Eredeti gyógyszerkutatás ELTE TTK vegyészhallgatók számára Dr Arányi Péter 2009 április, 5.ea. Szerkezet optimalizálás (II.) 1
In vivo speciális farmakológiai vizsgálatok (1/2) Célok In vitro hatások visszaigazolása állatmodellen Proof of mechanism, proof of concept Dózis hatás összefüggés Hatás időtartam Preventív vs kuratív hatékonyság Terápiás index meghatározása Ismételt adagolás 2
In vivo speciális farmakológiai vizsgálatok (2/2) Jellemzők Több állatfaj (rágcsáló, kutya stb) Validálás referens anyagokkal Akut és krónikus Többféle adagolási mód biohasznosíthatóság Problémák Prediktivitás (modell, speciesz különbségek) reprodukálhatóság 3
Hemosztázis enzimei 4
Racionális gyógyszertervezés 5
Racionális gyógyszertervezés 6
Racionális gyógyszertervezés 7
Racionális gyógyszertervezés 8
Racionális gyógyszertervezés 9
Racionális gyógyszertervezés 10
Ha van egy hatékony vegyületünk Gyógyszerkémia Stabilitás Szelektivitás Szabadalmaztathatóság Gyógyszer formázás Kompatibilitás Kioldódás Stabilitás 11
Ha van egy hatékony vegyületünk Analitika Hatóanyag tartalom Szennyező anyagok Enantiomer tisztaság Biztonságosság Biohasznosíthatóság Szöveti disztribúció Általános farmakológia metabolizmus 12
Szelektivitás vizsgálat Rokon targetek Ismert, nem rokon molekuláris célpontok és regulátor molekulák 13
Kioldódás vizsgálat 14
15
16
17
18
A gyógyszer metabolizmus fő Hidrolízis reakció típusai Oxidáció Fázis 1 Acetilálás Fázis 2 Glükuronidálás 19
CYP450 által katalizált rekciók 20
A cytochrom P450 ciklus 21
The P450 catalytic cycle 1: The substrate binds to the active site of the enzyme, in close proximity to the heme group, on the side opposite to the peptide chain. The bound substrate induces a change in the conformation of the active site, displacing a water molecule from the distal axial coordination position of the heme iron[1] changing the state of the heme iron from low-spin to high-spin[2]. This gives rise to a change in the spectral properties of the enzyme, with an increase in absorbance at 390~nm and a decrease at 420~nm. This can be measured by difference spectrometry and is referred to as the "type~i" difference spectrum (see inset graph in figure). Some substrates cause an opposite change in spectral properties, a "reverse type~i" spectrum, by processes that are as yet unclear. Inhibitors and certain substrates that bind directly to the heme iron give rise to the type~ii difference spectrum, with a maximum at 430~nm and a minimum at 390~nm (see inset graph in figure). If no reducing equivalents are available, this complex remains stable, allowing the degree of binding to be determined from absorbance measurements in vitro[3] 2: The change in the electronic state of the active site favours the transfer of an electron from NAD(P)H[4]. This takes place via the electron transfer chain, as described above, reducing the ferric heme iron to the ferrous state. 3: Molecular oxygen binds covalently to the distal axial coordination position of the heme iron. The cysteine ligand is a better electron donor than histidine, with the oxygen consequently being activated to a greater extent than in other heme proteins. However, this sometimes allows the bond to dissociate, the so-called "decoupling reaction", releasing a reactive superoxide radical, interrupting the catalytic cycle[1]. 4: A second electron is transferred via the electron-transport system, reducing the dioxygen adduct to a negatively charged peroxo group. This is a short-lived intermediate state. 5: The peroxo group formed in step 4 is rapidly protonated twice by local transfer from surrounding amino-acid side chains, releasing one mole of water, and forming a highly reactive iron(v)-oxo species[1]. 6: Depending on the substrate and enzyme involved, P450 enzymes can catalyse any of a wide variety of reactions. A hypothetical hydroxylation is shown in this illustration. After the product has been released from the active site, the enzyme returns to its original state, with a water molecule returning to occupy the distal coordination position of the iron nucleus. S An alternative route for mono-oxygenation is via the "peroxide shunt": interaction with single-oxygen donors such as peroxides and hypochlorites can lead directly to the formation of the iron-oxo intermediate, allowing the catalytic cycle to be completed without going through steps 3, 4 and 5[3]. A hypothetical peroxide "XOOH" is shown in the diagram. C: If carbon monoxide (CO) binds to reduced P450, the catalytic cycle is interrupted. This reaction yields the classic CO difference spectrum with a maximum at 450 nm. 22
23
gyógyszer- szubsztrát induktor gátlószer farmakoarány % genetika 24
Benzodiazepine scaffold Marketed drugs Diazepam Flunitrazepam Midazolam Lorazepam etc. (sedative, hypnotic, anticonvulsant) R 1 R 2 N R 4 N O R 3 (Plunkett, Ellman, 1995) 11,200 member library 25
Hydantoin scaffold Marketed drugs Phenytoin R 4 (antiepileptic) O N O 40 member library R 1 N R 2 R 3 (De Witt et al., 1993) 26
β-lactam scaffold Marketed drugs Penicillins Cephalosporins (antibiotics) 1760 member library R 3 R 2 R R 1 4 N O R 5 (Gordon et al, 1996) 27
Penicillin - cephalosporin Tiazolidin-βlactam Tiazin-βlactam 28
Benzofuran scaffold Marketed drugs Amiodarone (antiarrhytmic) HOOC O R (Fancelli et al, 1997) 29
Amiodarone - dronedaron 30
Indole scaffold Marketed drugs CONH 2 Indomethacine Etodolac (antipyretic, antiinflammatory) R 1 N R 2 (Zhang, Maryanoff, 1997) 31
Quinoline scaffold Marketed drugs Nalidixic acid Ciprofloxacine (antibacterial) R 1 N R 3 R 2 4000 member library O N H (Gopalsamy, Pillai, 1997, Pei et al,1997) O NH 2 32
Purine scaffold Marketed drugs Mercaptopurine (anthelmintic) Acyclovir (antiviral) Didanosine (anti-hiv) Vidarabine, etc (anti herpes) NHR 1 N R 2 HN N ((Gray et al, 1997) N N R 3 33
Pyrimidine scaffold Marketed drugs COX Zidovudine Stavudine (anti-hiv) Sorivudine (anti herpes) N R N R 1 2 (Obrecht et al, 1997) 34
Isoquinoline scaffold Marketed drugs Prasiquantel (anthelmintic) Papaverine Drotaverine (NoSpa) (smooth muscle relaxant) X O N R CONH 2 (Goff, Zuckermann, 1995) 35
Pyrazolone scaffold Marketed drugs R 2 R 1 Phenylbutazone Aminopyrine (analgesic, antiinflammatory) O N N (Tietze et al, 1997) 36
Clopidogrel P2Y12 antagonista trombocita aggregáció gátló tienopiridin 37
Nelfinavir (HIV proteáz gátló) (3S,4aS,8aS)-N-tert-butyl-2-[(2R,3R)-2-hydroxy-3-[(3-hydroxy-2-methylphenyl)formamido] 38-4-(phenylsulfanyl)butyl]-decahydroisoquinoline-3-carboxamide (MW=568)
HIV proteáz ritonavir komplex Ritonavir=1,3-thiazol-5-ylmethyl N-[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2 -{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido 39-1,6-diphenylhexan-2-yl]carbamate (MW=721)