1. 1 INTERNAL ACTIVITY OF BIOLOGICALLY ACTIVE COMPOUNDS. LIGAND-RECEPTOR RECOGNITION A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine

2. 2 Fisher Emil German (1852—1919) German organic chemist Chemistry of natural compounds, peptides, purines, carbohydrates Nobel Prize in Chemistry (1902) Erlich Paul (1854—1915) German chemotherapist and bacteriologist. Founder of modern chemotherapy (Salvarsan). Theory of receptors. Nobel Prize in Physiology and Medicine (1908)

3. 3 DEFINITIONS The term receptor formally is defined as a cellular macromolecule that is concerne with chemical signalling between or within cells. The term recognition site(s) refers to the fragment(s) on the receptor macromolecule to which agonist bind.

4. 4 Richards G, Schoch P, Haefely W: Benzodiazepine receptors: new vistas. Seminars in Neuroscience 1991, 3:191–203. Functional binding sites on the GABA A receptor

5. 5 RECEPTORS TYPES Principles of the agonist action on processes controlled by receptors I – direct influence on the ion channels penetrability (N-cholinoreceptors, GABA A – receptors) II – indirect influence (through the G-proteins) on the ion channels penetrability or on the activity of enzymes regulating formation of secondary transmitters (M-cholinoreceptors, adrenoreceptors) III – direct influence on activity of effector enzyme of tirosinkinase (insulin receptors, receptors of series of growth factors) IV – influence on the transcription of DNA (steroidal hormones, tireoidal hormones)

6. 6 GABA – RECEPTORS GABA– one of most important CNS neurotransmitters. GABA А R– are stimulated by GABA, muscimol and isoguvacine; are competitively inhibited by bicuculine, and non-competitively– by picrotoxin; are connected with chlorine channel. GABA В R– are stimulated by GABA, (-)-baclofen; are inhibited by faclofen; conjugate via secondary connecting systems with calcium and potassium channels. GABA С R– are stimulated by GABA, are indifferent to bicuculine and (-)- baclofen.

7. 7 GABA А R Targets for anxiolytics anticonvulsants miorelaxants hypno-sedative agents, etc. pharmacologically and clinically significant preparations. BDR - allosteric modulatory sites on GABA А R. MULTIPLICITY of GABA А R  - subunits, are labeled by 3 Н-flunitrazepam  - subunits, are labeled by 3 Н-muscimol  - subunits, are cloned using m-RNA Average molecular masses of  - and  -subunits are close (within 50-57 кD) Different subunits are coded by different DNA

8. 8 LOCALISATION OF GABA А SUBTYPES IN MAMMALIAN BRAIN Subtypes GABA А % of total BDR content Pharmacological effect Neuronal localisation 1 122122 60 Anticonvulsive (tonic), sedative, retrograde amnesia Cortex, hippocampus, pallidum, striatum, cerebellum, olfactory bulb, nucleus deep brain 2 222222 15-20Anxiolytic, myorelaxantCortex, hippocampus, striatum 3 3n23n2 10-15 Cortex, hippocampus, pallidum, striatum, cerebellum, olfactory bulb, nucleus deep brain 4 4n4n4n4n 5Gyrus dentatus 5  5  1/3  2 5Anticonvulsive (clonic)Cortex, hippocampus, spinal cord 6  6  2,3  2  6  n  5 Mediated by the effect of alcohol and barbiturates Cerebellum 7 22 80 Provide the affinity for BDR ligands 8 1+31+3 20

9. 9 Ligands of CBDR Midazolam (full agonist) Bretazenil (partial agonist) Flumazenil (antagonist) Ro15-4513 (partial inverse agonist) Ro 19-4603 (inverse agonist)

10. 10 Ligands of CBDR – derivatives of different heterocyclic systems Benzodiazepines  -Carbolines Cyclopirrolones Diazepam Abecarnil Zopiclon

11. 11 Ligands of CBDR – derivatives of different heterocyclic systems Imidazopiridines Imidazopirimidines Piridones Zolpidem Divaplon Ro 41-3696

12. 12 Scheme of GABA А receptor R + L ↔ RL, biological response - BD - GABA - Cl - GABA А R: supramolecular heteropentameric assembly which forms (Cl -, HCO 3 - ) anionic channel. Includes  -,  -,  -,  -,  -types of subunits.  -,  - and  - types have several isoforms. General number of variants of subunits– about 20. (1)2(2)22(1)2(2)22

13. 13 TWO-STATE MODEL Two-state model to explain the bidirectional modulatory effects of benzodiazepine (BZR) receptor ligands on GABA A receptor channel function. Fragments of two subunits with their interphase are shown. The two inerconvertible states, one with a positive allosteric influence, the other with negative allostericinfluence, oscillate with the rate constants  and . BZR agonists are shown to fit the positive modulatory state. BZR inverse agonists fit into the negative modulatory state. Antagonists bind equally well to both states. The bottom diagram indicates that GABA binding to its site (GA-R) shifts the BZR into the positive modulatory state, increasing the binding of agonists and decreasing that of inverse agonists without affecting that of pure antagonists (so-called GABA shift) (W.E. Haefely, The Challenge of Neuropharmacology, 1994, 15)

14. 14 INTERNAL ACTIVITY Internal activity (IА) – it is ligand ability to provoke receptor conformation changes, leading to signal transformation into physiological response. Antagonists: IA = 0 Inverse agonists: high negative IA Full agonists: high positive IA

15. 15 INFLUENCE OF GABA ON THE RECOGNITION OF BDR RECEPTORS

16. 16 INTERNAL ACTIVITY OF BENZODIAZEPINE RECEPTOR LIGANDS  Convulsants   Anxiogenes   Anticonvulsants   Sedation  Anxiolytics

17. 17 Effect of the concentration of acetycholine (neurotransmitter) on muscle contvaction. Dose-response curves are a means of measuring drug-receptor interactions and are standart method comparing the potencies of compounds that interact with particular recepor. Muscle Contraction, % -lgC ACh (M) KdKd

18. 18 Dose – response curve for an agonist Agonist produce the same maximal response as the neurotransmitter. Muscle Contraction, % -lgC agm (M)

19. 19 Dose – response curve for an antagonist compound shows no response. Muscle Contraction, % -lgC comp. (M)

20. 20 Influence of competitive antagonist on the response of the neurotransmitter 1.Without competitive antagonist (Com. Ant) 2.With Com.Ant 3.With 2 doses Com. Ant 4.With 3 doses Com.Ant If antagonist is added to the neurotransmitter (acetylcholine) effect of the neurotransmitter is blocked until a higher concentration of the neurotransmitter is added. Degree of antagonist is dependent on the relative concentrations of the agonist and antagonist of the same receptor. Muscle Contraction 12 3 4

21. 21 Influence of noncompetitive antagonist on the response of the neurotransmitter 1.Without Non Com. Ant 2.+ Non Com. Ant 3.+ 2 doses Non Com. Ant 4.+ 4 doses Non Com. Ant Degree of blocking of a noncompetitive antagonist of the amount of agonist present. On this case two different binding sites may be involved. Muscle Contraction, % -lgC AiCh (M) 1 2 3 4

22. 22 Dose – response curve for a partial agonist On a case of partial agonist some response is elicited, but not a full response, regardless of how high the concentration of ligand used. Muscle Contraction, % -lgC p.aq 45%

23. 23 Drug Affinity for (K і, nM) GABA- shift Cinazepam72.6±0,81.30±0.4 3-Hydroxyphenazepam2.481.54 Zopiclon125±10.61.97±0.05 The values Ki and GABA-shift of cinazepam 3-hydroxyphenazepam and zopiclon

24. 24 Functional activity 3,3′-Bis-[7-bromo-5-(o-chloro)phenyl- 1,3-dihydro-2Н-1,4-benzodiazepine-2- one]amine (3) Partial inverse agonist S.Yu. Makan, К.S. Andronati, ….2005 ethyl-  -carboline-3- carboxilate (β-CCE) (4) phenazepam (1) 3-hydroxyphenazepam (2)

25. 25 RELATIONSHIP BETWEEN ANTICONVULSIVE ACTIVITY OF BD AND THEIR AFFINITY FOR BDR Log(1/ED 50 ) = - 0,78log(K i ) +1,41; R=0,95

26. 26 Relationship between lipophilicity of dihydro-1,4-benzodiazepine-2-ones and their affinity for BDR

27. 27 L2L2 S1S1 S2S2 S3S3 H1H1 H2H2 Scheme of inclusive complex “ pharmacophore - BDR” H 1 and H 2 – Н-bonds of donor sites on receptor protein L 1 and L 2 – lipophilic fragments of ligand S 1 and S 2 – regions of steric repulsion in ligand-binding domain of receptor Q. Huang et al. Drug Design and Discovery, 1999, 16, 55

28. 28 Structure-activity relationship of dihydro-1,4-benzodiazepine-2-ones R 1 – groups > СН 3 are not favorable for increase of the activity >C=O – play important role: interact with cationic site of the receptor R 2 – groups > СН 3 – are not favorable =N – interact with cationic site of the receptor R 3 – electronoacceptor and hydrofobic groups promote increase of the activity R 4 – substituents are not favorable R 5 – cooperative are electronoacceptor, hydrophylic groups R 6 – substituents in positions 8 and 9 are not desirable

29. 29 5-HT RECEPTORS CHARACTERISTICS ReceptorsStructuresEffectorsAgonistsAntagonistsPharmacological activity 5-HT 1A 422aa,7TMG i/o 8-OH-DPATWAY100635anxiolytic, antidepressive 5-HT 1B 390aa,7TMG i/o sumatriptan L694247GR55562, SB216641antidepressive 5-HT 1D 377aa,7TMG i/o sumatriptan, L694247BRL15572antimigraine 5-HT 1E 365aa,7TMG i/o -- 5-HT 1F 366aa,7TMG i/o LY334370-antimigraine 5-HT 2A 471aa,7TMG q/11 α-Me-5-HTKetanserin, MDL100907anxiolytic, antidepressive, hypotensive 5-HT 2B 481aa,7TMG q/11 BW723C86, α-Me-5-HT SB200646, SB204741anxiolytic vasoconstrictor 5-HT 2C 458aa,7TMG q/11 α-Me-5-HThypnotic 5-HT 3 478aa, α-subunit, homopentamer cation channe l SR57227, m-chlorophenylbiguanide Mesulergine, SB242084, RS102221 neuroleptic, antiemetic, antidepressive, analgetic 5-HT 4 387aa,7TMGSGS BIMU8, RS67506, ML10302 Granisetron, ondasetron, tropisetron antiarrythmic, cognitive 5-HT 5A 357aa,7TMunknown-GR113808, SB204070, RS100235 5-HT 5B 370aa,7TMunknown-- 5-HT 6 440aa,7TMGSGS -RO046790Antipsychotic, sedative 5-HT 7 445aa,7TMGSGS -SB258719

30. 30 TRANSMEMBRANE TOPOLOGY OF 5-HT 1A R

31. 31 LIGANDS OF 5-HT 1A RECEPTORS

32. 32 AFFINITY TO 5-HT 1A AND D 2 RATS HEAD BRAINS RECEPTORS AND ANXIOLYTIC ACTIVITY OF N-(ARYLPIPERAZINYLALKYL)PHTALIMIDES NN comp RnAffinity (K i ), nMSelectivityAnxiolytic activity (number of punished water intakes) 5-HT 1A RD2RD2R 1H3746,2±31,7907,8±91,01,3 2H410,1±0,9187.8±11,818,652 ± 11,1 3o-Cl45,2±0,5226,7±20,043,654,1 ± 11,4 4o-CH 3 473,1±0,8693,3±57,29,537,3 ± 15,4 5m-CH 3 4128±10,6413,0±39,03,227,1 ± 7,4 6n-CH 3 4371,5±36,2710,0±70,01,91 7o-Cl56,9±0,5627,4±58,292,370 ± 17,3 8o-CH 3 513,1±0,9743,5±69,756,968,8 ± 19,7 9m-CH 3 5252±19,8300,0±21,01,022,8 ± 6,9 10n-CH 3 5163±13,5198,0±13,11,227,4 ± 6,6 11o-Cl614,3±1,3411,6±40,829,067,2 ± 15,4 12o-CH 3 691,5±7,2534,0±54,75,8 Buspirone16 ± 253 ± 17 Control11 ± 5 S.A. Andronaty, S.G. Soboleva, S.Yu. Makan Chem.-Pharm. Zhurn. – 2003. – Vol. 37. – N. 1, – P. 17-21

33. 33 THE ANXIOLYTIC ACTIVITY OF N-(ARYLPIPERAZINYLALKYL)PHTHALIMIDES AND THEIR AFFINITY FOR 5-HT 1A - AND FOR D 1 -RECEPTORS SubstancesRn Number of punished water intakes -log K i 5-HT 1A D1D1 1H1 32  13 4.704.51 2H2 35  10 5.024.71 3H4 52  11 8.015.82 4Cl4 54  11 8.296.05 5Cl5 68  17 8.176.58 6CH 3 5 65  20 7.896.44 7Cl6 67  15 7.867.03 Buspirone 53  17 7.834.93 Control 11  5 S.A. Andronaty, T.A. Voronina, V.M. Sava, G.M. Molodavkin, S.Yu. Makan, S.G. Soboleva. Molecular Recognition and Inclusion. – Ed. A.W. Coleman. – Kluwer Academic Publishers, Netherlands, 1998. – P. 245 – 249.

34. 34 THREE-POINT MODEL OF THE PHARMACOPHORE FOR BINDING BUSPIRONE ANALOGS AT 5-HT 1A R Z.Chilmonczyk,… Arch.Pharm.Med.Chem., 1997, 330, 146

35. 35 PHENYLPIPERAZINYLBUTYLBARBITURIC ACIDS Maximum affinity for 5HT 1A R (K i =1.26 nM) and anxiolytic activity (107  8.1 number of punished water intakes) Anticonvulsant activity (pentamethylentrazol) ED 50 = 135 (122,5 – 142.0)mg/kg Hypnotic activity (ED 99 = 80 mg/kg) S. Andronati, S. Makan …, Chem. Pharm. J., 2002 T. Karaseva…, Voprosi Biolog. i Med. Chem., 2005