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

Fisher Emil German(1852—1919)

German organic chemistChemistry of natural compounds,

peptides, purines, carbohydrates

Nobel Prizein Chemistry

(1902)

Erlich Paul (1854—1915)

German chemotherapist and bacteriologist.

Founder of modern chemotherapy(Salvarsan).

Theory of receptors.

Nobel Prizein Physiology and Medicine

(1908)

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.

4Richards G, Schoch P, Haefely W: Benzodiazepine receptors: new vistas. Seminars in

Neuroscience 1991, 3:191–203.

Functional binding sites on the GABAA receptor

5

RECEPTORS TYPES

Principles of the agonist action on processes controlled by receptors

I – direct influence on the ion channels penetrability (N-cholinoreceptors, GABAA – 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

GABA – RECEPTORS

GABA – one of most important CNS neurotransmitters.

GABAАR – are stimulated by GABA, muscimol and isoguvacine; are competitively inhibited by bicuculine, and non-competitively– by picrotoxin; are connected with chlorine channel.

GABAВR – are stimulated by GABA, (-)-baclofen; are inhibited by faclofen; conjugate via secondary connecting systems with calcium and potassium channels.

GABAСR – are stimulated by GABA, are indifferent to bicuculine and (-)- baclofen.

7

GABAАR Targets for anxiolytics

anticonvulsantsmiorelaxantshypno-sedative agents, etc.pharmacologically and clinicallysignificant preparations.

BDR - allosteric modulatory sites on GABAАR.

MULTIPLICITY of GABAАR

- subunits, are labeled by 3Н-flunitrazepam - subunits, are labeled by 3Н-muscimol

- subunits, are cloned using m-RNAAverage molecular masses of - and -subunits are close

(within 50-57 кD)Different subunits are coded by different DNA

8

LOCALISATION OF GABAА SUBTYPES IN MAMMALIAN BRAIN

Subtypes GABAА

% of total BDR content

Pharmacologicaleffect

Neuronal localisation

1 122 60Anticonvulsive (tonic), sedative,

retrograde amnesia

Cortex, hippocampus, pallidum, striatum, cerebellum, olfactory

bulb, nucleus deep brain

2 222 15-20 Anxiolytic, myorelaxant Cortex, hippocampus, striatum

3 3n2 10-15

Cortex, hippocampus, pallidum, striatum, cerebellum, olfactory

bulb, nucleus deep brain

44n4n

5 Gyrus dentatus

5 51/32 5 Anticonvulsive (clonic) Cortex, hippocampus, spinal cord

662,32

6n5

Mediated by the effect of alcohol and barbiturates

Cerebellum

7 2 80Provide the affinity for BDR ligands

8 1+3 20

9

Ligands of CBDR

N

N

NCH3

F

ClN

NO

Br

O

O

CH3

CH3CH3

H

N

N

N

F

O

O

CH3

OCH3

Midazolam(full agonist)

Bretazenil(partial agonist)

Flumazenil (antagonist)

N

NO

O

ONN

N+

-

Ro15-4513 (partial inverse agonist)

N

S

N

N

CH3

O

O

O

CH3

CH3 CH3

Ro 19-4603(inverse agonist)

10

Ligands of CBDR – derivatives of different heterocyclic systems

Benzodiazepines

-Carbolines

Cyclopirrolones

N

NO

Cl

C6H5

CH3

NH

N

COOCH(CH3)2C6H5CH2O

N

N

N

O CO N N

NCl

CH3

O

Diazepam

Abecarnil

Zopiclon

11

Ligands of CBDR – derivatives of different heterocyclic systems

Imidazopiridines

Imidazopirimidines

Piridones

N

NC6H4CH3

CH2CON(CH3)2

CH3

N

NCOC6H5

H5C2

CH3O

CH3

N

N

CON

Cl

O

C6H5

H5C2O

Zolpidem

Divaplon

Ro 41-3696

12

Scheme of GABAА receptor R + L ↔ RL, biological response

- BD - GABA - Cl -

GABAАR: supramolecular heteropentameric assembly which forms (Cl-,

HCO3

-) anionic channel. Includes -,-,-,-,-types of subunits. -,- and

-types have several isoforms. General number of variants of subunits– about 20.

(1)2(2)22

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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)

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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

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INFLUENCE OF GABA ON THE RECOGNITION OF BDR RECEPTORS

16

INTERNAL ACTIVITY OF BENZODIAZEPINE RECEPTOR LIGANDS

Convulsants Anxiogenes Anticonvulsants Sedation

Anxiolytics

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.

0

50

100

34567891011

Muscle Contraction,

%

-lgCACh (M)

Kd

18

Dose – response curve for an agonist

Agonist produce the same maximal response as the neurotransmitter.

0

50

100

34567891011

Muscle Contraction,

%

-lgCagm (M)

19

Dose – response curve for an antagonist compound shows no response.

0

50

100

34567891011

Muscle Contraction,

%

-lgCcomp. (M)

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

0

50

100

34567891011

1 2 3 4

21

0

50

100

34567891011

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,

%

-lgCAiCh (M)

1

2

3

4

22

0

50

100

34567891011

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,

%

-lgCp.aq

45%

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DrugAffinity for

(Kі, nM)GABA-

shift

Cinazepam 72.6±0,8 1.30±0.4

3-Hydroxyphenazepam 2.48 1.54

Zopiclon 125±10.6 1.97±0.05N

N

N

O CO N N

NCl

CH3

O

N

NH

Br

O

Cl

O

OOH

O

The values Ki and GABA-shift of cinazepam 3-hydroxyphenazepam and zopiclon

N

NH

Br

O

Cl

OH

24

0,5

1

1,5

2

1 2 3 4

GA

BA

-shi

ft

Functional activity

N

NH

Br

O

Cl

NNH

OC2H5

O

3,3′-Bis-[7-bromo-5-(o-chloro)phenyl-1,3-dihydro-2Н-1,4-

benzodiazepine-2-one]amine (3)

N

NH

Br

O

Cl

NHN

NH

Br

O

Cl

Partial inverse agonist

S.Yu. Makan, К.S. Andronati, ….2005

N

NH

Br

O

Cl

OH

ethyl--carboline-3-carboxilate (β-CCE) (4)phenazepam (1) 3-hydroxyphenazepam (2)

25

-1,5

-1

-0,5

0

0,5

1

1,5

0 1 2 3 4

lgKi

lg(1/ED50)

Br

NO2

Cl

CF3

H

OCHF2

NHAc

NH2 CH3

RELATIONSHIP BETWEEN ANTICONVULSIVE ACTIVITY OF BD AND THEIR AFFINITY FOR BDR

NH

N

O

R

Log(1/ED50) = - 0,78log(Ki) +1,41; R=0,95

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O

N

N

Br

R

R

Cl

1

2

R2 = 0,8677

0

1

2

3

2 3 4 5 6 7 8

logP

log(

Ki )

R1=H, R2=OCOC5H4N

R1=H, R2=OCOC6H5

R1=H, R2=OCOCH(C3H7)2

R1=H, R2=OCOCH3

R1=H, R2=OH

R1=CH3, R2=OH

Relationship between lipophilicity of dihydro-1,4-benzodiazepine-2-ones and

their affinity for BDR

27

L2

N

N

O

ClL2

L1

S1

S 2S

3

H1

H2

Scheme of inclusive complex “pharmacophore - BDR”

H1 and H2 – Н-bonds of donor sites on receptor proteinL1 and L2 – lipophilic fragments of ligandS1 and S2 – regions of steric repulsion in ligand-binding domain of receptor

Q. Huang et al. Drug Design and Discovery, 1999, 16, 55

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Structure-activity relationship of dihydro-1,4-benzodiazepine-2-ones

N

NO

RR

R

R

R

R1

2

3

4

5

6

R1 – groups > СН3 are not favorable for increase of the activity

>C=O – play important role: interact with cationic site of the receptor

R2 – groups > СН3 – are not favorable

=N – interact with cationic site of the receptor

R3 – electronoacceptor and hydrofobic groups promote increase of the activity

R4 – substituents are not favorable

R5 – cooperative are electronoacceptor, hydrophylic groups

R6 – substituents in positions 8 and 9 are not desirable

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5-HT RECEPTORS CHARACTERISTICS

Receptors Structures Effectors Agonists Antagonists Pharmacological activity

5-HT1A 422aa,7TM Gi/o 8-OH-DPAT WAY100635 anxiolytic, antidepressive

5-HT1B 390aa,7TM Gi/o sumatriptan L694247 GR55562, SB216641 antidepressive

5-HT1D 377aa,7TM Gi/o sumatriptan, L694247 BRL15572 antimigraine

5-HT1E 365aa,7TM Gi/o - -

5-HT1F 366aa,7TM Gi/o LY334370 - antimigraine

5-HT2A 471aa,7TM Gq/11 α-Me-5-HT Ketanserin, MDL100907 anxiolytic, antidepressive, hypotensive

5-HT2B 481aa,7TM Gq/11 BW723C86,α-Me-5-HT

SB200646, SB204741 anxiolyticvasoconstrictor

5-HT2C 458aa,7TM Gq/11 α-Me-5-HT hypnotic

5-HT3 478aa, α-subunit, homopentamer

cation channel

SR57227,m-chlorophenylbiguanide

Mesulergine, SB242084, RS102221

neuroleptic,antiemetic, antidepressive,

analgetic

5-HT4 387aa,7TM GS BIMU8, RS67506, ML10302

Granisetron, ondasetron, tropisetron

antiarrythmic,cognitive

5-HT5A 357aa,7TM unknown - GR113808, SB204070, RS100235

5-HT5B 370aa,7TM unknown - -

5-HT6 440aa,7TM GS - RO046790 Antipsychotic,sedative

5-HT7 445aa,7TM GS - SB258719

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TRANSMEMBRANE TOPOLOGY OF 5-HT1A R

31

LIGANDS OF 5-HT1A RECEPTORS

OHN(C3H7)2

N (CH2)4

O

O

N

NNN

N (CH2)4

O

NN

OCH3

F(CH2)3

O

N

NHN

C6H5

O

8-OH-DPAT

BUSPIRONE

WAY-100635

SPIPERONE

INVERSE AGONIST

ANTAGONIST

PARTIAL AGONIST

FULL AGONIST

32

AFFINITY TO 5-HT1A AND D2 RATS HEAD BRAINS RECEPTORS AND ANXIOLYTIC ACTIVITY OF N-(ARYLPIPERAZINYLALKYL)PHTALIMIDES

NNcomp

R n Affinity (Ki), nM Selectivity Anxiolytic activity (number of punished water intakes)

5-HT1AR D2R

1 H 3 746,2±31,7 907,8±91,0 1,3

2 H 4 10,1±0,9 187.8±11,8 18,6 52 ± 11,1

3 o-Cl 4 5,2±0,5 226,7±20,0 43,6 54,1 ± 11,4

4 o-CH3 4 73,1±0,8 693,3±57,2 9,5 37,3 ± 15,4

5 m-CH3 4 128±10,6 413,0±39,0 3,2 27,1 ± 7,4

6 n-CH3 4 371,5±36,2 710,0±70,0 1,91

7 o-Cl 5 6,9±0,5 627,4±58,2 92,3 70 ± 17,3

8 o-CH3 5 13,1±0,9 743,5±69,7 56,9 68,8 ± 19,7

9 m-CH3 5 252±19,8 300,0±21,0 1,0 22,8 ± 6,9

10 n-CH3 5 163±13,5 198,0±13,1 1,2 27,4 ± 6,6

11 o-Cl 6 14,3±1,3 411,6±40,8 29,0 67,2 ± 15,4

12 o-CH3 6 91,5±7,2 534,0±54,7 5,8

Buspirone 16 ± 2 53 ± 17

Control 11 ± 5

N OO

N

N

R

(CH2)n

S.A. Andronaty, S.G. Soboleva, S.Yu. Makan Chem.-Pharm. Zhurn. – 2003. – Vol. 37. – N. 1, – P. 17-21

33

THE ANXIOLYTIC ACTIVITY OF N-(ARYLPIPERAZINYLALKYL)PHTHALIMIDES AND THEIR

AFFINITY FOR 5-HT1A- AND FOR D1-RECEPTORS

N

NR

N OO

(CH2)n

Substances R nNumber of punished

water intakes

-log Ki

5-HT1A D1

1 H 1 32 13 4.70 4.51

2 H 2 35 10 5.02 4.71

3 H 4 52 11 8.01 5.82

4 Cl 4 54 11 8.29 6.05

5 Cl 5 68 17 8.17 6.58

6 CH3 5 65 20 7.89 6.44

7 Cl 6 67 15 7.86 7.03

Buspirone 53 17 7.83 4.93

Control11 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

THREE-POINT MODEL OF THE PHARMACOPHORE FOR BINDING BUSPIRONE ANALOGS AT 5-HT1A R

Z.Chilmonczyk,… Arch.Pharm.Med.Chem., 1997, 330, 146

35

PHENYLPIPERAZINYLBUTYLBARBITURIC ACIDS

N N

O

RRO O

R

NN

C6H5

NN C6H51 2

3 R = C2H5, C6H5

R = C2H5, C6H5,CH(CH3)C3H7

R = H, CH3, (CH2)4

Maximum affinity for 5HT1A R (Ki =1.26 nM) and anxiolytic activity (107 8.1 number of punished water intakes)

N N

O

C2H5H5C2

O O

H

NN

C6H5

Anticonvulsant activity (pentamethylentrazol) ED50 = 135 (122,5 – 142.0)mg/kg

Hypnotic activity (ED 99 = 80 mg/kg)

S. Andronati, S. Makan …, Chem. Pharm. J., 2002T. Karaseva…, Voprosi Biolog. i Med. Chem., 2005