receptors (delta-Receptor, mü-Receptor and κ-Receptor)
1994 OPRL1 (Opioid Receptor-like 1 gene) shares ~60% sequence identity with ‘classical’ opioid receptors.
1995 Orphanin FQ (Nociceptin)
→The discovery of Nociceptin is the first example of reverse pharmacology!
Classical approach
Functional activity
-> Ligand purification
Ligand/Receptor
-> Functional studies
Biological role/Pathophysiology
-> target disease
Compound screening
-> medicinal chemistry
Drug candidate
-> molecular targeting
molecular mechanism genomic research
Reverse pharmacology
bioinformatics/molecular genetics
-> full length cloning
receptor
-> ligand fishing
ligand
-> receptor/ligand
1st compound screening
-> HTS
lead compound
-> functional studies
functional activity, biological role/pathophysiology
-> target disease
2nd compound screening
-> medicinal chemistry
drug candidate
Forward pharmacology approach
Functional activity in vivo or in vitro (e.g. of extracts or natural products)
-> Lead compounds
-> Target identification
Reverse pharmacology approach
Identification of promising target proteins
-> Screening for compounds interacting with the target protein
-> Functional activity in vivo
Short History of GPCR-Deorphanization
1986 G-protein coupled receptors (GPCRs) share sequence similarities; orphan GPCRs can be found by homology srceening
1987 Report of the first reported orphan GPCR: G-21
1988 The first deorphanizations: G-21 as the 5-HT1A receptor
and RGB-2 as the dopamine D2 receptor
1989 Introduction of the PCR-based screening approach for the discovery of orphan GPCRs
1995 First novel transmitter as target of an orphan GPCR identified: orphanin FQ/nociceptin
1995 Various deorphanized GPCRs
The human genome as a source of new drugs?
The human genome sequence
➢ How big is the human genome?
➢ How many genes are there?
➢ How can potential candidates be identified?
New G-protein coupled receptors and ligands
➢ The orexin system
The decoding of the human genome
1990 Start of the Human Genome Project (HGP)
1998 Start of genome sequencing by US company Celera Genomics
2001 First publication of the human genome sequence
2004 Completion of the sequencing of the human genome (euchromatic fraction of the genome)
Genomic DNA -> hnRNA -> mRNA (ESTs, cDNA clones) -> proteins
The human genome
- 3.000.000 nucleotides
- 20.000 - 25.000 protein-coding genes
Genes encode different types of proteins
Structural proteins -> Collagen: skin, bones, teeth Elastin: bands, vascular walls
Transport proteins -> Hemoglobin: transports oxygen in the blood
Storage proteins -> Ferritin: stores iron in the liver
Filament proteins -> Myosin: forms muscles
Antibody proteins -> Immunoglobulins: recognize and bind foreign substances
Receptor proteins -> Rhodopsin: transmits light signals in the eye
Enzymes -> DNA polymerase: synthesizes DNA
Signaling proteins -> Tumor necrosis factor: signal to inactivate tumor cells
Membrane proteines -> Permeases: funneling particles across cell membranes
Targets for Drug Action (Drug Receptors)
A drug is a chemical applied to a physiological system that affects its function by binding specifically (chemically) to a receptor.
Four main kinds of regulatory proteins are commonly involved as primary drug targets, namely:
* Physiological receptors
* Ion channels
* Enzymes
* Carrier molecules (neurotransmitter/electrolyte transporters)
Percentage of biochemical structures used in therapies
GPCRs comprise the single largest group! -> Most drugs target GPCRs! (> 30 % of all pharmaceutical drugs approved by the FDA)
G-protein coupled receptors (GPCRs)
Galphas -> stimulates adenylate cyclase, modulates Ca2+ and Na+ channels.
Galphai -> inhibits the adenylate cyclase, activates cGMP phosphodiesterase, opens K+ channels, closes Ca2+ channels.
Galphaq/11 -> activates phospholipase C.
Galpha12/13 -> stimulates cell growth via Rho-GTPases.
Identifying new GPCRs
Molecular biological methods to identify nucleotide sequence homologies
▪ Hybridization at low stringency (“colony screening” cDNA libraries)
▪ Polymerase chain reaction (PCR) from cDNA or genomic DNA with degenerate primers
In silico analyses („database mining“)
▪ Comparison of amino acid or nucleotide sequences with databases (GenBank ect.)
▪ Identification of specific structural features using special algorithms (TMHMM)
Identification of specific structural features using special algorithms
- TMHMM Server v. 2.0
Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes - Input in FASTA format
- Output
GPCRs in the human genome
More than 800 different human genes ( or 4 % of the entire protein-coding genome) have been predicted as GPCRs from genome sequence analysis.
G-protein coupled receptors (GPCRs)
25.000 number of human genes
872 number of GPCRs
482 GPCRs (without olfactory GPCRs)
121 GPCRs with unknown function (“Orphan” receptors)
Ligand screening: cell-based reporter systems
Cloned “orphan” GPCR
-> Expression
-> Assay (known ligands, putative ligands, compound libraries, extras from tissues –> fractionation)
-> identification of “hits”
-> biological validation
Orphan receptor strategy I
Tissue extract
-> fractionation
-> isolation, determination of structure
-> cloned orphan GPCR
-> second messenger
-> discovers novel transmitter
The orphan receptor strategy II (reverse pharmacology)
Potential transmitters are randomly tested on a range of orphan GPCRs with the hope that one of the transmitters will activate a matching GPCR.
FLIPR technology for detection of intracellular calcium release
Compound addition: substance to be tested
Ligand addition: known agonist
Fluometric imaging plate reader (FLIPR): fluorescent dye Ca2+ indicators: Fluo-4. Fura-2
100% and 0% controls are overlaid as red- and blue- hatched lines.
Isolation of endogenous ligands for HFGAN72
200 g rat brain (150 brains)
-> HFGAN72 (Orphan GPCR in HEK-293 cells)
-> Ca2+
The orexin system
Orexins and Orexin Receptors: A Family of Hypothalamic Neuropeptides and G Protein-Coupled Receptors that Regulate Feeding Behavior
➢ The isolated peptides were named orexins, the orphan GPCR HFGAN72 was named orexin type-1 receptor (OX1R).
➢ A second orexin receptor was identified and called OX2R due to its homology to OX1R.
➢ ICV injection of orexins triggers feeding behavior in rats.
➢ Fasting stimulates orexin expression.
Pharmacological characterization of synthetic human orexins
COMPETITIVE RADIOLIGAND BINDING ASSAY
Displacement of [125I-Tyr17]orexin-A binding to cells expressing human OX1R (A) and OX2R (B) by increasing concentrations of unlabeled human orexin-A (open circles) and orexin-B (filled circles)
Concentration-response relationships of [Ca+]i transients evoked by orexin-A (open circles) and human orexin-B (filled circles) in CHO cells expressing human OX1R (C) and OX2R (D)
Orexins and narcolepsy
➢ In dogs, mutations of the OX2 receptor result in narcolepsy like symptoms.
➢ Orexin “knockout” mice exhibit narcolepsy like symptoms.
➢ In humans, the lack of orexin neurons leads to narcolepsy.
The central orexin system
- Neuroendocrine functions
- Sleep-wake rhythm
- reward system
- energy balance
- autonomic nervous system
Orexin receptor agonists and antagonists
Development of selective orexin receptor agonists and antagonists
* Treatment of narcolepsy and metabolic diseases (agonist)
* Treatment of insomnia and obesity (antagonists)
L-Alanine Scan of Orexin-B -> OX2 receptor selectivity
The Alanine scan is based on the systematic, sequential exchange of each individual amino acid for alanine. Alanine is sequentially exchanged by glycine.
D-Amino Acid Replacement of Orexin-B
Sequential substitution of each L-amino acid with the corresponding D-isomer (Glycine to d-alanine)
OX2 receptor selective agonists
Increased selectivity of modified orexin B for OX2 receptors by
-> L-Alanin scan
-> D amino acid exchange
TAK-925 (Danavorexton) is a potent, selective, and brain-penetrant small molecule OX2R agonist.
The first OX2 receptor selective antagonists
N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
Identification by high-throughput screening (HTS) and chemical modification
Almorexant (actelion pharmaceuticals ltd.)
ACT-078573 (Almorexant)
➢ A tetrahydroisoquinoline derivative: (2R)-2-{(1S)-6,7-dimethoxy-1-[2-(4-trifluoromethylphenyl)- ethyl]-3,4-dihydro-1H-isoquinolin-2-yl}-N-methyl-2-phenyl-acetamide
➢ Passes the blood-brain barrier
➢ The first oral orexin receptor antagonist
➢ IC50
OX1 Receptor: 16 ± 4 nM (Rat, n = 3), 13 ± 1 nM (Human, n = 8)
OX2 Receptor: 15 ± 2 nM (Rat, n = 3), 8 ± 1 nM (Human, n = 8)
→Dual OX1 / OX2 Receptor Antagonist (DORA)
Orexin receptor antagonists as hypnotic drugs?
- Orexin-RA-1: 68 % increase in REM-sleep
- Zolpidem: 43 % decrease in REM-sleep
➢ Almorexant (Actelion): development was discontinued in 2011 during phase III because of side effects (abnormally elevated liver enzymes).
➢ Further DORAs: Suvorexant (Belsomra®, MK-4305, Merck): approved 2014 in USA for the treatment of sleeping disorders Suvorexant has a half-life of about 12 hours;
Lemborexant (DayvigoTM, Eisai) FDA 2019; Daridorexant (QuviviqTM); Filorexant (discontinued); Seltorexant (phase III)
Some transmitters identified as ligands of orphan GPCRs
Nociceptin: Anxiety and pain
Ghrelin and MCH: Food intake and metabolism
Metastin (KiSS-1): Cell cycle control
Succinate: Renal function and hypertension
GPCRs are attractive targets for magic bullets
➢ The isolation of endogenous ligands for “orphan” receptors has revolutionized the discovery of new transmitters.
➢ It is expected that at least 50 more new transmitter systems are discovered.
➢ One aim of “reverse” pharmacology is the clarification their biological and pathophysiological functions.
