III. Conformational shifts at the benzodiazepine receptor related to the binding of agonists antagonists and inverse agonists

Life Sciences, Vol. 39, pp. 1947-1957 Printed in the U.S.A.

Pergamon Journals

CURRENT TOPICS: Benzodiazepine Receptor Function

i i i . CONFORMATIONAL SHIFTS AT THE BENZODIAZEPINE RECEPTOR RELATED TO THE BINDING OF AGONISTS ANTAGONISTS AND INVERSE AGONISTS R. Ian Fryer

~+

, Ch. Cook, N.W. Gilman and A. Walser

Pharmaceutical Research and Development, Hoffmann-La Roche Inc., Nutley, New Jersey 07110

The characteristic differences in in vivo activity for agonist, antagonist and inverse agonist ligands has been well c-ha~terized for compounds acting at the benzodiazepine receptor (BZR) component of the benzodiazepine-picrotoxinin-GABA receptor supramolecular complex LFIG. lJ (1). At the receptor level in

SPECTRA AGONIST

OF ACTIVITIES

ANTAGONIST =

INVERSE

AGONIST

Convulsant

Anticonvulsant

~

Antianxiety

~

Anxiogenic

Sedative

~

Stimulant

Amnestic

~

Cognition

Improver

?

Ataxic Ethanol

Potentiator

=

Muscle

Relaxant

~

=

2 2

FIG. 1 Biological Properties of Compounds Acting as Agonists or Inverse Agonists at the BZR. (Antagonists, by themselves, do not exhibit observable biological effects).

*To whom all inquiries should be addressed +Present address, Dept. of Chemistry, Rutgers, The State University of N.J., 73 Warren St., Newark, NJ 07102.

0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.

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vitro studies have shown that BZ agonists enhance the binding of GABA to its receptor, inverse agonists inhibit GABA binding while antagonists which reverse both of these effects have no influence on GABA binding (2). The reverse effect, that is binding of the ligands to the BZR in the presence or absence of GABA, follows the same pattern. The affinity of agonists is enhanced by GABA, the affinity of inverse agonists is decreased in the presence of GABA, and there is no effect of GABA on the affinity of antagonists (3,4). It has been postulated that these differences in activity are due to conformational changes effected at the BZR by the various ligands (5,6,7). We have previously proposed a model, in which there are two major sites, for binding of ligands at the BZR based a structural correlation of compounds (both benzodiazepines and non-benzodiazepines) which have IC50 values in the nM range [FIG. 2 and 3] (8,9).

H~C

CI

~Cl Cl

F

OaN

Midazolarn

Oiazepam H

ci H3 0 ~1

N

~N F

RO 11- 6 8 9 6

°TI F

~N

N\CH3

w2C=O N

Q;

-,

Ro15"1788

Ro22-8515

CH~ Zopiclone

~

© N H3

~

HN-N

0 ~1

CF3 CL218,B72

CGS B216

OEt

~

~"0~

H B-CCE

FIG. 2 Structures of Compounds Used in Correlations Shown in Fig. 3 This correlation was carried out for a large number of compounds by the superpositioning of one molecule on another, and was accomplished with the aid of computer graphics analysis. We examined both molecular volumes and surface potentials omitting from consideration as primary binding sites portions of structure that were shown to be unnecessary for binding activity (10). Only in vitro binding was considered as a prerequisite for inclusion in these studies and aK-tl s ~ o i n t , no attempt was made to make correlations based on in vivo profiles of activity. From this study it was shown that a three dimensional r e - T a t ~ h i p exists for all compounds examined to date, in which an aromatic or heteroaromatic ring is spatially related to a proton accepting group. The aromatic ring probably is involved in-IT, T stacking interactions with the aromatic rings of amino acid side chains within the receptor. If this ring (called 'A') is placed in the xy plane of a set of cartesian coordinates

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with its center at (0,0,0) the proton attracting group or groups for active compounds, always have position values for the x, y and z coordinates. While it is possible that only one proton donating

SCHEMATIC REPRESENTATION OF MODEL FOR RECEPTOR BINDING X

y

z

Mid A to ~'t

Midazolam Diazepam Roii-6896 Ro21-8515 Ro21-9187 Ro15- 8670 Ro15-8670 Ro15-1788

3.38 4.33 4.47 5.72 6.38 6.38 6.36 6.19

2.15 2.10 1.94 2.07 1.39 1.59 3.44 5.64

0.60 0.99 0.58 1.19 1.64 1.64 0.95 1.17

3.85 4.91 4.91 6.20 6.73 6.75 7.29 7.28

Zopiclone

5.45 5.67 5.98 6.50

2.52 2.13 1.18 3.72

0.36 0.49 0.61 0.61

6.02 6.07 6.12 7.51

5.57

2.00

0.90

6.13

COMPOUND

Three-dimensional representation of the binding sites for benzodiazepine ligandreceptor interaction. i

@

CL218,872 Distances shown are measuredCGs 8216 from the center of the aromatic ring (A) to the center CCE of the n"1-system. Average

FIG. 3 Structural Model for Ligand Binding to the BZR group ( S H , NH 2, OH, imidazo NH) is involved in hydrogen bonding with the~ portion of the ligand, it sometimes seems intuitively better to invoke two such interactions as indicated below for antagonists-inverse agonists. Although the absolute stereochemical requirements for a positive ' z ' value are always preserved, it is apparent that the relative distance (measured in A) f r o m the center of the 'A' ring to the proton accepting group(s) can change.

RELATIONSHIP AND

OF MID A / T r I D I S T A N C E (~)

CONVULSANT

ACTIVITY

Activity Agonists Anticonvulsant

.

Antagonists Inverse

Agonists, Convulsants

-ll-

x

× I

7-12 I I

I

I 2

I

I 5

I

I 4

t

J 5

MID A ~ ' r r

I

I 6

I

I 7

I

I 8

I (,~)

FIG. 4 Graphic Representation of in vivo Activity of Ligands vs Mid 'A' to -Dis--t-~ces (A)

I

I 9

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We have related this variability in distance to in vivo activity as shown in Fig. 4 (9). We are not aware of any compounds that exh~it antagonist or inverse agonist activity with a mid A to - ~ distance of less than 6A. Furthermore all of these compounds seem to have only one proton accepting ( ~ - , ) group. As this distance increases the in vivo activity profile shifts from gonist to antagonist to inverse agonist. Simile-? o-b-~ervations ave been made and reported independently by Codding et.al. (11). Distance overlaps, shown by the dotted lines in Fig. 4, indicate that compounds may exist which have either mixed agonist/antagonist or mixed antagonist/inverse agonist activity. Such compounds will exhibit an agonist (or inverse agonist) pharmacological profile for some of the activities shown in Fig. 1 and an antagonist profile for other activities. An example of a compound with mixed agonist/antagonist activity (a partial a g o n i s t ) i s the pyrazoloquinoline derivative CGS 9896 LFig. 5J. (11,12,13) An example of a compound with a mixed inverse agonist/antagonist profile (a partial inverse agonist) is the ~ -carboline methyl ester (CCM) (14). Such compounds can be rationalized on the basis that these structures are capable of binding to the receptor in more than one way (conformation).

×

HN---N

CIBA-GEIGY COMPOUND

X

PROFILE

CGS 9 8 9 6 CGS 8216 CGS 9 8 9 5

CI H OCH3

Agonist Antagonist Mixed

FIG. 5 Reported Activities of CGS Compounds This concept was an important provision of our predictive model for activity at the BZR (9). As an alternative we had also proposed that other molecules could achieve the same e f f e c t by allowing the ~-1 binding group sufficient rotational flexibility to bind independently to either one of two different proton sources or to one proton source in two different ways. In both cases the two A to "ill distances would be different, one corresponding to agonist and one to antagonist activity. These differences would then cause a conformational change within the receptor. An

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example of the second type of compound (rotational flexibility) is demonstrated by the compounds shown in Fig. 6. The amides related to Ro 21-9187, all exist in the "cis" configuration as shown, due in part to hydrogen bonding with the imidazole nitrogen.

H.N,.H

_/CH2CH3

,., r 1

Ro2 I-9187

Ro 15-8670

Ro 15-8670

"cis"

"cis"

"trons"

FIG. 6 'Cis' and ' T r a n s ' F o r m s of E s t e r s In addition there is steric interference in rotating the amide protons past the methylene protons of the 7-membered ring. This is not true of the ester in which there are no energy d i f f e r e n c e s that would preclude either ' c i s ' or 'trans' configuration. ( T h e mid A to -fit distances for both conformations of the ester Ro 15-8670 are shown in the table, Fig. 3). In t e r m s of in vivo activity, all amides that bind to the receptor and have in vivo activity are ago-fl-ist-~-,while the in vivo active esters can be agonist, antagonisT or mixed agonist/antagonists. Althougl:i-we---[iave not carried out the analysis, we would predict that the d i f f e r e n c e s in profile seen with the CGS compounds [Fig. 5] are probably due to the ability of these rigid molecules to bind in more than one conformation. A question still to be answered is whether the receptors for these different biological activities exist in different parts of the brain in which each different lipophilicity of the two f o r m s might be important or whether the receptor complex for different activities exists in a different mi]ieu in different membranes. The question of heterogeniety of receptors is probably not answerable by these studies. A testing of the hypothesis concerning the ' c i s ' and 'trans' compounds is currently under investigation and involves the synthesis of rigid analogs of both configurations followed by an examination of the biological profile. Evidence confirming the occurrence of a conformational shift within the receptor for agonist and antagonist has been given by an examination of the steric requirements for the two types of activity. By f a r the most subtle and yet the most convincing data are the substituent e f f e c t s at the 1-position of the imidazo ring [fig. 7]. Agonists will

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Conformational Shifts at the BZR

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support almost any substituent (type and size) at this position without deleterious effects in activity. Antagonists lose all in vitro binding activity and (of course) in vivo activity by substituting hydrogen for m - g t ~ r any other substituent.

THE EFFECT OF 1-ALKYL SUBSTITUENTS ON IMIDAZO- 1,4-BENZODIAZEPINES

~.~COOEt R

R"~,~N/~ - COOEr

N

~o"

NN~cHCIOE

cl

Binding Activity

R=H

R=CH3

R =H

R = CH3

yes Antagonist

no

yes Antagonist

no

R = H

yes Antagonist

R =

CH 3

no

FIG. 7 E f f e c t s of 1-methyl Substituent on Binding Activity of Antagonists A second difference is noted for the 4-5-6 portion of the molecule [Fig.8]. Antagonists can support almost any variation in structure in this region whereas agonists are sensitive to relatively small structural changes. For example, while it is

[3H]Diazepam Binding, IC5o (nM)

~ eam~

X

L CI NO2

I 7.4 1.8

I ~0 4.6

~

I" 51

1.45

H"I H 18.5 3.9

Diazepam, Midazolam

allowed

allowed

not

allowed

not allowed

not allowed

FIG. 8 E f f e c t s of Changes in the 4-5-6 l'ositions for Agonists and Antagonis~

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known that the 6-phenyl substituent is not a primary requirement for binding, when this ring is present, agonists will not support a 4'-substituent. For agonists it would appear that for steric reasons the receptor will allow only a proton at that site (e.g. Ro 22-8476 vs Ro 22-9395). Antagonists are not conformationally constrained in this region and 6-(4'-substituted phenyl) groups have no effect on binding or on activity, (e.g. Ro 15-1624 vs. Ro 22-9735) [Fig. 9]. EFFECT

OF p-SUBSTITUENT N N~COR

Y

Hoffmonn- Lo Roche COMPOUND

R

X

Ro22-9735 Ro 15-1624 Ro22-9395 Ro22-8476

OC2H5 OC2H5 NH 2 NH 2

Y

Z

IC5o (nM)

METRAZOLE

H Q H H H 0 H H

H H CI H

7.4 7.2 >1000 45

>100 >100 9

FIG. 9 E f f e c t of 4'-substituent on Agonist and Antagonist Activity Structural correlations for the inverse agonists have not been examined thoroughly due to the lack of a sizeable data base. However, as an extension of the three dimensional model we have predicted that compounds that have an extended A to IT1 distance would cause a different (third) conformational change within the

POSSIBLE CONFORMATIONAL RELATIONSHIP AT A BZ RECEPTOR (e.g. ANXIOLYTIC )

AGONIST

ANTAGONIST l

//

CONFORMATION 1 b¿ ENHANCESGABA I C)" ANXIOLYTIC

~

INVERSE AGONI~T\

[ 5 CONFORMATION a ) ~2 I] ~ la) [ C~ONFORMATO IN CHANGEFROMBZR [c) b) NOEFFECTON GABA [ [ b) INHIBITS GABA NOBIOLOGICALEFFECTJ It) ANXIOGENIC

FIG. 10 Proposed Model for Interactions of Agonist, Antagonist and Inverse Agonist Ligands at the BZR

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receptor, the e f f e c t of which would be the known inhibition of GABA binding to its receptor finally resulting in the inverse agonist activity. The interrelationship between these three conformational f o r m s of the receptor has been discussed earlier (9) and is shown schematically in Fig. 10. An insight into the function of the phenyl substituent on the diazepine ring ( I I 2 in Fig. 3), that of orientation of the molecule within the receptor cleft, has been given by a computer graphics analysis of two of the /3-carbolines shown in Fig. 11. It has been observed that the inverse agonist activity of compounds related to / ? carboline carboxylic acid ethyl ester (CCE), could be changed to an agonist profile, by the addition of a benzyloxy substituent at either the 5 or the 6 positions (ZK 91296 and ZK 93423 respectively) of the .8-carboline ring (13).

R3 COR4 R I ~ N H SCHERING/FERROSAN COMPOUND ZK ZK ZK ZK

9.3425 95426 90886 91296

FG 7142

R1

R2

OCH2C6H5 H H OCH(CH3) 2 H OCH3 H OCH2C6H 5 H

H

R3 CH2OCH3 CH3 CH2CH3 CH2OCH3 H

PROFILE

R4

OCH2CH3 Agonist OCH2CH3 Antagonist OCH2CH3 Inverse Agonist OCH2CH3 Partial Agonist NHCH3

Partial InverseAgonist

FIG. 11 Reported Biological Activity of f ~ - c a r b o l i n e Analogs (13) The molecular modelling study for the two CCE analogs was carried out using the Roche Interactive Molecular Graphics (RIMG) system (16), with Ro 21-8384 [the 6-(2'-chlorophenyl) analog of Ro 21-9187, Fig. 5] as the representative agonist benzodiazepine ligand. The atomic coordinates were taken f r o m the X-ray structure, but for this study the ( ~ 2 ) 6-phenyl ring was rotated by 130° to improve the fit for the two molecules. The c-oordinates for ZK 91296 and ZK 93423 were built from the X-ray structure of ..O-CCE using SYBYL and optimized using Allinger's MM2 program. The ethyl ester group for each carboline was changed to an amide in order to simplify the match between heteroatoms of the side chains. For this study, it was assumed that neither the d i f f e r e n c e s in side chain length nor hydrogen bonding of the side chains was essential, and thus, the difference between amide and ester groups would not significantly alter the graphical analysis. Matching was done by requiring that the amide nitrogen atoms for both molecules and also the ring nitrogen atoms of the /a-carbolines (N2) and the imidazo ring (N2) of the BZ overlap. This matching would account in a general way for possible hydrogen

Vol. 39, No. 21, 1986

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Shifts at the BZR

1955

bonding at the receptor. Flexibility of the side chains was allowed and a volume matching was carried out which required the ~-carboline to fit within the Van der Waal's envelope of Ro 21-8384 (which was held fixed). The resulting fit is shown in Fig. 12 and Fig. 13. In Fig. 12 the two fi~-carbolines are shown on the left while Ro 21-8384 is on the right. The orientations of each molecule are such that the best fit is depicted when one structure is superpositioned on the other.

FIG. 12 Matched Conformations of ZK Ro 21-8384 (right) in a Similar Bottom: Matched Conformation of ZK Ro 21-8384 (right) in a Similar Top:

91296 (left) with Orientation 93423 (left) with Orientation

This overlay is shown in Fig. 13 where the solid figure structure represents the /3carboline and the dashed-line structure depicts Ro 21-8384. Both matches show a high degree of overlap between the A and D rings of Ro 21-8384 and the six membered ring o f the carboline. The center of the A ring of Ro 21-8384 is 0.54 A f r o m the center of the corresponding ring of ZK 91296. For ZK 93423, this distance is only 0.34 A. The methylene carbon of the methoxymethyl moiety overlaps the methylene carbon of the seven membered ring. T h e r e is only a moderate overlap of the 31-2 ring and the phenyl of the benzyloxy side chain. The distances f r o m the centers of these rings are 1.09 A for ZK 91296 and 1.45 A f o r ZK 93423. In addition, the orientation o f the phenyl group in the carbolines is somewhat different f r o m the ~'2 ring of Ro 21-8384. The total Van tier Waal's volume occupied by the

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1986

two phenyl rings in this region was calculated and it was found that 65.7% of this volume was shared by the two rings for ZK 91296, 66.5% for ZK 93423. For a nonspecific hydrophobic interaction, the overlap of the two structures may be sufficient Although the degree of overlap in this region is only moderate, the non-overlapping

sl

#it

•

\X,,', FIG. 13(a) Superposition of ZK 91196 (solid line) with Ro 21-8384 (dashed line)

Z %4

FIG. 13(b) Superposition of ZK 93423 (solid line) with No 21-8384 (dashed line)

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1957

region of the benzyloxy sidechain does not extend into the region (forbidden for agonists) associated with substitution at the 4' position of the Tf 2 ring. The extension of the molecular volume in the region of the carboline-pyrrole nitrogen past that of Ro 21-8384 is in the 'allowed' region for substituents in the agonist conformation of the receptor. It is apparent from an examination of all of the above data that it is possible to interrelate all compounds that bind to the BZ-GABA receptor complex to fit a three dimensional dynamic model, which by changes in the spatial distance between binding sites, can force conformational shifts within the receptor, thus affecting GABA binding, the C1- channel and hence a biological (pharmacological) response as either an agonist, an antagonist or an inverse agonist. Some of the steric effects (requirements) of the conformational change from agonist to antagonist have been elucidated. REFERENCES 1. M. KAROBATH, P. SUPAVILAI and P.A. BOREA, in Benzodiazepine Recognition Site Ligands: Biochemistry and Pharmacology, G. Biggio and E. Costa, Eds. Raven Press, New York, pp. 37-45, 1983. 2. W. HAEFELY, Neuroscience Letters 47, 201-206 (1984). 3. C. BRAESTRUP, R. SCHMIECHEN, IVE.NIELSEN and E. N. PETERSEN, Science 216, 1241-1243 (1982). 4. H. MOHLER and J. G. RICHARDS, Nature 294, 763-765 (1981). 5. H. MOHLER, in Benzodiazepine Recognition Si~e Ligands: Biochemistry and Pharmacology, G. Biggio and E. Costa, Eds. Raven Press, New York, pp. 47-56 (1983). 6. H. MOHLER and J. G. RICHARDS, Psychopharmacology Bulletin 18, 10-12 (1982). 7_ P. POLE, E. P. BONETTI, R. SCHATTNER and W. HAEFELY, NaunynSchmiedeberg's Arch. Pharmacol. 321, 260-264 (1982). 8. R. I. FRYER in The Benzodiaz-g~nes, from Molecular Biology to Clinical Practice, E. Costa, Ed., Raven Press, New York, pp. 7-20, 1983. 9. R. I. FRYER, N. W. GILMAN, V. MADISON, and A. WALSER, in VIIIth International Symposium on Medicinal Chemistry, Proceedings, Vol. 2, R. Dahlbom and J. L. G. NILSSON, Eds, Swedish Pharmaceutical Press, Stockholm, pp. 265-279, 1985. 10. R. IAN FRYER, N. W. GILMAN and A. WALSER unpublished results. 11. P. W. CODDING and A. K. S. MUIR, Molecular Pharmacology 28, 178-184 (1985). 12. P. S. BERNARD, D. E. WILSON, W. BROWN, T. M. GLENN, Feder. Proceed. 42, 314 (1983). 13. C. BRAESTRUP, T. HONORE, M. NIELSEN, E. N. PETERSEN, and L.H. JENSEN, Biochem. Pharmacol. 33, 859-862 (1984). 14. C. L. BROWN and I. L. MARTINTEur. J. Pharmacol. 106, 167173 (1985). 15. L. PRADO de CARVALHO, P. VENAULT, E. CAVALHEIRO, M. KAIJIMA, A. VALIN, R. H. DODD, P. POTIER, J. ROSSIER and G. CHAPONTHIER, in Benzodiazepine Recognition Site Ligands: Biochemistry and Pharmacology, G. Biggio and E. Costa, Eds. Raven Press, New York, pp. 175-187 (1983). 16. The RIMG system was developed by Dr. K. Muller at Hoffmann-La Roche, Basle, Switzerland.