Acetylcholine receptor site: a proposed model

Acetylcholine receptor site: a proposed model Rex A Palmer, Jasmine H Tickle and Ian J Tickle Department

of Crystallography,

Birkbeck

College,

Malet Street,

An acetylcholine receptor site model is proposed, based on computer graphics fitting of non-depolarising neuromuscular blocking agents to the acetylcholine molecule. The receptor surface is formed by the complementary van der Waal’s surface of one molecule (the object molecule). Fitting of the second (target) molecule into this is investigated. It is proposed that binding of acetylcholine or antagonist to the receptor protein takes places at a primary (inner) binding site (P-I) receiving the cationic head, and a secondary (outer) binding site (S-O). This provides a donor for hydrogen bond formation for receiving the carbonyl oxygen of acetylcholine and certain neuromuscular blocking agents such as pancuronium, and is also capable of forming a JI - n interaction with other neuromuscular blockers such as (+)-tubocurarine and certain steroid derivatives. Keywords: computer graphics fitting, van der Waals surface, acetylcholine receptor site model, drug design received

24 May 1983, revised 8 September

1983

The natural neurotransmitter acetylcholine (ACH) (see Figure 1) which is released from cholinergic neurons, acts upon a variety of receptors, including those at skeletal muscle endplates, smooth muscles, secretory gland cells, autonomic ganglion cells, and some nerve cell types of the central nervous system. Neurotransmitters and their macromolecular receptors attract a great deal of attention in many research fields, including studies attempting to correlate structure and function and for the optimization of drug design. Acetylcholine itself has been subjected to intense scrutiny and because it is a chemically simple substance, these studies can be undertaken in great detail. For example, a recent ab initio quantum chemical study of the cationic head’ has characterized the detailed electronic charge distribution associated with coulombic attraction to the receptor site. Recent work on acetylcholine receptors includes an elegant low-resolution electron micrograph analysis which has revealed the arrangement of crystalline arrays in membrane-bound receptor assemblies*, thus providing a picture of the four-subunit assembly. Molecular genetics studies have led to the sequencing of the (Y subunit3.4 and the cloning of the y subunit5 is expected to lead to a successful sequencing in the near futureb. However, in spite of these elegant results it seems unlikely that detailed structural studies will be possible on actual ACH receptors for many years to come.

94

0263-7855/83/04094-13

$03.00 0

Butterworth

London

WClE

7HX, UK

Figure 1 (a). Acetyfcholine (ACH) atom identification schemes: from (above) Datta et alZ4and (below) Jagner and Jensen”. Torsion angles are also indicated, t3 = C6-Ol-CS-C4, being the most variable (Table I)

Figure 1 (b). Conformation of ACH in the tetraphenzylborate complex (entry 6, Table 1; see also entries 1, 4 and 5 of Table 1)

Figure 1 (c). Conformation of ACH in the iodide (entry 3, Table 1; see also entry 2, Table 1) We are concerned in this paper with the design of drugs which act upon nicotinic transmission sites to interrupt nerve impulses at the skeletal neuromuscular junction (neuromuscular blocking agents), and the topography of the corresponding receptor site. A neuromuscular blocking agent may be depolarizing or competitive (non-depolarizing) in its mode of action. For reviews of the subject see, for example references’-“‘. Depolarizing agents are agonists which, like acetylcholine itself, stimulate the postjunctional membrane by depolarization, thus causing a contractile muscular response, but with a more persistent action which is succeeded by muscle paralysis. Such molecules which are long, flexible bisonium derivatives are not hydrolyzed by the action of acetylcholinesterase, the enzyme which degrades, and therefore terminates the action of acetylcholine. Examples of these derivatives are decamethonium,

& Co (Publishers)

Ltd

Journal

of Molecular

Graphics

(CH~)3N+-(CN2)lo-N+(CN3)3 and succinylchofine. Non-depolarizing neuromuscular blocking drugs, the classical example of which is (+)-tubocurarine (Figure 2a), have an an&gonistic mode of action associated with strong affinity for acetyIchoIine receptor molecules located at the postjunctional membrane. Drug molecules in this category are bulky and contain rigid or semi-rigid ring systems. Like the depolarizing agents they are not hydrolyzed by acetylchojinesterase, and may remain bound to the ACZ-1receptor for some time. Both depolarizing and non-depolarizing neuromuscular blocking agents always possess at least one cationic onium site, many are bisonium derivatives, some monoquaternaryimonoterdary and like some, gallamine, contain more than two such groups.

(+)-Tubocurarine, although monoqu;it~ma~y”.‘~~~?has two cationic N” sites in the halide salts and presumably also at physiological pH. Two distinct conformations of (-t-)-tubocurarine are known, related by rotation about the linkage bonds between the rigid phenyl and semi-rigid isoquinoline ring componen@. Views of the two molecules perpendicular to the AB plane clearly show this difference (see Figure 3 (a) and (b)). In contrast many potent non-depolarizing neuromuscular blocking agents are known which were derived from more rigid steroid molecules. Examples are stercuronium”,‘S (Figure 2(f)) a derivative of conessine, and pancuronium , a derivative af androstanelb. Pancuronium bromide (Figure 2b)” has Ms

F@w 2. lnhibil~rs of ihe Acelylcholine recepwr: (a) (-I-) Tubacurarine dickkwide (TCL, .i=Ci) and dibromide {TBR, / = Br); (b/ pancuronium dibromide (PAM); (c) chnndonium di-iodide {HXZXO); (d,‘ ’ I7a hydroxyethyl chundonium di-iode (HS626); Ce) Dihydrochandonium (H.5692); and (f) Stercuronium iodide (S,TERC). Funcriomzl groups arc?shaded

Volume 1 Number 4 December 1983

95

a

b

d

Figure 3. FRODO graphics superpositioning of selected molecular pairs (see also Table 3). (a) ACHTPB fitted to the quaternary end of TCL, C=O locating on the Cl4 . . . . Cl5 bond of benzene ring C. (b) As (a) but for ACHTPB TBR. (c) ACHIfitted to the quaternary end of TCL, C=O locating on the Cl2 . . . . Cl 7 bond of benzene ring C. (d) As (c) for ACHI - TBR, C=O locating near Cl7 of benzene ring C. (e) ACHTPB fitted to PANC, A ring end. (f) ACHTPB fitted to HS626, A-B end, with C=O of ACHTPB near to the C5=C6 bond in ring B. (g) As (f) for ACHI -

been widely used to produce muscle relaxation with rapid onset and medium duration in clinical anaesthesia. Another series of synthetic steroidal neuromuscular blocking agents has been produced and tested by Singh and co-workers’“.‘Y. The series includes the compound HS310 (chandonium iodide) (Figure 2c) which is 3.5 5 times as potent as ( +)-tubocurarine”~20.*‘. Structural data derived from X-ray analysis are now potent non-depolarizing available on many neuromuscular blocking agents with a variety of characteristics. From their analysis of X-ray and model building data, Pauling and Petcher proposed a design for the ideal drug in this classzZ.This was an essentially rigid bisonium derivative requiring an N’ N’ separation of 10.8 (3) A bridging a hydrophylic surface for dipole-dipole interaction with the ACH receptor. In the light of more recent conformational analysis and activity studies, Pauling and Petcher’s model appears to require updating. Earlier, Beers and Reich reported the common factor between many nicotinic acetylcholine agonists and antagonists as being a quaternary nitrogen at about 5.9A from an esterophilic

96

oxygen atomz3, the former for coulombic interaction with a complementary site on the acetylcholine receptor and the latter for formation of a hydrogen bond to a secondary site. We report in this paper attempts to correlate the known structural features of a selection of neuromuscular blockers, to compare their molecular geometry and to model features of the ACH receptor site by computer graphics analysis using an Evans and Sutherland Picture System 2. The significance of the bisonium function is discussed. The graphics analysis has been supplemented by preliminary ab initio quantum chemical calculations to establish the charge distribution and polarization of functional groups of some model compounds. The results of the calculations will be published elsewhere. MATERIALS Acetylcholine The conformational behaviour of the acetylcholine ion in different environments is known from X-ray

Journal of Molecular Graphics

e h

’

HS626. (h) Superposition of the steroidal derivative HS626 and TBR from the quaternary sites N’31 (HS626) and N+20 (TBR) to double bond C5=C6 (HS626) and the ring C Cl5 . . . . Cl6 bond (TBR). (i) As (g) but bridging TBR across to the benzene ring F. The fit is not good and would not conform to the topography of the proposed acetylcholine receptor site

structures of various crystalline derivatives (Table 1). Two different atom numbering schemes are found in the literature as indicated in Figure 1 which also shows the four torsion angles ‘cr to ‘c4 defining the molecular conformation. The torsion angle values quoted in Table

Table 1. Crystalline derivatives of acetylcboline. Abbreviations used in the text are: acetylcholine iodide (ACHI) and acetylcholine tetraphenyl borate (ACHTPB)

See reference

Anion

no

29 30 and 31

Chloride Bromide Iodide (ACHI) Perchlorate @-resorcylate

25 32 33

Tetraphenyl (ACHTPB)

borate

24

1 Number

4 December

Volume

Torsion =I 11.4 175.5 -174.8 -174.1 168.3 (a)-179.9 (b)-175.5 173.7

1983

angles =2 84.7 78.4 78.5 89.0 73.7 84.3 76.7 66.9

(degree) t4 =3 -166.9 78.9 78.5 83.0 179.5 158.8 163.4 154.6

5.2 4.1 4.7 -0.9 0.8 1.9 0.0 -5.4

1 indicate that ts is the most variable, adopting a value around 180” (t) in the presence of heavy cations (bromide or iodide) and around 80” (g) in the presence of light cations. As examples of the two cases, we have used in our graphics work the atomic coordinates derived from the two accurately determined crystal structures, acetylcholine tetraphenylborate (ACHTPB) (t3 = 154.6”) and acetylcholine iodide (ACHI) (t4 = 83”) respectively. For the tetraphenylborate the atom numbering is that of Datta et alZJwhile for the iodide that of Jagner and Jensen5 is used. Studies other than crystallographic (ie where the molecule is not in the solid state) including NMR”,” and potential energy several for indicate preference calculations*8 conformations including the two in question. It seemed reasonable, therefore, to adopt the two coordinate sets above as the starting point of our studies.

(+)-Tubocurarine-dichloride, methylated diodide salts

dibromide and fully

The bisbenzyl isoquinoline alkaloid (+)-tubocurarine has been shown to adopt different conformations in the

97

aza-steroid synthetic bisquaternary series of compounds prepared by Singh and co-workers’8*‘9. It exhibits rapid onset and short duration of action compared with (+)-tubocurarine. In addition to the ammonium functions (on rings E and D) it possesses an unsaturated double bond in ring B (C5=C6). Its fully saturated congener HS692 (Figure 2e) exhibits greatly reduced neuromuscular blocking activity compared to chandonium itself’.

dichloride and dibromide salts’2.‘3(Figure 3a, b and 4 a, b). Our graphics studies have used both the chloride and bromide forms and, where necessary, atom numbers quoted are those of Reynolds and Palmer13. The fully methylated derivative of (+)-tubocurarine, in the form of the diiodide, has been shown to have a conformation similar to that of the dibromide3,” and similar N’ N’ separation is observed of 8.9A (Table 2)12, a consequence of the changes in rotation about the linkage bonds. These observations cast doubt on the requirement of a rigid molecule with N+ N’ = 10.8 (3)A as proposed by Pauling and PetcherZ2 for non-depolarizing neuromuscular blocking activity. Steroidal neuromuscular

Hydroxy

ethyl chandonium (HS626) (Figure 2d) like the parent compound chandonium, is bisquaternary with a pyrrolidine ring attached to C(3) of ring A and with a modified ring D, having N’ in position 17a. Further modification has been effected in the compound HS626 by the addition of a hydroxyethyl function on N17a as the next step towards an acetylcholine-line fragment at the D end of the molecule. Initial tests on this compound indicated enhanced activity36, but it is now thought that HS626 has about the same potency as HS310 itselp7,3R.In view of these differing results further tests on HS626 may be required39.

blockers

Table 2 also lists neuromuscular blockers derived from steroids used in our graphics work. Crystal structure coordinates have been published for each molecule as indicated in the references given in the table. Where it has been necessary to identify particular atoms or rings of the molecules concerned these are indicated in Figure 2, and correspond to the numbering schemes in the structure references. Pancuronium

Stercuronium

iodide. (MYC 1080) is a highly potent monoquaternary derivative of conessine having a second site (N22) which may become ionized at physiological pH. Undesirable side effects have precluded its use in clinical anaesthesia14. It is included studies as an example of a in our steroidal compound monoquaternary-monotertiary whose crystal structure has been well characterized”. It has a more extended region of partial unsaturation than chandonium, spanning bonds C4 = C5 and C6 = C7 in rings A and B respectively (Figure 2f).

bromide

is a bisquaternary ammonium steroidal derivative” widely used as a muscle relaxant of rapid onset and medium duration. It has the advantage of having little effect on the cardiovascular system, does not release histamine and is free from hormonal activity. The molecule consists essentially of two acetylcholine-like fragments (on rings A and D respectively (Figure 2(b)) supported and separated by a steroid nucleus. Chandonium

a

iodide (HS310)

(Figure 2c) is one of a

/

\

b

Figure 4. FRODO stereoviews of selected molecular pairs from Figure 3. (a) as 5(a) ACHTPB - TCL; (b) as 5(b) ACHTPB - TBR; (c) as S(f)ACHI - HS626; and (d) as 5(h) TBR - The primary (P) and secondary (S) binding sites are indicated (see text)

98

Journal of Molecular Graphics

Table 2. Neuromuscular blockers used in the present study to model an acetylcholiue receptor site Compound

(+)-tubocurarine

Pancuronium Chandonium (HS310) Hydroxyethyl chandonium (HS626) Stercuronium (MYC1080)

Approximate potency ratio (tubocurarine = 1.0) 1.00 (See reference 43) 9.41 (See references 44 and 45) 3.5 to 5.0 (See references 20, 21 and 47, 48) 3.5 to 10.0 (See references 36 and 38) 0.5 (See references 14 and 51)

Class or type of compound

Bisbenzylisoquinoline alkaloid Dipiperidino androstene

Nature of ammonium functions

Both sites N’ one quaternary one tertiary (See reference 11) Bisquaternary

Crystal structure reference

Dichloride (See reference 12) Dibromide (See reference 13) Fully methylated di iodide (See reference 34) (See reference 46) (See references 49 and 50)

Pyrolidino-aza androstene As., A4,, Conessine derivative

GRAPHIC STUDIES: METHODS USED Computer graphics analysis of the molecular models in 3D was undertaken on an Evans and Sutherland Picture System 2 using the following programs: Frodo”,4’ - this program allows two molecules to be inspected at any one time and includes the following facilities: 0 Fitting of target regions within limits controlled by the user. o Calculation of distances between regions of the oriented, superposed models, selected by the user. o Plotting of the models as viewed from any selected direction. Examples of Frodo pictures are given below. BILBO@ - this is similar in operation to Frodo. It does not, in its present form, calculate molecular geometry or contact distances (which must therefore be established independently by use of Frodo) but has the following additional facilities: 0 Simulation of hydrophilicity or charge, hydrophobicity by pictorial devices such as fur or dots (Figure 5). o Preparation of van der Waal’s molecular net (see Figure 6). This facility produces on the screen a surface at van der Waal’s distance from the object molecule projected in a given direction selected by the user. The result is similar to that of pushing a CPK model held in a given orientation through an elastic net. The surface thus produced simulates the inside of an active site cleft or receptor site for the object molecule.

Bisquaternary (See reference 39)

(See reference 15) Monoquaternary

The results of this fitting procedure are summarized in Table 3. They represent a quantification of the principle of Beers and ReichZ3 which is not feasible by model-building or computation alone. Examples of other molecular pairs fitted in this way are also included and discussed below. In the four examples given involving tubocurarine, N’ of ACH has been closely superposed on the quaternary N’20 in ring B of TBR or TCL. (In these preliminary studies little attention has been paid to the alignment of substituents on the N’ atoms.) The carbonyl oxygen may then be adjusted to be close to some part of ring C, selected as shown in the table. The latter distances can be fitted more closely by sharing the deviation between the two sites. However we consider this to be unnecessary and assume that the molecular activity operates through slight adjustments in either receptor or inhibitor molecule. Similar superpositioning is possible involving N’ in ring E combined with Phenyl ring F. In the case of acetylcholine and pancuronium, (Table 3 and Figure 3) the example chosen illustrates the very good fit between the quaternary N’ sites and the C7=02 bond of ACHTPB with the C33=02 bond of pancuronium. It is evident that slight torsion angle adjustments in either or both molecules will result in a really good correspondence. It is also possible then that small

RESULTS OF FRODO GRAPHICS IMPLICATIONS FOR RECEPTOR BINDING graphics computer molecular Examples of superpositions are given below. These studies were based primarily on fitting either acetylcholine in the tetraphenylborate form (ACHTPB) or acetylcholine in the iodide form (ACHI) to the selected molecules described earlier. The initial objective was to investigate the measure of fit between the quaternary N+ sites in the molecule pairs and the carbonyl oxygen of ACH (02 in ACHTPB or 03 in ACHI) with a corresponding group of the neuromuscular blocker.

Volume 1 Number 4 December 1983

Figure 5. Van der Waal’s surfaces produced by BILBO showing superpositioning of ACH and TBR. The matching sites are ACH (N’) with TBR (N’20) (upper right) and ACH (02) with TBR (Cl4 = Cl5 in benzene ring C) (upper left). Code = ACH ‘fur’, TBR ‘dots’

99

Figure 6. Formation of a van der WuatS net for HS310, simulating a receptor surface (R RR)

conformational changes in the acetylcholine receptor site would promote the binding to pancuronium. The inherent twofold pseudo symmetry of the pancuronium molecule provides a similar fitting sequence at the D-ring end of the steroid framework (Figure 2b). We consider this an important factor in determining its drug potency. Most of the graphics fitting of steroid molecules in the chandonium series has been undertaken with coordinates of the well refined HS626 structures (Table 2, Figure 2d). As can be seen in Figure 3 (f) and (g) and 4 (c) and (d), good correspondence can be achieved between the N’ sites of ACHTPB or ACHI with HS626, the acetylcholine molecule being aligned with C6=02 (ACHTPB) or C2=03 (ACHI) with the double bond C5=C6 of HS626. The separation of atoms superimposed is s.4A but this can be easily distributed between the atoms in question to make the maximum deviation much less. Slight conformational adjustment in ACH could also improve this figure. In view of molecular rigidity, which is far more severe for atoms in the steroidal framework, there is less room for movement in HS626 or other members of this series such as HS310. Reference to Table 2 and Figure 2 indicates the essential biomolecular features of HS310, HS626 and HS692. Several important factors may now be taken into consideration. HS692 is only half as potent as HS310 yet differs only in the C5-C6 bond, which is single in HS692 and double in HS310. Both HS692 and HS310 are bisquaternary and their N’ N’ distance conforms to the range, specified by Pauling and PetcherZ3 as being a prerequisite for potent neuromuscular blocking, yet HS692 has diminished activity. This suggests the existence of a potential n - n interaction between the double bond C5=C6 region in HS310 and the supposed esterophilic group on the acetylcholine receptor. This secondary site on the acetylcholine receptor is thus required to provide a hydrogen bond donor for the carbonyl oxygen of molecules such as acetylcholine or

100

pancuronium, or to form a x, - JC interaction with molecules such as ~+)-tubocurarine (to a benzyl group) or chandonium (to the CS = C6 double bond). It also suggests that the true role of the quaternary ammonium functions is associated with binding mainly to a single site on the acetylcholine receptor (one point theory of attachment, as apposed to two point). If this is so we may imagine the non-depolarizing blocker entering the active site with the binding N’ function head first and its esterophyllic group trailing behind. The secondary N’ site (N17a in HS310 for example) may enhance the activity of the drug molecule by providing a molecule entering the active cleft the wrong way round (ie D end first) with a further binding opportunity. Binding of the N17a site of HS310 would, however, be less effective than the N31 site since it lacks an esterophilic group at this end of the molecule. HS626 has an additional hydroxyethyl group on N17a, but this appears not to enhance the activity. This supports the view that the N’ group must enter the active site first and interact with a more deeply buried receiving group in the active site. The hydroxyethyl group on N17a of HS626 with respect to the N’ site is thus incorrectly positioned for binding to the receptor site, and could well hamper the process. These ideas are supported by observations on other drug molecules. Pancuronium (Figure 2 (b) and 3 (e)) has pseudo twofold symmetry. Both the A-ring and the D-ring end of the molecule have a x - JCbonding site. This is the acetyl group which we have shown for the A-ring end to fit ACHTPB and ACHI. It is possible, similarly, to fit acetylcholine to the D end of the pancuronium molecule. Similar comments apply to the fitting of acetylcholine with tubocurarine chloride and bromide, since tubocurarine also possesses pseudo twofold symmetry. The fitting can be achieved either at the N20 (AB) end of tubocurarine involving C15=C16 of benzene ring D for JC- JGinteraction (Figure 3c,d) or from the DE end with Nl as the ammonium function and C32=C33 in ring F as the x - JC function. The operative N’ binding end of the molecule entering the active site head first will be N20 or Nl respectively. Fitting of acetylcholine with N20 (or Nl) and ring F (or ring D) of tubocurarine, ie across the molecule, is far less promising (Figure 3i) and, as shown below, this would not suit our proposed model for an acetylcholine binding site. A model of the topography of the ACH binding site emerges from the above discussion: the primary inner site P-I, binding to a quaternary ammonium function, and the secondary outer site (S-O) forming a JC- JGor esterophilic interaction with the drug molecule. The required orientation of the entering drug molecule is thus specific, drug-receptor binding being enhanced by the presence of two or more symmetrically disposed P-I and S-O groups on the drug molecule. The appropriate P-I and S-O groups are labelled in Figure 7. The examples used in the above analysis, although representing a limited selection of molecules, include the classical tubocurarine, the clinically useful pancuronium and the well-characterized HS steroidal derivatives. Inclusion of the latter has been influenced by our own involvment in the X-ray structure analysis. Application of computer graphics to other neuromuscular blockers and neurotoxins is in progress

Journal of Molecular Graphics

Table 3. FRODO analysis of measure of fit between sites in selected molecular pairs* illustrated in Figure 3 Molecule 1 Primary or Secondary or quaternary N’ esterophyllic site Sli site Pl

Molecule 2 Primary or Secondary or quaternary N+ esterophyllic site P2 site S2*

(a) Nl (ACHTPB)02

(ACHTPB)

N20 (TCL)

E;;@CL)

0.02 Nl . . . N20

(b) Nl (ACHTPB)02

(ACHTPB)

N20 (TBR)

E;; (TBR)

0.01 Nl . . . N20

;*;; g;

: ’ ’ z;; . .

N20 (TCL)

$; WL)

0.02 N7 . . . N20 ‘d.2; g;

’ . ’ E;; . . .

(d) N7 (ACHI) 03 (ACHI) (e) Nl (ACHTPB)Ol (ACHTPB) (f) Nl (ACHTPB) 02 (ACHTPB)

N20 (TBR) N20 (PANC) N31 (HS626)

Cl7 (TBR) 02 (PANC) C5=C6 (HS626)

0.00 N7 . . . N20 0.33 03 . . . Cl7 0.41 Nl . . . N7 0.39 02 . . . 02 0.16 Nl . . . N31 0.20 02 . . . C6

(g) N7 (ACHI)

03 (ACHI)

N31 (HS626)

C5=C6 (HS626)

0.10 N7 . . . N31

(h) N31 (HS626)

C5=C6 (HS626)

N20 (TBR)

Cl5 . . . Cl6

0.68 N31 . . . N20 ;.;2c;6

(c) N7 (ACHI)

03 (ACHI)

dp (A)+

ds (A)’

= Pl . . . P2

= Sl . . . s2 .69 02 . . . Cl4 59 02 . . . Cl5

;‘;;

VW

(i) N31 (HS626)

C5=C6 (HS626)

N20 (TBR)

c30 . . . c35

0.68 N31 . . . N20 :‘;;

WW

$

’ . * E”6 . . . . ’ “AT5 . . .

E; ’ . . z;:, . . .

*All necessary and relevant pairs have been considered but it is not possible to show all combinations as these are too many. The examples given have been selected both as being of high priority and to illustrate the principles involved. It is hoped that other combinations can be envisaged by comparison t The superposition has sometimes been undertaken in favour of the primary N’ site, P, at the expense of the secondary site S. In most cases it would have been possible to produce a more equitable division of separations between the sites but this was not thought necessary for establishing the principle of site matching $ Where

the secondary

site is a ring, part of a ring or double

bond,

and will be reported elsewhere. Preliminary results indicate general agreement with the above proposals. The importance of the absolute orientation of the N’ group with respect to the esterophyllic region of the drug molecule is emphasized for example with reference to 3@, 17p - di(methyl -2’ acetoxyethylamino) - 5a-androstone dibromide”, a compound of low neuromuscular blocking potency. Although possessing two esterophilic or acetylcholine-like fragments, these are both on the wrong side of their respective quarternary nitrogens and are therefore incorrectly placed for binding to the acetylcholine receptor site. For such molecules the primary inner (P-I) N’ groups are effectively shielded from the ACH binding site by the presence of the supporting steroid molecules. Further details of this effect appear in the following section. RESULTS OF BILBO GRAPHICS Fitting of corresponding groups between pairs of molecules was undertaken using the program Bilbo in a similar manner to that described in the preceding section using Frodo. In addition, charged groups, or those with potential for interaction with the receptor site, were enhanced for one member of each molecular pair (the target molecule) by using a shading option of the program mentioned previously (see also Figure 5). This enhancement appears in most of the following graphics as ‘furry spheres’ of radius approximately equal to the van der Waal’s radius of the atom

Volume 1 Number 4 December 1983

both atoms

and corresponding

distances

are sometimes

quoted

concerned. It is intended to represent an approximate sphere of action, for example corresponding to a charge distribution in a rr - JI interaction on the target molecule. Although approximate, this representation is probably the best available in the abscence of detailed charge and molecular orbital data for the target molecule. For the other member of each molecular pair (the object molecule), the net facility in Bilbo has been used to generate a van der Waal’s surface projected in a selected direction. Both molecules are shown in each diagram, which thus shows the generated inside surface of the receptor site as modelled by the object molecule, and the degree of fitting to this surface of the target molecule. For ease of representation and in order to reduce the discussion to manageable proportions, the examples given below have mainly used the steroidal molecule HS310 as the target molecule. This is also justified in view of the relative rigidity of HS310 which provides a constant template for the active surface, and as shown below, this strategy also emphasizes the steric function of the C5=C6 bond in HS310. Figure 7(a) shows the net surface generated for HS310 with the target molecule ACHI fitting into the pocket. The fitting is similar to that shown in Figure 3(g). The net was generated by pushing the HS310 object molecule, N’31 head first, through the base net. As shown in Figure 7(a), in addition to formation of the inner primary pocket (P-I) a secondary bump or outer pocket (S-O) arises due to the C5=C6 bond of HS310, corresponding to the carbonyl oxygen 02 of ACHI. The spheres of action (fur) of ACHI also fit quite

101

c Figure 7(a). As main caption, showing object molecule HS310, target molecule ACHi

Fig ure 7(c). As main caption docking PANC with the functional groups at the A-ring end of the molecule interacting with the receptor site.

b

d Figure 7(b). As main caption, showing object molecule HS310, target molecule TBR docked with its quaternary N+ZO and benzene ring C interacting with the receptor binding sites. The graphics studies also showed that a similar fit of the HS310 molecule across the N’l tertiary TBR site and benzene ring F of TBR is feasible, thus indicating the approximate twofold symmetry of the TBR molecule

Figure 7(d). As main caption, showing alternative fitting of PANC A-ring end, considered to be a disallowed match. This orientation of PANC would require a receptor topography different from the previo~ modems and is therefore rejected. The target molecule, as shown, would protrude in the protein structure of the ACH receptor molecule (shaded)

snugly into the pockets P-I and S-O respectively. Figure 7(b) shows the fitting of (+)-tubocurarine bromide, TBR, into the HS310 receptor model in which the

quaternary,nitrogen, N20 of TBR is superimposed with N31 of HS310. Figure 7(b) corresponds to Figure 3(h). The alternative orientations of the TBR molecule fit

102

Journal

of Molecular

Graphics

Figure 7(g). As main caption, showing object molecule PANC, target molecule TCL

e Figure 7(e). As main caption, showing object molecule HS310, target molecule STERC

Figure 7(f). As main caption, showing object molecule PANC, target molecule HS310

well with both the P-I and the S-O receptor sites. Neither of the above combinations involves any significant protrusion of the TBR molecule into the body of the receptor surface. This is an important condition, especially at the inner P-I site where reorganization of the receptor structure would be unlikely to occur. The C5=C6 bond of HS310 again

Volume 1 Number 4 December 1983

Figure 7. Bilbo graphics modelling the acetylcholine receptor surface. In each case an object molecule is used in the production of the surface and a second target molecule is docked into the site thus produced by superpositioning with the object molecule as in Figure 6 and Table 2. The superposed quaternary ammonium sites are indicated by jl and the esterophilic sites by l . Figures 7(f) and (g) indicate the complimentary of HS310 and PANC, the receptor site modelled by PANC being similar to that modelled in the previous examples by HS310. The receptor protein surface has two pockets, an inner P and an outer S.

corresponds to one of the benzyl groups of TBR in each case, which are thus positioned for x - x interaction with the receptor surface. The OH39 group in this region may enhance this interaction in TBR. Figure 7(c) which should be compared with Figure 3(e) shows the fitting of pancuronium into the active site model. Again there is no protrusion of the target molecule PANC into the inner receptor surface. The D ring end of pancuronium is swung away from the receptor binding region into the solvent region of the active . cleft, which is therefore required to cover a relatively large volume. As shown in Figure 7(d) it is possible to fit incorrectly the target and object molecules involving prohibited protrusion of the target molecule into the active surface. Such a fit would be disallowed by our model. Figure 7(e) shows the fitting of stercuronium (P-I at the quarternary N’31 end) into the HS310 net. The double bond system of stercuronium, extending from C4 to C6, clearly fits into the esterophilic site at S-O. Finally, Figure 7(f) and (g) illustrate the use of pancuronium as object molecule for development of the receptor site model using Bilbo. The target molecules HS310 (Figure 7(f) and TCL both fit well into the receptor pockets P-I and S-O as in the above examples.

103

accommodate each separate half of all such studied. We propose that the molecules activity with associated enhancement of bisquaternary compounds is related to the greater opportunity for binding afforded by the presence of such groups. The significance of a specific N’ N’ separation, as proposed by Pauling and Petcher** is neither proved nor disproved by this model, but would require two (or more) active site crevices similar to the one we have described in approximate and may even exist in different juxtaposition, subunits of the receptor assembly. NATURE OF THE RECEPTOR SITE AND FURTHER STUDIES Further development of the computer graphics studies described here involves building into the receptor site suitably placed mainchain and side-chain protein components capable of binding to transmitter and inhibitor molecules. These include: 1 a primary anionic site P complementary to the cationic N’ acetvlcholine site. l 2 a secondary site S capable of providing 0 an H-bond donor for interaction with the C=O group (eg in acetylcholine or pancuronium) 0 a site capable of x - n interaction with a benzyl group (eg in (+) tubocurarine) or a C=C group (eg in HS310) l

b

The most suitable groups satisfying these conditions are as follows: 01

Acidic groups: 0 aspartic acid 0 glutamic acid o and possibly serine

Figure 8. BILBO stereoviews of (a) Figures 7(a), (b) Figure 7(b) and (c) Figure 7(c) respectively l

CONCLUSIONS A model for the nicotinic acetylcholine receptor site emerges from the above analysis consistent with the binding of both ACH itself and selected neuromuscular blocking agents. This involves: A primary inner site (P-I) for Coulombic interaction with the quaternary nitrogen (the only feature common to all molecules considered) A secondary site located further towards the entrance of the receptor site (S-O) at about 5-6A from the P-I site (as proposed by Beers and Reichz3). is envisaged that a degree of conformational flexibility exists both in the receptor molecule and in some of the inhibitor molecules which allows for slight adjustments to accommodate binding. The third important point is: l

High neuromuscular blocking activity is associated with molecules which possess approximate twofold symmetry. The receptor model proposed here can

104

l

(ASP) (GW

(SER) o threonine (THR) o carboxylate (C-terminus) o sulphydryl (SH) o or mainchain carbonyl (C=O) 2(a) H-bond donor groups: o serine 0 aspartic E!{ o asparagine (ASN) o threonine (THR) 0 glutamic (GIU) o glutamine 0 lysine (Et{ 0 arginine (ARG) o histidine (HIS) o tyrosine 0 amino (;Z:{ 2(b) Groups providing n - x interaction: o Rings: 0 phenylalanine o histidine $E.)) 0 tyrosine (TYR) o and amide groups (C=O NH)

The amide group has been included in this category since it is known to form such interactions in small mo1ecules5’. Groups satisfying both conditions 2(a) and 2(b) are thus HIS, TYR and amide (or amino). The number of suitable groups for formation either of site P or site S is quite limited and this should facilitate the

Journal of Molecular Graphics

building of the model receptor site in more detail. Continuation of the work will also include suitably displayed charges in colour graphics and we have begun calculation of charge density of suitable molecular fragments using both GAUSSIAN 76 and ATMOL 3 computations. Preliminary results of these studies indicate a polarization of charge in the C5=C6 bond of HS310 and HS626 which is not present in HS692. [This could account for the reactivity of this bond and the consequent increase in potency of HS310 and HS626 over HS692]. Further studies will also involve the docking of neurotoxin structures into the acetylcholine model. ACKNOWLEDGEMENTS We thank the MRC for support (JHT) course of this work (Grant No 8005588).

during the

REFERENCES Barrett, A N, Roberts, G G, Burgen, A S V and Clore, G M (In press) Kistler, J and Stroud, R M Proc. Nut. Acud. Sci. USA Vo178 No 6 (1981) pp 3678-3682 Noda, M, Takahashi, H, Tanabe, T, Toyosato, M, Furntani, Y, Hirose, T, Asai, M, Inayama, S, MUFF;+ T and Numa, S Nature No 299 (1982) pp 4 Sumikawa, K, Houghton, M, Smith, J G, Bell, L, Richards, B M and Barnard, E A Nucleic Acid Res. Vol 10 No 19 (1982) pp 5809-5822 5 Baliivet, M, Patrick J, Lee, J and Heinemann, S Proc. Nat. Acad. Sci. USA 79 (1982) pp 4466-4475 6 Stevens, C F Nature No 299 (1982) pp 776-777 7 Koelle, G B in Goodman, L S, and Gilman, A (eds) The

pharmacological

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Macmillan, NY, USA Vo14 (1970) pp 601-619 8 Zaimis, E J ‘Neuromuscular junction’ Handbook of experimental pharmacology Springer-Verlag, NY, USA Vo142 (1976) 9 Triggle, D J and Triggle, C R (eds) Chemical pharmacology of the synapse A P London, UK (1976) 10 Stenlake, J B Prog. Med. Chem. No 16 (1979) pp 257-286 11 Everett, A J, Lowe, L A and Wilkinson, S Chem. Comm. No 23 (1970) pp 1020-1021 12 Codding, P W and James, M H G Acta Cryst. (1973) Vol B29 pp 935-942 13 Reynolds, C D and Palmer, R A Acta Cryst. (1976) Vol B32 pp 1431-1439 14 Wieriks, J PhD Thesis University of Rotterdam, the Netherlands (1972) 15 Husain, J, Palmer, R A and Tickle, I J (1981) J. Cryst. Mol. Struct. No 11 pp 87-103 16 Buckett, W R Adv. Steroid Biochem. Pharmacol. (1972) No 3 pp 39-65 17 Buckett, W R, Hewett, C L and Savage, D S J. Med. Chem. (1973) No 16 pp 1116-1124 18 Sing, H, Paul, D and Parasha, V V J. Chem. Sot. Perkin I (1973) pp 1204-1206 19 Singh, H and Paul, D J. Chem. Sot. Perkin I (1974) pp 1475-1479 20 Gandiha, A, Marshall, I G, Paul, D and Singh, H f.

Volume 1 Number 4 December 1983

Pharm. Pharmacol. (1974) No 26 pp 871-877 21 Gandiha, A, Marshall, I G, Paul, D, Rodger, I W, Scott, W P and Singh, H Clin. Exp. Pharmacol. Physiol. (1975) No 2 pp 159-170 22 Pauling, P and Petcher, T J Chem. Biol. Interactions (1973) No 6 pp 351-365 23 Beers, W H and Reich, E Nature (1970) No 228 pp 917-922 24 Datta, N, Mondal, P and Pauiing, P Acta Cryst. (1980) Vol B36 25 Jagner, S and Jensen, B Actu Cryst. (1973) Vol B29 pp 161-167 26 Culvenor, C C J and Ham, N S Chem. Commun. (1966) pp 537-541 27 Partington, P, Feeney, J and Burgen, A S V Mol. Pharmacol. (1972) No 8 pp 269-277 28 Liquori, A M, Damiani and Coen, J L .De f. Mol. Biol. (1968) No 33 pp 445-4.50 29 Herdklotz, J K and Sass, R L Biochem. Biophys. Res. Commun. Vol40 (1970) pp 583-588 30 Svinning, T and Sorum, H Actu Cryst. Vol B31 (1975) pp 1581-1586 31 Datta, Mondal and Pauling unpublished work 32 Mahajan, V and Sass, R L J. Cryst. Mol. Struct. (1974) No 4 pp 15-21 33 Jensen, B Acta Chem. Stand. Ser. B (1975) No 29 pp 531-537 34 Sobell, H M, Sakore, T D, Tavale, S S, Canepa, F G, Pauling, P and Petcher, T J Proc. Natl. Acad. Sci. USA (1972) No 69 pp 2213-2215 35 Terrapong, P, Marshall, I G, Harvey, A L, Singh, H, Paul, D, Bhardwaj, T R and Ahna, M J J. Pharm. Pharntacol. (1979) No 31 pp 521-528 36 Marshall, I G, Harvey, A L, Singh, H, Bhardwaj, T R and Paul, D Excerpta Medica Abstract No 438 International Congress Series No 452, V European Congress of Anaesthesiology, (1978) p 243 37 Singh, H, Bhardwaj, T R and Paul, D J. Chem. Sot. Perkin I (1979) pp 2451-2454 38 Marsha& I G, Harvey, A L, Singh, H, Bhardwaj, T R and Paul, D J. Pharm. Pharmacol. (1981) No 33 pp 451-457 39 El-Shora, A I, Palmer, R A, Singh, H, Bhardwaj, T R and Paul, D J. C.S. R. No 12 (1982) pp 255-269 A J. Appl. Crystallogr. No 11 (1978) pp 40 eci7:

41 Tickle, I J Personal communication (1979) 42 Pearl, L H and Honegger, A J. ~of. graphics Vol 1 No 1 (March 1983) pp 9-12 43 Griffith, H R and Johnson, G E Anesthesiology No 3 (1942) pp 418-420 44 Bonta, I L and Goorissen, E M Eur. Jour. Pharmacol. (1968) No 4 pp 303-305 45 Buckett, W R in Briggs and Christie (eds) Advances in steroid biochemistry and pharmacology Academic Press No 3 (1972) pp 39-65 46 Savage, D S, Cameron, A F, Ferguson, G, Hannaway, C and Mackay, I R J. C. S. No 84 (1971) pp 2018-2020 47 Marshall, I G, Paul, D and Singh, H J. Pharm. Pharmacol. No 25 (1973) pp 1441-1446 48 Marshall, I G, Paul, D and Singh, H Eur. J. Pharmacol. No 22 (1973) pp 129-134 49 Mazid, M A, Palmer, R A, Singh, H and Paul, D Acta Cryst. Vol B33 (1977) pp 3641-3649 50 Palmer, R A, Kaiam, M A, Singh, H and Paul, D J.

iO5

Cryst. Mol. struct.

(1968) p 609

No 10 (1980) pp 31-53

51 BusfieId, D, Child, K J, Clarke, A J, Davis, B and Dodds, M G &it. J. Pharrnacol. Chemother. No 32

dP I

52 Moore, J C, Yeadon, A and Palmer, R A J. C. S. R. No 13 (1983) pp 279-291

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