Bioinformatics
Computational and Combinatorial
Chemisty
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Absorption
Distribution
Metabolism
Elimination
Pharmacokinetic
Bioavailability
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reasons that cause the failure of a potential drug candidate
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Bioavailablity of Drugs (I)
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Uptake of orally administered drug proceeds after the stomach passage via the small intestine.
In the liver, a series of metabolic transformation occurs.
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The super-family of cytochrome P450 enzymes has a crucial role in the metabolism of drugs.
Almost every drug is processed by some of these enzymes.
This causes a reduced bioavailability.
Cytochrome P450 enzymes show extensive structural polymorphism (differences in the coding region).
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During first liver passage: First pass effect extensive chemical transformation of lipophilic or heavy
(MW >500) compounds. They become more hydrophilic
(increased water solubility) and are therefore easier to excreat.
H
N
CH
3
COOH
O
COOH phase I phase II
Predominantly cytochrome P450 (CYP) enzymes are responsible for the reactions belonging to phase I.
Usually, the reaction is a monooxygenation.
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The substrates are monooxygenated in a catalytic cycle.
Drug-R + O
2
CYP
Drug-OR + H
NADPH NADP
2
O
The iron is part of a HEM moiety
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The cytochromes involved in the metabolism are mainly monooxygenases that evolved from the steroid and fatty acid biosynthesis.
So far, 17 families of CYPs with about 50 isoforms have been characterized in the human genome.
classification: CYP 3 A 4 *15 A-B family isoenzyme
>40% sequencehomology sub-family
>55% sequencehomology allel
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Human 14+
Molluscs 1
Plants 22
Insects 3
Fungi 11
CYP450
Yeasts 2
Bacteria 18
Nematodes 3
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Of the super-family of all cytochromes, the following families were confirmed in humans:
CYP 1-5, 7, 8, 11, 17, 19, 21, 24, 26, 27, 39, 46, 51
Function:
CYP 1, 2A, 2B, 2C, 2D, 2E, 3 metabolismus of xenobiotica
CYP 2G1, 7, 8B1, 11, 17, 19, 21, 27A1, 46, 51 steroid metabolism
CYP 2J2, 4, 5, 8A1 fatty acid metabolism
CYP 24 (vitamine D), 26 (retinoic acid), 27B1 (vitamine D), ...
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Flavin Monooxygenase Isoenzyme
Alkohol Dehydrogenase
Aldehyd Oxidase
Monoamin Dehydrogenase (MAO)
The redox activity is mediated by an iron porphyrin in the active center
Drug-R + O
2
CYP
Drug-OR + H
NADPH NADP
2
O
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Despite the low sequence identity between CYPs from different organisms, the tertiary structure is highy conserved.
Superposition of h CYP 2C9 (1OG5.pdb) and
CYP 450 BM3 (2BMH.pdb)
Bacillus megaterium
In contrast to bacterial CYPs, the microsomal mammalian CYPs possess an additional transmembrane helix that serves as an anchor in the membrane
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The structures of several mammalian CYPs have now been determined in atomistic detail and are available from the Brookhaven Database: http://www.pdb.mdc-berlin.de/pdb/
1DT6.pdb CYP 2C5 rabbit Sep 2000
1OG5.pdb CYP 2C9 human Jul 2003
1PO5.pdb CYP 2B4 rabbit Oct 2003
1PQ2.pdb CYP 2C8 human Jan 2004
They are suitable templates for deriving homology models of further CYPs
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The majority of CYPs is found in the liver, but certain CYPs are also present in the wall cells of the inestine
The mammalian CYPs are bound to the endoplasmic reticulum, and are therefore membrane bound.
CYP 2D6
2%
CYP 2A6
4%
CYP 1A2
13%
CYP 1A6
8%
CYP 2C6
6%
CYP distribution other
7%
CYP 2E1
13%
CYP 3
31%
CYP 2C11
16%
CYP 3
CYP 2C11
CYP 2E1
CYP 2C6
CYP 1A6
CYP 1A2
CYP 2A6
CYP 2D6 other
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Especially CYP 3A4, CYP 2D6, and CYP 2C9 are involved in the metabolism of xenobiotics and drugs. hepatic only
Metabolic Contribution
CYP 2C9
10%
CYP 1A2
2% other
3%
CYP 3A4
CYP 2D6
CYP 2C9
CYP 1A2 other
CYP 2D6
30%
CYP 3A4
55% also small intestine
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spezific substrates of particular human CYPs
CYP 1A2
CYP 2A6
CYP 2B6 verapamil, imipramine, amitryptiline, caffeine (arylamine N -oxidation) nicotine cyclophosphamid
CYP 2C9
CYP 2C19
CYP 2D6
CYP 2E1
CYP 3A4 diclofenac, naproxen, piroxicam, warfarin diazepam, omeprazole, propanolol amitryptiline, captopril, codeine, mianserin, chlorpromazine dapsone, ethanol, halothane, paracetamol alprazolam, cisapride, terfenadine, ...
see also http://medicine.iupui.edu/flockhart/
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Decision tree for human P450 substrates
CYP 1A2, CYP 2A-E, CYP 3A4
CYP 2E1
CYP 2C9 low high
Volume acidic pK a medium basic neutral
CYP 3A4
CYP 2D6
CYP 2B6
CYP 1A2, CYP 2A, 2B low planarity high medium
CYP 2A6
CYP 1A2
Lit: D.F.V. Lewis Biochem. Pharmacol. 60 (2000) 293
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„Every human differs (more or less) “
The phenotype can be distinguished by the actual activity or the amount of the expressed CYP enzyme.
The genotype, however, is determined by the individual
DNA sequence. Human: two sets of chromosomes
That mean: The same genotype enables different phenotypes
Depending on the metabolic activity, three major cathegories of metabolizers are separated: extensive metabolizer
(normal), poor metabolizer , and ultra-rapid metabolizer
(increased metabolism of xenobiotics)
Lit: K. Nagata et al. Drug Metabol. Pharmacokin 3 (2002) 167
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The polymorphismus of CYP 2D6 (debrisoquine 4hydroxylase) has been studied in great detail, as metabolic differences have first been described for debrisoquine and sparteine (antipsychotics) localized on chromosome 22
Of the 75 allels, 26 exprime CYP2D6 proteines see http://www.imm.ki.se/CYPalleles/cyp2d6.htm
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Lit: J. van der Weide et al. Ann. Clin. Biochem 36 (1999) 722
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MGLEALVPLAVIVAIFLLLVDLMHRRQRWAARYPPGPLPLPGLGNLLHVDFQNTPYCFDQ poor debrisoquine metabolism S R impaired mechanism of sparteine
LRRRFGDVFSLQLAWTPVVVLNGLAAVREALVTHGEDTADRPPVPITQILGFGPRSQGVF poor debrisoquine metabolism I
LARYGPAWREQRRFSVSTLRNLGLGKKSLEQWVTEEAACLCAAFANHSGRPFRPNGLLDK poor debrisoquine metabolism R
AVSNVIASLTCGRRFEYDDPRFLRLLDLAQEGLKEESGFLREVLNAVPVLLHIPALAGKV
LRFQKAFLTQLDELLTEHRMTWDPAQPPRDLTEAFLAEMEKAKGNPESSFNDENLRIVVA missing in CYP2D6*9 allele
DLFSAGMVTTSTTLAWGLLLMILHPDVQRRVQQEIDDVIGQVRRPEMGDQAHMPYTTAVI
P loss of activity in CYP2D6*7
HEVQRFGDIVPLGMTHMTSRDIEVQGFRIPKGTTLITNLSSVLKDEAVWEKPFRFHPEHF
LDAQGHFVKPEAFLPFSAGRRACLGEPLARMELFLFFTSLLQHFSFSVPTGQPRPSHHGV
FAFLVSPSPYELCAVPR
T impaired metabolism of sparteine in alleles 2, 10, 12, 14 and 17 of CYP2D6 see http://www.expasy.org/cgi-bin/niceprot.pl?P10635
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variability of debrisoquine-4-hydroxylation
H O H
CYP2D6
N
NH
2 N
NH
2
NH NH
= number of individuals (european population) homocygote extensive metabolizers homocygote poor metabolizers
= metabolic rate heterocygote extensive metabolizers
Lit: T. Winkler Deutsche Apothekerzeitung 140 (2000) 38
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CYP 1A2 individual: fast, medium, and slow turnover of caffeine
CYP 2B6 missing in 3-4 % of the caucasian population
CYP 2C9 deficit in 1-3 % of the caucasian population
CYP 2C19 individuals with inactive enzyme (3-6 % of the caucasian and 15-20 % of the asian population)
CYP 2D6 poor metabolizers in 5-8 % of the european,
10 % of the caucasian, and <1% of the japanese population. Over expression (gene duplication) among parts of the african and oriental population.
CYP 3A4 only few mutations
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Affymetrix (US) has developped microarrays (gene chips) using immobilized synthetic copies of P450 nucleotides, that allow the identification of all clinically relevant allelic variants.
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A series of xenobiotics have been identified, that lead to increased expression of enzymes of the CYP3A family.
Indinavir efavirenz cyclosporin carbamazepine atorvastatin tamoxifen antiviral antiviral immuno-suppressant antispychotic
HMG CoA Reductase Inhibitor anti-hormone
These bind to the pregnane X receptor (PXR) which is the transcription factor for the regulation of the CYP3A gene expression.
Lit: T.M. Wilson et al. Nature Rev. Drug Disc. 1 (2002) 259
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The PXR receptor functions together with the retinoid X receptor (RXR) as a heterodimer.
CYP3A induction leads to an increased metabolism of the administered substance due to upregulated enzymes.
This can cause adverse reactions, like inflammation of the liver (hepatitis).
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As a specific, endogen activator of RXR, 5 b
-pregnane-
3,20-dione has been identified.
In contrast, PXR is much less specific and is activated by glucocorticoids as well as by anti-glucocorticoids.
Conversely, the unspecific constitutive androgen receptor
(CAR) is found in the cytoplasm and dimerizes with PXR in the nucleus. Analog to PXR, the CYP2B gene is regulated.
Likewise high sequence homology has been found for the vitamine D receptor (VDR) that regulates CYP27, and for the arylhydrocarbon receptor (AHR) (dioxin receptor).
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These nuclear receptors all belong to a family of transcription factors. Each one possess a double zinkfinger DNA-binding domain (DBD), and a larger ligand binding domain (LBD) which is carboxy terminal.
They have been called orphan nuclear receptors as their ligands have been found later.
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Contribution to the human genome and number of marketed drugs
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H O
O
O
O hyperforin, a natural ingredient of
St. John‘s wort (Johanniskraut,
Hypericum performatum ) exhibits the highest measured affinity to
PXR (K d
= 27 nM) so far.
Application: remedy against cholestasis, mild antidepressant
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X-ray structure of PXR with bound hyperforin (1M13.pdb)
Lit: R.E. Watkins et al. Biochemistry 42 (2003) 1430
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CYP 1A2 omeprazole, insulin, aromatic hydrocarbons
(cigarette smoking, charbroiled meat) causes increased caffeine level in the plasma, if you quit smoking.
CYP 2C9
CYP 2C19
CYP 2D6
CYP 2E1 rifampicin, secobarbital carbamazepine, prednisone dexamethason ethanol, isoniazid
CYP 3A4 sulfadimindine, glucocorticoide, phenobarbitone, rifampicin, nevirapine, nevirapine, sulfinpyrazone, troglitazone
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CYP 1A2
CYP 2C9
CYP 2C19
CYP 2D6
CYP 2E1
CYP 3A4 cimetidine, ciprofloxacine, enoxacine...
grapefruit juice (naringin, 6‘,7‘-dihydroxybergamottin) chloramphenicol, amiodarone, omeprazole,...
fluoxetine, fluvastatin, sertraline,...
fluoxetine, paroxetine, quinidine, haloperidol, ritonavir,...
disulfiram, cimetidine,...
cannabinoids, erythromycin, ritonavir, ketokonazole, grapefruit juice see also http://medicine.iupui.edu/flockhart/
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