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 reakció típusai • Hidrolízis • 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. 22 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.
23
gyógyszer- szubsztrát arány %
induktor
gátlószer
farmakogenetika
24
Benzodiazepine scaffold Marketed drugs • • • •
Diazepam Flunitrazepam Midazolam Lorazepam etc. (sedative, hypnotic, anticonvulsant)
2
R
O N 3
1
R
R
N 4
R
(Plunkett, Ellman, 1995)
11,200 member library
25
Hydantoin scaffold Marketed drugs • Phenytoin (antiepileptic)
40 member library
R4 O R1 R2
N
O N R3
(De Witt et al., 1993) 26
β-Lactam scaffold Marketed drugs • Penicillins • Cephalosporins (antibiotics) 1760 member library
R2 R3 R1
R4 N
O
R5
(Gordon et al, 1996) 27
Penicillin - cephalosporin
Tiazolidin-βlactam
Tiazin- βlactam 28
Benzofuran scaffold Marketed drugs • Amiodarone (antiarrhytmic) HOOC
R O
(Fancelli et al, 1997) 29
Amiodarone - dronedaron
30
Indole scaffold Marketed drugs CONH2 • Indomethacine • Etodolac (antipyretic, antiinflammatory)
R1 N R2 (Zhang, Maryanoff, 1997)
31
Quinoline scaffold Marketed drugs • Nalidixic acid • Ciprofloxacine (antibacterial) 4000 member library
R2
N R1 R3 O
N H
NH2 O
(Gopalsamy, Pillai, 1997, Pei et al,1997) 32
Purine scaffold Marketed drugs • Mercaptopurine (anthelmintic) • Acyclovir (antiviral) • Didanosine (anti-HIV) • Vidarabine, etc (anti herpes)
NHR1 N
N R2HN
N
N R3
((Gray et al, 1997) 33
Pyrimidine scaffold Marketed drugs
COX • Zidovudine • Stavudine (anti-HIV) • Sorivudine (anti herpes)
N R1
N
R2
(Obrecht et al, 1997) 34
Isoquinoline scaffold Marketed drugs • Prasiquantel (anthelmintic) • Papaverine • Drotaverine (NoSpa) (smooth muscle relaxant)
O X
N
R
CONH2 (Goff, Zuckermann, 1995) 35
Pyrazolone scaffold Marketed drugs R2 • Phenylbutazone • Aminopyrine (analgesic, antiinflammatory)
O
R1 N
N
(Tietze et al, 1997) 36
Clopidogrel – P2Y12 antagonista trombocita aggregáció gátló
tienopiridin 37
Nelfinavir (HIV proteáz gátló)
38 (3S,4aS,8aS)-N-tert-butyl-2-[(2R,3R)-2-hydroxy-3-[(3-hydroxy-2-methylphenyl)formamido] -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 39 -{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido -1,6-diphenylhexan-2-yl]carbamate (MW=721)
