Note: Descriptions are shown in the official language in which they were submitted.
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Allosteric Sites on Muscarinic Recet~tors
Field of the Invention
The present invention relates to muscarinic receptors,
and in particular to compounds which are capable of
binding to an allosteric site on a muscarinic receptor
and modulating the binding of a primary ligand such as
acetylcholine to the receptor. The present invention
further relates to methods for aiding in the
identification of compounds which bind to the allosteric
site and their use in methods of medical treatment.
Background of the Invention
The five muscarinic receptors subtypes are designated M1-
MS and all are activated by the binding of acetylcholine
(ACh). These receptor subtypes are widely distributed in
the central nervous system and in the periphery where
they mediate a number of important physiological
functions. As a consequence these receptors are a
therapeutic target for the treatment of a variety of
conditions and potential therapeutic agents are both
agonists and antagonists. In the treatment of many
conditions it has been thought to be important that the
therapeutic agents have a selective action on one or a.
limited number of subtypes. However, there remains a
problem in the art that the muscarinic receptor subtypes
are structurally very similar as a consequence of the
identity of amino acids in the regions of sequence that
are considered to constitute the ACh binding site, i.e.
the site of binding of agonists and competitive
antagonists. Therefore, it has not been possible to
synthesize highly selective muscarinic antagonists and no
directly acting muscarinic agonists of any substantial
selectivity exist (Caulfield and Birdsall, 1998). A
further problem is that synthetic exogenously applied
agonists chronically stimulate receptors and this can
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2
result in desensitization and downregulation of the
receptor function as well as losing any information
content of the pulsatile endogenous ACh signalling
mechanism.
However, in addition to the primary site on these
receptors at which agonists and competitive antagonists
bind, muscarinic receptors are known to also contain an
allosteric site. Compounds binding at the allosteric
site mediate the binding of the ligands to the primary
binding site (GB 2 292 685 A and WO 96/03377). Thus, it
is possible that compounds binding at the allosteric site
may overcome some of these problems involved in selective
modulation of muscarinic receptor subtypes. By way of
example, it is known that when brucine and some of its N-
substituted analogues bind at the allosteric site, they ,
modulate the response of muscarinic receptors to the
primary ligand acetylcholine (ACh) or N-methylscopolamine
(NMS), a competitive antagonist of ACh. The modulation
caused by compounds binding at the allosteric site can be
positive, negative or neutral. A compound which has
neutral cooperativity with ACh at one muscarinic receptor
subtype binds to the receptor but has no action at any
concentration. In contrast, if the same-ligand has ..
positive or negative cooperativity at another subtype it
has an action at that subtype which is totally selective.
This form of selectivity based on cooperativity can be
termed 'absolute subtype selectivity'... Thus, the
allosteric agents cari modulate the interaction between
the muscarinic receptor and the primary ligand.
Summary of the Invention
Broadly, the present invention relates to the finding
that muscarinic receptors have a further allosteric site
which is characterised herein using compound la, and a
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series of related indolocarbazoles represented by formula
l, and compounds 2a and 2b, and a series of related
compounds represented by formula 2. These compounds are
capable of binding to the allosteric site to modulate the
binding of a primary ligand to the receptors, showing
positive, negative and neutral cooperativity and
selectivity for muscarinic receptor subtypes.
Compounds represented by formula 1:
R~
Rz Rs R.
8
Ra Rs
Rs /
~ ~ ~ ~ R ,o
R 6 N, N
R»
R
R ,z O R ,s
X
R ,a
(CHz)m
R ,s
wherein:
R1 is hydrogen, lower alkyl, aralkyl, iminoalkyl or
an imino protecting group such as an acyl group R19C0,
where R19 is alkyl or aralkyl;
R2 and R3 are independently an oxygen or two hydrogen
atoms;
R9-R11 are independently selected from hydrogen or
general aromatic substituents e.g. halo, nitro, cyano,
lower alkyl, haloalkyl, alkoxy, hydroxy, aralkoxy;
Rlz is H or lower alkyl;
X is CHz,
CH (0R16) , where R16 is hydrogen, lower -alkyl or
an O-protecting group,
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CH (ORIS) -CH (ORls) ,
CH (ORls) -CH (CHZ) ~-N (R1,) 2, where n is an integer
between 0-5 and Rl, is one or two of the following;
alkyl, aralkyl;
R14 is hydrogen, ORls, NHR1" where R1, is alkyl,
haloalkyl, aralkyl or an acyl group;
m is an integer between 0-8; and,
R15 is hydrogen, COzRle, CONHR18 or an isostere for an
ester or substituted ester, where Rl8 is hydrogen, alkyl
or substituted alkyl, aralkyl or substituted aralkyl.
Compounds represented by formula 2:
Ra
R~ R
s
RZ N N R6
R3
wherein:
X is N (H) or C (H) ;
Rl, RZ are independently hydrogen; general aromatic
substituents e.g..halo, nitro, cyano, lower alkyl,
haloalkyl, alkoxy, hydroxy, aralkoxy;
R3 is hydrogen, alkyl, iminoalkyl, or aralkyl;
R1 and RZ together and/or RZ and R3 together are a
fused aromatic or heterocyclic system optionally with
ring substituents;
Rq, R5, Rs are normal aromatic substituents;
or Rs, Rs together are a fused alicyclic system with
1-5 rings including steroid ring systems, preferably with
substituents at either or both 17a and 17(3 positions,
e.g. hydroxy, alkoxy, aralkoxy, alkyl, alkenyl, or
alkynyl, and R4 is a substituent compatible with the
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synthetic method;
or R5, R6 together are a fused alicyclic system with
1-5 rings and RQ is hydrogen or an alkyl group;
or R4, R5, R6 are part of a fused alicyclic system;
5 and a dotted line indicated either the presence or
the absence of a bond.
Preferably, the above generally defined substituents are
C1-Clo, or in the case of lower alkyl substituents, C1-C6;
and in either case, optionally including branching and/or
halogen substitution.
The formulae of compounds la, 2a and 2b are shown on
pages 54 to 56.
Accordingly, in a first aspect, the present invention
provides a compound represented by formula 1 or 2 for use
in a method of medical treatment.
In a further aspect, the present invention provides the
use of a compound for the preparation of a medicament for
the treatment of a condition mediated by the binding of a
primary ligand to a muscarinic receptor, wherein the
compound binds to an allosteric site of the muscarinic
receptor which is capable of binding to compound 1a
and/or 2a and thereby modulates the binding of the
primary ligand to the muscarinic receptor. Preferred
compounds include those represented by formula 1 or 2.
In a further aspect, the present invention provides a
method of modulating the response of a muscarinic
receptor to a primary ligand, the method comprising
contacting the muscarinic receptor with a compound which
binds to an allosteric site of the muscarinic receptor
which is capable of binding to compound la and/or 2a and
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which thereby modulates the binding of the primary ligand
to the muscarinic receptor. This method~may be carried
out in vitro, e.g. as part of a screening method or to
otherwise activate or modulate the response of the
receptor, or in vivo, e.g. in the treatment of a patient
suffering from one of the conditions described herein.
The primary ligand binding to the muscarinic receptor may
be an agonist or an antagonist of the receptor's
biological activity. Examples of primary ligands include
acetylcholine (ACh) or N-methylscopolamine (NMS). Other
primary ligands are well known to those skilled in the
art.
The use of a compound which binds to this allosteric
site, and in particular an allosterically acting compound
which has a positive or negative cooperative effect on
the binding of the primary ligand, can have the advantage
of selectively modulating the natural function of a
limited group of the muscarinic receptor subtypes, and
more preferably only a single muscarinic receptor
subtype. Thus, the invention helps to solve the problem
of selectively activating the function of specific
muscarinic receptor subtypes in a way which is difficult
to achieve using a primary ligand which binds to multiple
receptor subtypes, and opens up the possibility of
therapeutic treatment based on this selectivity.
Examples of these conditions are discussed below. While
it is generally preferred that the binding of the
allosteric compound to the muscarinic receptor enhances
the binding of the primary ligand (i.e. shows positive
cooperativity), compounds which decrease the binding of
the primary ligand (i.e. act antagonistically or show
negative cooperativity) can also have therapeutic
potential. Such compounds have the property of not
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blocking 100% of the receptor response when they bind to
the allosteric site of the receptor (as competitive
antagonists do). These compounds can be used in the
treatment of conditions including Alzheimer's disease,
motion sickness, depression, bronchitis, gastric and
duodenal ulcers, non ulcer dyspepsia, urinary bladder
incontinence and retention, sinus bradycardia,
Parkinson's disease, incontinence, asthma, chronic
obstructive pulmonary disease, irritable bowel syndrome,
excessive vagal drive, as a preanaesthetic, for cardiac
pacemaker regulation, or for the regulation of sleep.
The allosteric site defined herein is distinct from the
'common allosteric site' disclosed in the prior art (e. g.
GB 2 292 685 A) which binds to gallamine, strychnine,
brucine and N-substituted brucine analogues. In
contrast, the present inventors have found a new
allosteric site which binds compound la and a series. of
related indolocarbazoles having formula 1 and compound 2a
and a series of related compounds having formula 2.
Preferably, the muscarinic receptor is selected from the
M1, Mz, M3, Mg or MS receptors known in the art . The
receptors may be-human or an appropriate animal homologue
(rat, mouse etc). The generation of transfected cell
lines stably or transiently expressing one or more of the
M1-Ms receptor genes from any given species is well in the
art and relevant references are cited, for example, in
the reviews: Hulme et al, 1990 and Caulfield and
Birdsall, 1998.
Exemplary compounds which are. capable of modulating the
binding of a primary ligand to a muscarinic receptor by
interaction with the allosteric site described for the
first time herein include compounds la, 2a and 2b.
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In a further aspect, the present invention provides a
method for aiding in the identification of compounds
capable of modulating the binding of a primary ligand to
a muscarinic receptor by binding to an allosteric site of
the muscarinic receptor which is capable of binding to
compound la and/or compound 2a, the method comprising:
(a) contacting the muscarinic receptor and the
primary ligand with one or more concentrations of a
candidate compound; and,
(b) determining whether the candidate compound
modulates the binding of a primary ligand to the
muscarinic receptor by binding to the allosteric site of
the receptor which is capable of binding compound la
and/or compound 2a.
The method may comprise the further step of selecting a
candidate compound which modulates the binding of the
primary ligand to the muscarinic receptor.
In a further aspect, the present invention provides the
use of an allosteric site of a muscarinic receptor which
is capable of binding to compound la and/or 2a in
screening for compounds which are capable of modulating
the binding of a primary l.igand to a muscarinic receptor
by binding to the allosteric site.
The modulation of the binding of the primary ligand may
be achieved by a number of mechanisms. The allosteric
compound may have a positive or negative cooperative
effect at one or more of the muscarinic receptor
subtypes, and preferably no effect (neutral
cooperativity) at other receptor subtypes. Alternatively
or additionally, the binding of the allosteric compound
may affect the binding of an agent acting at a different
allosteric site such as the common allosteric site which
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binds brucine.
Preferably, candidate allosteric compounds are selected
if they have a positive or negative cooperative effect on
the binding of the primary ligand. Thus, in preferred
embodiments, step (b) involves determining whether the
candidate compounds bind to the allosteric site and
additionally determining how the binding modulates the
action of the primary ligand at its binding site. This
can be carried out using the assays described herein
including equilibrium and/or kinetic binding assays
and/or functional assays.
General assays which are suitable or can be adapted for
use in the present invention are described in Lazareno
and Birdsall (1995), Detection, quantitation and
verification of allosteric interactions of agents with
labelled and unlabelled ligands at G-protein-coupled
receptors: Interactions of strychnine and acetylcholine
at muscarinic receptors. Mol. Pharmacol. 48:362-378;
Lazareno et al (1998), Subtype selective positive
cooperative interactions between brucine analogues and
acetylcholine at muscarinic receptors: radioligand
binding studies. Mol. Pharmol. 53:573-589; Birdsall et
al (1999), Subtype selective. positive cooperative
interactions between brucine analogues and acetylcholine
at muscarinic receptors: functional studies. Mol.
Pharmacol. 55:778-786.
Examples of specific assays which can be employed are
described in the 'Materials and Methods' section below.
Firstly, the candidate compounds are selected as being
allosteric using the criteria defined in the above
general and specific assays. The compounds are further
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selected as binding to the novel site, e.g. the site
which binds compound 2a, using specific kinetic and
equilibrium assays. For example, any allosteric compound
which is not competitive with the binding of brucine,
5 gallamine.or strychnine and is competitive with the
binding of 2a to the novel allosteric site is a candidate
compound for further investigation or development as a
therapeutic. These methods are useful for finding
compounds which are positively, negatively or neutrally
10 cooperative with the binding of the primary ligand.
In preferred embodiments, the screening is carried out
using a receptor, one or more concentrations of a
candidate allosteric ligand (possibly including an assay
carried out in the absence of the candidate ligand by way
of control) and one or more primary ligands, in the
presence or absence of another allosteric ligand.
Preferably, the primary ligand employed in this screening
method is acetylcholine (ACh) and/or N-methylscopolamine.
(NMS), although other suitable primary ligands are well
known to those skilled in the art. In one embodiment,
the method involves as assay in which the binding of a
candidate compound to the allosteric site is determined
in using labelled NMS in the absence or presence of one
or.more concentrations of ACh. Alternatively or
additionally, the binding of a candidate compound to the
allosteric site is determined..using labelled NMS in
assays which determine the NMS dissociation rate constant
in the presence and absence of one or more concentrations
of the candidate compound. Another preferred assay
format is an assay of the effects of one or more
concentrations of an allosteric ligand on the
acceleration of the dissociation rate of NMS from
muscarinic receptors produced by 2a (described in Part II
below). A further assay is_to quantitate the effects of
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a test compound, which has been demonstrated in the
general assays to be allosteric, on the equilibrium
allosteric effects of ligands which are known to bind one
or other of the two allosteric sites described in Part I;
Figure 5).
In these methods, the candidate compound may be selected
if it enhances the binding of the primary ligand to the
muscarinic receptor (otherwise referred to as positive
cooperativity). .However, other compounds may be selected
if they reduce the binding of the primary ligand to the
muscarinic receptor (otherwise referred to as negative
cooperativity). Can.dida.te compounds having neutral
cooperativity are selected if they bind to one or more of
the muscarinic receptor subtypes but have no action on
the equilibrium binding of a primary ligand at any
concentration..
The allosteric site employed in the work described herein
is capable of binding compounds la and/or 2a. In
contrast to the first allosteric site disclosed in the
prior art, the site described herein does not bind to
brucine, gallamine and/or strychnine to any substantial
extent at concentrations up to 10-4M.
In a further aspect, the present invention provides a
method which comprises, having identified a candidate
compound by the above method, the further step of
manufacturing the compound in bulk and/or formulating the
compound as a pharmaceutical composition.
In a further aspect, the present invention provides the
use of a.compound as obtainable by the above method for
the preparation of a medicament for the treatment of a
conditions mediated by the binding of the primary ligand
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to the muscarinic receptor. Examples of these conditions
discussed below.
Embodiments of the invention will now be described by way
of example and not limitation with reference to the
accompanying drawings.
Brief Description of the Figures
Figure 1: Effect of staurosporine (1f) on the binding of
3H-NMS (210 pM) at M1 receptors in the absence and
presence of 2.2 mM ACh, all in the presence of 0.2 mM
GTP. The points are individual observations. The lines
show the fit to Equation 2 (see Methods), which yielded a
log affinity of 5.95 ~ 0.06, a slope factor of 1.01 ~
0.05, cooperativity with 3H-NMS of 1.51 ~ 0.06, and
cooperativity with ACh of 0.27 ~ 0.03. The affinity ratio
plots of these data are shown in Figure 2.
Figure 2: Affinity ratio plots of five indolocarbazoles
(la, 1b, 1e, if and 1i) at M1-Mq receptors. The points
were derived from duplicate observations of 3H-NMS binding
in the absence and presence of ACh, as described in
Methods....The parameter estimates pK (log affinity of the
test agent for tho free receptor), aNMS (cooperativity
with 3H-NMS) , and l3A~h (cooperativity with ACh) were
derived from nonlinear regression analysis with Equations
1 or 2 as appropriate (see Methods). The parameter
estimates from a number of similar assays a.re summarised
in Table 1.
Figure 3: Effect of la,lb, 1e, if and 1i on the
dissociation rate constant ( koff) of 3H-NMS at M1-MQ
receptors, expressed as a percent inhibition of the
control kofF. The points are the mean and range/2 of
duplicate observations. The lines show the fit to a
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logistic function, as described in Methods. The parameter
estimates from a number of similar assays are summarised
in Table 2.
Figure 4: Effect of various concentrations of KT5720 (la)
on the inhibition of 3H-NMS (50 pM) binding at M1
receptors by ACh in a volume of 3 ml. The points are the
mean and range/2 of duplicate observations. The. lines
show the fit to Equation 1 with the slope factor for
KT5720 binding set to 1. The parameter estimates were:
log affinity of KT5720 6.6 ~ O.l,~cooperativity with
3H-NMS 1.9 ~ 0.1, cooperativity with ACh 1.6 ~ 0.2. The
inset shows affinity ratio plots derived from these
parameters (see Methods). The -log ICSO values of ACh in
the presence of increasing concentrations of KT5720, from
independent logistic fits of the curves, were 5.28, 5.33,
5.40 and 5.42.
Figure 5: Inhibition by gallamine of 3H=NMS binding at M1
receptors in the presence of various concentrations of
(A) KT5720 (1a) and (B) staurosporine (1f). The points
are individual observations. The lines show the fit of
the data to Equation 3, where the cooperativity estimates
of gallamine with KT5720 and stauros-porine were not
significantly different from 1 and 0 respectively and
were set to those values. The slope factors for
gallam.ine, KT5720 and staurosporine.were not different
from 1 and were set at that value. From three such
assays, similarly constrained, KT5720 had a log affinity
of 6.22 ~ 0.17 and cooperativity with 3H-NMS of 2.39 ~
0.08. From three such assays, similarly constrained,
staurosporine had a log affinity of 5.75 ~ 0.11 and
cooperativity with 3H-NMS of 1.62.~ 0.13. From these six
assays gallamine had .a log affinity of 5.05 ~ 0.05 and
cooperativity with 3H-NMS of 0.11 ~ 0.01. The insets show
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the effect of the test agent on the -log ICSO of
gallamine, obtained from nonlinear regression analysis of
the individual curves.
Figure 6: Effect of KT5720 (la) on 3H-NMS dissociation
from M1 receptors, alone and in the presence of other
allosteric agents, measured at a single time point as
described in Methods. The points show the mean and s.e.m
of quadruplicate observations obtained in two assays,
except for 10-4M gallamine, and 3.10-SM and 3.10-9M brucine
,which show the mean and range/2 of duplicate
observations. The lines in the top panel show the fits of
the individual curves to a hyperbolic function, except
for those in the presence of staurosporine. The estimates
of the log ECso of KT5720 derive from those fits. The top
panel shows the data as o inhibition of the control koff
of 3H-NMS. The lower panel shows shows Ef, the 3H-NMS koff
values in the presence of KT5720 and a certain
concentration of test agent (gallamine, brucine or
staurosporine) as a fraction of the koff values in the
presence of that concentration of test agent alone.
Eigure-7: Concentration-dependent effects of the
compounds shown on pages 55 and 5.6 on the 3H-NMS-occupied
receptor. The percent.inhibition of the-dissociation
rate constant of 3H-NMS from M1-M9 receptors was measured
at a single time point. The legends indicate the ICSo (or
ECso) values obtained using nonlinear regression analysis
of these curves.
Figures 8 and 9: Concentration-dependent effects of
active compounds on the equilibrium-binding of 3H-NMS and
ACh. The effects are expressed .as 'affinity ratios', i.e.
the apparent affinity of the 'primary' ligand (3H-NMS or
ACh) in the presence of a particular concentration of
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test agent divided by its apparent affinity in the
absence of test agent. Affinity ratios were calculated
as described in 'Methods'. Affinity ratios > 1 indicate
positive cooperativity, affinity ratios < 1 indicate
5 negative cooperativity, and affinity ratios of 1 with one
primary ligand at concentrations of test agent which
modify the binding of the other primary ligand indicate
neutral cooperativity. The ICSO, or ECso, of a test agent
on the affinity ratio of either primary ligand
10 corresponds approximately to the Kd~of the test agent for
the free receptor. High concentrations of compounds
which show neutral or positive cooperativity with 3H-NMS
and which strongly inhibit 3H-NMS dissociation may inhibit
3H-NMS binding through a kinetic effect, i.e. lack of
15 equilibration of 3H-NMS binding.
Figure 10: Inhibition of.3H-NMS binding to M3 receptors by
ACh, alone and in the presence of three concentrations of
2c (WIN 62577): GTP (0.2 mM) was present. The data were
fitted to Equation 1 (see Methods) to yield a log
affinity of 2c of 5.31, cooperativity with 3H-NMS of 0.47,
and cooperativity with ACh of 1.41. The inset shows
affinity ratios (1/dose ratio) for ACh and 3H-NMS,
calculated from the parameters of the fit.
Figure 11: Dissociation of 3H-NMS over .t.ime, alone and in
the presence. of three concentrations of 2a. For each
receptor subtype, the parameter estimates~and standard
errors were derived from the nonlinear regression fits of
the entire dataset to a version of Equations 2 and 3,
where koff and .ko~fx are the dissociation rate constants of
3H-NMS from the free and.2a-liganded receptor
respectively, and pKo~~ is the log Kd of 2a for the 3H-NMS-
occupied receptor. The insets show the linearising
transformation ln(B/Bo) vs. time, where B is the specific
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binding remaining after a certain time and Bo is the
initial level of specific binding.
Figure 12: Concentration-effect curves for 2a on 3H-NMS
dissociation, alone and in the presence of one or three
concentrations of test agent, measured at a single time
point. The data were converted to dissociation rate
constants (see Methods) and expressed as a % of the
control dissociation rate constant. The curves show the
fits from nonlinear regression analysis to Equation 1.
For strychnine and gallamine, dissociation of 3H-NMS from
the dually or triply liganded receptor was not different
from 0 and the cooperative interaction with 2a was not
different from 1 (i.e. neutral cooperativity), so these
values were fixed. For 1a (KT 5720), if (staurosporine)
and 2b (WIN 51708), the cooperative interaction with 2a
was not different from 0 (i.e. competition), so this
value was fixed. The panel marked 'dose-ratio plots'
shows the log of the ratio ECso +a9e~t~ECso alone of 2a vs
log[agent]. Each assay was. repeated at least once, with
similar results.
Detailed Description ,_
Assays
As indicated above, the screening assays of the invention
are useful in the identification of compounds capable of
binding to the allosteric site disclosed herein and
modulating the binding of a primary ligand to a
muscarinic receptor. The precise format of these assays
can be readily devised by the skilled person using the
common general knowledge in the art. In one embodiment,
the assays will employ (a) a muscarinic receptor of one
of the M1 to MS subtypes, (b) one or more primary ligands
or ligand analogues, and (c) one or more candidate
compounds. Optionally, the assays may additionally
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employ (d) a compound known to act at the allosteric site
which is capable of competing with the candidate compound
being tested. The assays will generally involve
contacting the receptor, the primary ligands or ligand
analogues and one or more concentrations of the candidate
compound in vitro, under conditions in which the
candidate compound can bind or compete for binding at the
allosteric site. The results of the assays can be
determined by labelling one or more of the candidate
compound, the competitive allosteric compound or the
primary ligand or ligand analogue, and determining which
species interact in the assay system.
In an alternative embodiment, and especially in the
context of high throughput screening, it may be desirable
that the screening assays involve determining whether the
candidate compound binds to the allosteric site disclosed
herein in the absence of the primary ligand. These
assays could be followed with a separate determination of
whether or in what sense the compounds binding to the
allosteric site modulate the binding of a primary ligand
or ligand analogue to the receptor. These assays could
be carried out by contacting (a) a muscarinic receptor
and (b) one or more candidate compounds, and optionally,
(c) one or more compounds known to act allosterically,
under conditions in which compounds (b) and/or (c) can
bind or compete for binding to the allosteric site. The
binding of the candidate compound or competitive compound
to the receptor can be determined by labelling compounds
(b) and/or (c).
The above. assays may comprise carrying out controls, e.g.'
carrying out the assay in the absence or presence of the
candidate compound(s).
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In these assays, the muscarinic receptors can be present
in either a free form or alternatively immobilised, e.g.
on the surface of a cell expressing the receptor, or a
solid support. A preferred format uses cell surface
receptors.
The labelling of different types of agents is well known
in the art. Broadly, this involves tagging the agent
with a label or reporter molecule which can directly or
indirectly generate detectable,~and preferably
measurable, signal. The linkage of reporter molecules
may be direct or indirect, e.g. by a covalent bond or a
non-covalent interaction. Examples of commonly used
labels include fluorochrome, phosphor or laser dyes with
spectrally isolated absorption or emission
characteristics. Suitable fluorochromes include
fluorescein, rhodamine, luciferin, phycoerythrin and
Texas Red. Suitable chromogenic dyes include
diaminobenzidine. Other detectable labels include
radioactive isotopic labels, such as 3H, 14C, 32p, 3sS, lz6l
or 99'"Tc, and enzyme labels such as alkaline phosphatase,
(3-galactosidase or horseradish peroxidase, which catalyze
reactions leading to detectable reaction products and can
provide amplification of signal.
Other reporters include macromolecular colloidal.
particles or particulate material such as latex beads
that are coloured, magnetic or paramagnetic, and
biologically or chemically active agents that can
directly or indirectly cause detectable signals to be
visually observed, electronically detected or otherwise
recorded. These molecules may be enzymes which catalyze
reactions that develop or change colour or cause changes.
in electrical properties. They may be molecularly
excitable, such that electronic transitions between
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19
energy states result in characteristic spectral
absorptions or emissions.
In the context of high throughput screening, the methods
described herein can involve carrying out assays using
groups or pools of candidate compounds, rather than
individual compounds, to enhance the rate at which.
candidate compounds can be discarded. Individual groups
of compounds having positive results in an assay can then
be separated and screened to identify the compounds) in
the group which interact with the allosteric site and
modulate the binding of the primary ligand. Appropriate
measures should be taken to ensure that any one candidate
compound is assayed with different pools of other
candidate compounds. This protocol minimises the
possible interfering masking effects of competitive
antagonists or agonists which may be in one pool~but not
another.
The candidate compounds used may be natural or synthetic
chemical compounds used in drug screening programmes.
Mixtures of naturally occurring materials which contain
several characterised or uncharacterised components may
als.o.be used.
Other candidate compounds may be based on modelling the
3-dimensional structure of a polypeptide-or peptide
fragment and using rational drug design to provide
candidate compounds with particular molecular shape,
size, hydrophobicity, hydrophilicity and charge
characteristics.
The amount of candidate compound which may be added to an
assay of the invention will normally be determined by
3S trial and error depending upon the type of compound used.
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Typically, from about 0.01 to 100,000nM concentrations of
candidate compound may be used, for example 0.1 to 100uM.
In a further step; the method of the present invention
5 may involve quantifying the amount of a candidate
compound required to modulate the binding of the primary
ligand to the muscarinic receptor by more detailed
equilibrium or kinetic assays.
10 The screening methods of the invention may be followed by
isolation and/or manufacture and/or use of a candidate
compound selected in an assay, and/or further testing to
determine whether a candidate compound having a positive,
neutral or negative cooperative effect on the binding of
15 the primary ligand to the muscarinic receptor has a
biological property which makes it suitable for further
development as a lead compound. These include tests to
determine its activity in assays of function in
membranes, whole cells, whole tissues and/or in vivo, as
20 well as tests of its metabolic stability, bioavailability
and duration of action and for the presence of side
effects.
In a further aspect, the present invention provides the.
use of the above compounds in the design or screening for
mimetics of the compounds which share the property of
binding to the allosteric site of muscarinic receptors
which binds to compound la and/or compound 2b.
The designing of mimetics to a known pharmaceutically
active compound is a known approach.to the development of
pharmaceuticals based on a lead compound. This might be
desirable where the active compound is difficult or
expensive to synthesise or where it is unsuitable for a
particular method of administration. Mimetic design,
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21
synthesis and testing maybe used to avoid randomly
screening large number of molecules for a target
property.
There are several steps commonly taken in the design of a
mimetic from a compound having a given target property.
Firstly, the particular parts of the compound that are
critical and/or important in determining the target
property are determined. These parts or residues
constituting the active region of the compound are known
as its pharmacophore. Once the pharmacophore has been
found, its structure is modelled to according its
physical properties, e.g. stereochemistry, bonding, size
and/or charge, using data from a range of sources, e.g.
spectroscopic techniques, X-ray diffraction data and NMR.
Computational analysis, similarity mapping (which models
the charge and/or volume of a pharmacophore, rather than
the bonding between atoms) and other techniques can be
used in this modelling process.
In a variant of this approach, the three-dimensional
structure of the ligand and its binding partner are
determined or modelled. This can be especially useful
where the ligand and/or binding partner change
conformation on binding, allowing the model to take
account of this. the design of the mimetic.
A template molecule is then selected onto which chemical
groups which mimic the pharmacophore can be grafted. The
template molecule and the chemical groups.grafted on to
it can conveniently be selected so that the mimetic is
easy to synthesise, is likely to be pharmacologically
acceptable, and does not degrade in vivo, while retaining
the biological activity of the lead compound. The
mimetic or mimetics found by this approach can then be
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22
screened to see whether they have the target property, or
to what extent they exhibit it. Further optimisation or
modification can then be carried out to arrive at one or
more final mimetics for further testing or optimisation,
e.g. in vivo or clinical testing.
Pharmaceutical Uses
The compounds identified herein as being useful for
modulating the binding of a primary ligand to a
muscarinic receptor can be formulated and used as
pharmaceuticals.
The pharmaceutical compositions may comprise, in addition
to one or more of the compounds, a pharmaceutically
acceptable excipient, carrier, buffer, stabiliser or
other materials well known to those skilled in the art.
Such materials should be non-toxic and should not
interfere with the efficacy of the active ingredient...
The precise nature of the carrier or other material may
depend on the route of administration, e.g. oral,
intravenous, cutaneous or subcutaneous, nasal,
intramuscular, or intraperitoneal routes.
Pharmaceutical compositions for oral administration may
be in tablet, capsule, powder or liquid form. A tablet
may include a solid carrier.su.ch as gelatin or an
adjuvant. Liquid pharmaceutical compositions generally
include a liquid carrier such as water, petroleum, animal
or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other
saccharide solution or glycols such as ethylene glycol,
propylene glycol or polyethylene glycol may be included.
The pharmaceutical formulations can be prepared by mixing
the compounds of the present invention with one or more
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23
adjuvants, such as excipients (e. g. organic excipients
including sugar derivatives, such as lactose, sucrose,
glucose, mannitol or sorbitol; starch derivatives, such
as corn starch, dextrine or carboxymethyl starch;
cellulose derivatives, such as crystalline cellulose, low
hydroxypropyl-substituted cellulose, carboxymethyl
cellulose, carboxymethyl cellulose calcium or internally
bridged carboxymethyl cellulose sodium; gum arabic;
dextran; and Pullulan; inorganic excipients including
silicates, such as light silicic acid anhydride,
synthetic aluminium silicate or magnesium meta-silicic
acid aluminate; phosphates, such as calcium phosphate;
carbonates, such as calcium. carbonate; and sulphates;
such as calcium sulphate); lubricants (e. g. metal
stearates, such as stearic acid, calcium stearate or
magnesium stearate; talc; colloidal silica; waxes, such
as beeswax or spermaceti; boric acid; adipic acid;
sulphates, such as sodium sulphate; glycol; fumaric acid;
sodium benzoate; DL-leucine;-sodium salts of aliphatic
acids; lauryl sulphates, such as sodium laurylsulphate or
magnesium laurylsulphate; silicates, such as silicic acid
anhydride or sil.icic acid hydrate; and the foregoing
starch derivatives); binders (e..g. polyvinyl pyrrolidone,
Macrogol; and similar compounds to the excipients
described above); disintegrating agents (e. g. similar
compounds to the excipients described above; and
chemically modified starch-celluloses, such as
Crosscarmelose sodium, sodium carboxymethyl starch or
bridged polyvinyl pyrrolidone); stabilisers (e.g. ~-
hydroxybenzoates, such as methylparaben or propylparaben;
alcohols, such as chlorobutanol, benzyl alcohol or
phenylethyl alcohol; benzalkonium chloride; phenols, such
as phenol or cresol; thimerosal; dehydroacetic acid; and
sorbic acid); corrigents (e.g. sweeteners, vinegar or
perfumes, such as conventionally used); diluents and the
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like.
For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active
ingredient will be in the form of a parenterally
acceptable aqueous solution which is pyrogen-free and has
suitable pH, isotonicity and stability. Those of
relevant skill in the art are well able to prepare
suitable solutions using, for example, isotonic vehicles
such as sodium chloride injection, Ringer's injection,
lactated Ringer's injection. Preservatives, stabilisers,
buffers, antioxidants and/or other additives may be
included, as required.
Preferably, the pharmaceutically useful compound
according to the present invention is given to an
individual in a 'prophylactically effective amount' or a
'therapeutically effective amount' (as the case may be,
although prophylaxis may be considered therapy), this
being sufficient to show benefit to the individual.
Typically, this will be to cause a therapeutically useful
effect in the patient, e.g. using the compounds to
regulate the action of the primary ligand at a muscarinic
receptor, and preferably one of the muscarinic receptor
subtypes. The actual amount of the compounds
administered, and rate and time-course of administration,
will depend on the nature and severity of the condition
being treated. Prescription of treatment, e.g. decisions
on dosage etc, is within the responsibility of general
practitioners and other medical doctors, and typically
takes account of the disorder to be treated, the
condition of the individual patient, the site of
delivery, the method of administration and other factors
known to practitioners. Examples of the techniques and
protocols mentioned above can be found in Remington's
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Pharmaceutical Sciences, 16th edition, Oslo, A. (ed),
1980.
In particular, the compounds may be useful in the
5 treatment of conditions mediated by the action of ACh at
a muscarinic receptor. By way of example, these include
Alzheimer's disease, Parkinson's disease, motion.
sickness, Huntingdon's chorea, schizophrenia, depression,
anxiety, sedation, analgesia, stroke, preanaesthetic,
10 antispasmodic, irritable bowel syndrome, bladder-
incontinence or retention, peptic ulcer disease,
bronchitis/asthma/chronic obstructive airway disease,
sinus bradycardia, pacemaker regulation, glaucoma,
achalasia, symptomatic diffuse oesophageal spasm, biliary
15 dyskinesia, scleroderma, diabetes mellitus, lower
oesophageal incompetence, intestinal pseudo obstruction,
regulation of sleep, control of pupil diameter and non-
ulcer dyspepsia.
20 Depending on the type and severity of.condition, the
composition can be administered to provide an initial
dose of about 0.01 to 20 mg, more preferably 0.02 to 10
mg, of compound/kg.of patient weight. As mentioned
above, other dosing regimens and the. determination of
25 appropriate amount of the compounds for inclusion in the
compositions can be readily determined by those skilled
in the art.
Materials
3H-NMS (81-86 Ci/mmol) was from Amersham International,
UK, and 35S-GTPyS (1000-1400 Ci/mmol) was from NEN,
Boston. Brucine sulfate, gallamine triiodide and ACh
chloride were from Sigma Chemical Co., Dorset,, UK.
Staurosporine was from Sigma and from Alexis Corporation,
Nottingham, UK. Go 7874 (1i), Go 6976 (1h) and K-252c
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(1g) were from Calbiochem, Nottingham, UK. K-252a (1b)
and K-252b (lc) were from Alexis and from TCS Biologicals
Ltd, Buckingham,. UK. KT5823 (1e) and KT5720 (1a) were
from TCS, Calbiochem and Alexis. KT5926 (1d) was from
TCS and Calbiochem. WIN 51708 (2b) and WIN 62577 (2c)
were from RBI (Semat), St Albans, UK. Analogues of 2
were synthesised using literature methods according to
the general scheme:
R' Ra
HO
z ~N NH2 +
I
R3 O R6
with further chemical modification of the product as
necessary. Further assistance for the synthesis of the
compounds or related ones is provided in the papers by
Bajwa and Sykes (1978-1980) cited in the references
section.
Methods
Cell culture and membrane preparation:
CHO cells stably expressing cDNA encoding human
muscarinic M1-M9 receptors (Buckley et al, 1989) were
grown in alpha-MEM medium (GIBCO) containing 100 (v/v)
new born calf serum, 50 U/ml penicillin, 50 ug/ml
streptomycin and 2 mM glutamine, at 37° under 5o CO2.
Cells were grown to confluence and harvested by scraping
in a hypotonic medium (20 mM Hepes + 10 mM EDTA, pH 7.4).
Membranes were prepared at 0°C by homogenization with a
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Polytron followed by centrifugation (40,000 x g, 15 min),
were washed once in 20 mM Hepes + 0.1 mM EDTA, pH 7.4;
and were stored at -70°C in the same buffer at protein
concentrations of 2-5 mg/ml. Protein concentrations were
measured with the BioRad reagent using bovine serum.
albumin as the standard. The yields of receptor varied
from batch to batch but were approximately 10, l, 2.and 2
pmol/mg of total membrane protein for the M1, M2, M3 and Mq
subtypes respectively.
Radioligand binding assays:
Unless otherwise stated, frozen membranes were thawed,
resuspended in incubation buffer containing 20 mM Hepes +
100 mM NaCl + 10 mM MgClz (pH 7.4) and incubated with
radioligand and unlabelled drugs for two hours at 30°C in
a volume of 1 ml. Membranes were collected by filtration
over glass fibre filters (4Vhatman GF/B) presoaked in 0.1%
polyethylenimine, using a Brandel cell harvester (Semat,
Herts, UK), extracted overnight in scintillation fluid
(ReadySafe, Beckman) and counted for. radioactivity, in
Beckman LS6000 scintillation counters. Membrane protein
concentrations (5-50 ug/ml) were adjusted so that.not
more than about 150 of added radioligand was bound.
Nonspecific binding was measured in the presence of 10-6M
QNB (an antagonist with picomolar potency) and accounted
for 1-50 of total binding. GTP was present at a
concentration of 2x10-9M in assays containing unlabelled
ACh. Data points were usually measured in duplicate.
CHO cell membranes do not possess cholinesterase activity
(Gnagey and Ellis, 1996; Lazareno and Birdsall, 1993) so
ACh could be used in the absence of a cholinesterase
inhibitor. The compounds were dissolved in dimethyl
sulfoxide which, at the highest final concentration of
20, had no effect on binding.
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Experimental designs and data analysis:
General data preprocessing, as well as the 'affinity
ratio' calculations and routine plots of the
semiquantitative equilibrium assay, were performed using
Minitab (Minitab Ltd, Coventry, UK). The other assays
were analysed with nonlinear regression analysis using
the fitting procedure in SigmaPlot (SPSS Inc., Erkrath,
Germany). This procedure is relatively powerful in that
it allows the use of two or more independent variables,
e.g. concentrations of two drugs.
Equilibrium binding assays for estimation of the affinity
of an allosteric agent for the receptor and the magnitude
of its cooperativity with 3H-NMS and ACh:
The design and analyses have been described in detail
(Lazareno and Birdsall, 1995;Lazareno et al, 1998).
Briefly, specific binding of a low. concentration of 3H-NMS
(1-2 times the Kd) was measured in the presence of a
number of concentrations of test agent, all in the
absence and presence of one or more concentrations of
ACh. Specific binding of a high concentration of 3H-NMS
(5-l0 times Kd) was also measured. Nonlinear regression
analysis was used to fit the data to the equation:
Equation 1
Bmax.L.K~.(1 +a.(X.K )S)
BLAX- 1 +(XK )s+(A.KA)".(1 +~3.(X.K )S)+L.KL.(1 +a.(X.K )S)
where BLS is observed specific bound radioligand, L, A,
and X are concentrations of 3H-NMS, ACh and allosteric
agent respectively, KL, KA and KX are affinity constants
for the corresponding ligands and the receptor, a and I3
are allosteric constants of X with 3H-NMS and ACh
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29
respectively, n is a logistic slope factor to describe
the binding of ACh, and s is a 'Schild slope' factor to
describe the binding of X. According to the allosteric
model s should be 1.
Above a certain concentration, some allosteric agents,
especially those which exhibit neutral or positive
cooperativity with 3H-NMS, may slow the kinetics of 3H-NMS
binding so much that the binding does not reach
equilibrium. In most cases sufficient incubation time
was used to allow 3H-NMS binding in the presence of the
agent to reach equilibrium. In a few cases, however, the
highest concentration of agent would be predicted to slow
3H-NMS kinetics sufficiently to prevent binding
equilibrium from being reached, and in these cases the
data were better fitted to the equation:
Equation 2
-t.ko~ t.ko~.L.KL
2 0 Bcaxr-Bcax+(Bc~-Bcax).(exP( 1 +a.(X K )S + 1 +(X.K )s+(A.KA)".(1 +(3.(X.K
)S) ))
where BL~t is observed specific binding under
nonequilibrium conditions, BL,~,~ is the predicted
equilibrium binding defined in Equation 1, t is the
incubation time, koff is the dissociation rate constant of
3H-NMS, and BLO is the initial amount of bound
radioligand, set to zero in this case. This equation
assumes that the dissociation of 3H-NMS from the
allosteric agent-occupied receptor is negligible, and
that the binding kinetics of both ACh and the allosteric
agent are fast in comparison with the dissociation rate
of 3H-NMS.
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If only a single concentration of ACh was used, the data
were visualised with 'affinity ratio' plots, where the
affinity 'ratio is the apparent affinity of the 'primary'
ligand (3H-NMS or ACh) in the presence of a particular
5 concentration of test agent divided by the apparent
affinity of the primary ligand in the absence of test
agent. Theoretically, the ECso or ICSO of the affinity
ratio plot corresponds to the Kd of the test agent at the
free receptor, and the asymptotic level corresponds to
10 the cooperativity constant for the test agent and primary
ligand (Lazareno and Birdsall, 1995). Affinity ratios
were calculated from the specific binding data as follows
(Lazareno and Birdsall, 1999):
15 The affinity ratio of 3H-NMS in the presence of a single
concentration of test agent is given by the equation:
Equation 3
2 0 r = BLX'~BLI BL~
L BLI'BL'~1 q~ BLX'~BL R~'BLI
The affinity ratio of ACh the presence of a single
concentration of test agent is given by the equation:
Equation 4
BL.BLA.~BLI BL~'~BLX BLAX
r = _ _ _
A BLAX'~BL BLA~'~BLI'BL'~l 9) BLX~BLx~BL q.BL~~J
where BL is binding in the presence of the low [3H-NMSJ
alone; BLl is binding in the presence of the high [3H-
NMS]; BLA is binding in the presence of the low [3H-NMS]
and ACh; BLX is binding in the presence of the low [3H-
NMS] and a -particular concentration of test agent; BLAX is
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31
binding in the presence of the low [3H-NMS], ACh arid the
same concentration of test agent; L is the low 3H-NMS
concentration; LI is the high 3H-NMS concentration; and q
is the ratio of low and high 3H-NMS concentrations, L/L1.
With assays containing a number of ACh concentrations,
affinity ratio plots were calculated using the parameter
estimates from the fit of the dataset to Equation 1 or 2
as appropriate (Lazareno and Birdsall, 1995).
The affinity ratio of 3H-NMS and ACh, rL and rA
respectively, are given by the equations:
Equation 5
1 +a.X.K
r =
1 +X.K
x
and equation 6
1 +~3.X.K
rA 1 +X.K
where' the symbols are as described above.
'Off=rate assay to estimate the affinity. of an allosteric
agent for the 3H-NMS-occupied receptor:
A high concentration of membranes (2-4 mg protein/ml) was
incubated with a high concentration of 3H-NMS (5 nM) for
about 15 minutes. Then 10 u1 aliquots were distributed
to tubes which were empty or contained 1 ml of 10-6M QNB
alone and in the presence of a number of concentrations
of allosteric agent (typically n=4). Non-specific
binding was measured in separately prepared tubes
containing 10 ~i membrane and 2 u1 of 3H-NMS + QNB. Some
time later, about 2.5 dissociation half-lives (see Table
2), the samples were filtered. The data were transformed
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to rate constants, k°ff, using the formula:
k°ff=In (Bo/Bt) /t
where Bo is initially bound radioligand and Bt is bound
radioligand remaining after t minutes dissociation.
These values were finally expressed as o inhibition of
the true 3H-NMS dissociation rate constant (k°ff in the
absence of allosteric agent) and fitted to a logistic
function using nonlinear regression analysis.
Theoretically the curves should have slopes of 1, and
correspond to the occupancy curves of the allosteric
agents at the 3H-NMS-occupied receptors, regardless of
whether the inhibition of 3H-NMS dissociation is caused by
an allosteric change in the shape of the receptor or the
trapping of the 3H-NMS in its binding pocket by the bound
allosteric agent (Lazareno and Birdsall, 1995).
Initially the curve was fitted without constraints. If
the slope factor was not different from 1, and the
maximal inhibition ('Emax') did not exceed about 100x,
then the slope was constrained to 1 and the Emax was
fitted. If the fitted Emax-exceeded~100% (a physical
impossibility, apart from experimental variation or
error) then the Emax was constrained to 100.and the slope
fitted. With the compounds under study the Emax was
often less than 100, and in most such cases the data were
well fitted with the slope constrained to 1.
GTPYS binding assay:
Membranes expressing M1 receptors (5-20 ug/ml) were
incubated with 35S-GTPYS (0. 1 nM) , GDP (10-'M) and ligands
in incubation buffer in a volume.of-1 ml for 30-60
minutes at 30°C. Bound label was collected by filtration
over glass fibre filters prewetted with water.
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Part 1
Results
The structures of the compounds examined are shown on
page 54. Figure 1 shows effects of compound if
(staurosporine) on equilibrium 3H-NMS binding at M1
receptors in the absence and presence of a fixed
concentration of ACh. 3H-NMS binding was increased by
staurosporine concentrations up to 10 uM and was reduced
at 30 uM. The increase in 3H-NMS binding reflects a
decrease in the Kd of 3H-NMS rather than an increase in
Bmax (data not shown). The decrease in binding with 30
uM staurosporine is caused by the slowing of 3H-NMS
kinetics by high concentrations of staurosporine (see
below) and the consequent lack of equilibration of 3H-NMS
binding (Lazareno and Birdsall, 1995). The effect of
staurosporine on ACh binding is not clear from inspection
of Figure 1, but nonlinear regression analysis of the
data, which also takes into account the effects of high
concentrations of staurosporine on the kinetics of 3H-NMS,
provided a good fit to the data (lines in Figure 1) and
revealed a 4-fold negative cooperativity between ACh and
staurosporine. The independent effects of staurosporine
on 3H-NMS and ACh binding across the four receptor
subtypes are easier to visualise when the binding data
are transformed into affinity ratios (Lazareno and
Birdsall, 1995;Lazareno et al, 1998) (Figure~2). In
theory, the ECSO or ICso of the affinity ratio. plot
corresponds to the Kd of the test agent for the free
receptor, and the asymptotic value corresponds to the
cooperativity with the primary ligand. Staurosporine
(1f)~showed positive cooperativity with 3H-NMS at M1 and
MZ receptors, neutral coaperativity with 3H-NMS at MQ
receptors and was inactive or neutrally cooperative at M3
receptors. It had negative cooperativity with ACh at M1,
M2 and MQ subtypes and was neutral with ACh or inactive at
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34
M3 receptors. Staurosporine had Kd values for unoccupied
receptors in the ~M range (Figure 2, Table 1). In two
functional assays with M1 receptors measuring the
stimulation by ACh of 35S-GTPYS binding, 10 uM
staurosporine reduced basal activity and the Emax by 170
~ 7% and 25% ~ 4°s respectively, and also caused a 2.9 ~
0.9 fold decrease in the potency of ACh, which is
consistent with the 3.6-fold change predicted from the
3H-NMS binding studies (data not shown).
Staurosporine also inhibited 3H-NMS dissociation (Figure
3). All the curves had slope factors of 1.
Staurosporine was most potent and effective at M1
receptors, causing apparently complete inhibition of
3H-NMS dissociation with an ICSO of 1 ~M (Table 2). It
was 3-4-fold weaker at the other receptor subtypes, and
also caused submaximal inhibition of 3H-NMS'dissociation,
with th.e smallest effect, 67% inhibition, seen at M3
receptors. The ICso values for the inhibition of 3H-NMS
dissociation correspond in theory to the Kd values of
staurosporine for the 3H-NMS-liganded receptors, and the
values at M1 and M2 receptors are consistent with the
values predicted from the equilibrium binding studies
according to the allosteric model (Table 2). There was a
2-fold disparity between predicted and observed values at
MQ receptors, probably because of inaccuracies in
measuring the .small degree of negative cooperativity with
3H-NMS. In equilibrium binding studies at M3 receptors
staurosporine had little or no effect on the binding of
either 3H-NMS or ACh: the clear.inhibition of 3H-NMS
dissociation caused by staurosporine over the same
concentration range suggests that staurosporine was
neutrally cooperative with 3H-NMS and ACh at M3 receptors,
rather than inactive.
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Go 7874 (1i), a ring-opened analogue of staurosporine
still bearing a positive charge, showed weak negative
cooperativity with 3H-NMS and stronger negative
cooperativity with ACh at M1, MZ and Mq receptors, and the
5 reversed pattern at M3 receptors (Figure 2). It was
necessary to introduce a slope factor >1 into the binding
equation for Go 7874 in order to fit the data adequately
to the allosteric model (Table 1). Go 7874 caused
apparently complete inhibition of 3H-NMS dissociation at
10 M1, MZ and M9 receptors, and submaximal inhibition at M3
receptors. The slopes of the curves at M1, MZ and M9
receptors were also >1 (Figure 3, Table 2). The ternary
complex allosteric model does not predict slope factors
different from l, so it cannot provide a complete
15 mechanistic explanation of the data. Nevertheless, the
affinity values of Go 7874 for the 3H-NMS-occupied
receptor predicted by the model from the equilibrium
binding studies are in excellent agreement with the
observed values at M1 and Mq receptors (Table 2), and show
20 only a 3-fold discrepancy at MZ and M3 receptors, caused
possibly by a combination of inaccuracies in the
measurement of the small cooperative effects which
occurred in equilibrium studies at the M2 and M3 subtypes
(Table 1) and the small.inhibitory effect on 3H-NMS
25 dissociation from M3 receptors.
KT5823 (1e), a ring-contracted analogue of staurosporine
in which the methylamino group is replaced by a methyl
ester, caused a large increase in 3H-NMS binding at M1 and
30 M~ receptors, and showed neutral or small positive
cooperativity with ACh at these receptors. KT5823 was
inactive-or.neutrally cooperative with 3H-NMS and ACh at
M3 and MQ receptors (Figure 2). The positive
cooperativity with NMS at M1 receptors was confirmed in
35 functional studies in which 1mM KT5823 increased the
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potency of ACh 1.9 ~ 0.9 fold at.Ml receptors for
stimulating 35S-GTPyS binding, and caused a 3.3 ~ 1.7 fold
increase in the affinity of unlabelled NMS (n=2, data not
shown). KT5823 inhibited 3H-NMS dissociation completely
at M1 receptors, 80s at Mz receptors and 30-40% at M3 and
Mq receptors (Figure 3). The affinity of KT5823 for the
3H-NMS-occupied receptor estimated from equilibrium
studies at M1 and Mz receptors was very similar to the
values measured directly. The inhibition of 3H-NMS
dissociation seen at M3 and M9 receptors may indicate that
KT5823 is neutrally cooperative with 3H-NMS and ACh at
these receptors, rather than inactive.
KT5720 (1a), a hexyl ester analogue of KT5823, was
positively cooperative with both 3H-NMS and ACh at M1
receptors (Figure l, Table 1). The small (400) increase
in ACh affinity was confirmed in more detailed assays
(Figure 5). KT5720 had little or no effect at M3
receptors, and showed neutral cooperativity with 3H-NMS
and negative cooperativity with ACh at Mq recep.tors. The-
effects of KT5720 at MZ receptoxs are unclear: earlier
batches had small inhibitory effects with 3H-NMS and ACh
(Figure 2), while a later batch had small positive
effects with 3H-NMS (data not.shown):.no batch-dependent
effects were noted at the other subtypes. KT5720 caused
incomplete inhibition of 3H-NMS dissociation at M1, M3 and
Mq receptors, with little or no effect at MZ receptors
(Figure 3). The largest effect was seen with M1
receptors, and, at this subtype alone,. low concentrations
of KT5720 caused a small but.consistent increase in 3H-NMS
dissociation. This phenomenon was observed in l0~out of
11 single-time point assays, with the dissociation rate
constant (~Coff) of 3H-NMS increased by 11 ~ 1 0 (n=10) in -
the.presence of the most effective concentration between
10 and 300 nM KT5720, and in two full time course studies
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in which koff in the presence of 0.1 uM KT5720 was
increased by 16.3 ~ 0.5 0 (data not shown). The affinity
of KT5720 for the 3H-NMS-occupied receptor estimated from
equilibrium studies at M1 and M4 receptors was similar to
the values measured directly (Table 2).
K-252a (1b), in which the methoxy group of KT5823 is
replaced by a hydroxyl group, showed positive
cooperativity with 3H-NMS at M1 receptors and neutral or
small negative cooperativity with ACh (Figure 2, Table.
1). Little or no effect was seen in equilibrium binding
studies with the other subtypes. K-252a inhibited 3H-NMS
dissociation at M1 receptors, apparently by 100%. Slope
factors > 1 were required to fit the data adequately.
Only small, though consistent, effects on 3H-NMS off rate
were seen at the other subtypes (Figure 3, Table 2).
K-252b (1c), K-252c (1g), KT-5926 (1d) and Go 6976 (1h)
at concentrations up to 10 uM had little or no effect on
equilibrium binding of 3H-NMS and ACh and on 3H-NMS
dissociation (data not shown) and were not studied
further.
We have attempted to determine whether some of the
allosteric effects described above occurred through an
interaction at the same site on the receptor at which
other known allosteric agents act. Figure 5a shows the
interaction between KT5720 (la) and gallamine on
equilibrium 3H-NMS binding at M1 receptors. Gallamine had
its expected inhibitory effect on 3H-NMS binding, and
KT5720 showed the expected positive cooperativity with
3H-NMS. If gallamine and KT5720 were acting at the same
site then gallamine should have become less potent in the
presence of KT5720 and the nonlinear regression analysis
would have indicated strong negative cooperativity
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between the two agents. In fact, the analysis revealed
neutral cooperativity, i.e. in equilibrium binding
studies gallamine and KT5720 interact allosterically at M1
receptors through distinct and apparently non-interacting
sites. In similar experiments with staurosporine and
gallamine at M1 receptors, however, there was an
negatively cooperative or competitive interaction between
the compounds (Figure 5b).
In order to study the sites) on the M1 receptor at which
KT5720 acts to affect 3H-NMS dissociation, the
concentration-related effect of KT5720~was measured alone
and in the presence of two or three concentrations each
of gallamine, brucine and staurosporine. Very similar
results were obtained in two independent assays, and the
combined data are shown in Figure 6. The data in each
condition are shown in two forms: as percentage
inhibition of the overall control (i.e. 'true') koff
measured in the absence of any test agent, and, for each
curve, as a fraction of its own control koff measured in
the presence of test agent and the absence of KT5720.
This latter 'fractional effect' measure has useful
properties: if the interaction between KT5720 and the
test agent is competitive, then in the presence of test
agent the ECso will increase and the asymptotic
'fractional effect' will also change; if the interaction
is noncompetitive and noninteracting (i.e. with neutral
cooperativity), and if maximal concentrations of test
agent completely inhibit 3H-NMS dissociation, then in the
presence of test agent both the ECSO and asymptotic levels
are unchanged.
The lines in the top panel of Figure 6 (except in the
presence of staurosporine) are hyperbolic fits to the
data. The effect of low concentrations of KT5720 to
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increase 3H-NMS dissociation was apparent in all the
curves. When the data are expressed as a fractional
effect of own control, the curves for KT5720 in the
presence of various concentrations of gallamine or
brucine overlap, i.e. they have the same ECSO and
asymptotic level. There was a small concentrati:on-
related increase in potency in the presence of gallamine,
but this.is probably experimental noise, since a
positively cooperative interaction would result in
decreases in the asymptotic level of the 'fraction of own
control' plots. These data therefore demonstrate that
KT5720 acts at a different site from the site(s.) at which
gallamine and brucine act to inhibit 3H-NMS dissociation
from M1 receptors.
A quite different pattern of results was seen with
staurosporine. The stimulating effect of low
concentrations of KT5720 became more apparent, and the
curves tend to converge at high concentrations of KT5720
more than in the presence of gallamine or brucine. It
was not possible to measure ECso values accurately, but
inspection of the 'fractional effect' plot suggests that
staurosporine reduced the potency of KT5720. These
results indicate that staurosporine and KT5720 compete
for the site which mediates inhibition of 3H-NMS
dissociation. They strongly suggest that staurosporine
can act at different sites) from gallamine or brucine.
Discussion
Five of the nine indolocarbazoles which we have studied
act allosterically at muscarinic receptors. Of these,
four have similar structures.and a number of similarities
in their allosteric effects, while the fifth, Go 7874
(1i),.lacks the tetrahydrofuran/pyran ring system, which
may account for its somewhat different effects.
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In equilibrium binding studies, the four active
staurosporine-like compounds (staurosporine (1f), KT5823
(1e),. KT5720 (la) and K-252a (1b)) showed only positive
or neutral cooperativity with 3H-NMS, or were apparently
5 inactive, while positive, neutral and negative
cooperativity was observed with ACh. The four compounds
showed their highest affinity, and largest positive
effects with 3H-NMS, at the M1 receptor, while they were
inactive (o.r neutrally cooperative with 3H-NMS and ACh) at
10 M3 receptors. These compounds bound with slope factors of
1, except for KT5823 at M1 receptors, and this exception
may be partly accounted for by artefacts arising from the
strong (7-10 fold) positive cooperativity with 3H-NMS seen
with this compound. Go 7874 (1i), the other positively
15 charged ligand in addition to staurosporine, also showed
selectivity for the M1 receptor but, in contrast to the
other four compounds, it showed negative cooperativity
with 3H-NMS, and both neutral and negative cooperativity
with ACh, and it bound with slope factors greater than 1.
The four staurosporine-like compounds also showed
selectivity for the 3H-NMS-occupied M1 receptor, but this
was manifest more clearly in the magnitude of inhibition
of 3H-NMS dissociation than in the affinity. Again; these
compounds bound to the 3H-NMS-occupied receptor with
slopes of l, except for K-252a at M1 receptors. Go 7874
inhibited 3H-NMS dissociation completely from M1, M2 and MQ
receptors with slope factors significantly greater than
1.
There seems to be a relationship between the activity of
the compounds in equilibrium binding assays and the
maximum degree of inhibition of 3H-NMS dissociation: an ad
hoc correlation for the current data is that compounds
showing less than 50o inhibition of 3H-NMS dissociation at
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a particular subtype appear.inactive in equilibrium
studies, while those slowing 3H-NMS dissociation.by > 500
show activity in equilibrium studies. This rule works in
17/20 cases, the exceptions being staurosporine at M3; Go
7874 at M3 and KT5720 at M4 receptors. The positive
relationship between allosteric activity at equilibrium
and the degree of inhibition of 3H-NMS dissociation may
reflect the degree to which binding of the allosteric
agent perturbs the primary ligand recognition site on the
receptor. Those cases where the test agent inhibits
3H-NMS dissociation but appears to be inactive at'
equilibrium may actually reflect a lack of cooperative
effect, i.e. neutral cooperativity, rather than a lack of
binding of the test agent at equilibrium.
According to the allosteric model, the affinity of a test
agent for the 3H-NMS-occupied receptor may be estimated in
two independent ways: from direct measurement of effects
on 3H-NMS dissociation, and from the product of.affinity
for the free receptor and cooperativity with 3H-NMS,
measured at equilibrium. In this study there are 11
instances .where these measures have been determined with
sufficient precision to allow comparison. There was good
agreement between the measures:- three comparisons
differed by about 3-fold, one by about 2-fold, and the
rest (7) by 60o or less, and there was no obvious bias
since in 5 cases the equilibrium estimate was larger than
the directly measured value and in 7 cases it was
smaller. These results suggest that the data can be
accounted for by the allosteric model, even though the
steep slopes seen with Go 7874 (1i) and K-252a (lb).are
not predicted by the model.
The simple model also cannot account for the effects of
KT5720 on 3H-NMS dissociation at M1 receptors, with an
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initial speeding of dissociation by about~l5 o at
submicromolar concentrations, followed by submaximal
inhibition of dissociation at higher concentrations. In
the presence of staurosporine the speeding effect became
more prominent, while the potency of KT5720 for slowing
3H-NMS dissociation appeared to be reduced, suggesting:
that KT5720 may be exerting its effects at two distinct
sites, only one of which can also be occupied by
staurosporine. In contrast, the presence of gallamine or
brucine had no effect on the potency of KT5270 or its
fractional asymptotic effect, suggesting that, unlike
staurosporine, gallamine and brucine act at a different
site from the sites) by which KT5720 modulates 3H-NMS
dissociation, and that there is no interaction (i.e.
neutral cooperativity) between the binding of KT5270 and
that of brucine or gallamine.
A similar conclusion can be drawn from equilibrium
binding studies at M1 receptors, in which KT5720 showed no
interaction with gallamine. In contrast, similar
equilibrium binding studies at M1 receptors with
s.taurosporine and gallamine revealed a negatively
cooperative or competitive interaction. The different
interactions with gallamine shown by staurosporine
(negative) and KT5720 (neutral) may be related to the
fact that staurosporine, like gallamine, is a positively
charged molecule, whereas KT5720 is neutral.
These results demonstrate that KT5720, and possibly other
indolocarbazoles, bind to an allosteric site on
muscarinic receptors which is distinct from the 'common
allosteric site' to which gallamine and most other
allosteric agents bind. Previously reported allosteric
agents have a positively charged nitrogen which is
thought to be important for their action. Staurosporine
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and Go 7874 are also positively charged, but the other
active indolocarbazoles are neutral, which suggests that
there is no necessity for a positively charged nitrogen
at this new allosteric site. The observed affinities and
cooperativities are sensitive to small changes in the
chemical structure of the analogues. For example,
increasing the alkyl chain length of the ester function
of K-252a or methylation of its hydroxyl group increase
affinity 3-15-fold, whereas removal of the methyl group
on the ester of K-252a or the alkoxy substitution of the
indolocarbazole ring generate apparently inactive
compounds.
The agents studied here are known to be potent inhibitors
of various protein kinases, and in most cases the agents
have much higher affinity for these targets than for
muscarinic receptors, but it is worth noting that KT5720
has only about 6-fold higher potency for its preferred
target, protein kinase A (PKA), than for the M1 receptor
(log affinity of 7.2 at PKA vs. 6.4 at M1 receptors).
One of our aims has been the development of drugs which
enhance the affinity of ACh at M1 receptors while having
no effect on ACh binding and function at the other
subtypes. The detection of the allosteric properties of
KT5720 may be a step towards that goal. KT5720 was the
most potent compound at M1 receptors with a log affinity
for the free receptor of 6.4, and it showed a small (400)
but consistent positive cooperative effect with ACh. In
addition it had little or no effect on ACh affinity at
the other subtypes, so KT5720 is close to displaying an
'absol.ute subtype selectivity' for the M1 receptor, i.e. a
positive or negative interaction with ACh at one receptor
subtype and neutral cooperativity at the others, so that
whatever concentration of agent is administered only the
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one receptor subtype is affected functionally (Lazarerio
and Birdsall, 1995).
The results in this section show a quite potent
allosteric interactions of staurosporine and some other
indolocarbazole analogues at muscarinic receptors which,
at least in the case of KT5720, occur at a site distinct
from the 'common allosteric site'. The active
indolocarbazoles cause different maximal effects on 3H-NMS
dissociation, and the size of the maximal effect on 3H-NMS
dissociation is a good predictor of the activity detected
in equilibrium studies, suggesting a common mechanism for
the two effects. In general the results from equilibrium
and dissociation assays were mutually consistent with the
ternary allosteric complex model as the underlying
mechanism of the observed effects. Finally, KT5720 is
the most potent agent described so far showing positive
cooperativity with ACh at M1 receptors.
2 0 Part 2
Results
We also examined the interaction of a second family of
compounds represented by general formula 2 shown on pages
55 and 56. Figure 7 shows.representative data from off
rate assays for these structures, and the parameter
estimates are summarised in Table 3. Figures 8 and 9
show representative affinity ratio plots from equilibrium
assays, and the parameter estimates are summarised in
Table 4.
2b (WIN 51708) strongly inhibited 3H-NMS dissociation at
Mz and Mq receptors, with about ~ 10-fold MQ selectivity, it
caused submaximal inhibition at the M1 receptor and had no
effect at the M3 receptor. 2c (WIN 62577), containing a
5-6 double bond, which reduced the affinity for the
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3H-NMS-occupied MQ receptor by about 10-fold and led to a
smaller maximal effect at Mz receptors. These effects
were reflected in the equilibrium assays, where 2b showed
a small degree of positive cooperativity with 3H-NMS at MZ
5 receptors and a larger positive effect at MQ receptors,
while..2c, which was up to 5-fold less potent, showed only
small negative cooperativity with 3H-NMS. With respect to
ACh, 2b showed small negative cooperativity at M1 and M3
receptors and larger negative effects at Mz and M9
10 receptors, while 2c was also negative at MZ and Mq
receptors, was almost neutral at M1 receptors, and showed
a small (1.5-fold) positive.interaction with ACh at M3
receptors. This latter effect was confirmed in more
detailed assays (e. g. Figure 10). It is worth noting
15 that the potency and small degree of negative
cooperativity with 3H-NMS of both 2b and 2c at M3
receptors should result in some activity at M3 receptors
in the off rate assay, whereas no activity was observed.
20 ~ Removal of the bridgehead nitrogen from 2c gave 2a and
led to a 10-fold increase in affinity at M1 and Mz-
receptors, a 30-fold increase at M3 receptors, but little
or no change in affinity at MQ receptors. With a log
affinity of 6.5 it was the most potent of these compounds
25 at M1 receptors. It showed 2-5 fold negative
cooperativity with both 3H-NMS and ACh across the receptor
subtypes. In the off rate assay 2a had the unique effect
of speeding 3H-NMS dissociation. It caused a 2-3 fold
increase in 3H-NMS off rate at M3 receptors, with smaller
30 effects at the other subtypes, with the order of
effectiveness M3>M1>MQ>M2. This effect was confirmed in
full dissociation assays (Figure 11).
Replacement of the ethynyl and hydroxy groups of 2a with
35 a keto function, in 2f and 2i, resulted in a complete
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loss of activity in the off rate assay (Figure 7) and. the
equilibrium assay (data not shown).
Removal of the ethynyl substituent from 2b and 2c gave
rise to the 17-hydroxy analogues, 2d and 2e. The
compounds had slightly greater potency than their
corresponding analogues in the off rate assay, and larger
inhibitory effects at M1 receptors, but still little or no
effect at the 3H-NMS-occupied M3 receptor. In equilibrium
studies (Figure 8) 2d showed positive cooperativity with
3H-NMS at all subtypes, and its log affinity of 7 at the
free Mq receptor was the most potent interaction in this
study.' 2e showed small negative, neutral and positive
effects with 3H-NMS. Both compounds had negative
cooperativity with ACh, except 2e which showed a small
(30%) positive cooperativity with ACh at M3 receptors.
2g and 2j are analogues of 2b lacking two rings of the
steroid moiety of 2b. 2g is reported to be the trans
isomer and 2j is reported to be the cis:isomer. 2b
itself has the trans configuration, so it is very
surprising that 2g was virtually inactive in the off rate
assay (Figure 7) and equilibrium assays (data not shown),
while 2j showed strong activity in the off rate assay
(albeit 10-100 fold weaker than 2b), and in equilibrium
assays, where it was strongly negative with 3H-NMS and
ACh, and only 2-5 fold less potent than 2b.
21 is a truncated form of 2j, and no longer chiral. It
was also active, showing similar potency to 2j in the off
rate assay (and a bigger effect on M3 receptors), but less
potency in equilibrium assays, especially at Mz and Mq
receptors (Figure 9), leading to positive cooperativity
with 3H-NMS at these subtypes.
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2h is the pyrimidoimidazole analogue of 2c, lacking the
fused benzene ring. This change caused a reduction in
affinity for both free the 3H-NMS-occupied receptor of 2- .
20 fold, and stronger negative cooperativity with jH-NMS
and ACh.
2k is an analogue of 2h with the imidazoie ring attached
to a different portion of the pyrimidine ring. It had a
30-200 fold higher affinity than 2h at 3H-NMS-occupied
receptors, or more than 1000-fold at M3 receptors if the
small effects at this subtype have been correctly
interpreted. It also had 10-50 fold higher affinity for
the free receptor than 2h, with Kd values of less than 1
mM at all subtypes. It showed strong positive
cooperativity with 3H-NMS at M1 receptors (Figure 9), and
was positive with 3H-NMS at Mz and M9 receptors and weakly
negative at M3 receptors. It had 2-4 fold negative
cooperativity with ACh.
2t, an analogue of 2k but with a pyrimidoimidazole
substituent on the 16,17 positions of the steroid
backbone, was inactive, reinforcing the importance of
substitution in this region for activity.
Most compounds which bind to the primary or allosteric
sites of muscarinic receptors have a basic nitrogen,
though neutrally charged antagonists have been described.
The two steroid structures (2m and 2n),. which do not
contain nitrogen, correspond to the steroid portion of
2c, 2h, 2a and 2k. Surprisingly, they show activity in
both the off rate and equilibrium assays, and 2m seems to
be more potent than 2h in the off rate assay (Figure 7).
The 17-keto analogue, 2s, and the.,2-substituted
analogues, 2o-2r appeared to be inactive.
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With regard to slope factors, most of the compounds had
binding slopes of 1 in equilibrium or kinetic assays. The
relatively weak compounds 21, 2j and 2h however had
steeper slopes indicating a more complex interaction.
The effect of 2a to increase 3H-NMS dissociation provides
an opportunity to assess whether 2a binds to the same
site on the 3H-NMS-occupied receptor as other allosteric
agents. The dissociation rate constant (koff) of 3H-NMS
from the M3 receptor was measured at a single time point
alone and in the presence of a range of concentrations of
2a, alone and in the presence of one or more
concentrations of a second agent. The data were
expressed as % of control koff, as described in Methods,
and fitted to Equation 1. The results are shown in
Figure 12 and Table 6. Strychnine and gallamine reduced
the Emax of 2a, indicating that they were acting at a
different site from that occupied by 2a, but they did not
affect the ECso of 2a; indicating that there was no
cooperative interaction, i.e. they showed neutral
cooperativity. In contrast, 1a (KT 5720), if
(staurosporine) and 2b (WIN 51708) did not appear to
alter the Emax of 2a but reduced its potency, and the data
were well fitted assuming a competitive interaction,
although a strongly negative interaction cannot be ruled
out. These results demonstrate that there are two
allosteric sites on the M1 and M3 receptors. It is worth
noting that 2b (WIN 51708) clearly binds to the 3H-NMS-
occupied M3 receptor, even though it does not modify the
3O koff of 3H-NMS .
Discussion
This paper describes a new series of compounds which
interact allosterically with muscarinic receptors. The
initial lead was provided by two commercially available
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compounds, 2b (WIN 51708) and 2c (WIN 62577). These
compounds are potent antagonists at rat NK1 receptors, but
the affinity of these compounds, exemplified by 2b, is .
reduced 400 fold at the human NK1 receptor. In fact our
results show that 2b is up to 60 fold more potent on
human muscarinic receptors compared to human neurokinin
receptors.
These two compounds and the analogues described in this
paper exhibit positive, neutral and low negative
cooperativities with NMS and especially ACh. This latter
characteristic is important in that it suggests that the
allosteric enhancers at a specific muscarinic receptor
subtype may be synthesised in this series which are
neutrally cooperative with ACh (and therefore inactive at
any concentrations) at other subtypes. This form of
selectivity, based on cooperativity rather than affinity,
has been termed 'absolute subtype selectivity' (Lazareno
et al, 1998; Birdsall et al, 1999) and is a direct
consequence of the ternary complex allosteric.model.
(Lazareno and Birdsall, 1995) which underpins the
analyses of all our binding and functional data.
The ternary complex allosteric model implies that the
affinity of a compound for the 3H-NMS-occupied receptor
can be estimated in two ways: as the product of affinity
for the free receptor and cooperativity with 3.H-NMS from
equilibrium assays, and as.the reciprocal of the IC50 (or
EC50) from off rate assays. Table 5 shows a comparison
between the pKocc (f.rom equilibrium studies). and the
pKoff (from off rate studies) values of those compounds
for which there are at least two observations of each
type of measure. Of the 32 comparisons, 23 show a
discrepancy between the two measures of less than 0.3 log
units (2-fold), a value which is readily accounted for by
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experimental error. In most other cases the discrepancy
can be accounted for by inaccuracies either observed in
the data 'or predicted from the effect of the compound.
The discrepancies of 2b and 2c at M1 receptors may be
5 explained by the small effects in the off rate assay, 2d
has small effects at the M3 receptor in the off rate
assay, and at the MQ receptor lower concentrations should
have been used to define better the parameters. 2j has
strong negative cooperativity at MZ and MQ receptors which
10 cannot be measured accurately with the experimental
designs used here, and 2k has very small effects in the
off rate assay with M3 receptors. That leaves the 3-fold
discrepancies for both 2d and 2k at MZ receptors, for
which there are no obvious explanations. Two outliers
15 out of 32 observations may be within the expected
variability of such data, and overall the data pass this
rather stringent test and are therefore consistent with
the ternary complex allosteric model as the underlying
mechanism which is responsible for effects on both
20 equilibrium binding and on 3H-NMS dissociation.
2b, 2c and and the analogues examined can be considered
in simplistic terms as a fusion of a planar aromatic
heterocyclic system with an alicyclic ring system,
25 especially a steroid structure. Most surprisingly both
the steroid moiety.alone, for example 2n, (but not some
other analogues) and the heterocyclic ring system (21)
are individually capable of interacting allosterically
with 3H-NMS and with comparable affinities to each other.
30. This result implies that these compounds may interact
with different but contiguous subdomains of the same
pharmacophore.
Binding to the allosteric site is sensitive to the nature
35 of the heterocycle when the steroid ring is kept constant
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(compare 2h, 2a and 2k with 2c). Equally the nature of
the 17-substituent on the steroid ring seems important in
the fused systems. The analogues with a 17-keto function
appear inactive, whereas all compounds with a 17(3
hydroxyl group are active. The presence of the 17.-a
ethynyl group has more subtle effects (mainly on the M9
receptor) and these seem to be interrelated with effects
of the saturation status of the 5-6 bond.
Another surprising result is that 2j is active whereas 2g
appears to be inactive. Both these compounds are
racemates and they represent truncated analogues of 2b
with the AB rings of the steroid in the cis and trans
configurations respectively. Activity was expected to be
associated with the trans isomer rather than the cis
isomer.
Many of the compounds investigated in this study, in
contrast to most muscarinic allosteric agents, do not
inhibit the association and dissociation of 3H-NMS
completely at high concentrations. They often only
produce a 2-fold or less slowing effects on the kinetics
and in some instances, especially at M3 receptors, very
small effects indeed (Figure 7).
The remarkable finding is that 2a increases the
dissociation rate of 3H-NMS with the largest effect being
observed a.t M3.receptors and the smallest effect at. M2
receptors. It is noteworthy that the data from
equilibrium and off rate studies with 2a are entirely
consistent with the allosteric model, i.e. there are no
discrepancies between estimates of affinity for the
3H-NMS-occupied receptor from the two types of assay, and
the slope factors were 1 or close to 1. The agreement
occurs despite the enhancement of 3H-NMS dissociation by
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2a being the opposite to the inhibitory effect seen, with
all the other compounds in this study, and in every other
published study of allosteric agents at muscarinic
receptors.
This unique effect of 2a allowed us to assess whether
other allosteric agents inhibit 3H-NMS dissociation by
acting at the same site as 2a. We found that la
(KT5720), 1f (staurosporine) and 2b bind to the same site
as 2a on the 3H-NMS-liganded receptor, but gallamine and
strychnine bind to a different site and have no effect on
the binding of 2a, i.e. gallamine and strychnine show
neutral cooperativity with 2a.
In conclusion, the following general points can be made.
(1) allosteric agents can enhance, inhibit, or have no
effect on the dissociation rate of 3H-NMS. (2) There are
at least two nonoverlapping allosteric sites, the
'common' site and the 'WIN' site; (3) Both allosteric
sites can support a positive cooperative interaction with
ACh; (4) The use of 2a provides a test of whether another
allosteric agent binds to the 'WIN' site.
CA 02386149 2002-04-19
WO 01/29036 PCT/GB00/04064
53
References:
The references mentioned herein are all expressly
incorporated by reference.
Caulfield and Birdsall, International Union of
Pharmacology XVII: classification of muscarinic
receptors. Pharmacol. Rev., 50:279-290, 1998.
Hulme et al, Ann. Rev. Pharmacol., 30:633-73, 1990.
Lazareno and Birdsall, Br. J. Pharmacol., 109:1110-1119,
1993.
Lazareno and Birdsall, Mol. Pharmacol., 48:362-378, 1995.
Lazareno et al, Mol. Pharmacol., 53:573-589, 1998.
Lazareno and Birdsall, 'Measurement of competitive and
allosteric interactions in radioligand binding studies.'
In: G protein-coupled receptors. Haga and Berstein, eds,
CRC press, Boca Raton, 1-48, 1999.
Birdsall et al, Mol. Pharmacol. 55:778-786, 1999.
Buckley et al, Mol. Pharmacol., 35:469-476, 1989.
Gnagey and Ellis, Biochem. Pharmacol., 52:1767-1775,
1996.
Bajwa and Sykes, J.C.S. Perkin I, 1618-1620, 1978.
Bajwa and Sykes, J.C.S. Perkin I, 1816-1820, 1979.
Bajwa and Sykes, J.C.S. Perkin I, 3085-3094, 1979.
CA 02386149 2002-04-19
WO 01/29036 PCT/GB00/04064
54
Bajwa and Sykes, J.C.S. Perkin I, 481-486, 1980.
Bajwa and Sykes, J.C.S. Perkin I, 1019-1030, 1980.
Bajwa and Sykes, J.C.S. Perkin I, 1859-1861, 1980.
CA 02386149 2002-04-19
WO 01/29036 PCT/GB00/04064
R1 R2 R3 H
0 N
1 a KT5720 OH COO(CHZ)5CH3 H
1b K-252a OH COOCH3 H R3 I \ ~ ~ I \
1 c K-252b OH COOH H ~ N~ ~N
,,,~0~
~~"'~Me
1 d KT5926 OH COOCH3 O(CH2)ZCHa H
R1
1e KT5823 OCH3 COOCH3 H R2
H
0 N
1f Staurosporine ~ / ~N
H o,.1_ 0
HMe
H
R1 R2
1 g K-252c H H
1 h Go 6976 CHs CH2CH2CN
H
AI
OMe
1 i Go 7874 HCI I
Me
NMet
SUBSTITUTE SHEET (RULE 26)
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56
23 MeoH 2b WIN 51708 ~ ZIC WIN 62577
.,"C-CH Me ",C=CH Me "uC-CH
Me a , Me
I ~ I ~ I ~ N ~ H I ~ N ~ H
i ~ i ~ N~' i
H H
2d a 22 ~ off 2f Me o
Me Me Me
N ~
I ~ N N I ~ ~~ ~ I ~ N I
H
2g traps 2h MeoH 2i a o
...,C-CH
H Me a - NCH
I ~ N~ ~i~ ~ v
0
' I ~~ i Ii N I , i
H TV N
H
2j cis 2k MeOH~~
H Me
N ~
I ~ N~/ '~~J~ N I i
N H ~N
N
2l 2m MeoH 2~ I"~oH
."~C-CH .,,.C.CH
Me a
H ~.o ~ HO ,
I i NJ~~ ~ i 0 i
SUBSTITUTE SHEET (RULE 26)
CA 02386149 2002-04-19
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57
20 2p
off
Me OH,C-CH Me .u~C=CH
N_-~ N=l Me
HN N Me HN~N.N
p ~ 0 / 0 OH ~ i
2q 2r
Me O.HC-CH
N
Me N~,N~ / Me
N~N.N
0
0
25 2f Me
Me 0 Me
I
Me N ~'~~ y%~ ~~=N
i ~N'
0 ~ N
2u
Me
I
~~N 0
CA 02386149 2002-04-19
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58
Table 1
Equilibrium binding parameters of indolocarbazoles with 3H-NMS and Ach at
muscarinic receptors. Assays such as those shown in Figures 2 and 5 were
fitted to
Equation 1 or 2 as appropriate (see Methods). The results are from at least 3
assays,
except * n=2. Empty cells indicate that it was not possible to obtain at least
2 sets of
parameter estimates.
#Some, but not all, of the values were obtained from analyses in which the
affinity for
the 3H-NMS occupied receptor was fixed at the mean value obtained from offrate
assays and shown in Table 2.
M, M~
cooperativity cooperativity
name pK slope 'H-NMS pK slope 'H-NMSACh
ACh
laKT 5720 mean 6.42"1.00 1.94 1.395.29 1.051.56 0.82
sem 0.09 0.00 0.14 0.090.33 0.050.60 0.45
IbK-252a mean 5.13'"1.00 2.62 0.86
sem 0.14 0.00 0.68 0.13
1eKT 5823 mean 5.70 1.44 8.11 1.315.67' 1.003.27 1.34
sem 0.05 0.14 1.61 0.440.15 0.000.19 0.03
1 staurosporinemean 5.91 1.01 1.53 0.195.13 I.001.49 0.42
f
sem 0.03 0.01 0.06 0.040.05 0.000.06 0.04
1 Go 7874 mean 5.77'1.60 0.54 0.075.03' 1.910.66 0.24
i
sem 0.16 0.17 0.04 0.000.16 0.080.00 0.11
M~ M.
cooperativity cooperativity
name pK slope 'H-NMS ACh pK slope 'H-NMS
ACh
1 KT 5720 mean 6.42' 1.000.92 0.55
a
sem 0.25 0.000.01 0.08
1b K-252a mean
sem
1e KT 5823 mean
sem
l staurosporinemean 5.31 1.000.88 0.15
f
sem 0.09 0.000.02 0.03
1 Go 7874 mean 5.12' 1.50 0.32 0.72 5.71' 1.470.80 0.10
i
sem 0.01 0.50 0.09 0.05 0.13 0.210.05 0.00
SUBSTITUTE SHEET (RULE 26)
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Table 2
inhibition of 3H-NMS dissociation from muscarinic receptors by
indolocarbazoles.
Curves such as those shown in Figure 4 were fitted to a logistic equation as
described
in Mothods. The 'estd pK' is the product of affinity for the free receptor and
cooperativity with 3H-NMS derived from the equilibrium binding assays
summarised
in Table 1. The 'diff' is the difference between the observed pK (-log ICSO)
and 'estd
pK'. Empty cells indicate that it was not possible to obtain at least 2 sets
of parameter
estimates. The 3H-NMS dissociation rate constants (minutes ~) observed in this
study
are (mean ~ sem (n)): M1 0.058 ~ 0.002 (26); MZ 0.34 ~ 0.01 (12); M3 0.054 ~
0.002
( 10); M4 0.057 ~ 0.002 ( 10).
M, M=
name pK slopeEmaxestd d;~j'pK slopeEmax estd
pK pK
I KT 5720 mean G.18 1.00SG.796.70 _p"52 5.36
a
sem 0.1 0.003.810.09 0.2G
S
n 7 G S
I K-2S2a mean S.S4 1.40100.00 5.651.0035.05
b
sem 0.01 0.070.00 0.000.002.35
n 2 2
IcKTS823 mean 6.40 1.00103.556.58 _O,~g6.211.0077.856.18 0.02
sem 0.01 0.003.150.08 0.050.001.95 0.18
n 2 4 2 2
I staurosporinemean 6.01 1.00104.736.10 -0.095.401.0090.775.31 0.09
f
sem 0.08 0.005.280.04 0.020.008.34 0.06
n 3 S 3 '
3
IiGo7874 mean 5.70 1.34100.00S.SO 0,~95.301.31100.004.85
0.45
sem 0.02 0.010.000.19 0.010.010.00 0.16
n 2 2 2 2
M~ M,
name pK slopeEmax estd slopeEmax estddfJf
pK d;~- pK
pK
I KT 5720 mean 6.661.0038.71 6.331.0024.706.38_0.0f
a
sem 0.150.001.52 0.080.000.21 0.24
n 3 3 2
I K-2S2a mean S.SS1.0037.10 5.371.0048.95
b
sem 0.000.004.20 0.020.005.75
n 2 2
I KT 5823 mean 6.45I 29.80 5.881.004
c .00 I
.SO
sem 0.080.005.70 0.040.004.40
n 2 2
I staurosporinemean 5.481.0067.10 5.611.0088.005.250.36
f
sem 0.070.002.77 0.080.00G.20 0.08
n 3 3 3
I Go 7874 mean 5.051.0039.504.G0 5.711.64100.005.600,~/
l p_qs
sem 0.280.0014.000. I 0.030.350.00 0.10
S
n 2 2 2 2
SUBSTITUTE SHEET (RULE 26)
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7~M 00 Ov Ov O M M O -~ et O
~ N N f~ f~ t~ d Ov O
' ~
O t~ M f~ V V o0 v1 OW Y1 O
~v1 O 00 .--.t . M N 1 O
~V v1 G~ M 1 M f~ -. O
00 d e~i I~ Ov vi Ni -
b -- O
W~O O~ (~ O G~ G1 00 O~ O O~
.-.
N
a~ - - - N f~ M ~ - ~ O~
Cy.y O O O O .- N M O f~ d
O n1 vW OO r~1
O -- n O O
N N - ~ - --: .-
o o o o o
C o0 M 00 Ov M N O 00 ~
YOv 'V v0 M d 00 I~ d O
O 1~ ~O O~ -- ~ ~ M t~ ~ N
O O O O O O ~ O O
~O ~D ~1 ~D ~D d ~ 'V d
O 0 0 O 0 0 0 O 0
~U ~
O ,.C
O
,~_, ~ XW Ov O o0 r f~ t~ ~D
caOv Ov O d N I~ ~ eh
M t~ O v1 ~ f~ Ow1
Os O V 1~
~p O~ o0 - O O N
I~ V ~1 ~ O ~
ILl~ N d O d
~
a~ - ~ .- .~ t~ ~ ~
O O O O O 'V O
I~
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~
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M N
N 00 N O ~O O Y1
~ '~~V 0 N O -. N ~O
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r O
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1tO ~D O O~ N O O N I~ O ~O
et ~O O ~O v0 O O Ov 00 v1
a1M v0 Oy N .-. O .-:v~ ~O N t~
N .-~Ov N N .~ O: oo 00
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~ .~ O~ t~ O~ N N ~D o0 .-~.-
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N Ov Ov ~O O O ~D
N O O O O O O
~ O O ~ ~ ~ ~
O O O O O O O
Ov M .-.00 00 ~ v1 .-.d ~ M
N V1 M O~ "' Y1 o0 Ov
O O O O O
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00 Cy r1 h M N M
-- O - O O
O -~ O~ v1 N l~ O N oo N
O 00 ~O d t~ O ~O N
~
Oy n a0 N v1 ~O O O t
00 v1 et A1 I~ v0 st N
V1 ~.-. t1 ~ (V 00 -- Os --
p M ~ -- C
a
N WOv d ~O Ov 00 O O Ov M
- N
a.-. - -- - ~ t~ d o~ O. .-
O O O N O oo O~ -- oo
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- O O O O
p -- - .- O -
p O C O G
y~O ~n v~ v~ N oo oo ~O I~
d N N vo O d d N
.- v1 N oo M 0 ~ oo I~ ~
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N O O O O O O O O
O
~ _
II C C C C C C G C C C C
C! ~ ~ v d N ~ N d N v d d v
~ ~ ~ ~ ~ ~ ~ ~ ~ ~
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N N N N N N
N N N N N
v~ N an o - d M vo ~0 00 ~n M N
00 Ov 01 N N o0 N o0 N o0 00
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'" ~
~r
. r h
h ~
z z
a.
v 3 3
M
N N N N N N N~~VN N N N
~i W ~r
SUBSTITUTE SHEET (RULE 26)
CA 02386149 2002-04-19
WO 01/29036 PCT/GB00/04064
61
n O W (D <O ~ O ~ O M N
N O f0 M ~ O N O N
N O ~ O O ~t O
O ~ O O O O
G C O O C C O C
O C C C O C C
O
N
tn N 01 tD ~ 11DN N fD CO 10
~ O N ~ n N O tD 01 ~
~t ~ n ~ ~D N O ~ ~ V n
O N O N M ~- O O O
Z O M O N ~ C O ~ ~- ~ O
~ O O O O O O O C O
O
t0
(O ~ ~ tD M p ~ 1n M ~ ~
(O O O ~ 0I N 07 N M O
- -
N M 00 r ~ a0 M
N O N O . M
p ~ O O ~ ~ O ~
O O O O O O O
N
N COO N OD ~ N M N ~ ~
01 M n ~ ~ 00 M In In N
.- ~ O) O a7 O ,n ~ p ~
O O ~ ~ .- o O o O
w coui n ~ci,~ ,~j~ti~ ~ .~
c o c c o d o c c o
c
M stGO n n N M O N O n
~ n O a0 N .- ~ n N N ~
~ n h M N O N ~ O ~fl
O ~ In O ~ O O O
O O ~ O ~ O O O O O O
O O O O O O O O O O
O
N
O (OOD O n O N ~ O O a0
N p at O O O N O n CD
O sfM h M O M tD N
O ~ fh N O O O N
O O O ~ O O O O O
O O O O O O O O O
Z
n
O
N
n M O r' ~ N 00 ~ (D ~- '-
~O M ~t O O n M O O O
M ch~ sf ~ (D
~ M N N N O
~ ~ ~ ~ ~ ~
O O O O O O
N
U
O
t
l!
U O ~OM OJ n N ~y tD ' ~ ~
t0 M n W tD tD N n N
U (O 'Qr' N a0 O ~ N ~ p ~
O r'~ N M O O O O N
, ~tiuiiciui v ~ ,~jco ~ ~.,j~
o d c o o o c o o o
~ Y
U
et N ~ c0 1n N M ~O N In fD
~ N M CD O N N ~ N M CO
~ O N M ~ O O ~ O N
O ~ O O O O O O O
U O G O C C O O O O O O
O O O O C O O C O O
'd W
tn O N M M N n n e- n N
N N M O Of M tf~n n CO M
~O f tD M n O O c'~t~ O
O ~ ~ O O O ~ O
O ~ O ~ O O O ~ ~ O ~
O ~ O O O O O O O O
O
U
m ~ N ~ n ~ ~ M M ~ ~ ~
N O M O ~ O c0 ~ O
O O O
N O
O N CO O O7
O N ~
O ~ ~ O ~ ~ ~ CV
p O O C O O O
~M
H
O
,~ p M In IA n N (O n Of ~
O t0(O O ~ ~ N In ~ ~ ~
O O ~ O M
~
N O)N ~ O
~ O N N ~
ao Sri~citc Sri~ ,~jcc .~ ~ ~
c c c o c c o c c c
4-~
O n O tD o ~ a0 aD aD M O
~t ~ c0 Os N M t0 N O N O~
N M ~ ~ M O ~
O O ~ ~ ~ O
(~S N ~ O n O O C C O C O O
O O O C C G C O O O
O O O
O O
~ O A
CD a0 u7O ~ O (O sf ~ 01 O O
N ~ t0 c0 ~ (O ~ M ~ O
I
~, U N Z N N N 1~ (O N O ~ ~
O O O N O N O
Cd t*.~ 4--~ ~ O O O ~ O O O a0 O
O O O O O G O ~ O
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j~
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.
. ~ f ~ M ~ M M 07 N r'
,~ M ~ O ~ O N O O
y.
~ N n M N n
~ ~ ~ N N
O
O
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W O O O O O
.,...i _
b0 N ~
U
O II ~[ t0 n a0 n CD ' ~ N N ~ '
n n CO M M N et O
O
Ci N et n tn t0 t0 ap N O n ~ M
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U U (O t(7M ~ M ~ ~j ~O ~ ~ ~
O O O O C O O O O O
N O ~'
N
O c c c c c c c c c c c
c~ O .~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
E E E E E E E E E E E
"" *' E E E E E E E E E E E
N N N N N N N m ~ N H
n O ~ ~ M (O (O CO tA M N
U N ~ ~ ~ ~ ~ ~ ~ m ~ ~ m
~
N N N X i cND
cad O E Z Z
' .~
. ~ o ~ c'i z
~
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E~ ~ f~ ~ GIa n m a c~ v o t N N (V N N
N N ~
N N N N
SUBSTITUTE SHEET (RULE 26)
CA 02386149 2002-04-19
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62
Table 5
Comparison estimates at
of 3H-NMS-occupied
affinity receptors
from equilibrium
(pKocc)
and
offrate
(pKoff)
assays.
'diff
is
the
difference
between
pKocc
and
pKof
n>=2
M, M2
Name pKocc sem sem
pKocc pKoff dill
sem
pKoff
sem
diff
2a 987 5.92 0.09 6.10-0.186.03 0.12 5.81 0.22
0.04 0.19
2b WIN 5.14 0.13 5.55-0.416.15 0.06 5.93 0.22
51708 0.12 0.02
2c WIN 4.63 0.37 5 -0.3 5.02 0.36 5.31 -0.29
62577 0.22 0.15
2d 924 5.90 0.16 5.850.05 6.67 0.23 6.08 0.59
0.05 0.03
2e 923 5.46 0.05 5.320.13 5.43 0.17 5.58 -0.16
0.10 0.01
2h 986 4.29 0.04 4.34 -0.05
0.09
2j 926 3.97 0.32 4.51 -0.55
ds 0.05
2k 988 6.91 0.06 6.860.05 6.70 0.12 6.21 0.49
0.04 0.05
21 925 4.60 0.08 4.77-0.164.73 0.05 4.84 -0.11
0.02 0.01
2n 982 4.96 0.35 4.33 0.63
0.09
M3 M.
Name pKocc sem semdill pKocc sem sem difl'
pKoff pKoff
2a 987 6.06 0.04 6.080.06 -0.035.77 0.09 6.04 -0.27
0.09
2b WIN 51708 6.67 0.07 6.78 -0.1a
0.06
2c WIN 62577 5.80 0.18 5.63 0.17
0.06
2d 924 5.52 0.22 6.100.12 -0.577.35 0.15 6.98 0.37
0.03
2e 923 6.06 0.05 6.19 -0.13
0.04
2h 986 4.40 0.28 4.63 -0.22
0.08
2j 926 3.85 0.02 4.73 -0.88
ds 0.12
2k 988 5.77 0.18 6.710.16 -0.946.31 0.06 6.30 0.01
0.07
21 925 4.58 0.12 4.600.02 -0.024.69 0.09 4.78 -0.09
0.04
2n 982 4.34 0.17 4.24 O.1G
0
Table 6
Estimates of log affinity values for the 3H-NMS-occupied M3 receptor and
maximal
inhibition of 3H-NMS dissociation rate, derived from competition assays with
2a
analysed with the allosteric model (Equation 1 in Appendix 2). The
cooperativity
values were fixed at 0 (competitive or strong negative cooperative
interaction) or 1
(neutral cooperativity, noninteracting). Slope values for the test agent were
fixed at 1,
except for staurosporine (slope=1.31 ~ 0.16, n=3).
Name n pK Emax % cooperativity
gallamine 2 3.38 t 0.02 100 1
strychnine 4 4.06 t 0.01 100 1
KT 5720 3 6.11 ~ 0.10 50 ~ 4 0
staurosporine 3 5.79 t 0.17 43 f 1 0
2b WIN S 1708 3 5.78 ~ 0.06 0 f 20 0
2k 988 2 no effect
SUBSTITUTE SHEET (RULE 26)
