Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-01-08
2003-05-20
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007210, C436S501000
Reexamination Certificate
active
06566080
ABSTRACT:
The invention relates to methods of identifying and using a compound that is an agonist to a peptide hormone receptor.
Peptide hormone receptors are important targets for drug research because a considerable number of diseases and other adverse effects result from abnormal receptor activity. One peptide hormone of interest, cholecystokinin (CCK), is a neuropeptide with two distinct receptors: CCK-A and CCK-B/gastrin (Vanderhaeghen et al.,
Nature
, 257:604-605, 1975; Dockray,
Nature
, 264:568-570, 1976; Rehfeld,
J. Biol. Chem
., 253:4022-4030, 1978; Hill et al.,
Brain Res
., 526:276-283, 1990; Hill et al.,
J. Neurosci
., 10:1070-1081, 1990; Woodruff et al.,
Neuropeptides
, (Suppl.) 19:57-64, 1991). The peripheral type receptor, the CCK-A receptor, is located in discrete brain nuclei and, in certain species, the spinal cord, and is also involved in gallbladder contraction and pancreatic enzyme secretion. The CCK-B/gastrin receptor is most abundant in the cerebral cortex, cerebellum, basal ganglia, and amygdala of the brain, in parietal cells of the gastrointestinal tract, in ECL cells, as well as in kidney cells. CCK-B receptor antagonists have been postulated to modulate anxiety, panic attacks, analgesia, and satiety (Ravard et al.,
Trends Pharmacol. Sci
., 11:271-273, 1990; Singh et al.,
Proc. Natl. Acad. Sci. U.S.A
., 88:1130-1133, 1991; Faris et al.,
Science
, 219:310-312, 1983; Dourish et al.,
Eur. J. Pharmacol
., 176:35-44, 1990; Wiertelak et al.,
Science
, 256:830-833, 1992; Dourish et al.,
Science
, 245:1509-1511, 1989).
SUMMARY OF THE INVENTION
Applicants have developed a systematic screening assay for identifying an agonist specific for a peptide hormone receptor, e.g., a peptide, peptoid, or non-peptide agonist. The assay is based on applicants' recognition that a peptide hormone receptor able to amplify the intrinsic activity of a ligand, e.g., a constitutively active peptide hormone receptor, is useful as a screening vehicle for identifying a receptor-specific agonist. A receptor with a signaling activity higher than the corresponding human wild-type basal level of signaling activity is further useful for detecting the reduction in signaling activity induced by an inverse agonist. In both cases, the receptor amplifies the signal generated when the ligand interacts with its receptor, relative to the signal generated when the ligand interacts with a human wild-type receptor. Thus, forms of a receptor with the ability to amplify receptor signaling are useful for efficiently screening positive and inverse agonists to the corresponding human wild-type form of the receptor.
Accordingly, the invention features a method for determining whether a candidate compound is an agonist of a peptide hormone receptor. In this method, a candidate compound is exposed to a form of the peptide hormone receptor which has a greater, or an enhanced, ability to amplify the intrinsic activity of an agonist (hereafter an ‘enhanced receptor’). The second messenger signaling activity of the enhanced receptor is measured in the presence of the candidate compound, and compared to the second messenger signaling activity of the enhanced receptor measured in the absence of the candidate compound. A change in second messenger signaling activity indicates that the candidate compound is an agonist. For example, an increase in second messenger signaling activity indicates that the compound is either a full or partial positive agonist; a decrease in second messenger signaling activity indicates that the compound is an inverse (also termed a ‘negative’) agonist. The method can further comprise using the agonist to treat or to prevent a physiological disorder involving a peptide hormone receptor by administering to a mammal the identified agonist in an agonist-effective amount.
By “intrinsic activity” is meant the ability of a ligand to activate a receptor, i.e., to act as an agonist. By ‘amplify’ is meant that the signal generated when the ligand interacts with the enhanced receptor is either higher for a positive agonist, or lower for an inverse agonist, than the signal produced when the same ligand interacts with a corresponding non-enhanced receptor, e.g., a wild-type human receptor. A ‘non-enhanced receptor,’ for the purposes of this invention, is a wild-type human receptor for the peptide hormone of interest. By “corresponding” is meant the same type of peptide hormone receptor albeit in another form, e.g., a constitutively active mutant receptor. By way of example, the corresponding wild-type form of a constitutively active mutant CCK-B/gastrin receptor would be a wild-type CCK-B/gastrin receptor; the human CCK-B/gastrin receptor is the corresponding human form of the rat CCK-B/gastrin receptor.
An “agonist,” as used herein, includes a positive agonist, e.g., a full or a partial positive agonist, or a negative agonist, i.e., an inverse agonist. An agonist is a chemical substance that combines with a receptor so as to initiate an activity of the receptor; for a peptide hormone receptor, the agonist preferably alters a second messenger signaling activity. A positive agonist is a compound that enhances or increases an activity, e.g., a second messenger signaling activity, of a receptor. A “full agonist” refers to an agonist capable of activating the receptor to the maximum level of activity, e.g., a level of activity which is induced by a natural, i.e., an endogenous, peptide hormone. A “partial agonist” refers to a positive agonist with reduced intrinsic activity relative to a full agonist. As used herein, a “peptoid” is a peptide-derived partial or full agonist (Horwell et al.,
Eur. J. Med. Chem
., 30 Suppl.:537S-550S, 1995; Horwell et al.,
J. Med. Chem
., 34:404-14, 1991).
An “inverse agonist,” as used herein, has a negative intrinsic activity, and reduces the receptor's signaling activity relative to the signaling activity of the wild-type receptor measured in the absence of the inverse agonist. In contrast, an “antagonist,” as used herein, refers to a chemical substance that inhibits the ability of an agonist to increase or decrease receptor activity. A ‘full,’ or ‘perfect’ antagonist has no intrinsic activity, and no effect on the receptor's basal activity (FIG.
1
). Peptide-derived antagonists are, for the purposes herein, considered to be non-peptide ligands.
A diagram explaining the difference between full and partial agonists, inverse agonists, and antagonists is shown in
FIG. 1
(see also Milligan et al.,
TIPS
, 16:10-13, 1995). In
FIG. 1
, the position of the equilibrium between an inactive state R and an active state R* varies with individual receptors and is altered by the presence of receptor ligands. Agonists function by stabilizing R* while inverse agonists preferentially stabilize R. A continuum of ligands between full agonists (at the extreme right-hand side of the see-saw as these move the equilibrium furthest to the right) and full inverse agonists (at the extreme left-hand side of the see-saw) is expected to exist. Antagonists, which do not alter the position of the equilibrium, define the position of the fulcrum. An antagonist is, e.g., a competitive or a non-competitive inhibitor.
Examples of peptide hormone receptor specific peptide and non-peptide agonists useful in the screening assay of the invention are described below. Non-peptide ligands include, but are not limited to, benzodiazepines and derivatives thereof, e.g., azabicyclo[3.2.2]nonane benzodiazepine (“L-740,093”; see Castro Pineiro et al., WO 94/03437; Castro Pineiro et al., U.S. Pat. No. 5,521,175). L-740,093 S and L-740,093 R refer to the S-enantiomer and the R-enantiomer of L-740,093, respectively. Where the peptide hormone receptor is a CCK-A receptor or a CCK-B/gastrin receptor, useful peptide agonists include, but are not limited to, gastrin (e.g., sulphated (“gastrin II”) or unsulphated (“gastrin I”) forms of gastrin-17, or sulphated or unsulphated forms of gastrin-34), or cholecystokinin (CCK) (e.g., sulfated CCK-8 (CCK-8s), unsulphated CCK-8 (CCK-8d), CCK-4, or pent
Beinborn Martin
Kopin Alan S.
Clark & Elbing LLP
New England Medical Center
Ulm John
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