JP4257322B2 - Nucleic acid molecules encoding calcium receptors - Google Patents

Description

  This invention interferes with the design, development, composition and use of novel calcium-like molecules that can act in a manner similar to extracellular calcium ions on cells, the activity of extracellular calcium ions on cells Calcium antagonists and methods for their production and identification.

  The invention also relates to inorganic-ion receptors, in particular calcium receptors, nucleic acids encoding such receptors, cells, tissues and animals containing such nucleic acids, antibodies to receptors, receptors And related to the inorganic-ion receptor, including methods for all of the above and methods for all of the above.

  The following is a summary of the disclosure relating to the present invention. None of the disclosures described herein are admitted to be prior art to the present invention, nor are any specific or otherwise cited publications admitted to be prior art to the present invention.

Certain cells in the body not only respond to chemical signals, but also to ions such as extracellular calcium ions (Ca ²⁺ ). Changes in extracellular Ca ²⁺ concentration (hereinafter referred to as [Ca ²⁺ ]) change the functional response of these cells. One such specific cell is a parathyroid cell that secretes parathyroid hormone (PTH). PTH is an important endocrine factor that regulates Ca ²⁺ homeostasis in blood and extracellular fluid.

PTH acts on bone and kidney cells to increase Ca ²⁺ concentration in the blood. The increase in [Ca ²⁺ ] then serves as a negative feedback signal that suppresses PTH secretion. The interrelationship between [Ca ²⁺ ] and PTH secretion is an important mechanism for maintaining Ca ²⁺ homeostasis in the body.

Extracellular Ca ²⁺ acts directly on parathyroid cells and regulates PTH secretion. The existence of parathyroid cell surface proteins that sense changes in [Ca ²⁺ ] has been suggested. This protein is a receptor for extracellular Ca ²⁺ (“Ca ²⁺ receptor”), and is thought to sense changes in [Ca ²⁺ ] and initiate a functional cellular response. For example, the role of extracellular Ca ²⁺ and cellular function in the regulation of extracellular Ca ²⁺ receptors and extracellular Ca ²⁺ is reviewed in Nemes et al., 11 Cell Calcium 319, 1990: Vesicular Perimeter And the role of Ca ²⁺ receptors in parathyroid cells is discussed in Nemes et al., 11 Cell Calcium 323, 1990. The role of Ca ²⁺ receptors on bone osteoclasts is discussed in Zeiji, 10 Bioscience Report 493, 1990.

Other cells in the body, specifically osteoclasts in the bones, glomerular proximate and proximal tubule cells in the kidney, keratinocytes in the outer skin, vesicular cells in the thyroid gland, and trophoblast cells in the placenta [ It has the ability to sense changes in Ca ²⁺ ]. Cell surface Ca ²⁺ receptors are also present on these cells, giving them the ability to initiate or enable responses to changes in [Ca ²⁺ ].

In parathyroid cells, osteoclasts, periplasmic follicle cells (C-cells), keratinocytes, glomerular adjacent cells and trophoblast cells, an increase in [Ca ²⁺ ] is the intracellular free Ca ²⁺ concentration (“[Ca ²⁺ ] _i )) increase. This increase can be caused by influx of extracellular Ca ²⁺ or by movement of Ca ²⁺ from intracellular tubules. Changes in [Ca ²⁺ ] _i are easily observed or quantified using a fluorometric indicator such as fura-2 or indo-1 (Molecular Probes, Eugene, OR). The measurement of [Ca ²⁺ ] _i provides a measurement method for assessing the ability of molecules to act as agonists or antagonists of the Ca ²⁺ receptor.

In parathyroid cells, an increase in the concentration of extracellular Ca ²⁺ results in a rapid and temporary increase in [Ca ²⁺ ] _i , followed by a low but still persistent [Ca ²⁺ ] _i The increase continues. A transient increase in [Ca ²⁺ ] _i is induced from the movement of intracellular Ca ²⁺ , while a low sustained increase is caused by the influx of extracellular Ca ²⁺ . After intracellular Ca ²⁺ migration, the production of inositol-1,4,5-triphosphate (IP ₃ ) and diacylglycerol is increased, and these two biochemical indicators are found in various other cells. ^Is associated with receptor-dependent movement of intracellular Ca ²⁺ .

In addition to Ca ²⁺ , various other divalent and trivalent cations such as Mg ²⁺ , Sr ²⁺ , Ba ²⁺ , La ²⁺ and Gd ²⁺ are also cells in parathyroid cells. Causes movement of internal Ca ²⁺ . Mg ²⁺ and La ²⁺ also increase the production of IP ₃ ; all these inorganic chemical cations suppress the secretion of PTH. The hypothetical Ca ²⁺ receptor on parathyroid cells is therefore ^irrelevant . This is because various extracellular divalent or trivalent cations are sensed.

The properties of various compounds resembling extracellular Ca ²⁺ in vitro are described in Nemes et al. (Spermine and Spermidine) “Calcium-binding proteins in health and disease”, 1987, Academic Press, Inc., pages 33-35. : Brown et al. (E.g. neomycin) 128 "Endoclinology", 3047, 1991; Chang et al. (Diltiazem and its derivatives, TA-3090), 5 Journal of Bone and Mineral Research, 581. 1990; and Zeiji et al. (Verapamil) 167 Biochemical and Biophysical Research Communication, 807, 1990.

Brown et al., 6 Journal Bone and Mineral Research, 11, 1991, examines the current theory of the effect of Ca ²⁺ ions on parathyroid cells and includes receptor-like mechanisms such as: It is proposed that the results can be explained by both and receptor-independent mechanisms.

Multivalent cations (eg, divalent and trivalent cations) have various effects on parathyroid function such as inhibition of parathyroid hormone (PTH) secretion and cAMP accumulation, stimulation of inositol phosphate accumulation and cytosol (Cytosolic) increase in calcium concentration, etc. These actions are thought to be transmitted by a “receptor-like” mechanism. Inhibition of agonist-stimulated cAMP accumulation by divalent and trivalent cations is prevented, for example, after perincubation with pertussis toxin. That is, putative multimers cation receptor, inhibitory guanine nucleotide control (G) protein, is thought to lead to inhibition of adenylate cyclase by G _i.

  We have recently found that the polyvalent cation antibiotic, neomycin, acts like divalent and trivalent cations in several properties of parathyroid function. In order to know if these actions are specific to this reagent or show a more generalized action of multivalent cations, the dispersal bull epithelium of very basic peptides, polyarginine and polylysine and protamine The effect on the same parameters was tested in body cells. The results show that parathyroid cells respond to various polycations and multivalent cations, possibly via similar biochemical pathways. These results are discussed in relation to the recently asserted "receptor-independent" regulation of G proteins by polycations in other systems.

While Ca ²⁺ receptors have been considered similar to other G protein-coupled receptors [eg, glycoproteins], recent studies on other cell types have found that polycations are other or additional The possibility that the function of the cell can be adjusted by the mechanism has emerged. In breast cells, various amphipathic cations, including, for example, mastoparan, peptides from bee venom, 48/80, synthetic polycations and polylysine, are enhanced in secretion by the pertussis toxin-sensitization mechanism, Suggests the relationship. None transmit these various agents to standard cell surface receptors. Furthermore, these same compounds are known to activate purified G-protein directly in solution or in artificial phospholipid vesicles. Based on these findings, amphipathic cations have been proposed to activate G-proteins and then activate mammary cell secretion by a “receptor-independent” mechanism.

Polycations have also been shown to interact strongly with acidic phospholipids. Various lengths of chain polylysine (20-1000 amino acids) are bound to artificial phospholipid vesicles with dissociation constants ranging from 0.5 nM to 1.5 μM. The binding affinity is directly related to the length of the polylysine chain, a polymer of 1000 amino acids has a K _{d of} 0.5 nM, a shorter polymer has a higher K _d value, and lysine interacts to a significant extent. Does not work. The correlation between activity and chain length is similar to that observed for the effects of polylysine _10.200 , polylysine ₃₈₀₀ , and lysine on parathyroid function.

  The binding of polycations to biochemical membranes can cause some of their biochemical effects. In certain types of cells, it is claimed that the permeability of plasma membranes induced by various pore formers including polycations is mediated by their interaction with phosphatidylserine-like structures . Furthermore, “receptor-independent” activation of purified G-proteins by amphiphilic cations is enhanced when these proteins are incorporated into phospholipid vesicles.

Calcium ions also cause remarkable changes in the membrane structure in the millimolar range. In certain cases, calcium can either interact and antagonize or enhance membrane lipid and polycation interactions. These considerations suggest that the action of both multivalent cations and polycations on parathyroid cells involves a receptor-independent mechanism that does not require standard cell surface G-protein-coupled receptors. It raises gender. However, further studies are required to elucidate the molecular principles of Ca ²⁺ sensitization by this or other types of cells [citation omitted].

Showback and Chang (6 (Supplement 1) Journal of Bone and Mineral Research, 1991, S135) and Rakke et al. (6 (Supplement 1) Journal of Bone and Mineral Research) 1991, S118) discloses an experiment to say that Ca ²⁺ receptors or Ca ²⁺ sensors are present in parathyroid cells. Messenger RNA isolated from such cells can be expressed in the oocyte and can give the oocyte a phenotype that can be explained by the presence of the Ca ²⁺ receptor protein.

WO93 / 04373

The present invention includes the following:
(A) a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 1;
(B) a nucleic acid molecule capable of hybridizing under high stringency conditions to a nucleic acid molecule complementary to the molecule of (a); and (c) high stringency to a nucleic acid molecule comprising the nucleotide sequence of the cDNA insert contained in ATCC75416. Provided is a purified nucleic acid molecule selected from the group consisting of nucleic acid molecules that can hybridize under conditions.

The present inventors have now shown that a Ca ²⁺ receptor protein can respond to a specialized cell involved in Ca ²⁺ metabolism in the body by sensing changes in the concentration of extracellular Ca ^2+. Indicated. While these receptors have certain general characteristics, they can be selectively influenced by various pharmacological agents. As described in detail below, certain molecules have been found to have selective effects on the parathyroid cells, osteoclasts and C-cell Ca ²⁺ receptors.

Ca ²⁺ receptors are similar to the action of extracellular Ca ²⁺ (“calcimimetics”) or antagonize (“calcium antagonists”) for a new class of molecules Constitute separate molecular targets. This receptor is present on the cell surface and has a low affinity for extracellular Ca ²⁺ (apparent Kd is generally greater than about 0.5 mM). This receptor may include free or bound effector mechanisms as defined by Cooper, Bloom and Ross, Biological Basis of Neuropharmacology, Chapter 4. That is, this receptor is different from intracellular Ca ²⁺ receptors such as calmodulin and troponins. A calcium-like substance is, for example, a substance that selectively acts on a Ca ²⁺ receptor, directly or indirectly suppresses the function of parathyroid cells or osteoclasts, and stimulates the function of C-cells. is there. The calcium-like substances and calcium antagonists of the present invention enable new treatments for hyperparathyroidism, osteoporosis and other Ca ²⁺ related diseases. This treatment targets the respective Ca ²⁺ receptors of these three and other cell types that sense and respond to changes in [Ca ²⁺ ].

The present invention is the first to show Ca ²⁺ receptor protein in parathyroid cells, and pharmacologically binds to Ca ²⁺ receptor in other cells such as C-cells and osteoclasts. First differentiated. The present invention also makes it possible to identify molecules that have an effect on these Ca ²⁺ receptors and to discover, develop, design, modify and / or find effective calcium-like or calcium antagonists active on Ca ^2+. It is the first to disclose a method that can be used as a leading molecule in construction. These calcium-like substances and calcium antagonists are controlled or influenced by being active on polypeptides, such as hormones, enzymes or growth factors, one or more Ca ²⁺ receptors, for example. And / or effective in the treatment of various disease states characterized by abnormal concentrations of one or more components such as release. Furthermore, the identification of various Ca ²⁺ receptors in various types of cells and the specific response to specific receptors for various leading molecules is dependent on the specific disease affected by the action at the Ca ²⁺ receptor. It allows the design and construction of specific molecules active in treatment. For example, the abnormal degree of parathyroid hormone secretion can be influenced by this specific molecule without affecting the degree of secretion of other Ca ^{2+ -regulated} hormones and the like.

The identification of this leading molecule has been hampered by the lack of prior art for efficient screening methods for discovery of active molecules and organized databases for designing effective drug candidates. These disorders are by cloning the parathyroid cell Ca ²⁺ receptor and functionally related receptors, and now also activate the thus cloned Ca ²⁺ receptor and functionally related receptors. It was removed by systematically examining the structural features of some leading molecules. Cloning of the Ca ²⁺ receptor also allows the development of transformation cell lines suitable for high efficiency screening of natural products or molecular libraries and synthetic molecules. This, along with the structure-activity studies discussed below, provides the necessary technology to develop new calcium-like and calcium antagonists.

The present invention enables such an operation. The bull parathyroid cell calcium receptor cDNA has been cloned and deposited with the ATCC under accession number ATCC75416. With this clone, inorganic-ion receptors in other tissues and similar species are readily obtained. For example, human parathyroid cell Ca ²⁺ receptor cDNA can be cloned by screening a nucleic acid library in Xenopus oocytes or by screening for functional expression, and Ca ²⁺ The structural characteristics of the organic molecule required for activity in the receptor activity are determined by testing of selected natural products or other molecular libraries and subsequent structural activity testing.

Specifically, a first aspect, the present invention is induced by causing an increase in [Ca ^2+] _i in a cell, or similar to the activity of extracellular Ca ^2+, or by extracellular Ca ²⁺ Features a pharmaceutical composition comprising a molecule that either prevents the increase in [Ca ²⁺ ] _i . The molecule has an ED ₅₀ of less than or equal to 5 μm and is not protamine.

By "similar", molecules to the extracellular Ca ²⁺ responses possible cells, it refers to having one or more specific actions of extracellular Ca ^2+. This predicate does not require that all of the extracellular Ca ²⁺ biological functions are similar, as long as at least one of these functions is similar. Furthermore, it is not necessary for the molecule to bind to the same site of the receptor to which the extracellular Ca ²⁺ receptor is bound (see, eg, the novel compound NPS467 and its action in Example 20 below). “Interfering” means that one of these actions of Ca ²⁺ is reduced or prevented by the molecule. The ED ₅₀ is measured by the following test method, in which a similar action is measured, and the concentration of molecules that are similar by half of the maximum similar effect is the ED ₅₀ . Conversely, the IC ₅₀ of a calcium antagonist is an amount that interferes with half of the maximum activity. Preferably, this test measures [Ca ²⁺ ] _i increase and confirms that it is specific for the Ca ²⁺ receptor, ie its equivalent, by the following method.

In a preferred embodiment, the bioassay described herein is such that the increase in [Ca ²⁺ ] _{i in} the cell is transient, lasts less than 1 minute, and the increase in [Ca ²⁺ ] _i is rapid. Occurs within 30 seconds; the molecule also causes (a) a sustained increase in [Ca ²⁺ ] _i (greater than 30 seconds); (b) inositol-1,4,5-triphosphate An increase in acid and / or diacylglycerol concentration occurs, for example, within 60 seconds, and (c) inhibits dopamine- or isopreterenol-stimulated cyclic AMP formation. Furthermore, a temporary increase in [Ca ²⁺ ] _i is prevented by pre-treatment of the cells with 10 mM sodium fluoride for 10 minutes, or a temporary increase is observed in protein kinase C, eg phorbol milli It can be reduced by a simple pretreatment (less than 10 minutes) with an activator such as start acetate (PMA), mezerein or (-) indolactam V.

Molecules that are active in all of the above test methods in parathyroid cells are particularly effective in the present invention. This is because the action of these molecules is specific to the Ca ²⁺ receptor of the cell. This is especially true for the PMA pretreatment described above.

Further, in a preferred embodiment, the cell is a parathyroid cell and the molecule inhibits parathyroid hormone secretion from the cell; other preferred embodiments are distal tubule kidney cells, thick Helen traps. Cells of the ascending limb and / or collecting duct, keratinocytes of the epidermis, vesicular peripheral cells in the thyroid (C-cell), intestinal cells, trophoblast cells in the placenta, platelets, vascular smooth muscle cells, cardiac atrial cells, gastrin and Contains molecules that induce an increase in Cl ^- conductance in Xenopus oocytes injected with mRNA from glucagon secreting cells, kidney glomerular cells and breast cells.

In another preferred embodiment, the molecule causes intracellular Ca ²⁺ migration and an increase in [Ca ²⁺ ] _i ; the cell is a C-cell or an osteoclast and the molecule is bone in vivo. Inhibits resorption; the cell is an osteoclast and the molecule inhibits bone resorption in vitro; or the cell is a C-cell and stimulates calcitonin secretion in vitro or in vivo; most preferably, the molecule is The EC ₅₀ or IC ₅₀ at 5 μM or less, more preferably 1 μM of the Ca ²⁺ receptor is 100 nmol, 10 nmol or 1 nmol or more. Such a low EC ₅₀ or IC ₅₀ is advantageous because it allows for lower concentrations of molecules used in vivo or in vitro for therapy or diagnosis. The discovery of such low EC ₅₀ and IC ₅₀ molecules also allows for the design and synthesis of powerful and effective molecules.

A “calcium-like” molecule refers to having one or more activities of extracellular Ca ²⁺ , preferably similar to the activity of Ca ^{2+ at} the Ca ²⁺ receptor. For example, when used with parathyroid cells, it has one or more of the following, preferably all characteristics measured in vitro by techniques well known to those skilled in the art when tested on parathyroid cells: Molecule.

1. Molecules produce a rapid (time to peak <5 seconds) and temporary increase in [Ca ²⁺ ] _i that is unresponsive to inhibition by 1 μM La ³⁺ or Gd ³⁺ . [Ca ^2+] increases in _i is sustained in the absence of extracellular Ca ^2+, it is blocked by pretreatment with ionomycin (in the absence of extracellular Ca ^2+):
2. The molecule promotes the increase in [Ca ²⁺ ] _i induced by submaximal concentrations of extracellular Ca ²⁺ .
3. The increase in [Ca ²⁺ ] _i induced by extracellular Ca ²⁺ is not inhibited by dihydropyridines.
4). The increase in [Ca ²⁺ ] _i caused by the molecule is blocked by pretreatment with 10 μM sodium fluoride for 10 minutes:
5. The transient increase in [Ca ²⁺ ] _i produced by the molecule is preceded by an activator of protein kinase C (PKC) such as phorbol myristate acetate (PMA), mezerein or (−) indolactam V. It can be reduced by processing.
The overall effect of protein kinase C activator is to shift the concentration-response curve of the molecule to the right without affecting the maximum response:
6). The molecule produces a rapid (<5 seconds) increase in the production of inositol-1,4,5-triphosphate and / or disilylglycerol:
7). The molecule inhibits dopamine- or isoproterenol-stimulated cyclic AMP formation:
8). The molecule inhibits PTH secretion:
9. Pretreatment with pertussis toxin (100 ng / ml,> 4 hours) interferes with the molecular inhibitory effect on cyclic AMP formation, but [Ca ²⁺ ] _i , inositol-1,4,5-triphosphate or Does not affect the increase in disilylglycerol and the decrease in PTH secretion:
10. The molecules were detected in [Ca ²⁺ ] as detected by an increase in Cl ⁻ conductance in Xenopus oocytes injected with, for example, poly (A) ⁺ rich mRNA from bull or human parathyroid cells. _Induces an increase in _i but does not affect Xenopus oocytes injected with water or rat brain or liver mRNA:
11. Similarly, cloned receptors from parathyroid cells are used to elicit responses in Xenopus oocytes introduced with specific cDNA or mRNA molecules encoding the receptor or synthetic sense RNA (cRNA). .

The "calcium antagonists" molecule, preferably by acting as an antagonist to the Ca ²⁺ receptor, extracellular Ca ²⁺ - interfere with one or more activities of extracellular Ca ²⁺ sensitized cells It means to do. For example, when used in connection with parathyroid cells, when testing parathyroid cells in vitro, the molecule has one or more, preferably all, of the following characteristics measured by techniques well known to those skilled in the art:
1. The molecule, either partially or completely, prevents the ability of increased extracellular Ca ²⁺ concentration to:
a) Increase in [Ca ²⁺ ],
b) intracellular Ca ²⁺ movement,
c) increased production of inositol-1,4,5-triphosphate,
d) reduced dopamine- or isoproterenol-stimulated cyclic AMP formation,
e) Inhibition of PTH secretion:
2. At low [Ca ²⁺ ] _i , ie 0.5 mM, the molecule itself does not change [Ca ²⁺ ] _i :
3. The molecule increases Cl ^- conductance in Xenopus oocytes introduced with poly (A) ⁺ -mRNA from bull or human parathyroid cells induced by extracellular Ca ²⁺ or calcium antagonist compounds But not in Xenopus oocytes introduced with water or rat brain or liver mRNA:
4). Similarly, cloned receptors from parathyroid cells are used to prevent the response of Xenopus oocytes introduced with specific cDNA or mRNA molecules encoding the Ca ²⁺ receptor.

A parallel definition of effective calcium-like substances and calcium antagonists at the Ca ²⁺ receptors of other types of cells is apparent from the examples below.

Ca ²⁺ receptors can sense and respond to certain inorganic polycations and polycation organic molecules. For example, parathyroid cells cannot distinguish between the addition of these organic polycations and an increase in extracellular Ca ²⁺ concentration. Perhaps because these organic molecules act on the Ca ²⁺ receptor like extracellular Ca ²⁺ . The calcium-like substance of the present invention is a particularly good agonist of the Ca ²⁺ receptor, and can be used as a drug that changes the function of selected cells, for example, secretion of PTH from parathyroid cells. Unlike Ca ²⁺ , many of these molecules act on one, but not all, one or more Ca ²⁺ receptors, ie specifically target one Ca ²⁺ receptor. Have the ability to

These molecules include [Ca ²⁺ ] _i such as hyperparathyroidism, osteoporosis, budget disease, hypertension, kidney disease, heart disease, cardiovascular disease, endocrine disease, abnormal water metabolism and cancer. And has a leading structure for the development of newer therapies effective in the treatment of various diseases in which Ca ²⁺ plays a role.

Calcium-like substances and calcium antagonists are effective to control the concentration of extracellular free Ca ^{2+ in} patients and to mimic the effect of extracellular Ca ²⁺ on cells selected from the above group when administered to patients. Can be formulated as a pharmaceutical composition such as Before the present invention has the disease or normal Ca ²⁺ receptors caused by abnormalities in the operation or control of the Ca ²⁺ receptor can not be treated by activating or inactivating the Ca ²⁺ receptor It is effective in the treatment of animal diseases. Such a molecule that acts on the Ca ²⁺ receptor has not been known.

Furthermore, in other preferred embodiments, parathyroid cells, bone osteoclasts, glomerular proximal kidney cells, proximal tubule kidney cells, distal tubule kidney cells, thick Helen trapping ascending limbs and / or collecting ducts Cells, keratinocytes of the epidermis, vesicular peripheral cells in the thyroid (C-cells), intestinal cells, trophoblast cells in the placenta, platelets, vascular smooth muscle cells, cardiac atrial cells, gastrin and glucagon secreting cells, kidney glomerular cells And, if not all, selected from the group consisting of breast cells, have one or more cells having an ED ₅₀ of 5 μM or less.

  The clarification of the action of molecules that are particularly useful in the present invention allows for specific in vitro or in vivo treatment and diagnosis and discovery of other calcium-like or calcium antagonists.

In a preferred embodiment, the molecule is positively charged at physiological pH and is selected from the group consisting of branched or cyclic polyamines, positively charged polyamino acids and arylalkylamines. For example, the branched polyamine has the formula: H ₂ N— (CH ₂ ) _j — (NR _i — (CH ₂ ) _j ) _k —NH ₂ (wherein k is an integer from 1 to 10; Are the same or different and are integers of 2 to 10, and each R _i is the same or different and is selected from the group consisting of hydrogen and — (CH ₂ ) _j —NH ₂ , where j is as defined above And at least one R _i is not hydrogen).

In another embodiment, the molecule has the formula
[Wherein X is independently H, CH ₃ , CH ₃ O, CH ₃ CH ₂ O, Br, Cl, F, CF ₃ , CHF ₂ , CH ₂ F, CF ₃ O, CH ₂ S, Selected from the group consisting of OH, CH ₂ OH, CONH ₂ , CN, NO ₂ and CH ₃ CH ₂ ; Ar is a hydrophobic material; R is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl , Isobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, indenyl, indanyl, dihydroindolyl, thiodihydroindolyl, 2-, 3- or 4-piperidyl (ni) yl; Y is CH, Selected from the group consisting of nitrogen and unsaturated carbon: Z is oxygen, nitrogen, sulfur,
(Wherein n is independently 1 to 4 and m is independently 0 to 5)
Selected from the group consisting of
Indicated by Most preferably, the molecule is either calcium-like or calcium antagonist.

In preferred embodiments, the hydrophobic material is from the group consisting of phenyl, 2-, 3- or 4-pyridyl, 1- or 2-naphthyl, 1- or 2-quinolinyl, 2- or 3-indolyl, benzyl and phenoxy. The molecule is R-diphenylpropyl-α-phenethylamine derivative, and the molecule is of the formula:
(X is preferably independently selected from the group consisting of Cl, F, CF ₃ , CH ₃ and CH ₃ O)
It is.

In a preferred embodiment of the invention, the formula:
Wherein alk is a linear or branched alkylene consisting of 1 to 6 carbon atoms; R ₁ is a lower alkyl consisting of 1 to 3 carbon atoms or 1 to 7 halogen atoms A substituted lower haloalkyl consisting of 1 to 3 carbon atoms; R ₂ and R ₃ are independently selected from carbocyclic aryl or cycloalkyl groups and are either monocyclic or bicyclic, Independently, lower alkyl consisting of 1 to 3 carbon atoms, lower haloalkyl consisting of 1 to 3 carbon atoms substituted with 1 to 7 halogen atoms, consisting of 1 to 3 carbon atoms Lower alkoxy, halogen, nitro, amino, alkylamino, amide, lower alkylamide consisting of 1 to 3 carbon atoms, cyano, hydroxy, consisting of 2 to 4 carbon atoms Sil, optionally substituted with 1 to 5 substituents selected from 1 to 3 carbon atoms lower hydroxyalkyl or 1 to 3 carbon atoms lower thioalkyl It is a member ring]
The novel phenyl-α-phenethylamine homologues and derivatives are provided. Preferred carbocyclic aryl groups have one or two rings, at least one of which has aromatic character and includes carbocyclic aryl groups such as phenyl and bicyclic carbocyclic aryl groups such as naphthyl. . As is apparent from the above formula, the compounds contained therein may exist as racemic mixtures and as various stereoisomers. Particularly preferred is an R-phenylpropyl-α-phenethylamine derivative, which is believed to exhibit a serum ionizing calcium lowering promoting activity.

Preferred compounds include those where alk is n-propylene. Also preferred are compounds wherein R ₁ is methyl. Also preferred are compounds wherein R ₂ and R ₃ are optionally substituted phenyl.

Particularly preferred compounds include those where R ₂ is mono-substituted phenyl, more preferably meta-substituted phenyl. Particularly preferred R ₃ groups include unsubstituted or monosubstituted, preferably ortho-substituted phenyl. Preferred substituents for R ₂ include halogen, haloalkyl, preferably trihalomethyl and alkoxy, preferably methoxy. Preferred substituents for R ₃ include halogen, more preferably chlorine.

In a second related aspect, the present invention is characterized by one or more cells or abnormal [Ca ²⁺ ] or [Ca ²⁺ ] _{i in} blood or plasma or extracellular fluid. Features a method for treating a patient that is a disease or condition. This method is similar to the activity of extracellular Ca ²⁺ caused by an increase in intracellular [Ca ²⁺ ] _i , or induced by extracellular Ca ²⁺ in a therapeutically effective amount in the patient [ Administering one or more molecules that either interfere with an increase in Ca ²⁺ ] _i .

“Unusual” means that the patient has a different Ca ²⁺ metabolite compared to the general population, which is one or more proteins (eg, hormones) or cells in the blood or extracellular fluid It is influenced by other molecules that affect the concentration of extracellular and / or intracellular Ca ²⁺ . That is, this disease includes hyperparathyroidism, osteoporosis, mineral-related diseases and the like (for example, described in standard medical books such as “Harrisons Principals of Internal Medicine”). Such diseases in the present invention resemble or interfere with the effect of one or more Ca ²⁺ , thereby directly or indirectly affecting the concentration of proteins or other molecules in the patient's body. Treated with the given molecule.

“Therapeutically effective amount” means an amount that reduces to some extent one or more symptoms of a patient's disease or condition. Furthermore, “therapeutically effective amount” means an amount that partially or completely restores a physiological or biochemical parameter associated with or causative of a disease or condition. In general, the amount is between about 1 nmol to 1 μmol of the molecule, depending on its EC ₅₀ and the age, weight and disease of the patient.

In a preferred embodiment, the molecule has an EC ₅₀ of less than or equal to 5 μM and not protamine; most preferably it interacts with the Ca ²⁺ receptor as a calcium-like or calcium antagonist. Most preferably, the molecule is selected from one of the above.

In other preferred embodiments, the patient suffers from a disease characterized by an abnormal concentration of one or more components, the concentration being controlled by the activity of one or more Ca ²⁺ receptors or Affected molecules are parathyroid cells, osteoclasts in bone, glomerular proximate cells in kidney, proximal tubule cells in kidney, distal tubule kidney cells, thick Helen trap ascending limbs and / or aggregates Vascular cells, epidermal keratinocytes, vesicular peripheral cells in thyroid (C-cells), intestinal cells, trophoblast cells in placenta, platelets, vascular smooth muscle cells, cardiac atrial cells, gastrin and glucagon secreting cells, kidney thread Active on the Ca ²⁺ receptor of cells selected from the group consisting of sphere cells and breast cells.

In still other preferred embodiments, the molecule is sufficient to bring the concentration of parathyroid hormone in the patient's plasma to a level found, for example, in normal individuals or to cause a decrease in plasma Ca ^2+. This molecule is provided to a degree sufficient to have a therapeutically appropriate effect on the patient.

In a third aspect, the present invention identifies the number and / or location (and / or functional integrity) of one or more Ca ²⁺ receptors in a patient, and the index number as a disease or symptom thereof. And / or a method of diagnosing a patient's disease or condition by comparing position (and / or functional integrity) with that observed in healthy individuals.

In a preferred embodiment, the method is immunoassay using antibodies against Ca ²⁺ receptor to determine the number and / or position and / or functional integrity of the Ca ²⁺ receptor, or measurement The method includes providing a labeled calcium-like or calcium antagonist molecule that binds to the Ca ²⁺ receptor; the disease to be diagnosed is a cancer, such as an ectopic parathyroid cancer, or a number of bone osteoclasts as described above Symptoms characterized by increased levels of activity in bone osteoclasts.

In a fourth aspect, the invention features a method for identifying a molecule useful as a therapeutic molecule. This method potentially has the ability to resemble the activity of extracellular Ca ²⁺ within the cell or to block the increase in [Ca ²⁺ ] _i induced by extracellular Ca ^2+. Screening for effective molecules, and determining whether a molecule has an EC ₅₀ or IC ₅₀ of less than or equal to 5 μM.

In other embodiments, the invention relates to recombinant Ca ²⁺ receptors, cells containing recombinant Ca ²⁺ receptors, purified nucleic acids encoding Ca ²⁺ receptors, biological activity and use of the molecule NPS019, NPS459, NPS467. , NPS551 and NPS568 (see FIGS. 44-63) and a first Ca ²⁺ receptor but not a second receptor, eg, by use of a recombinant Ca ²⁺ receptor Features a method of identifying useful calcium-like or calcium antagonist molecules by identifying molecules that resemble or interfere with one or more activities of Ca ²⁺ .

In other embodiments, cells containing recombinant Ca ²⁺ receptors from the kidney are employed as test methods for screening antibiotics (eg, aminoglycoside antibiotics) for activity at calcium receptors that cause nephrotoxicity in humans. Is done.

By "recombinant" is produced by recombinant DNA techniques, the position from the Ca ²⁺ receptors naturally occurring, that includes any Ca ²⁺ receptor as distinguished by either purity or structure Meaning. In general, such receptors are present in cells in amounts different from those normally observed in nature.

“Purification” means that an antibody or nucleic acid is distinguished from a naturally occurring antibody or nucleic acid, separated from a naturally found antibody or nucleic acid, and used for recombinant Ca ²⁺ receptor expression, eg, in a vector system. means. Preferably, the antibody or nucleic acid is provided as a homogeneous preparation by standard techniques.

Such cloned receptors can be expressed in the desired cells and isolated and crystallized for structure determination. Such a structure allows the design of useful molecules of the invention that can bind to the Ca ²⁺ receptor. In addition, equivalents of such receptors can be cloned using the first clone as a probe for other cell, cDNA or genomic library clones.

Antibodies for cloned receptors can be isolated, and the number and / or location and / or functionality of Ca ²⁺ receptors as therapeutic agents of the invention or for diagnosis of Ca ²⁺ -related diseases or conditions It can be used as a diagnostic tool to measure integrity. Such antibodies can also be used in vivo by intravenous administration as calcium-like or calcium antagonists.

That is, in general, the invention relates to parathyroid cells, osteoclasts in bone, glomerular proximate cells in kidney, proximal tubule cells in kidney, distal tubule cells, thickened limbs in Henle's loop and / or Or collection tube, keratinocyte of epidermis, vesicular peripheral cell (C-cell) of thyroid gland, trophoblast cell in placenta, platelet, vascular smooth muscle, cardiac atrial cell, gastrin and glucagon secreting cell, renal vascular membrane Calcium-like or calcium antagonists that can act as either selective agonists or antagonists at the Ca ²⁺ receptor of one, but not all, cells selected from the group consisting of cells and breast cells It is characterized by. Such compositions can include any pharmaceutically acceptable carrier known to those of skill in the art to provide pharmaceutical compositions.

The invention also features the adjustment of the number of Ca ²⁺ receptors in a patient by standard techniques, such as antisense and related techniques (eg, ribozymes) as a treatment for disease states.

As will be clarified below, the present invention provides a method for identifying a molecule that affects the activity of a Ca ²⁺ receptor using a detection method for detecting a calcium-like substance and / or a calcium antagonist. In addition, molecules that appear to be effective in reducing or increasing Ca ²⁺ receptor expression at the transcriptional or translational level can be defined for therapeutic use by the use of detection methods or antibodies or other techniques described below.

  The above abstraction was largely related to the preferred embodiment for calcium receptors in general. Further in that respect, the present invention relates to novel superfamily polypeptide receptor / sensor molecules. The first such clone disclosed herein and described as a properly deposited cDNA clone, BoPCaR1, encodes a receptor / sensor that is a member of this superfamily. For the purposes of the present invention, receptors / sensors belonging to this superfamily are referred to as inorganic ion receptors.

  Novel superfamily inorganic ion receptors include a variety of such molecules that are related to each other by similar amino acid sequences, by structure and / or by action. Overall, these are also attributed to prominent members of the superfamily of receptors from all receptors / sensors generally known in the art. Members of this superfamily of receptors detect and respond to changes in the level of inorganic cations such as calcium, magnesium, potassium, sodium or hydrogen ions and sense such ions to They are essentially functionally distinguished by their amazing ability to draw change. Such changes can include changes in second messenger levels or changes in ion flux inside and outside the ionic membrane as appearing in known G-protein coupled receptors. Some members of this superfamily also sense inorganic anions, such as phosphate or chloride ions. All such receptors are within the scope of the invention described herein. Furthermore, all isolated naturally occurring or synthesized ligands of receptors / sensors belonging to superfamily inorganic ion receptors are within the scope of the present invention. In another aspect, the invention features the therapeutic use of any such natural or synthetic ligand for inorganic ion receptors.

  It is the next aspect of the present invention that receptors belonging to superfamily inorganic ion receptors are activated by stimuli other than ligand binding. For example, some members of the superfamily of receptors are activated by physical factors such as stretch factors that act on the membranes of cells that express such receptors. All such receptors are within the scope of the present invention.

  Inorganic ion receptors can be used in methods such as those described above involving preferred receptors. Recombinant receptors expressed in various tissue types, including human tissue types, can be used to screen drug discovery (including high efficiency screens) in a manner known to those skilled in the art. Ion-like molecules and ion antagonist molecules can be easily identified. The method can be adapted to the individual effects of the receptor. For example, calcium activated chloride streams can be utilized with inorganic ion receptors that form calcium channels or neutralize intracellular calcium. Ligand gate ion channels are known in the art. Inorganic ion receptors that bind to the second messenger system allow for assays such as those described above, including measurement of cyclic AMP and inositol phosphates. Cells with recombinant receptor bound to an easily assayable reagent can also be used for G-protein coupling of the receptor to a dye suspension in melanocytes as described in the literature. Accordingly, the present invention includes inorganic ion receptor-like molecules or antagonist molecules and those preferably selected from such molecules and antagonist molecules as described in detail above. It fully enables methods for discovering reagents that are such molecules and antagonist molecules themselves. Similarly, the invention includes the diagnosis and treatment of inorganic ion receptor related diseases or conditions as described above in connection with preferred embodiments. For example, diseases associated with elevated levels of inorganic ion receptors can be diagnosed based on such elevated level assays. The therapeutic molecule can be identified by screening for reagents that mimic the activity of the associated autogenous ions or modify the potency or ability of the associated autogenous ions. Tissue specific expression and expression levels can be evaluated.

  Thus, in this aspect of the invention, a novel superfamily of isolated inorganic ion receptors are provided, as well as unique fragments thereof. Preferably, the isolated receptor is a human receptor, and most preferably the isolated receptor is parathyroid, defect, kidney, epidermis, thyroid, osteoclast, intestine, breast, trophoblast, Calcium receptors expressed in tissues or cells selected from the group consisting of platelets, gastrin secretory glands, heart and brain and unique fragments thereof. The present invention further features polypeptide fragmentation of prior inorganic ion receptors with desirable activity. For example, a fragment contains a binding site or a site that binds to a mimic, agonist or antagonist. Other useful fragments include those having only a protein outside the receptor, a spanning protein or an intracellular protein. In addition, these fragments are useful for forming chimeric receptors with other receptor fragments, as described in more detail below. The invention also features isolated receptor muteins or homologues or other derivatives. Accordingly, the present invention includes not only naturally occurring proteins but also derivatives thereof.

  The invention also features nucleotide sequences that encode the foregoing inorganic ion receptors and fragments and derivatives thereof. Such nucleotide sequences can be obtained via a variety of methods, and the disclosure of the present invention allows one skilled in the art to obtain cDNA or genetic clones encoding such receptors. For example, a hybridization probe can be made based on the nucleotide sequence of BoPCaR1. If any genomic or cDNA library is screened with such a probe with low stringency, hybridization probes encoding other members of the superfamily are obtained. Furthermore, antibodies that bind to clones or isolated inorganic ion receptors can be readily prepared. Such antibodies can also be used to isolate other receptors of the invention by expression cloning techniques known to those skilled in the art. Target gene walking can also be applied to identify and clone members of superfamily inorganic ion receptors. Further clones can be obtained by expression cloning methods as described above. These methods can be adapted to the individual action of the inorganic ion receptor in question. For example, a calcium active chloride stream can also be applied to the cloning of inorganic ion receptors that form calcium channels or mobilize intracellular calcium. As noted above, ligand-gated ion channels are known in the art. For example, serotonin gate ion channels have been cloned this way. However, the receptors of the present invention are quite different from other known ligand-gated ion channels in their amino acid sequences and in that they sense inorganic ions such as calcium, magnesium, hydrogen ions, phosphate ions, and the like.

  It is also possible that each clonal receptor obtained in this way provides an antibody probe that can be used to identify new such DNA or still additional clones that themselves encode inorganic ion receptors. It will be evaluated by the vendor. In addition, obtaining one or more sequences of the receptor as outlined above is relevant for local sequence conservation where the information is still useful to obtain additional clones that encode other members of the superfamily. It will be appreciated that it is possible to do. Such conserved sequences can also be derived therefrom, as analysis of the overall structure of BoPCaR1 conveniently includes the extracellular domain, the transmembrane domain and the intracellular domain.

  Accordingly, isolated nucleic acid cloned inorganic ion receptors and unique fragments thereof are provided. Preferred receptors are calcium receptors expressed in tissues and cells selected from the above group. More preferably, the nucleic acid encodes a human inorganic ion receptor or a specific fragment thereof. The present invention further provides endogenous regulatory elements that modulate the expression of the aforementioned inorganic ion receptors, and also identifies reagents that can agonize or antagonize the activity of these regulatory elements. sell.

  The present invention further provides a recombinant cell that expresses the nucleic acid of the present invention. In such cells, the nucleic acid can be under the control of its genomic regulatory element or under an exogenous regulatory element that includes an exogenous promoter. By “exogenous” is meant a promoter that does not normally bind transcriptionally in vivo to an inorganic ion receptor coding sequence.

  Purified antibodies against the aforementioned inorganic ion receptors are provided as well as antibodies against the allosteric sites of such receptors and the idiotypes of such receptors.

  Also provided are inorganic ion receptors that bind reagents conjugated to toxins. Such a reagent can be an antibody, for example an immunotoxin. Such immunotoxins can be used, for example, to kill cells that express inorganic ion receptors in vitro or in vivo.

  The present invention further provides a transgenic non-human animal comprising a transgene encoding an inorganic ion receptor or a specific fragment thereof. Such transgenes can be useful for affecting or altering the expression of inorganic ion receptors, or for inactivating the expression of inorganic ion receptors, eg, by homologous recombination. Other transgenes can be provided in such mammals, including those that encode proteins capable of altering the expression of antisense or natural inorganic ion receptor genes. Such changes can be up-regulation, down-regulation or complete inactivation.

  Accordingly, a method comprising contacting a cell with a reagent that binds to a cellular component for the purpose of influencing the expression of an inorganic ion receptor is provided. Such reagents can bind to nucleic acids encoding promoters, other regulatory reagents that act on the promoter, reagents capable of binding to the receptor, and receptors or specific fragments thereof.

  Other features and advantages of the invention will be apparent from the following description of preferred embodiments thereof and from the claims.

Calcium-like and calcium antagonist molecules Useful calcium-like and calcium antagonist molecules of the present invention are generally described above. These molecules can be readily identified using screening methods that reveal molecules that mimic or antagonize the activity of Ca ²⁺ at the Ca ²⁺ receptor. Examples of such methods are provided below. These examples are not intended to limit the invention, but merely to illustrate methods that are readily used or applied by those skilled in the art.

In general, calcium-like and calcium antagonist molecules are identified by screening molecules that are modified according to those described below (referred to as derived molecules). As can be seen below, there are several specific calcium-like and calcium antagonists that are useful for various Ca ²⁺ receptors. Derived molecules are easily designed by standard methods and tested in one of many protocols known to those skilled in the art. Many molecules can be easily screened to determine what is most useful for the present invention.

Organic cationic molecules that mimic or antagonize the action of Ca ^{2+ in} other systems contain the necessary structure for activity at the Ca ²⁺ receptor. Other useful molecular theoretical designs include the study of molecules known to be calcium-like or calcium antagonists and modifying the structure of the known molecules. For example, polyamines are potentially calcium-like because spermine mimics the utilization of Ca ^{2+ in} some in vitro systems. The results show that spermine actually changes [Ca ²⁺ ] and PTH secretion reminiscent of those induced by extracellular di- and trivalent cations (see below). Conversely, Ga ³⁺ antagonizes the action of Gd ^{3+ at} the bovine parathyroid calcium receptor. The experiments outlined below are therefore aimed at showing that this phenomenology obtained with spermine involves the same mechanism used by extracellular Ca ²⁺ . To do this, the effect of spermine on various physiological and biochemical parameters that characterize Ca ²⁺ receptor activation was evaluated. Those molecules having the same effect are effective in the present invention, and can be expressed by selecting or producing a molecule having a structure similar to that of spermine. Once other useful molecules are discovered, the selection method can be easily repeated.

For clarity, the following are specific to identify useful molecules that are active on parathyroid cell Ca ²⁺ receptors or act as agonists or antagonists of cellular responses to changes in [Ca ²⁺ ]. A series of screening protocols is provided. Equivalent assays to mimic or antagonize cellular functions that are active or otherwise regulated by [Ca ²⁺ ] or other ions to other Ca ²⁺ receptors, or other inorganic ion receptors, Can be used for molecules. These assays are examples of methods that are useful for finding molecules comprising the calcium-like molecules of the present invention. An equivalent method can be used to find calcium antagonist molecules by screening for molecules that most antagonize the action of ions including extracellular Ca ²⁺ . In vitro assays can be used to characterize the selectivity, saturability and reversibility of these calcium-like and calcium antagonists by standard techniques.

Screening Method Generally, parathyroid cells fed with hula-2 are first suspended in a buffer containing 0.5 mM CaCl ₂ . Add a small amount (5-15 μl) of test substance to the cuvette and watch for any changes in the fluorescence signal. A cumulative increase in the concentration of the test substance is made in the cuvette until some predetermined concentration is reached or a change in fluorescence is observed. If no change in fluorescence is seen, the molecule is considered inactive and is not tested further. For example, in initial studies with polyamine type molecules, the molecules were tested at concentrations as high as 5 or 10 mM. Since more effective molecules are now known (see below), lower the ceiling concentration. For example, new molecules are tested at concentrations up to 500 μM or less. If no change in fluorescence is observed at this concentration, the molecule can be considered inactive.

Molecules that cause an increase in [Ca ²⁺ ] are subject to additional testing. Two essential features of the molecule that are important for its consideration as a calcium-like molecule are the movement of intracellular Ca ²⁺ and its sensitivity to PKC activators. It has always been found that the molecules causing intracellular Ca ²⁺ movement in the PMA-sensitive method are calcium-like molecules and inhibit PTH secretion. If necessary, additional testing was conducted to strengthen this idea. Typically, all the various tests for calcium-like or calcium antagonist activity (see above) are not performed. Rather, if a molecule undergoes intracellular Ca ²⁺ migration in a PMA-sensitive manner, it is promoted to screening on human parathyroid cells. For example, the measurement of [Ca ²⁺ ] _i can be used to determine EC ₅₀ and PTH secretion in human parathyroid cells obtained from patients undergoing surgery for primary or secondary hyperparathyroidism. To measure the ability of the molecule to block. The lower the EC ₅₀ or IC ₅₀ , the more effective the molecule as a calcium-like substance or calcium antagonist.

Measurement of [Ca ²⁺ ] by Hula-2 provides a very rapid method for screening new organic molecules for activity. In a single afternoon, 10-15 molecules can be tested to assess their ability to (or not) migrate intracellular Ca2 ⁺ . The sensitivity of any observed increase in [Ca ²⁺ ] _i to a decrease by PMA can be assessed. Furthermore, single cell preparations can provide data on [Ca ²⁺ ] _i , cyclic AMP levels, IP and PTH secretion. A typical method is to give the cells fura-2 and then split the cell suspension in two. Most cells are used to measure [Ca ²⁺ ] _i and the rest are incubated with molecules to assess their effects on cyclic AMP and PTH secretion. Because of the sensitivity of the radioimmunoassay for cyclic AMP and PTH, both variables can be measured in a single incubation tube containing 0.3 ml cell suspension (approximately 500,000 cells). The measurement of inositol phosphate is a time-saver aspect of the screening. However, ion exchange columns that elute with chloride (or rather formate) do not require rotary vacuum evaporation (which takes about 30 hours), thus providing a very quick method of screening the IP ₃ format. To do. This method allows for the processing of nearly 100 samples in a single afternoon. These molecules of interest as assessed by measurement of [Ca ²⁺ ] _i , cyclic AMP, IP ₃ and PTH were then tested for the formation of various inositol phosphates and their isomerism by HPLC. Subject to more rigorous analysis by assessing shape.

The molecules of interest detected in these protocols can then be tested for specificity by, for example, testing their effects on [Ca ²⁺ ] in, for example, the calcitonin-secreting C-cells using the rat MTC6-23 cell line. evaluate.

  The following are examples of methods useful for these screening methods. Examples of typical results for various test calcium-like or calcium antagonist molecules are given in FIGS.

The parathyroid cell preparation parathyroid glands, obtained from freshly slaughtered calves at a local abattoir (12-15 weeks old), (mM): NaCl, 126: KCl, 4: MgCl 2, 1 : Na-HEPES, 20: pH7.4 ; glucose, 5.6, and variable amounts of CaCl _2, and transferred to the laboratory for example ice-cold parathyroid cell buffer containing 1.25mM in (PCB). Human parathyroid glands obtained from patients undergoing surgical removal of parathyroid tissue for primary or uremic hyperparathyroidism (HPT) were treated in the same manner as bovine tissue. The gland removed excess fat and connective tissue, then chopped into 2-3 mm appropriate swords with sharp scissors. The separated cells were then purified by centrifugation in Percol buffer. The resulting parathyroid cell preparation was substantially free of red blood cells, adipocytes and capillary tissue as assessed by relative ratio microscopy and Sudan Black B staining. Isolated and purified parathyroid cells were present as small masses containing 5-20 cells. Cell viability was 95% as usual, as indicated by the exclusion of trypan blue or ethidium bromide.

Although the cells can be used for experimental purposes at this point, the physiological response (potential suppression of PTH secretion and resting levels of [Ca ²⁺ ] _i ) is better after overnight culture of the cells. Primary cultures can also label cells with isotopes close to isotopic equilibrium, as required for studies involving measurement of inositol phosphate metabolism (see below). After purification by Percoll gradient, cells were washed several times with a 1: 1 mixture of Hams F12-Dulbecco modified Eagles medium (GIBCO) supplemented with 50 ug / ml streptomycin, 100 U / ml penicillin, 5 ug / ml gentamicin and ITS ⁺ . ITS ⁺ is a premix solution (Collaborative Research, Bedford, MA) containing insulin, transferrin, selenium and bovine serum albumin (BSA) -linolenic acid. Cells were then transferred to plastic flasks (75 or 150 cm ² ; Falcon) and incubated at 37 ° C. in a humidified atmosphere of 5% CO ₂ . Serum is not added to overnight cultures because its presence causes cells to attach to plastic and undergo proliferation and dedifferentiation. Cells cultured under the above conditions are easily removed from the flask by tilting and show the same viability as freshly prepared cells.

Measurement of cytosolic Ca ²⁺ Purified parathyroid cells were resuspended in 1.25 mM CaCl ₂ -2% BSA-PCB containing 1 μM fura-2-acetoxymethyl ester and incubated at 37 ° C. for 20 minutes. Cells were then pelleted and resuspended in the same buffer lacking ester and incubated for an additional 15 minutes at 37 ° C. Subsequently, the cells were washed twice with PCB containing 0.5 mM CaCl ₂ and 0.5% BSA and kept at room temperature (about 20 ° C.). Immediately before use, cells were diluted 5-fold with pre-warmed 0.5 mM CaCl ₂ -PCB to give a final BSA concentration of 0.1%. The concentration of cells in the cuvette used for fluorescence recording was 1-2 × 10 ⁶ / ml.

The fluorescence of the cells provided with the indicator was measured by a spectrofluorometer equipped with a thermostatic cuvette holder and a magnetic stirrer using the excitation and emission wavelengths of 340 and 510 nm (Biomedical Instrumentation Group, University of Pennsylvania, Philadelphia, PA). This fluorescence shows the level of cytosolic Ca ²⁺ . The fluorescence signal is calibrated using digitonin (50 ug / ml, final) to obtain maximum fluorescence (Fmax), EGTA (10 mM, pH 8.3, final) to obtain minimum fluorescence (Fmin), and a dissociation constant of 224 nM. did. Dye leakage is temperature dependent and occurs most often within the first 2 minutes after warming the cells in the cuvette. Dye leakage then increases only very slightly. To correct for dye leakage calibration, the cells were placed in a cuvette and agitated for 2-3 minutes at 37 ° C. The cell suspension was then removed, the cells pelleted and the supernatant returned to a clean cuvette. The supernatant was then treated with digitonin and EGTA as described above and obtained as an assessment of dye leakage. It is typically 10-15% of the total Ca ²⁺ dependent fluorescence signal. This evaluation was subtracted from the apparent Fmin.

Measurement of PTH secretion In most experiments, cells given fura-2 were used to study PTH secretion. Parathyroid cells providing fura-2 do not alter their secretory response to extracellular Ca ²⁺ . Cells were suspended in a PCB containing 0.5 mM CaCl ₂ and 0.1% BSA. Incubations were performed in plastic tubes (Falcon 2058) containing 0.3 ml cell suspension with or without small amounts of CaCl ₂ and / or organic polycations. After incubation at 37 ° C for various times (typically 30 minutes), the tubes were placed on ice and the cells were pelleted at 2 ° C. The supernatant sample was adjusted to pH 4.5 with acetic acid and stored at -70 ° C. This protocol was used for both bovine and human parathyroid cells.

For bovine cells, the amount of PTH in the sample supernatant was determined by homogeneous radioimmunoassay using GW-1 antibody or equivalent at a final dilution of 1 / 45,000. ¹²⁵ I-PTH (65-84: Inkstar, Stillwater, MN) was used as a tracer and fractions were separated by dextran-activated carbon. Sample and data reduction counts were performed on a Packard Cobra 5005 gamma counter.

  For human cells, the commercially available radioimmunoassay kit (INS-PTH: Nichols Institute, Loss) recognizes intact and N-terminal human PTH since the GW-1 antibody recognizes little human PTH. -Angels, CA) was used.

Cyclic AMP measurement cells are incubated as described above for PTH secretion studies and at the end of the incubation a 0.15 ml sample is taken and transferred to 0.85 ml hot (70 ° C.) hot water at this temperature. Heated for 10 minutes. The test tube was then frozen and thawed several times, and cell debris was sedimented by centrifugation. A portion of the supernatant was acetylated and the cyclic AMP concentration was measured by radioimmunoassay.

Measurement of Inositol Phosphate Formation Membrane phospholipids were labeled by incubating parathyroid cells with 4 μCi / ml ³ H-myo-inositol for 20-24 hours. Cells were then washed and resuspended in PCB containing 0.5 mM CaCl ₂ and 0.1% BSA. Incubations were performed in microfuge tubes for different times in the absence or presence of various concentrations of organic polycations. The reaction was stopped by the addition of 1 ml chloroform / methanol / 12N HCl (200: 100: 1; v / v / v). Then phytic acid hydrolyzate (200 μl: 25 μg phosphate ester / test tube) water was added. The tube was centrifuged and 600 μl of the aqueous phase was diluted in 10 ml water.

Inositol phosphates were separated by ion exchange chromatography using AG1-X8 in the chloride or formate form. When measuring only the IP ₃ level, the chloride form was used, while the formate form was used to elucidate the main inositol phosphates (IP ₃ , IP ₂ and IP ₁ ). For IP ₃ only measurements, the diluted sample was applied to a chloride-form column, the column was washed with 10 ml 30 mM HCl, then 6 ml 90 mM HCl, and IP ₃ was eluted with 3 ml 500 mM HCl. The final eluate was diluted and counted. For the determination of all major inositol phosphates, diluted samples were applied to a formate-form column and IP ₁ , IP ₂ and IP ₃ were eluted one after another by increasing the concentration of formate buffer. The eluted sample from the formate column was evaporated on a rotary vacuum and the residue was taken into a cocktail and counted.

The same IP ₃ variant was evaluated by HPLC. The reaction was stopped by the addition of 1 ml 0.45 M perchloric acid and kept on ice for 10 minutes. It was then centrifuged and the supernatant was adjusted to pH 7-8 with NaHCO ₃ . The extract was then applied to a Particill SAX anion exchange column and eluted with a linear distribution of ammonium formate. The various fractions were then desalted by Dowex and subjected to rotary vacuum evaporation before being subjected to a liquid scintillation counter with a Buckard Tricurve 1500 LSC.

  Appropriate controls using credible standards were used to determine whether organic polycations were hindered by separation for all inositol phosphate separation methods.

  If so, the sample was treated with a cation exchange resin to remove the detrimental molecules prior to inositol phosphate separation.

Measurement of cytosolic Ca ²⁺ in C-cells Tumor C-cells derived from rat bone marrow thyroid cancer (rMTC6-23) obtained from American Type Culture Collection (ATCC No. 1607) were treated with Dulbecco's modified Eagle medium ( DMEM) plus 15% horse serum was cultured as a monolayer in the absence of antibody. For the measurement of [Ca ²⁺ ] _i , cells were collected with 0.02% EDTA / 0.05% trypsin, washed twice with PCB containing 1.25 mM CaCl ₂ and 0.5% BSA, Somatic cells were given fuller 2 as described above. The measurement of [Ca ²⁺ ] _i was performed by appropriate correction for dye leakage as described above.

Measurement of [Ca ²⁺ ] _{i in} rat osteoclasts Osteoclasts were obtained from 1-2 day old Sprague-Dury rats using aseptic conditions. Rat children were sacrificed by bouncing their necks, their hind paws were removed, the thighs were quickly removed of soft tissue, and pre-warmed F-12 / DMEM medium (DMEM was 10% fetal bovine serum and antibiotics (penicillin). -Streptomycin-gentamicin; 100 U / ml-100 ug / ml-100 ug / ml). Bones from two children were cut longitudinally and placed in 1 ml culture medium. Bone cells were obtained by gentle crushing of bone fragments with a plastic pipette and diluted with culture medium. Bone fragments were allowed to settle and an equal portion (about 1 ml) of media was transferred to a 6-well culture plate with a 25 mm glass cover glass. Cells are clarified at 37 ° C. for 1 hour in a humidified 5% CO ₂ -air atmosphere. The cover glass was then washed 3 times with a new culture medium to remove non-adherent cells. The measurement of [Ca ²⁺ ] _{i in} osteoclasts was performed within 6-8 hours after removing non-adherent cells.

Cells attached to the coverslips lack serum and are incubated for 30 minutes at 37 ° C. with 5 μM indo-1 acetoxymethyl ester / 0.01% pluronic F28 in F-12 / DMEM instead containing 0.5% BSA. Gave indole 1. The cover glass was then washed and further washed at 37 ° C. in F-12 / DMEM lacking ester before being transferred to a supermelting chamber placed on a Nikon Diahoto inverted microscope stage equipped with a microfluorometric method. Incubated for 15 minutes. Osteoclasts were easily identified from their large shape and the presence of multiple nuclei. Cells (emitted by excitation at 340 nm) are superfused after buffering (typically a PCB containing 0.1% BSA and 1 mM Ca ²⁺ ) at 1 ml / min with or without the presence of a test substance. did. The fluorescence emitted by excitation at 340 nm was sent to a 440 nm dichroic mirror via a microscope video port, and the fluorescence intensities at 495 and 405 nm were collected by a photomultiplier tube. The production from the photomultiplier tube is amplified, digitized and stored in 80386 PC. The ratio of fluorescence intensities was used to evaluate [Ca ²⁺ ] _i .

In oocytes expressing additional studies using Xenopus cells injected with mRNA from bovine or human parathyroid cells in screening protocols, Cl ^- Conductance meters as an indirect means of monitoring increases in [Ca ^2+] _i did. The following are examples that test the effect of neomycin.

Oocytes were injected with poly (A) ⁺ -enhanced mRNA from human parathyroid tissue (parathyroid gland from the secondary HPT example). Three days later, the oocytes were tested for their response to neomycin. Neomycin B is Cl stopped superfusion with drug-free saline ^- to attract vibration amplification conductance (see Figure 26). Response to neomycin B was observed at concentrations between 100 μM and 10 mM. In order to confirm that the reaction elicited by neomycin B was incidental by injection of parathyroid mRNA, the effect of neomycin B on current was measured in water-injected oocytes. In each of the five oocytes tested, neomycin B (10 mM) did not cause any change in current. About 40% of the oocytes are known to respond to carbachol, and the effect was transmitted by endogenous muscarinic receptors. Of the five oocytes tested, one shows an inward current in response to carbachol, which is shown in the bottom graphic of FIG. That is, neomycin B does not elicit a response in cells expressing muscarinic receptors coupled to an increase in [Ca ²⁺ ] _i and Cl ⁻ conductance. This indicates that the response to neomycin B depends on the expression of a specific protein encoded by parathyroid mRNA. In intact cells, neomycin B acts directly on the Ca ²⁺ receptor, very strongly suggesting that it changes parathyroid cell function.

Drug Design from Most Important Molecules Some organic molecules mimic or antagonize the action of extracellular Ca ²⁺ by acting at the Ca ²⁺ receptor, as shown herein. However, the molecules tested are not necessarily suitable as drug candidates, but they serve to show that the treatment based on the Ca ²⁺ receptor on which the hypothesis is based is correct. These molecules can be used to determine structural features that allow them to act on Ca ²⁺ receptors and thus select molecules useful in the present invention.

  An example of one such analytical method is as follows. This example is described in detail in the examples below, but here to illustrate the rationale that can be used to design useful molecules of the invention from the most important molecules discussed herein. Use. One skilled in the art will recognize that the analysis steps revealed in the Examples can be performed on other molecules of similar importance, and until the most desirable calcium-like substance or calcium antagonist is revealed. You will recognize.

Other examples are also provided below. In summary of the existing data, the most important molecules useful are aromatics that are preferably substituted at one or more positions and may optionally have a branched or linear substituted or unsubstituted alkyl group. Indicates that it will have a group. In addition, it is important to select the correct stereospecific molecule that ensures a higher affinity for the desired Ca ²⁺ receptor. These data thus teach those skilled in the art the most important molecules suitable that can be derived to find the best desired molecules of the present invention, as described in more detail below.

Although structurally different, the molecules being tested can have general characteristics that can be studied. In this example, the correlation between the net positive charge and the possibility of moving intracellular Ca ²⁺ was tested. Protamine (+21: EC ₅₀ = 40 nM) was more effective than spermine (+4; EC ₅₀ = 150 μM) in causing [Ca ²⁺ ] _i migration in parathyroid cells. EC ₅₀ = more effective than in 20 μM human parathyroid cells and in 40 μM bovine parathyroid cells). These results raise the question of whether only a positive charge determines the potential or whether there are other structural features that contribute to activity on the Ca ²⁺ receptor. This is important to first determine that it greatly affects the point of view that Ca ²⁺ receptors can target effective and specific therapeutic molecules. Thus, neomycin B and various other organic polycations related to spermine can be studied to determine the relationship between the net positive charge of the molecule and its potential to migrate intracellular Ca ²⁺ .

The first series of molecules studied were aminoglycosides. The molecule was studied in bovine parathyroid cells and the EC ₅₀ for their intracellular Ca ²⁺ migration was measured. For aminoglycosides, the positional order of ability to induce cytosolic Ca ²⁺ transients was neomycin B (EC ₅₀ = 20 or 40 μM)> gentamicin (150 μM)> bekanamycin (200 μM)> streptomycin (600 μM). Kanamycin and lincomycin had no effect when tested at a concentration of 500 μM. The net positive charge of these aminoglycosides at pH 7.3 is neomycin B (+6)> gentamicin (+5) = bekamycin (+5)> kanamycin (average +4.5)> streptomycin (+3)> lincomycin (+1) . Then within the aminoglycoside series there is a relationship between the net positive charges, but this is not absolute and kanamycin, which was expected to be more effective than streptomycin, is inactive.

Testing various polyamines revealed an additional and more significant discrepancy between net positive charge and potency. Attempts were made to structurally classify these polyamines: (1) linear, (2) branched, and (3) cyclic. The structure of the tested polyamine is given in FIGS. 1-6. Among the linear polyamines, spermine (+4: EC ₅₀ = 150 μM) is more effective than pentaethylenehexamine (+6: EC ₅₀ = 500 μM) and tetraethylenepentamine (+5: EC ₅₀ = 2.5 μM). Even if the molecule has a larger net positive charge, it is strong.

We have synthesized several branched polyamines with varying numbers of secondary and primary amino groups, thus changing the net positive charge. These two molecules, NPS381 and NPS382, were tested for the effect of [Ca ²⁺ ] _i in bovine parathyroid cells. NPS382 (+8: EC ₅₀ = 50 μM) was about twice as effective as NPS381 (+10: EC ₅₀ = 100 μM), even though it contained two less positive charges.

A similar contradiction between positive charge and effectiveness was expressed in experiments with cyclic polyamines. For example, hexacyclene (+6: EC ₅₀ = 20 μM) was more potent than NPS 383 (+8: EC ₅₀ = 150 μM). The results obtained with these polyamines indicate that positive charge is not the sole factor contributing to efficacy.

Further research has provided insight into the structural features of the molecules that share the activity of the parathyroid cell Ca ²⁺ receptor. One structurally important feature is the intermolecular distance between the nitrogens (which carry a positive charge). Therefore, spermine causes an increase in [Ca ²⁺ ] _i in bovine parathyroid cells 50 times stronger than triethylenetetramine (EC ₅₀ = 8 mM), but both molecules carry a positive positive charge of +4. . The only structural difference between these two polyamines is the number of methylenes separating the nitrogen, which is 3-4-3 for spermine versus 2-2-2 for triethylenetetramine. It is. This slight change in the appearance of the spacing between nitrogens has a profound relationship to effectiveness, suggesting that the conformational relationship of nitrogen in the molecule is critical. This is supported by the results obtained with hexacyclene and pentaethylenehexamine. The former molecule is simply the latter cyclic analog and contains the same number of methylenes between all nitrogens, but the presence of a ring structure increases effectiveness by a factor of 25. These results indicate that its own positive charge is not a critical factor in determining the activity of organic molecules at the Ca ²⁺ receptor.

Another series of experiments reveals the importance of aromatic groups in determining the activity of the Ca ²⁺ receptor. The results were obtained with two allylalkylamines isolated from the spider, Argiope robota venom. These molecules, argiotoxin 636 and argiotoxin 659 have identical polycationic moieties attached to different aromatic groups (FIG. 30). Argiotoxin 659 caused an instantaneous increase in [Ca ²⁺ ] _i in bovine parathyroid cells when tested at concentrations of 100-300 μM. Conversely, Argiotoxin 636 had no effect when tested at similar concentrations (FIG. 30). The structural difference between these two allylic alkylamines is only the aromatic part of the molecule: Argiotoxin 659 contains a 4-hydroxyindole moiety, whereas Argiotoxin 636 Contains a 2,4-dihydroxyphenyl group. The net positive charge of these two allylalkylamines is the same (+4), so their different effectiveness must arise from different aromatic groups. This indicates that only the net positive charge does not determine effectiveness. However, the real importance of finding these is the discovery that aromatic groups contribute greatly to the ability of molecules to activate Ca ²⁺ receptors.

Both Argatoxin 489 (NPS017) and Argatoxin 505 (NPS015) cause intercellular Ca ²⁺ mobilization in parathyroid cells, with EC ₅₀ , 6 and 22 μM, respectively. The only structural difference between these molecules is the hydroxy group of the indole moiety (Figures 1-6). This indicates that substitution on the aromatic region of the molecule can affect efficacy. This indicates that the lead molecule being studied will include molecules in which the aromatic moiety is substituted.

  Structural features that are systematically different from the lead molecules described here are (1) effective positive charge, (2) number of methylenes separating nitrogen, and (3) changes in, for example, methylene spacing and effective positive charge. Includes cyclic polyamines with and without. In addition, systematic changes in structure and position of aromatic groups can be tested, for example, in various allylalkylamines isolated from wasps and spider venoms; commercially available aromatic moieties A synthetic molecule can be prepared by conjugating with an algitoxin polyamine moiety. Argiotoxin polyamine moieties can be readily combined with any aromatic moiety containing a carboxylic acid. Therefore, it is simple to systematically screen the hydroxy and methoxy derivatives of phenylacetic acid and benzoic acid, as well as the hydroxyindole acetic acid system. Analogs with heteroaromatic functionality can also be prepared and evaluated for activity.

  Comparison of efficacy and potency between the molecules will reveal the optimal structure and position of the aromatic group with a constant positive charge.

One of the structural diversity in the polyamine motif that appears to increase efficacy is the presence of a cyclic parent of the linear parent molecule. Budmunchiamine A is a plant found in the plant Albizia Amara.
amara), which is a cyclic derivative of spermine (FIGS. 1-6). Addition of badomuthiamine A to bovine parathyroid cells persisted in the absence of extracellular Ca ²⁺ and caused a rapid and instantaneous increase in [Ca ²⁺ ] _i that was blunted by pretreatment with PMA. Thus, it probably causes intercellular Ca ²⁺ fluidization in parathyroid cells, possibly by acting on the Ca ²⁺ receptor. It is almost as potent as spermine (EC ₅₀ ca. 200 μM), but carries a positive charge (+3) one lower than spermine.

  The results obtained from Bad Munthiamine A represent the predictive power of structure activity studies and the new structural information obtained by testing natural products. Therefore, the screening of natural products that are reasonably selected based on structural information is easily performed, for example, making molecules into a very well-established chemical separation principle using appropriate databases such as Napralate. You can make a selection based on this. For example, macrocyclic polyamine arkanoids derived from papillionoid legumes related to Albizia, such as Pisecorobium, and molecules derived from other plants can be screened.

Figures 44-63 provide a second example of a series of molecules that are screened to determine useful molecules for the present invention. These molecules are generally derived from phendiline and were tested to determine their respective EC ₅₀ . Furthermore, by testing related molecules such as NPS447 and NPS448, the stereospecific effects of the molecular structure are revealed. Most of the active compounds that have been tested and obtained data are novel compounds, designated NPS467 and NPS568, with EC ₅₀ values of 5 μM or less. One skilled in the art can determine other suitable derivatives that can be tested in the present invention by examining this series of molecules.

  These examples show general settings and screening methods useful in the present invention, where additional compounds and natural product libraries are desirably screened in the art, and other useful lead molecules or novel molecules of the present invention are identified. Can be determined.

  As noted above, examples of molecules useful as calcium-like substances include branched or cyclic polyamines, positively charged polyamino acids, and allylalkylamines. In addition, other positively charged organic molecules, including naturally occurring molecules and their analogs, are useful calcium-like substances. These naturally occurring molecules and their analogs preferably have a positive charge-to-mass ratio and correlate with these ratios for the molecules exemplified herein. (Examples include substances isolated from marine mammals, arthropod poisons, terrestrial plants and fermentation broths obtained from bacteria and fungi.) Preferred naturally occurring molecules and analogs useful as calcium-like substances One group is expected to have a positive charge: molecular weight (in dalton) ratio of about 1:40 to 1: 200, preferably about 1:40 to 1: 100. More detailed examples of the molecules are given below.

Polyamines Polyamines useful as calcium-like substances in the present invention can be either branched or cyclic. Branched or cyclic polyamines have potentially higher calcium-like activity than their linear analogues. That is, branched or cyclic polyamines tend to have a lower EC ₅₀ than their corresponding linear polyamines with similar effective charges at physiological pH (see Table 1).

As used herein, a “branched polyamine” is a chain consisting of short alkyl bridges or alkyl groups joined together by amino linkages and having points where the chain branches. It is a molecule. These “branch points” can be located at either the carbon atom or the nitrogen atom, preferably at the nitrogen atom. The nitrogen atom branch point is typically a tertiary amine, but may be quaternary. Branched polyamines may have 1 to 20 branch points, preferably 1 to 10 branch points.

  In general, alkyl bridges and alkyl branches in branched polyamines are 1 to 50 carbon atoms in length, preferably 2 to 6 carbon atoms. The alkyl branch is interrupted by one or more heteroatoms (nitrogen, oxygen or sulfur) or halo: hydroxy: nitro: acyloxy (R′COO—) containing fluoro, chloro, bromo or iodo, Acylamide (R'CONH-), or alkoxy (-OR '), where R' may be substituted with a functional group such as one that may contain from 1 to 4 carbon atoms. The alkyl branch may be substituted with a group that is positively charged at physiological pH, such as amino or guanide. These functional substituents may impart or change physiological properties such as solubility that increase the activity, delivery, or bioavailability of the molecule.

  Branched polyamines may have three or more chains and branch end points. These end points are methyl groups or amino groups, preferably amino groups.

One group of preferred molecules has the formula:
_{H 2 N- (CH 2) j} - (NR i - (CH 2) j) k -NH 2
Wherein k is an integer from 1 to 10, j is the same or different, and each is an integer from 2 to 20, R _i is the same or different, and hydrogen and — (CH ₂ ) _j —NH ₂ And j is selected from the group consisting of those defined above, and at least one R _i is not hydrogen)
Is a group of branched polyamines having

Particularly preferred branched polyamines according to the invention are the molecules N ¹ , N ¹ , N ⁵ , N ¹⁰ , N ¹⁴ , N ¹⁴ -hexakis (3-aminopropyl) spermine and N ¹ , N ¹ , N ⁵ , N ¹⁴ , N ¹⁴ -tetrakis- (3-aminopropyl) spermine, which are NPS381 and NPS382 in FIGS. 1-6, respectively.

As used herein, a “cyclic polyamine” is a heterocycle containing two or more heteroatoms (nitrogen, oxygen or sulfur), of which at least two are nitrogen atoms. . Heterocycles are generally about 6 to about 20 atoms around, preferably about 10 to about 18 atoms around. Nitrogen heteroatoms are separated by 2 to 10 carbon atoms. The heterocycle may be substituted with an aminoalkyl or aminoallyl group (NH ₂ R—) at the nitrogen site, where R is aminoallyl or lower alkyl of 2 to 6 carbon atoms.

  Particularly preferred cyclic polyamines of the present invention are shown in FIGS. 1-6 as hexacyclene (1,4,7,10,13,16-hexaaza-cyclooctadecane and NPS383.

Polyamino acids Polyamino acids useful in the present invention may contain two or more positively charged amino acid residues at physiological pH. These positively charged amino acids include histidine, lysine and arginine. These polypeptides may vary in length within the range of 2 to 800 amino acids, more preferably 20 to 300 amino acids in length. These polypeptides may consist of a single repeat of amino acid residues and may have natural protein or enzyme variants.

  An amino acid residue consisting of a polyamino acid may be some of the 20 natural amino acids or other alternative groups. Alternative groups can also include, for example, elements derived from molecules or ions that the composition of the present invention can act as labels. A wide variety of labeling moieties can be used, including radioisotopes, chromophores and fluorescent labels. In particular, radioisotope labels can be easily detected in vivo. Radioisotopes can be bound by coordination as cations in the porphyrin system. Useful cations are technetium and indium. In the composition, positively charged molecules can be bound to or paired with the label.

Synthetic Methods Polyamine synthesis and modification strategies involve the use of various amine protecting groups (phthalimide, BOC, CBZ, benzyl and nitrile) that can be selectively removed to create functionalized molecules. The synthetic methods involved are tailored to the methods used to make algiopine 636 and 659 and other allylalkylamines derived from spider venom.

Chain extension of 2-4 methylene was typically performed by alkylation with the corresponding N- (bromoalkyl) phthalimide. A 1: 1.2 mixture of amine to bromoalkylphthalimide was refluxed in acetonitrile in the presence of 50% KF over celite. Chain extension was also performed by alkylation of the resulting amine with acrylonitrile or ethyl acrylate. The progress of the reaction was followed by a silica gel purified intermediate using a combination of TLC and dichloromethane, methanol, and isopropylamine. The final product was purified by cation exchange (HEMA-SB) and RP-HPLC (Vidac C-18). Purity and structure confirmation were performed by H ¹ -and ¹³ C-NMR and high resolution mass spectrometry (EI, CI and / or FAB).

  The BOC protecting group was added by treating the amine (1 ° or 2 °) with di-tert-butyl dicarbonate in dichloromethane in the presence of a catalytic amount of dimethylaminopyridine. The benzyl protecting group can be obtained in two ways: (1) condensation of 1 ° amine with benzaldehyde followed by sodium borohydride reaction, or (2) alkylation of 2 ° amine with benzyl bromide in the presence of KF. One method was applied. Amide bonding and cyclization were typically performed by reaction of the amine (1 ° or 2 °) with the N-hydroxysuccinimide ester of the resulting acid. This (in the case of cyclization) was done directly by treating the “amino acid” with dicyclohexylcarbodiimide under dilution conditions.

  Deprotection of phthalimide functionality was performed by reducing with hydrazine while refluxing methanol. Deprotection of BOC functionality was performed in anhydrous TFA. Deprotection of the benzyl, nitrile, and CBZ protection functionality was performed by reduction in glacial acetic acid under 55 psi hydrogen in the presence of a catalytic amount of palladium hydroxide on carbon. Nitrile functionality was selectively reduced under hydrogen (in the presence of benzyl and CBZ groups) in the presence of sponge Raney nickel.

Specifically, branched polyamines are typically formula NH ₂ - are prepared from (CH ₂₎ simple polyamines such as _n simple diaminoalkanes of -NH ₂ or Superumiimido or spermine. One of the two (terminal) primary amines such as BOC (t-butyroxycarbonyl), phthalimide, benzyl, 2-ethylnitrile (Michael condensation production product of amine and acrylonitrile) or amide, etc. Protect or “mask” with protecting groups. A typical reaction is the addition of a BOC protecting group by treatment with di-t-butyl-dicarbonate (anhydrous BOC):
The monoprotected product is separated from the unprotected and diprotected products by simple chromatographic or distillation techniques.

Thereafter, the free amine remaining in the monoprotected product is selectively alkylated (or acylated) with an alkylating agent (or acylating agent). In order to ensure mono-alkylation, the free amine is partially protected by condensation with benzaldehyde followed by sodium borohydride reduction to form the N-benzyl derivative:
Thereafter, the N-benzyl derivative is reacted with an alkylating agent. A typical alkylating agent is N- (bromoalkyl) phthalimide, which reacts as follows:
For example, the chain is extended or branched with 4 methylene units using N- (bromobutyl) phthalimide. As an alternative, reaction with acrylonitrile followed by reduction of the cyano group would extend the chain with 3-methylene and amino groups.

The resulting chain extender molecule protecting groups can then be selectively cleaved to yield new free amines. For example, trifluoroacetic acid is used to remove the BOC group: catalytic hydrogenation is used to reduce nitrile functionality, benzyl group is removed; hydrazine is used to replace the phthalimide group as follows: Remove to:

New free amines may be further alkylated (or acylated) as described above to increase the length of the polyamine. This process is repeated until the desired chain length and number of branches are obtained. At the final stage, the product is deprotected, resulting in the desired polyamine. However, further modifications before deprotection may be brought about at the protected end by the following method:
For example, prior to BOC deprotection, the polyamine is acylated with N-hydroxysuccinimide ester of 3,4-dimethoxyphenylacetic acid to give the diprotected polyamine:
This will ultimately yield an allylpolyamine. The BOC group can then be selectively removed with trifluoroacetic acid and exposed to other amino termini that can be extended as described above.

Certain branched polyamines may be obtained by simultaneously alkylating or acylating free primary and secondary amines in the polyamines formed above. For example, treatment of spermine with excess acrylonitrile followed by catalytic reduction yields:
Cyclic polyamines may be prepared as described above, starting with a starting material such as hexacyclene (Aldrich Chemical).

  Polyamino acids within the scope of the invention may be made by recombinant techniques known to those skilled in the art or may be synthesized using standard solid phase methods known to those skilled in the art. Solid phase synthesis is initiated using an α-amino protected amino acid from the carboxy terminus of the peptide. BOC protecting groups can be used for all amino groups, even if other protecting groups are stable. For example, BOC-lys-OH can be esterified into a chloromethylated polystyrene resin support. The polystyrene resin support is preferably a copolymer of styrene and has about 0.5 to 2% divinylbenzene as a cross-linking agent that completely dissolves the polystyrene polymer in some organic solvent. Stewart et al., Solid Phase Peptide Synthesis (1969), W. H. See Freeman Company, San Francisco; and Merrifield, Journal of American Chemical Society (1963), 85, 2149-2154. These and other methods of peptide synthesis are described in US Pat. S. Patents 3,862,925; 3,842,067, 3,972,859; and 4,105,602 are also exemplified.

  Polypeptide synthesis can be done using manual techniques or, for example, Abride Biosystems 403A Peptide Synthesizer (Foster City, CA) or BioSearch SAMII Automatic Peptide Synthesizer (Biosearch Inc., Sun. Rafael, California), and it follows the instructions given in the user manual provided by the manufacturer.

  The allylalkylamines of the present invention are natural products isolated by known techniques, or Jachys et al., Tetrahedron Letter (1988), 29, 6223-6226, and Naison et al., Tetrahedron. -Synthesized as described in Letter (1989), 30, 2337-2340.

One general protocol for the preparation of fendiline (or the fendiline analog shown in FIGS. 44-63) is as follows. In a 10 ml round bottom flask equipped with a magnetic stir bar and rubber septum, 1.0 mmol 3.3'-bisphenylpropylamine (or primary alkylamine) in 2 ml ethanol was added 1.1 mmol phenol and 1.0 mmol. Treated with acetophenone (or substituted acetophenone). To this was added 2.0 mmol MgSO ₄ and 1.0 mmol NaCNBH ₃ . This was stirred at room temperature (about 20 ° C.) for 24 hours under a nitrogen atmosphere. The reaction was poured into 50 ml ether and washed three times with 1N NaOH and once with brine. The ether layer was dried over anhydrous K ₂ CO ₃ and reduced under vacuum. The product is then produced by column chromatography or HPLC incorporating CH ₂ Cl ₂ -methanol-isopropylamine (typically 3% methanol and 0.1% isopropylamine in methylene chloride) coupled silica stationary phase. did.

A preferred method for preparing fendiline or fendiline analog (as represented in FIGS. 44-63) is to use titanium (IV) isopropoxide, Journal of Organic Chemistry, Volume 55. , 2552 (1990), which is partly modified. For the synthesis of NPS544, titanium tetrachloride (a method described in Tetrahedron Letter, Vol. 31, page 5547 (1990)) was used in place of titanium (IV) isopropoxide. The reaction scheme is shown in FIG. 70a. In FIG. 70a, R, R ′ and R ″ represent hydrocarbon groups. According to one embodiment, in a 4 ml vial, 1 mmol of amine ( 1 ) (typically a general amine) and a ketone or 1 mmol of aldehyde ( 2 ) (generally acetophenone) is mixed and then treated with 1.25 mmol of titanium (IV) isopropoxide ( 3 ) and left at room temperature for 30 minutes with occasional stirring. As an alternative, secondary amines may be used in place of ( 1 ) Note: Some reactions will result in heavy precipitates or solids but may be warmed / heated (to their melting point) is several times stirred / mixed over the course of the reaction. the reaction mixture was treated with 1ml ethanol containing 1 mmole sodium cyanoborohydride (4), after which the resulting mixture at room temperature Leave for about 16 hours with occasional stirring, then stop the reaction by adding about 500 μl of water, then dilute the reaction mixture to a total volume of about 4 ml with ethyl ether and then centrifuge. The phase is removed and reduced with a rotovap, and the resulting product ( 6 ) is filtered in normal phase using silica (dichloromethane: methanol: isopropylamine with silica, or 0.1% TFA with acetonitrile or methanol. Chromatography on a short column of silica (or alternatively by using preparative TLC on silica) in dichloromethane: methanol: isopropylamine (typically 95: 5: 0.1) combination is partially purified.

  If appropriate or desired, chiral resolution may be performed using methods such as those described in Example 21.

Formulation and Administration As indicated herein, the molecules of the invention: mimic or antagonize one or more effects of extracellular Ca ²⁺ ; (b) individually at extracellular free Ca ²⁺ levels Affected: (c) Can be used to treat diseases such as hyperparathyroidism, esteroprosis and hypertension. In general, a disease or condition associated with an inorganic-ion receptor can now be treated to be considered, diagnosed, and / or advantageous. These molecules have been shown to generally act on parathyroid cells, whereas they also show osteolytic cells, paraglomerular kidney cells, proximal tubule kidney cells, distal tubule kidneys Cells, thick ascending limbs and / or collecting duct cells, keratinocytes of the epidermis, vesicular cells in the thyroid (C-cells), intestinal cells, trophoblast cells in the placenta, platelets, vascular smooth muscle cells, It may also regulate Ca ²⁺ receptors on other cells, including cardiac atrial cells, gastrin and glucagon secreting cells, kidney glomerular cells and breast cells.

  These molecules will typically be used to treat human patients, but other isothermals such as other primates, farm animals such as pigs, cattle and poultry, sports animals such as horses, dogs and cats. It can also be used to treat similar or identical diseases in animal species.

  In therapeutic and / or diagnostic applications, the molecules of the invention can be formulated in a variety of modes of administration, including systemic and local or intensive administration. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences, Mac Publishing Company, Easton, Pennsylvania.

  For systemic administration, oral administration is preferred. Alternatively, for example, intramuscular, intravenous, intraperitoneal, and subcutaneous injection may be used. For injection, the molecules of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the molecules may be formulated in solid form and immediately re-dissolved or suspended before use. Freeze-dried forms are also included.

  It can be administered systemically by transmucosal or transdermal methods, or the molecule can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known to those skilled in the art and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents may be used to facilitate penetration. Transmucosal administration may be, for example, spraying to the nose or suppository. For oral administration, the molecule is formulated in a suitable oral dosage form such as capsules, tablets, and tonics.

  For topical administration, the molecules of the invention are formulated in ointments, salves, gels or creams as generally known to those skilled in the art.

  As shown in the examples below, the amount to be administered of the various compounds of the invention can be determined by standard techniques. Generally between about 1 and 50 mg / kg per kg of animal to be treated.

Ca ²⁺ receptor natural product screening has traditionally provided a clue to the development of a variety of therapeutic molecules. However, high-throughput screening of natural product libraries or other molecular libraries for activity at the Ca ²⁺ receptor has not previously been possible. To enable this, it is best to clone the Ca ²⁺ receptor cDNA and then make a transfected cell line that is stable for high-throughput screening. The receptor structure can be used to gain additional insight into the molecular binding structure of the ligand binding site, and such information can be used to expand rational drug design plans as discussed above. I can do it. Limited structure activity studies and testing of selected natural product molecules will provide the first structural database necessary to guide rational natural product screening and drug design.

  The discovery of the superfamily of genes provides the same benefits in relation to all inorganic-ion receptors. Although the following discussion sometimes only refers to a methodology for cloning calcium receptors, those skilled in the art will appreciate that this methodology is generally applicable to all inorganic-ion receptor cloning. Will be done.

  These recombinant receptors enable for the first time the screening of -like and -antagonist molecules, including calcium-like and calcium antagonist molecules. For example, by combining receptor and test method. This is particularly useful for screening using human receptors to identify therapeutically useful compounds.

Bovine and human parathyroid cell Ca ²⁺ receptor cDNA can be cloned by functional expression in Xenopus oocytes. By measuring the current through the endogenous Ca ²⁺ activated Cl ⁻ channel, it is possible to indirectly follow the increase in intercellular Ca ²⁺ in Xenopus oocytes. Amplification of the response provided by this signal transduction pathway allows for the detection of receptor proteins encoded by mRNA at very low levels. This allows the detection of receptor-specific cDNA clones without the need for highly relaxed ligands, specific antisera or protein or nucleic acid sequence information. An example of the method is as follows.

  Adult female Xenopus laevis was obtained from Xenopus I (Ann Arbor, MI) and maintained according to conventional methods. The round ovary protrusion was removed from a hypothermically anesthetized toad. Oocyte tresses were transferred to modified Bath saline (MBS). Individual oocytes were obtained by incubating for 2 hours at 21 ° C. in MBS containing 2 mg / ml collagenase (Sigma, type 1A) and selected for injecting V-VI stage oocytes.

  Pull the glass capillary tube (diameter 1 mm) to a thin tip and fold it by hand to make the tip diameter about 15 μM. A small drop of mRNA (diethyl pyrocarbonate (DEPC)-1 ng / nl in treated water) was placed on parafilm and sucked up with a capillary tube. The capillary tube was then connected to a picospritzer (WPI Instruments) and the volume of air-pulsed droplets was adjusted to yield 50 ng of mRNA (typically 50 nl). A 35 mm culture dish with a nylon stocking applied to the bottom was used to keep the oocyte from moving during the injection of mRNA into the animal pole. The injected oocytes were placed in a 35 mm culture dish containing MBS, 100 ug / ml penicillin and 100 U / ml streptomycin and incubated at 18 ° C. for 3 days.

  Following incubation, oocytes were placed in a 100 μl plastic chamber and poured into MBS using a peristaltic pump at a flow rate of 0.5 ml / min. Test molecules or non-organic polycations were added by quickly transferring the tube to another buffer. The recorded and galvanic electrodes were assembled from thin wall capillary tubes pulled with a resistance of 1-3 mom and filled with 3MKCl. Axon, which is used to measure the current passed to maintain the holding potential by puncturing both electrodes under the microscope (at the animal pole) and aligning to the holding potential (-70 to -80 mV) Connected to an Instruments AxoClamp 2A voltage-clamp amplifier. The current was recorded directly on a strip chart recorder.

Tissue was obtained by surgically removing parathyroid bodies from calves or secondary HPT patients for mRNA preparation. It is not necessary to prepare purified cells; the entire gland was used to prepare mRNA that governs Ca ²⁺ receptor expression in Xenopus oocytes. Glands that were homogenized from total cellular RNA were prepared by acid guanidinium thiocyanate / phenol extraction. Poly (A) ⁺ mRNA was selected by standard methods using oligo dT cellulose chromatography. A glycerol gradient centrifugation was used to align the size of the mRNA fractions. The mRNA was denatured with 20 mV methylmercuric hydroxide and placed on a linear 15-30% glycerol gradient prepared in a Beckman TLS55 tube (50-100 ug at a concentration of 1 mg / ml). Subsequently, centrifugation was performed at 34,000 rpm for 16 hours, and 0.3 ml gradient fractions were collected and diluted with an equal volume of water containing 5 mM beta-mercaptoethanol. The mRNA was then recovered by two methanol precipitations. If desired, mRNA (poly (A) ⁺ 50-100 ug) may be separated on a 1.2% agarose / 6.0 M urea separation gel along the range of RNA size markers. Subsequently, mRNA was visualized by ethidium bromide staining, and a gel slice containing RNA in a 1.5-2.0 kb size step was scraped. mRNA is recovered from agarose gel sections using RNAid binding matrix (according to the supplier's standard protocol: Stratagene Incorporated) and the recovered mRNA fraction is eluted in DEPC-treated water did.

The amount of recovered mRNA was measured by UV absorption measurement. The size range of mRNA contained within each fraction was determined by formaldehyde / agarose gel electrophoresis using a small amount (0.5 ug) of each sample. The integrity of the mRNA was evaluated by translating each sample in vitro. 0.05-0.5 ug of each mRNA fraction was translated using reticulocyte lysate (commercially available kit; BRL). The resulting ³⁵ S-labeled protein was analyzed by SDS-PAGE. Intact mRNA was able to dominate protein synthesis in the full size range and roughly corresponded to the size of individual mRNA fractions.

A cDNA library was constructed in the vector λZAPII according to a modified Gabler and Hoffman technique. RNA from the fraction giving the optimal response in the oocyte assay was used as starting material. First strand cDNA synthesis was primed with oligo dT / Not I primer-linker. Second strand synthesis was performed by the RNase H / DNA polymerase I self-priming method. Double-stranded cDNA was blunted with an Eco RI adapter blunt end ligated to the cDNA with T4 DNA polymerase and T4 ligase. Following Not I digestion to cleave the linker, the full length of the cDNA was size selected by exclusion chromatography on Sephacryl 500HA. First strand cDNA was radiolabeled with α- ³² P-dATP and all synthesis and recovery steps were subsequently followed by incorporation of radioactivity. The full length of the cDNA recovered from the sized column was ligated to the Eco RI / Not I digested λZAPII hand. The ligation mix is packaged with a commercially available high efficiency packaging extract (Stractagene, Inc.) and the appropriate host strain (XL1-Blue) Put it on. The percentage of recombinant phage was measured by the ratio of white plaques to blue plaques when the library was placed on IPTG and X-gal.

The average insert size was determined from 10 randomly selected clones. The phage DNA “mini-preps” was digested with Eco RI and Not I to release the insert and the size was determined by agarose gel electrophoresis. The library consisted of> 90% recombinant phage and the insert size ranged from 1.5 to 4.2 kb. Recombinant ligation was packaged on a large scale, yielding 800,000 primary clones. The packaging mixture was titrated and covered with 50,000 plaques per 15 cm plate. Each pool of 50,000 clones was eluted in SM buffer and stored individually.

A stock of each plate lysate from the clone pool was used for small scale phage DNA preparation. Phage particles were concentrated by polyethylene glycol precipitation and phage DNA was purified by proteinase K digestion followed by phenol: chloroform extraction. Twenty micrograms of DNA were digested with Not I and used as a template for transcription of sense strand RNA in vitro. In vitro transcription uses T7 RNA polymerase and the 5 ′ cap analog m ⁷ GpppG in a total reaction volume of 50 μl according to standard protocols. Following DNase I / proteinase K digestion and phenol / chloroform extraction, RNA was concentrated by ethanol precipitation and used for oocyte injection.

Synthetic mRNA (cRNA) derived from 16 library subpools each consisting of 50,000 independent clones was injected into the oocytes. After 3-4 days of incubation, the ability of 10 mM neomycin to induce Ca ²⁺ -dependent Cl-current in the oocytes was evaluated. The pool named 6 contains positive clones and thus contains cDNA clones that encode functional calcium receptors. In order to reduce the complexity of pool 6, i.e. to purify the calcium receptor clones contained in this pool, pool 6 phage were re-plated at ~ 20,000 plaques per plate and 12 plates were recovered. DNA was produced from each of these subpools and cRNA was synthesized. The oocytes were again injected with cRNA, and after 3-4 days, the ability of 10 mM neomycin to induce Ca ²⁺ -dependent Cl-current was evaluated. Subpool 6-3 was positive and this pool was the subject of a further series of platings that reduced the complexity to approximately 5,000 clones per pool. Pools were again measured by cRNA production and injection into oocytes. Subpool 6-3.4 was positive. To facilitate further purification of positive clones in pool 6-3.4, phage DNA from this pool was rescued as plasmid DNA by superinfection with helper phage, ExAssist (Stratagene). Transformation of the rescued plasmid into the bacterial strain DH5alphaF ′ resulted in transformed bacterial colonies on ampicillin plates. Each was collected as a pool of 900 clones. Plasmid DNA was then prepared from each subpool and cRNA synthesis and evaluation was performed by conventional methods. Subpool 6-3.4.4 was positive. Bacteria containing plasmid subpool 6-3.4. 4 were each continuously plated in subpools of ~ 50 clones. It is expected that a single clone encoding a functional calcium receptor will be obtained by repetition of this method.

Another assay can be used to detect calcium receptor expression in oocytes. For example, ⁴⁵ Ca ⁺⁺ can be added to an oocyte and then treated with a calcium-like molecule. Movement of ⁴⁵ Ca ⁺⁺ from intermolecular storage results in an increase in the total amount of ⁴⁵ Ca ⁺⁺ efflux that can be easily measured. A fluorescent Ca ⁺⁺ indicator may also be introduced into the oocyte. In this case, the oocyte expressing the calcium receptor exhibits an enhanced fluorescent color when activated by a calcium-like molecule. These test methods use the cloned calcium receptor instead of the electrophysiological test method for calcium-induced Cl-current described in the above examples. Furthermore, the calcium-like molecular ligand used in the cloning step need not be neomycin as described above, but instead may be, for example, Gd ⁺⁺ , Ca ⁺⁺ , Mg ⁺⁺ or other calcium-like molecular compounds. If it is.

The first experiment used Xenopus oocytes injected with water or mRNA (50 ng) rich in bovine parathyroid body-derived poly (A) ⁺ -. Three days later, the ability of oocytes to increase intracellular Ca ²⁺ in response to increasing extracellular di- or trivalent cation concentrations was evaluated. Oocytes were pierced with recording and energizing electrode rods, and [Ca ²⁺ ] _i was assessed indirectly by measuring the current through the endogenous Ca ²⁺ -activated Cl ⁻ channel. In oocytes injected with poly (A) ⁺ -rich mRNA from bovine (or human, FIG. 32) parathyroid bodies, the concentration of extracellular Ca ²⁺ from 0.7 to 3, 5 or 10 mM increase of, Cl oscillates about the next higher basal conductance ^- caused a rapid and transient increase in current. Increasing the concentration of extracellular Mg ²⁺ from 1 to 10 mM similarly caused an oscillatory increase in Cl ⁻ current. Cl ^- responses to extracellular Mg ²⁺ currents persisted when the extracellular Ca ²⁺ concentration was reduced to <1 [mu] M (Figure 31).

The transient trivalent cation Gd ³⁺ (600 μM) also caused an oscillatory increase in Cl ⁻ current (FIG. 31). Such an increase in Cl ⁻ current, which is oscillatory and persists in the apparent absence of extracellular Ca ²⁺ , is expressed in oocytes by other Ca ²⁺ -flow receptors and proper coordination. It was found to be promoted in the offspring (eg Substance K, FIG. 31). In these examples, the increase in Cl ⁻ current reflects the fluidization of intracellular Ca ²⁺ . These initial studies also show that extracellular multivalent cations allow intracellular Ca ²⁺ to flow in parathyroid mRNA-injected oocytes.

Oocytes injected with water showed no change in Cl ⁻ current when exposed to extracellular Ca ²⁺ (10 mM) or Mg ²⁺ (20 or 30 mM). In a series of experiments, the oocytes were injected with mRNA encoding the substance K receptor. In these oocytes, extracellular Mg ²⁺ (20 mM) caused no current, but the cells responded actively to the addition of substance K (FIG. 31). These experiments suggest that oocytes are not intrinsically sensitive to extracellular Ca ²⁺ or Mg ²⁺ .

A similar experiment was performed using oocytes injected with mRNA rich in poly (A) ⁺ -produced from human parathyroid bodies (hyperplastic tissue derived from second stage HPT). In these oocytes, an increase in extracellular Ca ²⁺ concentration resulted in a reversible increase in oscillating Cl ⁻ current (FIG. 32). The addition of 300 μM La ³⁺ also caused a vibrational increase in Cl ⁻ current. An increase in extracellular Mg ²⁺ from 1 to 10 mM caused an increase in Cl ⁻ current that persisted in the absence of extracellular Ca ²⁺ . Additional experiments suggest that the response to extracellular Ca ²⁺ is concentration dependent. Thus, in 3 mRNA-injected oocytes, Cl ⁻ current increased with 3 mM up to 111 ± 2 nA and 10 mM extracellular Ca ²⁺ up to 233 ± 101 nA.

The results obtained in Xenopus oocytes show the presence of mRNA (s) in parathyroid cells that encode proteins that can confer extracellular Ca ²⁺ sensitivity in normally non-reactive cells. Prove that. Furthermore, extracellular Mg ^2+, Cl extracellular Ca ²⁺ absence ^- ability to cause oscillatory increases in current, Cl ^- current, rather than the influx of extracellular Ca ²⁺ intracellular Ca ²⁺ Prove that it depends on flow. The results obtained with La ³⁺ also indicate that the expressed protein is associated with intracellular Ca ²⁺ flux. In summary, these data indicate that expressed proteins act as cell surface receptors rather than channels. These studies provide indispensable evidence for the presence of the Ca ²⁺ receptor protein on parathyroid cells and the Xenopus oocyte line to accomplish molecular cloning of the Ca ²⁺ receptor cDNA. Indicates the possibility of using.

In another series of studies, parathyroid cell mRNAs modified with methyl mercury hydroxide were size fractionated by centrifugation through a glycerol gradient. Fractions were collected. Each group was injected into Xenopus oocyte line, and after 3 days incubation, the oocytes were evaluated for Ca ²⁺ receptor expression. Oocytes injected with fractions 4-6 showed the largest and most consistent increase in Cl ⁻ current in response to extracellular Ca ²⁺ . These results indicate that the Ca ²⁺ receptor is encoded by mRNA of size 2.5-3.5 kb. This suggests that a method using the direct invention of RNA synthesized from a transformation vector cDNA library is possible. This type of size fractionation experiment was conducted, and similar results were obtained in three different fractionation experiments.

The mRNA fraction obtained and characterized in the above experiment can be measured by injecting it into the oocyte. In each mRNA fraction, 50 ng RNA is injected into 10-20 oocytes as a 1 ng / nl aqueous solution. Injected oocytes are maintained for 48-72 hours in 18 ° C., then Cl ^- measuring the expression of Ca ²⁺ receptor using a current measurement. In each group of injected oocytes, the number of positives for receptor expression and the magnitude of the measured Ca ²⁺ -dependent Cl ⁻ current are measured. As negative subjects, oocytes are injected with mRNA, yeast RNA or water rich in rat liver poly (A) ⁺ -.

  An mRNA in the range of 2.5-3.5 kb is expected to encode the receptor. Large size mRNA may be necessary for cloning studies based on hybrid removal of parathyroid mRNA prior to injection into oocytes. The success of this method does not depend on the production of full length cDNA clones. If receptor expression is not obtained with a single sized fraction of mRNA, the oocytes are injected with a mixed size fraction to determine the combination that elevates functional receptors. If it becomes clear that multiple subunits are required for the formation of functional receptors, a hybrid excision expression cloning method is used. In this study, clones are selected based on their ability to deplete specific mRNA species from the total mRNA population. Clones encoding a single subunit are identified by their ability to block active multi-subunit complex formation. Thorough screening allows identification of clones that encode all of the required subunits.

This study allows the isolation of clones that encode the individual subunits required to form a functional receptor complex. Synthetic RNA from a pool of clones is evaluated by a technique similar to that used to measure the basal mRNA fraction due to its ability to induce Ca ²⁺ receptor expression in Xenopus oocytes. Essentially, 10 pools each representing 100,000 initial clones are tested. A pool of clones showing a positive reaction is screened with a low (typically 4 to 10 times) formulation, and the positive pool is again subdivided and screened. This method in library subfractionation continues until individual positive clones are identified. As a negative target for measuring oocyte expression, antisense transcripts are produced by T7 transcription of these template DNAs that induce a positive reaction. Antisense transcripts cannot express standard receptors and control any non-specific positive signal generated by injection of synthetic RNA. Another important thing is the fact that the synthetic RNA in the oocyte can sometimes be translated "toxic" by an unidentified mechanism. To control this possibility, synthetic RNAs that give a negative response are co-injected with various parathyroid cell mRNAs at various dilutions to determine whether they interfere with non-specific inhibition of Ca ²⁺ receptor expression. To do.

When individual clones encoding the Ca ²⁺ receptor are identified, the inserted cDNA is excised from the λ vector and used for large scale synthesis of the synthetic RNA. This single RNA species oocyte injection allows for a precise measurement of the characteristics of the expressed receptor.

If the size of the mRNA encoding the Ca ²⁺ receptor is too large for direct transcription cloning and expression, or even if multiple subunits are included, a hybrid removal technique of the pool of screened clones is used. cDNA insert DNA is produced from a pool of clones from a size-selective parathyroid cell cDNA library. This DNA is hybridized to parathyroid cell mRNA under conditions that allow the formation of DNA / RNA duplexes. Non-annealed, hybrid-removed RNA is recovered and used for oocyte injection. Alternative methods for hybrid removal or insertion can be used separately and methods known to those skilled in the art. DNA from a pool of clones containing sequences representing Ca ²⁺ receptor mRNA is removed from mRNA from this whole parathyroid cell mRNA population, and expression of this receptor is reduced or eliminated upon oocyte injection. The subfractionation method results from a decrease in the complexity of the pool of clones, and at each step, the cloned DNA is measured by removing the Ca ²⁺ receptor encoding mRNA from the total parathyroid mRNA population. The use of internal control during the hybrid removal measurement ensures that the hybrid removal RNA is intact and used for transformation into oocytes.

Human parathyroid cells express beta-adrenergic receptors bound to adenylate cyclase. This receptor can be expressed in oocytes where it is responsible for agonist-induced activation of endogenous adenylate cyclase. During hybrid removal screening of Ca ²⁺ receptor clones, oocytes injected with hybrid removal mRNA are measured as isoproterenol-induced adenylate cyclase activation. A positive response in this measurement serves to suggest that any observed inhibition of the Ca ²⁺ receptor response is specific and not due to general inhibition of the entire mRNA population.

  The hybrid removal screening method allows isolation of clones that do not contain the complete protein coding region. Positive clones isolated by this screening method are sequenced in order to determine protein-encoding ability. Northern blot analysis of human parathyroid RNA allows measurement of the size of the complete mRNA corresponding to a specific clone. If the positive clone does not appear to be full length, the cloned cDNA is used as a hybridization probe to screen a parathyroid cDNA library for the complete cDNA.

In various cell lines, linked exogenous expression receptors for endogenous functional receptors are possible. Many of these cell lines (eg, NIH-3T3, HeLa, NG115, CHO, HEK, 293 and COS7) can be tested to confirm that they lack the endogenous Ca ²⁺ receptor. These systems lacking a response to external Ca ²⁺ can be used to establish stable transduced cells expressing the cloned Ca ²⁺ receptor.

The production of these stable transfectants involves transfection of an appropriate cell line with a eukaryotic expression vector such as pMSG in which the coding sequence of the Ca ²⁺ receptor cDNA has been cloned into the multiple cloning site. Achieved by: These expression vectors contain a promoter region such as the mouse mammary tumor virus promoter (MMTV) that drives high levels of transcription of cDNA in various mammalian cells. Furthermore, these vectors also contain a gene for selecting cells that stably express the cDNA of interest. A selectable label (marker) in pMSG encodes an enzyme called xanthine-guanine phosphoribosyl transferase (XGPRT) that confers resistance to metabolic inhibitors added to the culture to kill untransfected cells. Yes. Usually, various expression vectors and selection methods are evaluated to determine the optimal conditions for producing a Ca ²⁺ receptor-expressing cell line used in high throughput screening assays.

The most effective method for transfecting a eukaryotic cell line with plasmid DNA depends on the given cell type. The Ca ²⁺ receptor expression construct is introduced into cultured cells by an appropriate technique, ie, Ca ²⁺ phosphate precipitation, DEAE-dextran transfection, lipofection or electroporation.

Cells stably incorporating the transfected DNA are identified by their resistance to the selective media described above, and a clonal cell line is produced by expansion of resistant colonies. The expression of Ca ²⁺ receptor cDNA by these cell lines is assessed by solution hybridization and Northern blot analysis. The functional expression of the receptor protein is determined by measuring intracellular Ca ²⁺ fluidization as a response to an externally added Ca ²⁺ receptor agonist.

Cloning of the Ca ²⁺ receptor allows both structural and functional studies of this novel receptor. Recombinantly produced receptors can also be crystallized for structural studies. Stablely transfected cell lines expressing this receptor can be used for high-throughput screening of natural products and other compound libraries. Molecules with essential potency and specificity can be labeled (radioactively or fluorescently). The ability of a test molecule / extract to displace such a labeled molecule forms the basis of a high throughput assay for screening.

  Another method for cloning inorganic ion receptors in oocytes is as follows.

The following is a general description of the frog oocyte isolation, defoldation and injection procedure. Briefly, an Xenopus laevis frog is anesthetized by soaking in 0.17% tricaine, and the ovarian lobes are removed and broken into small pieces. After incubating ovarian tissue for 90 minutes at room temperature in calcium-free buffer, individual V-VI oocytes are isolated using L-type capillaries. Next, the isolated oocytes [usually 200 to 300 per experiment] are further incubated for 90 minutes in collagenase solution [Sigma, type II; 2 mg / ml], and then the vesicular layer is removed with fine tweezers. Can do. Defolated oocytes are incubated in frog Ringer [ND96] at 17 ° C. overnight to remove denatured oocytes [usually <2 to 3% of total]. Next, H ₂ O 50nl [control] or poly (A) ⁺ RNA to surviving oocytes; after injection of [15~50ng / oocyte in H ₂ O50nl], 2~4 days at 17 ° C. incubation To do. (Froglingel [ND96] contains the following components (mM): NaCl (96), KCl (2), CaCl ₂ (0.5), MgCl ₂ (0.5), HEPES (5), pyruvate (2.5)).

Total RNA is extracted from tissues [kidney, osteoclasts, etc.] with guanidinium thiocyanate and separated with a CsCl cushion. The poly (A) ⁺ RNA is then isolated by oligo (dT) cellulose chromatography [pass through this column twice]. The integrity of poly (A) ⁺ RNA is assessed by agarose gel electrophoresis. Evaluate the ability of 20-50 ng poly (A) ⁺ RNA [ie as evidence of Ca ²⁺ receptor activity] to produce Ca ²⁺ -dependent Cl ⁻ channel activity when exposed to extracellular Gd ³⁺ . The next step is to locate the mRNA that expresses Ca ²⁺ receptor activity in a small size range (˜1 kb). For this, approximately 100 μg of poly (A) ⁺ RNA is separated by a sucrose gradient and 40 size fractions are collected from the gradient. Alternatively, preparative continuous flow agarose gel electrophoresis [Hediger, M .; A. : Anal. Biochem. 159: 280-86 (1986)], about 200-300 μg of poly (A) ⁺ RNA can be fractionated and 70-90 fractions can be collected. A pool of fractions [3-5 fractions / pool] of the poly (A) ⁺ RNA [0.2-0.5 ng / oocyte] fractions obtained from these pools is designated X. These pools are evaluated for their ability to be injected into laevis oocytes and produce Ca ²⁺ receptor activity. This poly (A) ⁺ RNA pool that injects individual fractions obtained from the pool that gives the highest Ca ²⁺ receptor activity into the oocyte and best expresses the Ca ²⁺ receptor in the oocyte. Clarify the fractions inside.

A directed cDNA library is constructed from this poly (A) ⁺ RNA pool using the SuperScript Plasmid system [pSPORT1; BRL]. CDNA is made using superscript, MuMLV-RT, reverse transcript [many of which will be the full length of the coding region]. The cDNA is size fractionated by gel electrophoresis and the appropriate cDNA size region [based on poly (A) ⁺ RNA size range] is extracted using GENECLEAN II [Bio101]. Next, this size-selected cDNA is ligated in a pSPORT1 plasmid vector using a NotI primer at the 3 ′ end and an adapter for SalI at the 5 ′ end. The resulting plasmid is introduced into ELECTROMAX DH18B cells [BRL] by electroporation. Transformed bacteria are grown on nitrocellulose filters [500-800 colonies / filter] and the original filters are stored at 4 ° C. [short term] or −70 ° C. [long term]. A replication filter is grown, plasmid DNA is isolated from the filter, linearized by restriction digestion, and then used as a template for sense cRNA synthesis by in vitro transcription [T7 promoter in the presence of Cap analog]. The pool of cRNA was injected separately into oocytes, which, Cl ^- assayed for expression of Gd ³⁺ induced [1~100μM] activation flow. Standard sister selection methods are used to identify clones that express Ca ²⁺ receptor activity in oocytes.

A restriction map of the Ca ²⁺ receptor cDNA is created and the appropriate restriction fragment is subcloned into pSPORT1. The subcloned cDNA is sequenced bi-directionally using standard methods [Sequenase Polymerase 2 ^R 2, USB]. Use sequencing primers (18-mer) when you need to sequence within or between subclones, or when you need to analyze compressed or unclear sequences To do. The coding region of the Ca ²⁺ receptor protein is preferably determined from the longest open reading frame with a start site that is homologous to the Kozak consensus sequence. A topology for the Ca ²⁺ receptor protein is created using hydropathy and other protein algorithms. The nucleotide sequence of the cDNA, the amino acid sequence of the Ca ²⁺ receptor protein and the protein topology are compared with those of other Ca ²⁺ receptors and other known cDNAs and proteins present in the database. A homologous region may represent a domain involved in cation binding or regulation.

  Currently preferred methods for isolating inorganic ion receptor nucleic acids are based on hybridization screening.

  Initiating DNA synthesis and PCR amplification by using region-specific primers or probes derived from BoPCaR1 and using known methods (Imis et al., PCR Protocols, Academic Press, San Diego, Calif. (1990)) Colonies containing cloned DNA encoding receptor family members can be identified.

  When using a primer derived from a nucleic acid encoding an inorganic ion receptor, by using high stringency conditions, annealing at 50-60 ° C., a sequence homologous to that primer is about 76% or more. One skilled in the art will recognize that it will be amplified. By using annealing at lower stringency conditions, 35-37 ° C, sequences that are about 40-50% or more homologous to the primer will be amplified.

  High stringency conditions, 50-65 ° C., 5 × SSPC, hybridization with 50% formamide, 50-65 ° C., 0 when DNA probes derived from inorganic ion receptors are used for colony / plaque hybridization. By using washing with 5 × SSPC, a sequence with a region of about 90% or more homology to the probe can be obtained, with lower stringency conditions, 35-37 ° C., 5 × SSPC, 40- Those skilled in the art will recognize that using hybridization with 45% formamide, washing at 42 ° C., SSPC will yield a sequence having a region of 35-45% or more homology to the probe. Let's go.

  Any tissue may be used as a source of genomic DNA encoding a member of the inorganic ion receptor family. However, in the case of RNA, the most preferred source is tissue where the expression level of the desired inorganic ion receptor family component is elevated. In the present invention, oocyte injection and two-electrode whole oocyte voltage clamping were used to identify expression from such tissue sources. However, by using the sequences described herein, it is now possible to identify such cells using inorganic ion receptor sequences as probes in Northern blots or in situ hybridization. The need for the procedure used to clone the first member of this family has been eliminated, and the need to obtain RNA from tissues with elevated levels of inorganic ion receptor expression has also been eliminated.

  The present invention further provides a method of identifying a cell or tissue that expresses a member of the inorganic ion receptor family. For example, by using a probe consisting of a DNA sequence encoding BoPCaR1, a fragment thereof or a DNA sequence encoding another member of the inorganic ion receptor protein family as a probe or amplification primer, a message homologous to that probe or primer is expressed Cells to be detected can be detected. One skilled in the art can readily adapt currently available nucleic acid amplification techniques and nucleic acid detection techniques to use probes or primers based on sequences encoding members of the inorganic ion receptor family.

Given the appropriate cells or tissues that express other receptors, these receptors can be cloned in a manner similar to that described above for the parathyroid cell calcium receptor. For example, mRNA obtained from human osteoclastoma tissue encodes an osteoclast calcium receptor (FIG. 42). Therefore, to isolate human osteoclast receptor clones, it is only necessary to isolate mRNA from osteoclast tissue as described above, prepare a cDNA library, and assay / fractionate subpools. Furthermore, preferred receptors for drug screening are of human origin. By using clones that encode certain receptors, corresponding human cDNA clones can be obtained by cross-hybridization well known to those skilled in the art. In addition, receptor parathyroid cells and other cell receptor clones allow the isolation and expression of genes encoding similar inorganic ion sensing proteins in other cells. This can be achieved by various methods. Southern blot analysis of human genomic DNA using Ca ²⁺ receptor cDNA as a hybridization probe gives an indication of the number of related sequences encoded in the genome, and hybridization at various stringencies is related to these related sequences. It will give an indication of the degree of divergence between. This will provide information on the potential number of genes encoding the relevant receptor proteins. Northern blot analysis using Ca ²⁺ receptor cDNA as a probe will determine whether the same or related transcripts are present in various tissues. If relevant transcripts that are homologous to the parathyroid cell Ca ²⁺ receptor are detected, obtain a clone of these mRNAs by screening an appropriate cDNA library or by polymerase chain reaction (PCR) technology. Is a relatively simple problem.

  Targeted gene walking (TGW) is a variation of the standard polymerase chain reaction (PCR) that allows amplification of unknown DNA sequences adjacent to a short portion of known sequence. Parker et al., Nucl. Acids Res. 19: 3055 (1991). Unlike conventional PCR techniques that amplify the DNA sequence between two known primer sites, the TGW can amplify DNA adjacent to one such site. Thus, TGW can serve as an alternative to conventional cloning and library screening methods to isolate sequences upstream or downstream of known sequences. By using this technique, a gene can be isolated from a starting DNA template with a limited amount of sequence information.

  First, several standard PCR reactions are performed in parallel using one “target primer” and a different “walking primer”. The target primer is a sequence-specific primer that is exactly complementary to a known sequence on the DNA molecule of interest and is directed to an unknown flanking sequence. A walking primer is a non-specific sequence that is not complementary to DNA near the target primer. The walking primer can be any oligonucleotide unrelated to the target primer sequence. In the first series of PCR, a product is produced only when the walking primer anneals to a complementary DNA strand that is adjacent to the strand to which the target primer is hybridizing. The PCR product of interest is preferably in the size range of 5 kilobases. The amplification product is produced with about 60% mismatched nucleotides in the walking primer relative to the DNA template. Full base pairing is only required for the first 2 nucleotides 3 'of the walking primer, but otherwise partial homology is allowed. Annealing temperature is an important variable in determining the number of PCR products identified by agarose gel electrophoresis.

Second, an oligomer extension assay is performed using an “internal detection primer”. This primer represents a known sequence between the previous two primers and adjacent to the target primer. The internal detection primer is kinased with ³² P-gamma-ATP and then used in one PCR cycle using the DNA obtained from the first PCR as a template. This extension identifies the product in the first PCR adjacent to the target primer. These new products are identified by agarose gel electrophoresis and autoradiography. Any product that does not hybridize to the internal detection primer represents a discontinuous amplification product produced by a subset of the primers.

  Finally, the band identified by the oligomer extension assay is excised from the gel and reamplified by standard PCR using the target primer and the walking primer that originally produced the band. The new PCR band is then directly sequenced to obtain sequence information that was not known.

  To extend the information in the opposite direction, a complement is created from the target and the internal detection primer and their order is reversed in the above operation.

  The following points should be considered when performing hybridization screening and cloning: Due to the degeneracy of the genetic code, there are multiple codons for almost all amino acids (except tryptophan and methionine). Furthermore, the frequency of use of specific codons differs in non-humans compared to humans. Oligonucleotides are synthesized taking into account codon degeneracy and human preferred codons. In addition, oligonucleotides with various possible codon substitutions are also synthesized. To avoid huge numbers, it is not necessary to synthesize all possible sequences resulting from the degeneracy of the code. Rather, select the subset of codons with the highest expression frequency.

  The novel receptor clones thus obtained can be functionally evaluated by expression in oocytes or transfected cell lines. Transfected cell lines that express cell-specific inorganic ion receptors can then be screened for high-throughput molecules that specifically act on ion-sensing mechanisms such as osteoclasts and paraglomerular cells. The means can be provided.

Alternatively, the calcium receptor can be cloned by expression in eukaryotic cells. For example, a cDNA library can be prepared from parathyroid mRNA and cloned into a eukaryotic expression vector such as pcDNA1. Transfection of subpools obtained from this library into eukaryotic cells such as COS7 and HEK293 cells can provide a relatively high level of transient expression of the encoded cDNA sequence. Cells transfected with a functional calcium receptor clone will express a calcium receptor that can be activated by calcium, neomycin or other calcium-like compounds. If cells are first loaded with a fluorometric indicator for [Ca ²⁺ ] _i , activation of the calcium receptor results in an increase in fluorescence. Thus, library subpools containing calcium receptors are identified by their ability to induce a calcium or calcium-like specific increase in fluorescence when transfected into eukaryotic cells. This fluorescence can be detected using a fluorometer or a fluorescence activated cell sorter (FACS).

The present inventors have also developed a “calcium capture assay” for detecting COS7 cells that express G protein-coupled receptors. In this assay, COS7 cell monolayers are transfected with cDNA clones derived from a bovine parathyroid cell cDNA library (eg, a subfraction or pool from a library prepared in pcDNA1), and an agonist of Ca ²⁺ receptor. Their ability to capture radioactive ⁴⁵ Ca ²⁺ in response to treatment with. Emulsion autoradiography is performed on this monolayer, and cells that have captured ⁴⁵ Ca ²⁺ are identified by the presence of grain bunches on the photograph under a dark field microscope. The library pool that gives a positive signal is then subdivided until a single cDNA that gives this signal is identified.

Calcium receptors appear to be functionally related to a group of receptors that utilize so-called “G” proteins to bind to ligands that bind to intracellular signals. Such “G-coupled” receptors can induce an increase in intracellular cyclic AMP upon stimulation of adenyl cyclase by receptor-activated “Gs” protein, or else are receptor-activated. Inhibition of adenyl cyclase by the “Gi” protein can induce a decrease in cyclic AMP. Other receptor activated G proteins lead to changes in inositol triphosphate levels, resulting in the release of Ca ⁺⁺ from intracellular stores. This latter mechanism applies particularly well to the calcium receptor. All known G-coupled receptors are structurally related and have seven conserved transmembrane domains. A number of such receptors have been cloned based on sequence homology to previously cloned receptors. One particularly useful approach is to use synonymous primers homologous to the conserved transmembrane main coding region and amplify the DNA regions encoding these sequences using polymerase chain reaction (PCR). . That is, such an oligonucleotide primer is mixed with genomic DNA or cDNA or RNA isolated from a selected tissue, and PCR is performed. Some experimental methods may require specific amplification of novel G-coupled receptor sequences from selected tissues, which are not necessarily identical to known G-coupled receptors. This is because there is not. This is well understood by those skilled in the art [see, for example, Buck, L. et al. And Axel, R .; (1991) Cell, 65: 175-187].

  In addition, the calcium receptor can be cloned by the PCR method as described above. Two examples of synonymous oligonucleotide primer pairs that can be used to PCR amplify sequences encoding calcium receptors are given below. The first pair is based on the cross-homology exhibited by the majority of G-coupled receptors in the sequences encoding transmembrane main II and VII, respectively. The second primer pair is based on the cross-homology exhibited by a more branched subgroup of G-coupled receptors including calcitonin, secretin, PTH and GLP receptors. This pair corresponds to a conserved sequence encoding transmembrane main III and VII. When one or both of these two primer pairs are mixed with cDNA derived from parathyroid cells or osteoclast tissue, for example, PCR amplification results in the production of amplified DNA of approximately 500-800 base pairs. . These DNAs can be isolated and analyzed for the presence of calcium receptor sequences. For example, each amplified sequence can be used as a probe to isolate a full-length cDNA clone, which is then evaluated by one or more of the assays shown above to encode an inorganic ion receptor. It can be determined whether or not.

Primer pair 1:

Primer pair 2:

  In another method, inorganic ion receptors can be cloned by use of monoclonal antibodies raised against the receptors. Monoclonal antibodies provide a powerful means for immunoaffinity purification of specific proteins. When purified, limited amino acid sequence data can be obtained from the desired protein and can be used to design oligonucleotide sequence probes for screening clones of the complete cDNA sequence.

An example involving a calcium receptor will be described. To prepare hybridomas, whole bovine parathyroid cells are used as an immunogen. Purified and dispersed cells are obtained and live or fixed cell preparations are injected intraperitoneally into an appropriate mouse strain according to established methods. Follow normal protocols for immunization regimens and hybridoma preparation. Hybridomas that secrete monoclonal antibodies that recognize the Ca ²⁺ receptor are identified using a two-step screening method. The initial screen identifies monoclonals that recognize parathyroid cell surface antigens. The hybridoma supernatant is then screened for the presence of mouse antibodies that bind to the surface of parathyroid cells using immunohistochemical methods. This screening can be performed using fixed sections of parathyroid tissue or using primary cultured dispersed cells. Methods for this assay are well established in the literature.

This screen will identify hybridomas that produce monoclonal antibodies against various cell surface determinants, and monoclonals specific for the Ca ²⁺ receptor would be expected to contain only a small subset of these. In order to identify monoclonal antibodies that bind to this Ca ²⁺ receptor, hybridoma supernatants that tested positive in the initial screen were tested for those that blocked the response of cultured parathyroid cells to Ca ²⁺ receptor agonists. Test for ability. The portion of the antibody that binds to the extracellular domain of this receptor is expected to inhibit or activate ligand binding, or otherwise interfere with or affect receptor activation.

Positive monoclonal antibodies in both screens are characterized by Western blot, immunoprecipitation and immunohistochemistry. This makes it possible to determine the size of the recognized antigen and its tissue distribution. A suitable monoclonal antibody is then used to purify the Ca ²⁺ receptor protein by immunoaffinity chromatography according to conventional methods. A sufficient amount of protein is obtained that allows limited amino acid sequencing. A synonymous oligonucleotide probe is then designed based on the peptide sequence information. These probes are then used to screen a parathyroid cell cDNA library for full length clones of the Ca ²⁺ receptor. The resulting clones are characterized by DNA sequencing and functional expression in oocyte lines and cultured mammalian cell lines.

Furthermore, using these antibodies, an expression library (for example, a cDNA library in λgt11 or an equivalent thereof) can be screened to determine a clone that expresses a reactive protein as an antigen. Such clones can then be sequenced to determine if they encode proteins that may be Ca ²⁺ receptors.

  Furthermore, those skilled in the art will appreciate that phage display libraries can be used to clone and analyze calcium receptors instead of monoclonal antibodies. In these libraries, antibody variable regions or random peptides are shotgun cloned into a phage expression vector so that antibody regions or peptides are displayed on the surface of the phage particles. Phage displaying antibody regions or peptides that can specifically bind to calcium receptors bind to cells that display these receptors (eg, parathyroid cells, C cells, osteoclasts, etc.). Let's go. One hundred million of such phage can be screened against cell types that preferentially select phage that can bind to these cells (including phage that bind to calcium receptors). In this way, the complexity of the library can be greatly reduced. Repeating this method will result in a pool of phage that binds to the cell type used. The screens described above for monoclonal antibodies can then be used to isolate phage that display calcium receptor binding antibodies or peptide regions, and these phage can be used to identify calcium receptors for structural identification and cloning purposes. It can be isolated. Kits for preparing such phage display libraries are available from commercial sources [eg, Stratacyte or Cambridge Antibody Technology Limited]. In addition, recombinant phages having such calcium receptor binding properties can be used in place of monoclonal antibodies in various assays of calcium receptors. In addition, such phages can be used in high-efficiency binding-competition screening to functionally bind to calcium receptors, which can serve as structural indicators for the development of human therapeutics that act at calcium receptors. Organic compounds can be identified.

In another method, the affinity of a radioligand to its receptor is cross-linked using the literature [Pilch & Czech, Receptor Biochem. Methodol. 1 161, 1984], the receptor protein can be isolated. The covalent binding of the radioligand allows thorough washing to remove nonspecific binding. For example, high affinity molecules such as random copolymers of arginine and tyrosine (Mw = 22K; arg tyr ratio = 4: 1) that migrate intracellular Ca ²⁺ with an EC ₅₀ of about 100 nM or less are iodinated at ¹²⁵ I. Crosslink. Protamines are preferred for cross-linking studies due to their relatively small size, and are described in the literature [Dottavio-Martin & Ravel, 87 Analyt. Biochem. 562, 1978] can be reductively alkylated.

  Non-specific labeling is kept to a minimum by crosslinking in the presence of unlabeled polycations and di- and trivalent cations. When these molecules are at high concentrations, non-specific interactions between the label and the cell surface will be reduced.

  The present invention provides an isolated nucleic acid sequence encoding an inorganic ion receptor and the isolated receptor itself. The present invention also provides the unique fragments described above. The term “isolated” refers to a nucleic acid sequence as follows: (i) a nucleic acid amplified in vitro, eg, by polymerase chain reaction (PCR); (ii) a nucleic acid synthesized, eg, by chemical synthesis; (Iii) nucleic acid prepared recombinantly by cloning; or (iv) nucleic acid purified by cleavage and gel separation. The term “isolated” when used in describing a polypeptide or amino acid sequence refers to a polypeptide obtained from an expression system using the isolated nucleic acid sequence of the invention, as well as synthesized by, for example, chemical synthesis methods. By polypeptide, it is meant a polypeptide that has been separated from natural biological material and subsequently substantially purified using conventional protein analysis methods.

  Also included within the scope of the present invention are unique fragments of inorganic ion receptors. The term “unique fragment” refers to the portion of the receptor that was found to correspond to no known sequence as of the day of the present invention. These polypeptide fragments can be readily identified as unique by scanning a database of proteins known at the time of the present invention for the same peptide. In addition to creating receptor fragments by expression of cloned partial sequences of receptor DNA, fragments can be created directly from intact proteins. The protein is specifically cleaved by a protein hydrolase (including but not limited to trypsin, chymotrypsin or pepsin). Each of these enzymes is specific for the type of peptide bond it attacks. Trypsin catalyzes the hydrolysis of peptide bonds whose carbonyl group is derived from a basic amino acid (usually arginine or lysine). Pepsin and chymotrypsin catalyze the hydrolysis of peptide bonds derived from aromatic amino acids, specifically tryptophan, tyrosine and phenylalanine. Another set of cleaved polypeptide fragments is created by preventing cleavage at sites that are sensitive to protein hydrolases. For example, reaction of the lysine ε-amino group with ethyl trifluorothioacetate in mild basic solution produces blocked amino acid residues whose adjacent peptide bonds are no longer sensitive to hydrolysis by trypsin [Goldberger et al. Biochem. , 1: 401 (1962)]. Thus, when such a polypeptide is treated with trypsin, it is cleaved only at the arginyl residue. The polypeptide can also be modified to create peptide bonds that are sensitive to hydrolysis catalyzed by protein hydrolases. For example, alkylating a cysteine residue with β-haloethylamine produces a peptide bond that is hydrolyzed with trypsin [Lindley, Nature, 178: 647 (1956)]. In addition, chemical reagents that cleave polypeptide chains at specific residues can be used [Witcop, Adv. Protein Chem. 16: 221 (1961)]. For example, cyanogen bromide cleaves polypeptides at methionine residues [Gross & Witkip, J. et al. Am. Chem. Soc. 83: 1510 (1961)]. Thus, by treating inorganic ion receptors (eg, human calcium receptor or fragments thereof) with various combinations of modifying reagents, protein hydrolases and / or chemical reagents, a large number of distinct sizes can be obtained. Duplicate peptides are created. These peptide fragments can be isolated and purified from such digests by chromatographic methods.

  Alternatively, fragments can be synthesized using suitable solid phase synthesis methods (Steward and Yomg, Solid Phase Peptide Synthesis Freemantle, San Francisco, CA (1968)).

  These fragments can be used in assays as ion binding reagents in the preparation of antibodies and the like. These fragments can also be selected to have the desired biological activity. For example, the fragment can include a site that binds exactly its binding site or agonist or antagonist (eg, of calcium), as described herein. Such fragments are readily identified by those skilled in the art using routine methods for detecting specific binding to the fragment. For example, in the case of a calcium receptor, the test fragment can be expressed from a gene fragment encoding the recombinant receptor using recombinant DNA methods. The fragment is then contacted with calcium or other chemical under appropriate binding conditions to determine whether calcium binds to the fragment. These fragments are useful in calcium agonist and antagonist screening assays, as well as in therapeutic screening assays that are useful in removing calcium from serum or other body tissues.

  Other useful fragments include those having only the outer part of the receptor, the membrane-spanning part or the intracellular part. These portions are readily identified by comparing the amino acid sequence of the receptor to the sequence of a known receptor or by other conventional methods. These fragments are useful for forming chimeric receptors with fragments of other receptors, thereby allowing cells lacking the receptor to become intracellular moieties that perform the desired function within the cell, and ions or It can be produced with an extracellular moiety that causes the cell to respond to the presence of an agonist or antagonist as described herein. When properly configured, these chimeric receptor genes are useful gene therapy for various diseases involving receptor dysfunction, or for various diseases in which modulation of receptor function has the desired effect on the patient. It is. In addition, it is activated by the interaction of the extracellular moiety with its natural ligand (eg, calcium) or agonists and / or antagonists of the present invention and can be readily detected by colorimetric, emission, luminescence, spectroscopic or fluorescence assays Chimeric receptors can be constructed such that intracellular domains are involved in the enzymatic process. Cells expressing such chimeric receptors are the basis for easy screening for discovery of the novel agonists and / or antagonists of the present invention.

  The present invention also provides mutants or homologues and other derivatives of the isolated receptors described herein. Accordingly, the present invention encompasses not only naturally occurring proteins but also such derivatives. These derivatives have the desired receptor activity described herein and are generally identified by the methods described herein. Examples of such proteins and the genes encoding them are given, but these examples are not intended to limit the present invention and only illustrate mutations that may be associated with such genes and proteins. Generally, the gene has the amino acid sequence of the natural receptor, but can be varied at one or more amino acid sites that do not significantly affect the receptor activity of the receptor protein produced. Thus, for example, amino acids may be deleted, added or substituted in non-conserved regions of calcium receptor proteins (ie, regions that are not essential for receptor activity). A more conserved region is necessary for receptor activity, but the amino acids in that region may be more conservatively substituted. For example, one or more amino acid residues in this sequence can be replaced with another amino acid of similar polarity that serves as a functional equivalent. A substitution with an amino acid in this sequence may be selected from other amino acids within the class to which the amino acid belongs. Nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

  One skilled in the art can use standard techniques to determine conserved and non-conserved regions of proteins by in vitro mutagenesis or deletion analysis, and all such derivatives are equivalent to those described below. It will be understood that.

  Further within the scope of the present invention are receptor proteins, or unique fragments or derivatives thereof that are modified separately during or after translation, such as phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, antibodies Those with modifications such as binding to molecules, membrane molecules or other ligands can be included [Ferguson et al., 1988, Am. Rev. Biochem. 57: 285-320].

  Furthermore, the processing or expression of the receptor sequence can be altered by manipulating the recombinant nucleic acid sequence of the invention. For example, the coding sequence can be linked to the promoter sequence and / or ribosome binding site by well-characterized methods, thereby improving expression and bioavailability, but is not limited to this. .

Also, certain recombinant coding sequences can be mutated in vitro or in vivo to mutate within the coding region and / or generate new restriction endonuclease sites or previously existing restriction sites. Further in vitro modifications can be made, such as destroying. Techniques for mutations known to those skilled in the art include in vitro site-specific mutations [Hutchinson et al., 1978, J. MoI. Biol. Chem. 253: 6551], use of TAB ^R linker [Pharmacia], PCR-directed mutations, and the like.

  Furthermore, the codon can be modified so that the codon encodes the same amino acid, but is a preferred codon for the selected expression system.

  The present invention further provides unique fragments of nucleic acids encoding inorganic ion receptors. The term “unique fragment” means a portion of a nucleic acid for which no identical counterpart is found in the sequences known at the time of the invention. These fragments can be easily identified by analysis of a nucleic acid database for detecting a counterpart existing at the time of the invention. Unique fragments are particularly useful in cloning procedures and assays.

  The invention further provides receptor binding agents such as antibodies and / or fragments thereof that can be conjugated to the toxin moiety or expressed with the toxin moiety as a recombinant fusion protein. This toxin moiety binds to and enters the target cell by interaction of the binding agent with the corresponding target cell surface receptor. The toxin moiety to which such a binding agent, antibody and / or fragment is conjugated can be a protein, such as a pokeweed anti-viral protein, ricin, gelonin, aprin, diphtheria exotoxin or pseudomonas exogenous Such as toxins. The toxin moiety may be a high energy emitting radionuclide such as cobalt-60. The chemical structure of the toxin moiety is not intended to limit the scope of the invention in any way. One skilled in the art will appreciate that a wide variety of potential moieties can be combined with the binder. See, for example, “Conjugate Vaccines”, “Contributions to Microbiology and Immunology,” M.M. Cruse and R.C. E. See Lewis, Jr (eds.), Carger Press, New York, (1989). It will also be appreciated that many toxin moieties, such as those listed above, are pharmaceutically acceptable.

  Conjugation of the binding agent with other moieties (eg, bacterial toxins) can be accomplished by any chemical reaction that can bind both molecules as long as the two molecules retain their respective activities. This binding includes many chemical mechanisms such as, for example, covalent bonding, affinity binding, insertion (intercalation), coordination bonding and complexation reactions. However, the preferred bond is a covalent bond. Covalent bonds can be made either by direct condensation of existing side chains or by incorporation of external cross-linking molecules. Many bivalent or multivalent binders are useful for coupling a protein molecule such as an antibody to another molecule. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylenediamines. This list is not intended to be exhaustive of the various classes of coupling agents known to those skilled in the art, but merely exemplifies the more common coupling agents [see below. : Killen and Lindstrom 1984, "Specific Killing of Physiologically Impaired Physiologically Impaired Lymphocyte Caused by the Toxin-acetylcholine Receptor Conjugate" "Jour. Immun. 133: 1335-2549; Jansen, F .; K. , H .; E. Blythman, D.M. Carriere, P.M. Casella, O .; Gros, P.M. Gros, J. et al. C. Laurent, F.A. Paulucci, B.C. Pau, P.M. Poncelet, G.M. Richer, H .; Vidal, and G.G. A. "Voisin, 1982;" Immunotoxins: Hybridmolecules combining high specificity and potential cytotoxicity, "Immunorecitative et al.

  By using recombinant DNA techniques, the present invention provides target cells such as mammalian target cells that have been engineered to express inorganic ion receptors. For example, if such a receptor gene cloned by the above method is inserted into a cell that naturally expresses the receptor, the recombinant gene can be expressed at a very high level.

  The present invention also provides an experimental model system for studying the physiological role of receptors. In this model system, inorganic ion receptors, fragments or derivatives thereof may be supplied to the system or produced in the system. Such a model system could be used to study the effects of receptor over or depletion or cell function. Using this experimental model system, effects on cells or tissue cultures, whole animals, or individual cells or tissues within whole animals or tissue culture systems, or their effects over a specific time interval (eg embryogenesis) Can study. Preferred embodiments are those useful for assays involving the cloned receptor or a portion thereof, for example, high flow-put drug screening assays.

  In a further aspect of the invention, the use of recombinant genes can inactivate endogenous genes by homologous recombination, thereby creating inorganic ion receptor-deficient cells, tissues or animals. For example, recombinant genes can be engineered to contain insertion mutations (eg, the neo gene) that inactivate receptor transcription when inserted into the genome of a recipient cell, tissue or animal. Not. Such a construct can be introduced into cells such as embryonic stem cells by techniques such as transfection, transduction (transduction), and injection. With stem cells lacking an intact receptor sequence, transgenic animals lacking this receptor can be generated.

  A “transgenic animal” is an animal having cells that contain DNA that has been artificially inserted into a cell and that is part of the genome of the animal that grows from that cell. Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats. The transgenic DNA can encode a human inorganic ion receptor. In a further aspect of the invention, natural expression in an animal can be reduced by providing an amount of antisense RNA or DNA effective to reduce receptor expression.

  Various methods can be used to create transgenic animals relevant to the present invention. DNA can be injected into the pronucleus of the fertilized egg before the male and female pronuclei fuse or can be injected into the nucleus of the embryonic cell (eg, the nucleus of a two-cell embryo) after the start of cell division [ Brinster et al., Proc. Natl. Acad. Sci. , U. S. A. 82: 4438-4442 (1985)]. Embryos can be infected with viruses, particularly retroviruses, that have been modified to carry the inorganic ion receptor nucleotide sequences of the present invention.

  Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to contain the nucleotide sequences of the present invention. Transgenic animals can be made from such cells by transferring them to blastocysts transplanted in the foster mother and bringing them to the full term.

  Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River [Wilmington, MA], Taconic [Germantown, NY], Harlan Sprague Dawley [Indianapolis, IN]. .

  Manipulation of murine embryos and microinjection of DNA into the pronuclei of zygotes is well known to those skilled in the art [Hogan et al., Supra]. Microinjection techniques for fish, amphibian eggs, and birds are described in detail in Houdebine and Chourout, Expertia, 47: 897-905 (1991). Another technique for introducing DNA into animal tissue is described in US Pat. No. 4,945,050 (Sandford et al., June 30, 1990).

As an example only, superovulation was induced in female mice to create transgenic mice. Female animals are placed together with male animals, mated female animals are killed by CO ₂ asphyxiation or cervical dislocation and embryos are collected from excised oviducts. Remove surrounding cumulus cells. The pronuclear embryo is then washed and stored until the time of injection. Randomly cycling adult female mice are mated with vasectomized male animals. Recipient female mice are mated at the same time as donor female mice. The embryo is then surgically transferred.

  The technique for producing the transgenic rat is the same as that of this mouse. Hammer et al., Cell, 63: 1099-1112 (1990).

  Methods for creating transgenic animals by culturing embryonic stem cells (ES) cells and then introducing DNA into the ES cells by methods such as electroporation, calcium phosphate / DNA precipitation and direct injection are well known to those skilled in the art. It is. See, for example, Teratocarcinomas and Embryonic Stem Cells, Abstract Approach, E.M. J. et al. See Robertson, Ed., IRLPless (1987).

  In the case of random gene integration, a clone containing the sequence (s) of the invention is cotransfected with a gene encoding resistance. Alternatively, a gene encoding neomycin resistance is physically linked to the sequence (s) of the present invention. Transfecting and isolating the desired clone can be done by any of several methods well known to those skilled in the art [E. J. et al. Robertson, supra].

  DNA molecules introduced into ES cells can also be integrated into the chromosome by a homologous recombination process. Capecchi, Science 244: 1288-1292 (1989). Methods for positive selection of recombination events (ie neo resistance) and binary positive-negative selection (ie neo resistance and gancyclovir resistance), and subsequent identification of desired clones by PCR are described in Capecchi, Cited above and in Joyner et al., Nature 338: 153-156 (1989), the teachings of which are incorporated herein. The final stage of the operation involves injecting the targeted ES cells into blastocysts and transferring the blastocysts into pseudopregnant female animals. The resulting chimeric animals are mated and their progeny are analyzed by Southern blotting to identify individuals carrying the transgene.

  Techniques for producing non-murine mammals and other animals are discussed separately. Houdebine and Chourout, supra; Pursel et al., Science 244: 1281-1288 (1989); and Simms et al., Bio / Technology, 6: 179-183 (1988).

Early use hyperparathyroidism (HPT) is characterized by hypercalcemia and increased levels of circulating PTH. One of the major defects in HPT appears to be a decrease in the sensitivity of parathyroid cells to negative feedback control by extracellular Ca ²⁺ . Therefore, in tissues derived from patients with early HPT, the “fixed point” for extracellular Ca ²⁺ is shifted to the right, and therefore a higher than normal extracellular Ca ²⁺ concentration is necessary to suppress PTH secretion. Furthermore, in early HPT, PTH secretion inhibition is often only partial, even if the extracellular Ca ²⁺ concentration is high. In the second stage (uremia) HPT, it is observed that the fixed point for extracellular Ca ²⁺ is similarly elevated even though P2 ⁺ secretion suppressed by Ca ²⁺ is at a normal rate. Changes in PTH secretion are parallel to changes in [Ca ²⁺ ] _i : the fixed point of extracellular Ca ²⁺ -induced [Ca ²⁺ ] _i increases to the right and the degree of such increase decreases. Furthermore, staining of the tissue with monoclonal antibodies indicates that Ca ²⁺ receptors are reduced in adenoma-like and hyperplastic parathyroid cells.

Ca ²⁺ receptors constitute a discontinuous molecular body for pharmacological intervention. Molecules that have similar or opposite effects of extracellular Ca ²⁺ are beneficial for the long-term management of both early and second stage HPT. Such molecules provide the additional stimuli necessary to suppress PTH secretion that cannot be achieved by hypercalcemic conditions alone. Such molecules with greater effects than extracellular Ca ²⁺ may conquer the obvious non-suppressible factor of PTH secretion that is particularly troublesome in adenoma-like tissues. Alternatively or additionally, such molecules may inhibit PTH synthesis, such that long-term hypercalcemia in bovine and human adenomatous parathyroid tissues indicates suppression of preproPTH mRNA levels. Long-term hypercalcemia also inhibits proliferation of parathyroid cells in vitro, so calcium-like substances are also effective in limiting parathyroid hyperplasia specific to second-stage HPT It can be.

Other somatic cells can respond directly to physical changes in extracellular Ca ²⁺ concentration. Calcitonin secretion from thyroid follicular cells (C-cells) is regulated by changes in extracellular Ca ²⁺ concentration. Renin secretion from renal paraglomerular cells is suppressed by an increase in the concentration of extracellular Ca ²⁺ , like PTH secretion. Extracellular Ca ²⁺ causes the intracellular Ca ²⁺ flux of these cells. Other kidney cells respond to calcium as follows: Ca ²⁺ elevation inhibits the formation of 1,25 (OH) ₂ vitamin D by proximal tubule cells and the calcium-binding protein in distal tubule cells Stimulates production, inhibits tubule reabsorption of Ca ²⁺ and Mg ²⁺ , inhibits the action of vasopressin on medullary Helen trap thick ascending limb (MTAL) cells, reduces vasopressin action in cortical collecting duct cells, It affects the smooth muscle cells of the blood vessels of the glomeruli of the kidney. Calcium promotes the differentiation of intestinal microsphere cells, breast cells and skin cells. It also inhibits atrial calcium excretion increasing peptide secretion from the heart atrium, decreases cAMP accumulation in platelets, alters gastrin and glucagon secretion, acts on vascular smooth muscle cells, and secretes vasoactive factor cell secretion Adjust. Isolated osteoclasts respond to an increase in the concentration of extracellular Ca ²⁺ to an increase in [Ca ²⁺ ] _i resulting to some extent from intracellular Ca ²⁺ flux. An increase in [Ca ²⁺ ] _{i in} osteoclasts is associated with inhibition of a functional response (bone resorption) similar to PTH secretion in parathyroid cells. Release of alkaline phosphatase from bone-forming osteoblasts is stimulated directly by calcium. Thus, in addition to its ubiquitous role as an intracellular signal, there are sufficient indications that Ca ²⁺ may also function as an extracellular signal that controls certain specific cellular responses. The molecules of the invention can be used to treat diseases associated with a disrupted Ca ²⁺ response in these cells.

Cloning of parathyroid cells and other cells' Ca ²⁺ receptors allows the presence of homologous proteins in other cells to be evaluated directly. A group of structurally homologous Ca ²⁺ receptor proteins can be obtained in this way. Such receptors allow an understanding of how these cells detect extracellular Ca ²⁺ and are effective therapies for the treatment of HPT, osteoporosis and hypertension as described herein. Enables the evaluation of the mechanism as the active site of and the novel therapy of other bone and mineral related diseases.

Other uses are described above. For example, recombinant Ca ²⁺ receptor protein is used in therapy and is introduced by standard methods such as transduction of a nucleic acid encoding this protein. Further, such proteins are useful for the measurement of calcium-like molecules of the present invention.

The following examples illustrate the invention without limiting its scope.
The studies described herein have shown that various organic molecules flow intracellular Ca ²⁺ and suppress PTH secretion in parathyroid cells. Although these molecules are structurally diverse, many have a net positive charge at physiological pH. The cationic nature of organic compounds plays a useful role, but is not the only factor in determining activity.

Example 1: Screening of calcium-like molecules in bovine parathyroid cells Isolated bovine parathyroid cells were purified on a Percoll gradient and cultured overnight in serum-free medium. Cells were continuously introduced with fura-2 and free intracellular Ca ²⁺ concentration was measured fluorescently. Changes in [Ca ²⁺ ] _i were used to screen for molecules active on the Ca ²⁺ receptor. To be considered calcium-like, the molecule had the normal effect of increasing extracellular Ca ²⁺ and was required to be triggered by activation of the Ca ²⁺ receptor. 1) molecule, [Ca ^2+] it must induce an increase of _i; [Ca ^2+] _i increase is obtained continuously in the absence of extracellular Ca ^2+, and / or molecules of extracellular Ca induced by ²⁺ may strengthen increase in [Ca ^2+] _i.
2) The molecule must cause an isoproterenol-stimulated decrease in cyclic AMP formation that is blocked by pertussis toxin:
3) The molecule must inhibit PTH secretion resulting in an increase in [Ca ²⁺ ] _i to the same extent as the concentration: and 4) Concentration response curves of the molecule for increases in [Ca ²⁺ ] and PTH secretion Must move to the right with a PKC activator such as phorbol myristate acetate (PMA).

Several structurally different groups of molecules were tested: polyamines, aminoglycoside antibiotics, protamine and lysine or arginine polymers. The structures of these molecules are depicted in Figures 1-6. Included in FIGS. 1-6 are the net positive charge of the molecule, the EC ₅₀ for induction of intracellular Ca ²⁺ flux in bovine parathyroid cells.

In general, the greater the net positive charge of a molecule, the greater its effectiveness in generating intracellular Ca ²⁺ flux. However, some significant exceptions to this apparent regulation were discovered as follows.

As can be seen from the figure, spermine, neomycin B and protamine induce a rapid and transient increase of [Ca ²⁺ ] _i in fura-2-introduced bovine parathyroid cells (FIGS. 11, 12, 17). However, although they are human parathyroid cells (FIG. 25), they do not produce a sufficient steady increase of [Ca ²⁺ ] _i in bovine parathyroid cells (FIGS. 11, 17). In this regard, they are similar to the cytosolic Ca ²⁺ response induced by extracellular Mg ²⁺ in bovine cells and cause fluidization of intracellular Ca ²⁺ without the influx of extracellular Ca ^2+. (FIG. 17b). The transient increase in [Ca ²⁺ ] _i induced by spermine, neomycin B or protamine is not blocked by low concentrations (1 μM) of La ³⁺ or Gd ³⁺ (FIG. 17f, g). Cytosolic Ca ²⁺ transients induced by multivalent cation molecules persisted in the absence of extracellular Ca ²⁺ but were blocked when Ca ²⁺ cellular accumulation was depleted by pretreatment with inomycin ( Figure 12: 17h, i). All these molecules thus give rise to intracellular Ca ²⁺ flux in parathyroid cells.

Multivalent molecules of intracellular Ca ^2+, the same pool was suggested by adding fluidizing as used by extracellular Ca ^2+. Thus, increasing the concentration of extracellular Ca ²⁺ progressively inhibits the transient increase in [Ca ²⁺ ] _i induced by spermine (FIG. 11). Conversely, maximally effective concentrations of spermine or neomycin B (FIG. 7) block transients but are not a steady increase in [Ca ²⁺ ] _i induced by extracellular Ca ²⁺ .

Importantly, spermine, neomycin B and protamine inhibited PTH secretion to the same extent as extracellular Ca ²⁺ . These inhibitory effects on secretion were obtained at concentrations that caused intracellular Ca ²⁺ fluidization (FIGS. 13, 14, and 19). These findings relate to an understanding of the mechanism that contributed to the suppression of PTH secretion by extracellular Ca ²⁺ . Since various inorganic multivalent cations inhibit the secretion of all, only the extracellular Ca ²⁺ is still sustained, causing steady increase in [Ca ^2+] _i, such [Ca ^2+] _i increase in It cannot be critically involved in the control of secretion. Flow rather intracellular Ca ²⁺ than extracellular Ca ²⁺ influx is an essential mechanism associated with PTH secretion inhibition. This is important because it defines a sufficient mechanism to be affected when the molecule affects PTH; molecular stimulation selective extracellular Ca ²⁺ influx is relatively ineffective at suppressing PTH secretion. In comparison, molecules that simply produce Ca ²⁺ flux are just as effective as extracellular Ca ^{2+ in} suppressing PTH secretion.

Those induced by polyvalent cation molecules, such as intracellular Ca ²⁺ fluidization induced by extracellular Ca ^2+, were suppressed by PMA. A surrogate experiment showing the preferential inhibitory effect of PMA on spermine-induced cytosolic Ca ²⁺ transients is shown in FIG. The cytosolic Ca ²⁺ transient induced by ATP was ineffective even when using almost the maximum concentration of ATP. The effect of PMA on the cytosolic Ca ²⁺ transient induced by multivalent cations was parallel to its effect on the response to extracellular Ca ²⁺ : in both cases the concentration response curve shifted to the right (Fig. 21). The inhibitory effect of PMA on [Ca ²⁺ ] _i was associated with the effect of increasing potency in secretion conquered with high concentrations of organic multivalent cations (FIG. 22).

Intracellular Ca ²⁺ flux induced by multivalent cation molecules was associated with increased inositol phosphate formation. For example, protamine caused a rapid (within 30 seconds) increase in IP ₃ formation associated with increased IP ₁ levels. Both these effects were dependent on the concentration of extracellular protamine (FIG. 23). Furthermore, pretreatment with PMA blunted the formation of inositol phosphates induced by multivalent cation molecules. Representative results obtained with spermine are shown in FIG.

Spermine, neomycin B and protamine inhibited isoproterenol-induced cyclic AMP increase. Like the inhibitory effect of molecular extracellular Ca ²⁺ on cyclic AMP formation, that induced by multivalent cation molecules was blocked by pretreatment with pertussis toxin (Table 2).

Pertussis toxin (PTx) blocks the inhibitory effect of multivalent cations on extracellular Ca ²⁺ and cyclic AMP formation. Bovine parathyroid cells were cultured for 16 hours in the presence or absence of 100 ng / ml pertussis toxin. The cells were then washed and incubated with 10 μM isoproterenol for 15 minutes in the presence or absence of the indicated amount of extracellular Ca ²⁺ or multivalent cation molecules. Total cyclic AMP (cells + supernatant) was measured by RIA, and results are expressed as a percentage of the level obtained with 0.5 mM Ca ²⁺ (112 ± 17 pmol / 10 ⁶ cells). Each value is the mean ± SEM of 3 experiments.

In human parathyroid cells, extracellular Mg ²⁺ induced a sustained, steady [Ca ²⁺ ] _i increase in addition to a rapid transient increase (FIG. 16). Just as bovine parathyroid cells respond to extracellular Ca ²⁺ , the Mg ²⁺ -induced [Ca ²⁺ ] _i steady increase in human parathyroid cells is due to Ca ²⁺ influx through voltage-insensitive channels. It is a result (FIG. 16a). The effect of Mg ²⁺ on the [Ca ²⁺ ] _i steady-state increase in human parathyroid cells was seen in both adenoma-like and hyperplastic tissues.

Neomycin B and spermine were tested for the effects of [Ca ²⁺ ] _{i on} human parathyroid cells prepared from adenoma-like tissue. Representative results for neomycin B are shown in FIG. Neomycin B caused not only [Ca ²⁺ ] _i transients but also a steady increase in human parathyroid cells (FIG. 25a). Thus, in human cells, changes in [Ca ²⁺ ] _i induced by extracellular Ca ²⁺ , Mg ²⁺ or neomycin B are very similar.

The cytosolic Ca ²⁺ transient induced by neomycin B persisted in the presence of La ³⁺ (1 μM) and in the absence of extracellular Ca ²⁺ . Neomycin B therefore causes intracellular Ca ²⁺ flux in human parathyroid cells. Neomycin B inhibited human parathyroid cell PTH secretion at concentrations that produced intracellular Ca ²⁺ flux (FIG. 19). However, there were certain differences in the response of human and bovine parathyroid cells to neomycin B. The EC ₅₀ of neomycin B on intracellular Ca ²⁺ flux is 40 μM in bovine parathyroid cells and 20 μM in humans (see FIGS. 19 and 21), whereas spermine is effective in human and bovine parathyroid cells. Similar (EC ₅₀ = 150 μM). Thus, bovine cells can be used for screening studies of molecular activation tests, but it is important to follow up with human parathyroid bodies.

To assess the effect of multivalent cation molecules on C-cells, neoplastic cells derived from rat spinal thyroid tumors (γMTC6-23 cells) were used. Both spermine (10 mM) and neomycin B (5 mM) had no effect on basal [Ca ²⁺ ] _i in these cells. Neither of the molecules had an effect on the reaction with the continuous addition of extracellular Ca ²⁺ . A representative result reporting the lack of effect of neomycin B is shown in FIG. Neomycin B (1 mM) or spermine (1 or 5 mM) failed to induce any increase in [Ca ²⁺ ] _{i in} osteoclasts (FIG. 29). As the record shows, there appears to be some effect on the continuous increase in extracellular Ca ²⁺ , but this is not a consistent finding. In the other second cell, spermine (5 μM) again has no effect on basal [Ca ²⁺ ] _i , resulting in a slight inhibition (about 15%) of extracellular Ca ²⁺ -induced [Ca ²⁺ ] _i increase. I let you. In the third cell, neomycin B had no effect on basal [Ca ²⁺ ] _i and no effect on extracellular Ca ²⁺ -induced [Ca ²⁺ ] _i increase. The relative situation that has evolved from these studies is that spermine and neomycin B are ineffective at the basal or stimulatory level of osteoclast cytosolic Ca ²⁺ levels.

Defects in the influence of the Ca ²⁺ -sensitive mechanism of multivalent cations on C-cells or osteoclasts act specifically on parathyroid cell Ca ²⁺ receptors, or parathyroid bodies on [Ca ²⁺ ] _i Demonstrate the ability to discover or design new precursors that alter the function of one or more of the normal reactions.
The screening of various other molecules is detailed below and the results are summarized in Table 1.

Example 2
Screening for polyamines Linear polyamines (spermine, spermidine, TETA, TEPA and PEHA) and two of their derivatives (NPS381 and NPS382) are screened as in Example 1. All of these molecules were found to mobilize intercellular Ca ²⁺ in bovine parathyroid cells. Their order of efficacy is as follows, with the net positive charge shown in parentheses.

Putrescine (+2) and cadaverine (+2) are inactive at 2 mM.
Another linear polyamine, DADD, behaves somewhat differently than other polyamines and is described in Example 7.

Example 3
Two cyclic polyamines, hexacyclene and NPS 383 are screened as in Example 1. Hexacyclene (+6, EC ₅₀ = 20 μM) is 7 times more effective than NPS 383 (+8, EC ₅₀ = 150 μM). This opposite result appears to be based solely on the net positive charge as a structural feature of Ca ²⁺ receptor activity.

Example 4
Aminoglycoside antibiotic screening Six antibiotics are screened as in Example 1. The EC ₅₀ (rank order of potency) obtained for mobilization of intercellular Ca ²⁺ is as follows.

Kanamycin (+4.5) and lincomycin (+1) were not effective at a concentration of 500 μM. Within the aminoglycoside system, there is a correlation between net positive charge and potency. However, neomycin is significantly more effective than various polyamines (NPS381, NPS382, NPS383, PEHA) with equal or larger positive charges. Since this type of amidoglycoside antibody has nephrotoxicity that may be associated with interaction with calcium receptors in the kidney, such a screen may be used to screen for toxicity in the development of new amidoglycoside antibodies.

Example 5
Peptide and polyamino acid screening Protamine and lysine or arginine polymers with varying peptide lengths were screened in the same manner as in Example 1 to examine their ability to mobilize intercellular Ca ²⁺ . The EC ₅₀ (rank order of potency) obtained for mobilization of intercellular Ca ²⁺ is as follows.

The net positive charge of these polymers increased with increasing Mw. That is, of this series of polyamino acids, for aminoglycosides, there is a direct correlation between net positive charge and potency. Protamine is substantially polyArg with a net positive charge of +21.

Example 6
Allylalkylamine Screening A molecule selected from the venom and a class of allylalkylamine toxicants obtained from spiders is screened as in Example 1.

  Philanthotoxin-433 (+3) is not effective at a concentration of 500 μM. This is a structure similar to the argiotoxin described below.

Argiotoxin-636 (400 μM) does not cause an increase in [Ca ²⁺ ] _i but is effective in the cytosolic Ca ²⁺ response to subsequent addition of intercellular Ca ²⁺ . This is a common feature of all molecules that activate the Ca ²⁺ receptor and is seen for various intercellular divalent cations. This will be discussed in detail in Example 7.

In contrast to Argiotoxin-636, Argiotoxin-659 induces an increase in [Ca ²⁺ ] _i with an EC _{50 of} 300 μM. Argiotoxin-659 differs from Argiotoxin-636 in that it has a hydroxylated indole rather than a dihydroxyphenyl group. This is the only structural difference between the two of these molecules. That is, potency differences exist in the properties of aromatic groups, not in polyamine chains carrying positive charges.

Example 7
Screening Ca ²⁺ channel blockers Ca ²⁺ channel blockers, i.e. to cut off the flow through voltage-sensitive Ca ²⁺ channel of extracellular Ca ^2+. These molecules are screened as in Example 1. There are three structural classes of Ca ²⁺ pathway blockers: (1) dihydropyridine (2) phenylalkylamine (3) benzothiadipine.

Dihydropyridines tested (nifedipine, nitrendipine, BAY K8644 and (−) 202-791 and (+) 202-791) increased [Ca ²⁺ ] _i induced by extracellular Ca ²⁺ when tested at 1 μM. Or it shows no basic effect on [Ca ²⁺ ] _i . As previous studies have shown, parathyroid cells lack the voltage-sensitive Ca ²⁺ pathway, but have a voltage-insensitive Ca ²⁺ pathway regulated by the Ca ²⁺ receptor.

The phenylalkylamines investigated were verapamil, D-600 (a methoxy derivative of verapamil), TMB-8, and a TMB-8 family, NPS384. The first three molecules are tested at a concentration of 100 μM. Phenylalkylamines behave differently than other molecules studied. They show no change in [Ca ²⁺ ] _i even when added to cells soaked in low concentrations of extracellular Ca ²⁺ (0.5 mM) containing buffer. However, verapamil, D-600 and TMB-8 allow mobilization of intercellular Ca ²⁺ induced by extracellular divalent cations, and they additionally block extracellular Ca ²⁺ influx. To do. At intermediate levels of extracellular Ca ²⁺ (1-1.5 mM), these molecules are only slightly induced, but the increase in [Ca ²⁺ ] _i resulting from mobilization of intercellular Ca ²⁺ is strong. is there.

Phenylalkylamines work differently than organic polycations such as neomycin. Data suggests that verapamil, D-600 and TMB-8 were either partial agonists or allolytic activators at the Ca ²⁺ receptor. This was in contrast to the other partial investigated being full agonists.

The molecule NPS384 (concentration 300 μM) does not induce an increase in [Ca ²⁺ ] _i but blocks extracellular Ca ²⁺ influx. Tests at high concentrations indicate the ability of the molecule to cause movement of Ca ²⁺ between cells.

The ability of these molecules to block influx is interesting but not surprising, while it is the ability of these molecules to induce a significant increase in [Ca ²⁺ ] _i resulting from intercellular Ca ²⁺ mobilization. It is. As shown by a considerable number of experiments with [Ca ²⁺ ] _i measurements in parathyroid cells, the temporary increase in [Ca ²⁺ ] _i is almost unchanged due to the movement of intercellular Ca ^2+. Therefore, it shows activation of the Ca ²⁺ receptor.

  The investigated benzothiadipine and diltiazem are similar in all respects to verapamil, and D-600 is also effective at 100 μM.

Note that, with the exception of phenylalkylamine, all active molecules tested above elicit an increase in [Ca ²⁺ ] _i , similar to that induced by the maximum effective concentration of extracellular Ca ^2+. It is the maximum. This indicates that such molecules have similar potency as extracellular divalent cations. This is also in contrast to the activity of phenylalkylamines that appear to act only as partial agonists.

Some phenylalkylamines exhibit interesting structure-activity relationships. The different potencies of molecules such as TMB-8 and NPS384 are important. TMB-8 allows a temporary increase in [Ca ²⁺ ] _i at 100 μM, while NPS 384 does not increase even at 300 μM, but these molecules carry the same net positive charge. In the following, it will be explained that certain other structural features unrelated to the net positive charge impart greater potency to TMB-8.

Example 8
Molecular screening for human parathyroid cells Spermine and neomycin are tested for the effect of [Ca ²⁺ ] _{i on} human parathyroid cells prepared as in Example 1 obtained from surgically removed parathyroid bodies. In human parathyroid cells, spermine was found to cause only a slight increase in [Ca ²⁺ ] _{i when} tested at a concentration of 300 μM.

On the other hand, neomycin induces a large increase in [Ca ²⁺ ] _i in human parathyroid cells when tested at a concentration of 20 μM. The maximum response elicited by neomycin is similar to that elicited by the maximum effective concentration of extracellular Ca ²⁺ .

Example 9
Molecular Screening for Xenopus Ocytes Oocytes that express Ca ²⁺ receptors when injected with mRNA from human parathyroid cells and also have a variety of extracellular inorganic di- or trivalent cations. It moves Ca ²⁺ between responding cells. This screen can be used to test direct effects on the Ca ²⁺ receptor. Oocytes that express Ca ²⁺ receptors also respond to several molecular activities on intact parathyroid cells when screened as follows. Hexacyclene causes intercellular Ca ²⁺ migration at a concentration of 135 μM. Neomycin (100 μM) and NPS382 (5 mM) are also effective. This is a fairly compelling evidence, indicating that such molecules act on the Ca ²⁺ receptor or several other proteins closely related to its function.

For example, we can detect the expression of Ca ²⁺ receptors in oocytes by measuring ⁴⁵ Ca ²⁺ migration. In these experiments, oocytes are injected with bovine parathyroid mRNA or water and exposed to serum or 10 mM neomycin 72 hours later. Prior to stimulation, ⁴⁵ Ca ²⁺ is injected into the oocyte. Stimulation with serum for 20 minutes resulted in intercellular [Ca ²⁺ ] _i release, indicating a 45% increase compared to sham antigen administration with buffer. 20 minutes of neomycin 10 mM challenge results in a 76% increase in [Ca ²⁺ ] _i release. The assay is sufficiently sensitive to the use of cloning of the Ca ²⁺ receptor and has significant advantages over the electrophysiological measurement of Ca ²⁺ activated Cl current.

In another experiment, human osteoclastoma tissue was obtained from bone viability test tissue. The oocyte is injected with mRNA isolated from this cell and challenged with Ca ²⁺ 30 mM. Controls do not respond, while 8 out of 12 oocytes injected with osteoclastoma tissue mRNA respond unambiguously (FIG. 42). According to these experiments, the Ca ²⁺ response of osteoclasts to extracellular Ca ²⁺ is in fact genetically encoded. Also, according to this result, the osteoclast Ca ²⁺ receptor can be cloned by expression in Xenopus oocytes.

Example 10
Molecular screening for rat osteoclasts The Ca ²⁺ receptors are different, suggesting the various susceptibility of parathyroid cells and osteoclasts to extracellular Ca ²⁺ . Parathyroid cells respond to extracellular Ca ²⁺ at concentrations between 0.5 and 3 mM, whereas osteoclast extracellular responds only when the level of extracellular Ca ²⁺ exceeds 5 mM. This fairly high Ca ²⁺ concentration is nevertheless physiological for osteoclasts. Because they are absorbed by osteoclasts, the local extracellular Ca ²⁺ concentration can reach as high as 30 mM.

Molecular screening using rat osteoclasts is performed as follows. Osteoclasts are obtained from the long bones of newborn rats. [Ca ²⁺ ] _i is measured in a single cell using the fluorometric indicator India-1. Spermine, spermidine, neomycin and verapamil were tested, but they do not cause a large [Ca ²⁺ ] _i increase in osteoclasts (slight response is detected).

At a concentration of 1 mM, spermidine causes a slight [Ca ²⁺ ] _i increase (approximately 10% increase caused by the maximum concentration of extracellular Ca ²⁺ ). Neomycin (10 mM) or spermine (10 or 20 mM) does not cause an increase in [Ca ²⁺ ] _i in rat osteoclasts. Neomycin (10 mM) does not block the increase in [Ca ²⁺ ] _i concentration caused by subsequent addition of 25 mM extracellular Ca ²⁺ . On the other hand, pretreatment with spermine (20 mM) attenuated the response to extracellular Ca ²⁺ . Verapamil (100 μM) does not cause a detectable increase in [Ca ²⁺ ] _i but blocks the response to extracellular Ca ²⁺ .

Comparison between osteoclasts and parathyroid cells shows that the activity of such molecules against the latter is relatively ineffective in osteoclasts. This indicates that pharmaceuticals that target specific Ca ²⁺ receptors are easily developed without affecting these receptor types present in other Ca ²⁺ sensing cells. Similarly, pharmaceuticals useful with two or more such Ca ²⁺ receptors may be developed.

Screening for calcium-like and calcium antagonism at the osteoclast calcium receptor Compounds having activity at the osteoclast calcium receptor measure [Ca ²⁺ ] _i in single rat osteoclasts as described above Can be discovered. The improved assay allows for all of the compound relaxation-to-high levels. The new method is based on the use of rabbit osteoclasts and can be obtained in high yield (10 ⁵ per animal) and purity (95% of the cells are osteoclasts). Rabbit osteoclast preparation allows the measurement of [Ca ²⁺ ] _i to be performed on a solid population of cells. Because the recorded oral signal is an average solid group response, intracellular variability is minimized and the accuracy of the assay is greatly increased. This in turn allows more compounds to be screened for activity.

Rabbit osteoclasts are prepared from 6 day old rabbits. The animals are sacrificed by pinching and the long bones are removed and placed in osteoclast medium (OC medium: alpha-minimal basic medium with 5% fetal calf serum and penicillin / streptomycin). The bone is cut into sections with a scalpel and placed in 2 ml of OC medium in a 50 ml conical centrifuge tube. The bone slice is subdivided with scissors until a fine uniform suspension of bone particles is obtained. The suspension is then diluted with 25 ml of OC medium and the preparation is gently agitated (“vortex”) for 30 seconds. The bone particles are removed from the supernatant, added to a 50 ml centrifuge tube, and left for 2 minutes. Bone particles are obtained by resuspending, stirring and sedimenting in OC medium as described above. The supernatants from the two acquisitions are combined and centrifuged, and the resulting cell pellet is resuspended in Percol. The suspension is then centrifuged to remove the white viscous band just below the meniscus and washed with OC medium. The Percol centrifugation step provides a significant improvement in purity, and osteoclasts are coated at high density, suitable for measuring [Ca ²⁺ ] _i in a solid population of cells by one of the methods described below. Become so. If necessary, the purity of the preparation can be improved. In this case, the cells are cultured overnight and then rinsed with Ca ²⁺ and Mg ²⁺ free buffers. The cell monolayer is then immersed in a Ca ²⁺ and Mg ²⁺ free buffer containing 0.02% EDTA and 0.001% pronase for 5 minutes. The buffer is then removed and replaced with OC medium and the cells are recovered for 1-2 hours, after which the cells are given a fluorometric indicator and [Ca ²⁺ ] _i is measured as described below.

In one embodiment, the technique allows measurement of [Ca ²⁺ ] _i in a solid group of osteoclasts using a fluorescence microscope. Purified osteoclasts are attached to a 25 mm glass coverslip and then given India-1. Store the coverslip in the overmelt chamber and place it on the stage of the fluorescence microscope. A field of view containing 10 to 15 osteoclasts is visualized by the use of a low magnification objective (× 4). In one variation, the fluorescence of each cell in the field of view can be recorded simultaneously and stored separately for later analysis. The [Ca ²⁺ ] _i charge of each cell can be determined and the average response of all cells in the field of view can be calculated. In other variations, fluorescence from the entire field of cells can be recorded and processed immediately. In another variation, the final data is in the form of an average response from cells present in the microscopic field. This minimizes intracellular variability and greatly increases the accuracy of the assay. This method is capable of 10-20 compounds per week to screen for osteoclast calcium receptor activity.

In a more preferred embodiment, the present technology allows the measurement of [Ca ²⁺ ] _i in a solid group of osteoclasts using a conventional fluorescence analyzer. Purified osteoclasts can adhere to a rectangular glass cover slip. In one variation, a standard quartz cuvette (1 cm ² ) is used and the glass cover slip is 2 × 1.35 cm. In another variant, a micro cuvette is used (0.5 cm ² ) and the glass cover slip is 1 × 0.75 cm. In another example, the cells provide fura-2 or other suitable fluorescent indicator for measuring [Ca ²⁺ ] _i . The fluorescence of the cells of the indicator-donating cells is recorded as described above for bovine parathyroid bodies. This method allows for higher throughput than fluorescence microscopy and allows 10-50 compounds per week to assess osteoclast calcium receptor activity.

In the most preferred embodiment, the technique can be used to measure [Ca ²⁺ ] _i of osteoclasts in 96 well plates. Purified osteoclasts are placed at high density into each well of a 96 well plate and then given the appropriate fluorescent indicator. Record the fluorescence of each well using a fluorimetric plate reader. This method has the potential to be fully automated using robotics and would allow for high throughput screening that can screen 50 to 100 compounds per week.

Other Ca ²⁺ Receptor Examples The following experiments demonstrate that there are Ca ²⁺ receptor subtypes that can be differentially affected by drugs just as the molecular ligand receptor subtypes exist. To do. The parathyroid cell Ca ²⁺ receptor is sensitive at a level of about 1.5 mM extracellular Ca ^2+, while the Ca ²⁺ receptor on osteoclasts responds to a level of about 10 mM (FIG. 28). Neomycin or spermine, which activates parathyroid cell Ca ²⁺ receptor, does not affect the Ca ²⁺ receptor on C-cells or osteoclasts (FIGS. 27 and 29). These data constitute the first evidence for pharmaceutically distinguishable subtypes presence of Ca ²⁺ receptors and act selectively to these data specific type of Ca ²⁺ receptor agents Can be used for the design and development of In fact, lead molecule testing proves such cell-specific effects. For example, NPS449 is elicits increases in [Ca ^2+] _i in osteoclasts, no effect on [Ca ^2+] _i in osteoclasts. Conversely, NPS447 activates parathyroid cell Ca ²⁺ receptor, but is effective in activating osteoclast Ca ²⁺ receptor only at a 10-fold higher concentration. Finally, agatoxin 489 is not very effective at activating C cell Ca ²⁺ receptor (EC ₅₀ = 150 μM), but is very effective as an activator of parathyroid cell Ca ²⁺ receptor. Yes (EC ₅₀ = 3 μM). Lead molecules currently under development selectively affect specific types of activity of Ca ²⁺ sensitive cells in vivo.

Drugs with poor specificity are inevitably not therapeutically desirable. That is, inhibiting osteoclast activity and stimulating calcitonin secretion are two different methods for inhibiting bone resorption. These drugs targeting the Ca ²⁺ receptors of both cells are very effective for osteoporosis. Since PTH also includes regulation of bone metabolism, agents that act on parathyroid cell Ca ²⁺ receptors are also useful in the treatment and / or prevention of osteoporosis.

The results for several test molecules are shown below. Table 6 shows a comparative test of calcium-like substance molecules. Bovine parathyroid cells and C cells (rMT6-23 cells) are filled with hula-2, rat osteoclasts are filled with indo-1, and the indicated molecules for the movement of intracellular Ca ²⁺ Potency is determined by generating a cumulative concentration-response curve.

Example 11
Structure activity studies using the parathyroid Ca ²⁺ receptor lead molecules polyamines and allylalkylamines lead to the testing of molecules structurally similar to NPS456. NPS456 is an effective activator of parathyroid cell Ca ²⁺ receptor. This molecule is unique because it has only one positive charge but is more effective than many polybasic molecules. Pretreatment with PMA for a short time (2 minutes) shifts the concentration response curve of NPS456 to the right hand. This indicates that NPS456 acts through the same mechanism used by extracellular Ca ²⁺ . NPS456 induces intercellular Ca ²⁺ migration in Xenopus oocytes expressing parathyroid cell Ca ²⁺ receptor, demonstrating a direct effect on the Ca ²⁺ receptor (FIG. 41). . In addition, since it contains NPS456 chiral carbon, there are two isomeric forms. Both isomers were synthesized and examined for activity. The R-isomer, NPS447, is 12 times more effective than the S-isomer, NPS448 (Figure 34). This is the first proof that the Ca ²⁺ receptor can recognize organic molecules in a stereospecific manner.

Since NPS447 is a structurally simple molecule and exhibits a selective and effective action on the parathyroid cell Ca ²⁺ receptor, structural activity studies on this lead molecule are simple. The purpose of these studies is to form sequences of related molecules with various properties from which the final development candidates can be selected. This effort discloses some of the structural regions of NPS447 that already contribute to activity and efficacy. For example, the novel compound NPS459 is an analog of NPS447 that is small (molecular weight <240) but has potency almost equivalent to that of the parent molecule. On the other hand, some other analogs are relatively inactive. The most interesting molecules from this analog project are placed in vivo to test their effects on PTH secretion and serum Ca ²⁺ levels (see Examples 15, 16, 17, 18, 23).

The new compound NPS467 is a slightly smaller molecule than NPS447, but the former is about three times more effective than the latter in causing an increase in [Ca ²⁺ ] _i in parathyroid cells. Like NPS456, NPS467 is a racemic mixture. Resolution of NPS467 to its enantiomer provides a more potent isomer than the racemic mixture (see Example 16). Furthermore, structure activity studies on molecules related to NPS447, NPS467 and NPS568 are expected to produce pure isomers that are more effective than their racemic molecules.

NPS456 results obtained using as (Fig. 41) is shown, its is Cl at a concentration 100 [mu] M ^- induces amplitude increase in current. NPS456 is the most effective molecular activator for Xenopus oocytes expressing the parathyroid cell Ca ²⁺ receptor. The results obtained with this expression system using neomycin and NPS456 demonstrate that these molecules act directly on the Ca ²⁺ receptor.

Example 12
The strategy used to elucidate the mechanism of action of extracellular Ca ^{2+ on} osteoclast Ca ²⁺ receptor lead molecule osteoclasts is the same as demonstrating the effectiveness of parathyroid cells. The first experiment examines the effect of La ³⁺ on [Ca ²⁺ ] _i in single rat osteoclasts loaded with fluorimetric indicator India-1. As described above, trivalent cations such as La ³⁺ are temporary and block Ca ²⁺ influx. Low micromolar La ³⁺ partially inhibits extracellular Ca ²⁺ -induced increase in [Ca ²⁺ ] _i (FIG. 35). Proof of La ³⁺ anti-increase for [Ca ²⁺ ] _i provides evidence of intercellular Ca ²⁺ migration. Results from experiments similar to those obtained in parathyroid cells suggest that a similar mechanism is used by extracellular Ca ²⁺ to regulate [Ca ²⁺ ] _i in both cell types.

Another series of experiments show that permanent Mn ²⁺ induces a transient increase in [Ca ²⁺ ] _i (see FIG. 36) in the absence of extracellular Ca ²⁺ (FIG. 37). I will show you that. These results are similar to those suggesting the movement of Ca ²⁺ between cells. Mn ²⁺ can penetrate certain cells, but does not resemble such behavior in osteoclasts. This is because Mn ²⁺ stops Indian-1 fluorescence. That is, when Mn ²⁺ enters between cells, a decrease, that is, an increase in fluorescence is not observed.

The results obtained by the use of various divalent or trivalent cation, all, Ca on the surface of osteoclasts leading inflow through voltage-sensitive passage of migration and extracellular Ca ²⁺ in intracellular Ca ²⁺ Consistent with the presence of the ²⁺ receptor. The results show evidence of genetic material in human osteoclasts encoding the Ca ²⁺ receptor protein (see below). The [Ca ²⁺ ] _i transient increase due to intercellular Ca ²⁺ migration is sufficient to suppress osteoclastic bone resorption in vitro. That is, as in the case of using parathyroid cells, activation of the Ca ²⁺ receptor seems to be a variable means of suppressing osteoclast activity.

NPS449 is currently the lead molecule for the calcium-like drug of this receptor. This is a small molecule (Mw <425) that moves intercellular Ca ²⁺ in rat osteoclasts with an EC _{50 of} 200 μM (FIGS. 38 and 39). NPS449 has a relatively low potency, simple structure and only one positive charge, and appears to have the desired pharmacological and dynamic pharmacological properties.

NPS449 is tested for its ability to inhibit bone resorption in vitro. This is done by biomorphic analysis of bovine cortical bone debris pit formation using a scanning electron microscope. Rat osteoclasts are incubated for 24 hours in bone debris in the presence or absence of various concentrations of NPS449. NPS449IC ₅₀ 10 μM causes a concentration-dependent inhibition of bone resorption. The first evidence that molecules acting at this novel site can inhibit osteoclast bone resorption is an unexpected result. More effective analogs of NPS449 are obtained using synthetic chemistry and tested and analyzed by the methods described herein.

Example 13
Activation of C Cell Ca ²⁺ Receptor Lead Molecule C Cell Ca ²⁺ Receptor Stimulates the secretion of calcitonin that acts on osteoclasts and suppresses bone resorption. It is useful for the treatment of the calcium-like drug osteoporosis that selectively affects C cells.

The movement of intercellular Ca ²⁺ is used as a functional index of Ca ²⁺ receptor activity. C cell screening can be facilitated by the use of cultured cell lines that express the C cell phenotype (eg, rat medullary dry dairy cancer cells, rMTC 6-23 cells). Three alkylamine molecules are selected in early experiments. There are two natural ones (Agatoxin 489 and Agatoxin 505) and the other (NPS019) is a synthetic analogue. Agatoxin 505 was found to suppress the Ca ²⁺ -induced increase for [Ca ²⁺ ] _i with an IC ₅₀ = 3 μM. The inhibitory effect is due to the blockade of L-type voltage sensitive Ca ²⁺ passage present in such cells. In contrast, agatoxin 489 was found to migrate intercellular Ca ²⁺ in rMTC cells (EC ₅₀ = 150 μM). This is the first disclosed organic molecule that has been found to activate the C cell Ca ²⁺ receptor. The synthetic analog, NPS019, is more effective and mobilizes intercellular Ca ²⁺ (EC ₅₀ = 5 μM) (FIG. 40). It is important that the structural difference between NPS019 and agatoxin 489 is only whether or not it has a hydroxy group. This supports the development of highly effective and selective activators of the Ca ²⁺ receptor.

NPS019 is a small molecule (Mw <500), which is a lead molecule for the development of calcium-like substances in the C cell Ca ²⁺ receptor and can test its ability to stimulate calcionin in vitro. . In vivo tests then determine the ability of this molecule to stimulate calcionin secretion and inhibit bone resorption. These in vivo studies are conducted in rats. The results obtained in these studies are unexpected and provide the first evidence that small organic molecules acting on novel receptors stimulate calcionine secretion and inhibit bone resorption.

Example 14
The compound appears to be a calcium antagonist activity calcium antagonist of NPS021 for parathyroid cells, it must be blocking the action of the working or calcium antagonist compound of extracellular Ca ²⁺ to extracellular Ca ²⁺ sensitive cells. A specific example of the calcium antagonist compound is NPS021, and its structure is shown in FIGS. In bovine parathyroid cells filled with hula-2, NPS021 induced by extracellular Ca ²⁺ blocks the increase in [Ca ²⁺ ] _i . IC ₅₀ of NPS021 for this response blockade be about 200 [mu] M, at a concentration of about 500 [mu] M, an increase in [Ca ^2+] _i elicited by extracellular Ca ²⁺ is stopped. Importantly, NPS021 itself does not cause any change when tested at low [Ca ²⁺ ] (0.5 mM, FIG. 64). ³⁺ is also calcium antagonistic to Xenopus oocytes expressing the cloned Ca ²⁺ receptor: Gd ³⁺ itself is a Cl ⁻ current activated by Gd ³⁺ Will not be affected. Calcium-like molecules, however pretreatment with Gd ^3+, to block Gd ^3+.

Example 15
Reduction of serum ionized calcium of NPS 467 Compounds that have been shown to activate bovine parathyroid cell Ca ²⁺ receptors in vitro were tested for hypocalcemia activity in vivo. Male Sprague-Dury rats (200 g) were kept on a low calcium diet for one week prior to receiving test substance or vehicle as a control. Blood was collected from the tail vein 3 hours after intraperitoneal administration of NPS467. Ionized Ca ²⁺ in whole blood or serum is measured using a Ciba-Corning 634 analyzer according to the instructions attached to the device. Serum total calcium, albumin and phosphate are measured by conventional methods.

NPS467 results in a dose-dependent decrease in serum or whole blood Ca ²⁺ (FIG. 65). The decrease in blood Ca ²⁺ at this point is in parallel with the proportional decrease in blood total calcium levels. There was no change in serum albumin or phosphate levels at any of the doses examined. In a preliminary study, NPS467 at a blood Ca ²⁺ reducing effective dose results in a dose-dependent decrease in circulating levels of PTH (FIG. 66). The hypocalcemic effect of NPS467 is maximal within 3 hours and returns to control levels after 24 hours (FIG. 67).

NPS467 (Example 16) is also effective in reducing serum ionized Ca ^{2+ in} rats maintained on normal, calcium-repeated diets. A single dose of NPS467 (10 mg / kg ip) results in a rapid decrease in serum levels of ionized Ca ²⁺ . Such a level is maximal at 1 hour (22% reduction from the control level) and is suppressed near this level until 6 hours.

Example 16
Reduction of serum ionized calcium by stereospecific methods of NPS467 NPS467 is a racemic mixture. Resolution of NPS467 into its two enantiomers was performed by resolution on a chiral column. The R-isomer is about 100 times more effective than the S-isomer, as assessed by the ability of the enantiomer to induce an increase in [Ca ²⁺ ] _{i in} parathyroid cells (FIG. 68). Activates bovine parathyroid cell Ca ²⁺ receptor in vitro. Similarly, a similar resolution of the novel compound NPS568 into its enantiomers is that the R-isomer is approximately 40 times more effective than the S-isomer and results in intercellular Ca ²⁺ migration in bovine parathyroid cells. (See Table 6 above).

NPS467 isomers were tested for effects on serum Ca ²⁺ as in Example 15. The R-isomer of NPS 467 proved to be more effective than the S-isomer in reducing serum Ca ²⁺ in vivo or NPS 467 is consistent with the in vitro results (FIG. 69). Each compound is tested at a concentration of 5 mg / kg body weight).

Example 17
Serum ionized calcium reduction in the NPS467 secondary hyperparathyroidism in vivo model is widely accepted and used as an animal model for secondary hyperparathyroidism resulting from chronic renal failure. 6 Nephrectomy rats. When animals undergoing such surgery are first hypocalcemic to maintain serum Ca ²⁺ levels, compensatory hyperplasia of the parathyroid bodies and increased levels of circulating PTH occur. Male Stubrag-Dury rats (250 g) are 5/6 nephrectomized and allowed to recover for 2 weeks. At this point, it is standard calcemia (due to elevated levels of serum PTH). Administration of NPS R-467 (10 mg / kg ip) caused a rapid (within 2 hours) reduction of serum ionized Ca ²⁺ levels to 83% of controls in an animal model of secondary hyperparathyroidism. Bring. This suggests that: This type of compound effectively suppresses PTH secretion in patients with secondary hyperparathyroidism and proliferative parathyroids.

Example 18
In order to determine the primary target tissue on which NPS467 acts to cause a hypocalcemic response that does not reduce serum ionized calcium concentration in NPS467 deparatomized animals, parathyroid bodies in rats are removed surgically. Animals from which the entire parathyroid body has been excised are hypocalcemic and are highly dependent on calcium diet to maintain a serum Ca ²⁺ homeostasis state. Parathyroid depleted animals have a serum ionized Ca ²⁺ level of 0.92 mM after 6 hours of fasting, which gradually decreases to 0.76 mM. Administration of a single dose of NPS R-467 (10 mg / kg ip) did not give any change in serum ionized Ca ²⁺ levels over a 6 hour period. These results demonstrate that intact parathyroid bodies are necessary for the hypocalcemic effect of NPS R-467. The data additionally demonstrates that NPS R-467 can target parathyroid bodies in vivo. The results are consistent with the following view: NPS R-467 acts on parathyroid cell Ca ²⁺ receptors in vivo to suppress the secretion of PTH, thereby reducing the serum level of ionized Ca ²⁺ .

Example 19
Increased intercellular calcium in human parathyroid bodies of NPS467 Dissociated parathyroid cells are prepared from parathyroid adenomas obtained surgically from patients with primary parathyroid hyperplasia. Cells were filled with hula-2 and [Ca ²⁺ ] _i was measured as described above. Both NPS R-467 and NPS R-568 result in a concentration-dependent increase in [Ca ²⁺ ] _i . NPS R-467 and NPS R-568 have EC ₅₀ of 20 and 3 μM, respectively. Both of these compounds can increase [Ca ²⁺ ] _i in pathological human tissues and appear to decrease serum levels of PTH and Ca ^{2+ in} patients with primary hyperparathyroidism.

Example 20
Mechanism of action of NPS467 at the parathyroid cell calcium receptor The dissociated bovine parathyroid action was used to further investigate the mechanism of action of NPS467 at the receptor level. In the presence of 0.5 mM extracellular Ca ²⁺ , NPS R-467 results in a rapid and primary [Ca ²⁺ ] _i increase. This increase is maintained in the presence of 1 μM La ³⁺ and is partially suppressed by pretreatment with PMA (100 nM × 2 min). All these results are consistent with the action of NPS R-467 on the Ca ²⁺ receptor. However, the response of cytosolic Ca ²⁺ to NPS R-467 is stopped when parathyroid cells are suspended in a Ca ²⁺ -free buffer. This suggests that NPS R-467 cannot itself cause intercellular Ca ²⁺ mobilization. This suggests that dominance of a partial Ca ²⁺ binding site is necessary for NPS R-467 to elicit a response. To test this hypothesis, parathyroid cells are suspended in a Ca ²⁺ -free buffer and exposed to submaximal concentrations of neomycin. Neomycin is used for the following reasons: This is almost in all respects similar to the effect of extracellular Ca ^{2+ on} parathyroid cells and Xenopus oocytes (expressing parathyroid Ca ²⁺ receptors). Because. The addition of 10 μM neomycin by itself does not cause an increase in [Ca ²⁺ ] _i under these conditions. However, NPS subsequent addition of R-467 (30μM) to induce primary increase in [Ca ^2+] _i, but this is because the extracellular Ca ²⁺ is absent, the movable intercellular Ca ²⁺ Must arise from When cells immersed in Ca ^{2+ -free} buffer are exposed to 30 μM NPS R-467, there is no increase in [Ca ²⁺ ] _i . The concentration of NPS R-467 is maximally effective in increasing [Ca ²⁺ ] _{i in} the presence of 0.5 mM extracellular Ca ²⁺ . However, subsequent addition of 10 μM neomycin induces a primary [Ca ²⁺ ] _i increase. Presumably, neomycin binds to the same site as extracellular Ca ^{2+ and} is therefore functionally substituted. A submaximal concentration (which itself does not cause a response) is used to achieve partial Ca ²⁺ binding site control and allow activation of Ca ²⁺ receptors by NPS R-467.

Additional work will be done to additionally define the mechanism of action of NPS R-467. Cells are once again suspended in calcium-free buffer to ensure that any observed increase in [Ca ²⁺ ] _i is due to mobilization of intercellular Ca ²⁺ . In these experiments, however, a maximum effective concentration of 100 μM neomycin was used. In the absence of extracellular Ca ²⁺ , 100 μM neomycin induces a rapid and primary [Ca ²⁺ ] _i increase. Subsequent addition of 30 μM NPS R-467 does not cause an increase in [Ca ²⁺ ] _i . In the reverse experiment, 30 μM NPS R-467 is added before 100 μM neomycin. As expected, NPS R-467 does not cause any increase in [Ca ²⁺ ] _i . However, it does not affect the increase in [Ca ²⁺ ] _i induced by subsequent addition of 100 μM neomycin. These results obtained with the maximum effective concentration of NPS R-467 suggest that these two compounds do not act at the same site. Rather, the results assume sites on two separate Ca ²⁺ receptors, namely those that bind extracellular Ca ²⁺ and neomycin and NPS R-467 and structure related compounds (such as NPS R-568). Explain enough. A ligand that binds to the former site can lead to full activation of the Ca ²⁺ receptor, whereas a ligand that binds to the latter site dominates to some extent, but not limited, the extracellular Ca ²⁺ binding site. May occur only when it is done and / or may be functionally appropriate. Ligands that bind to the extracellular Ca ²⁺ binding site can be exposed to the previously blocked NPS R-467 binding site. The NPS R-467 binding site appears to be an allosteric site that increases receptor activation in response to ligand binding at the extracellular Ca ²⁺ binding site.

The data demonstrate that the parathyroid cell Ca ²⁺ receptor has an organic ligand at at least two distinct sites. One site binds a physiological ligand, extracellular Ca ²⁺ , and certain organic polycations such as neomycin. Binding at this site results in complete Ca ²⁺ receptor activation, an increase in [Ca ²⁺ ] _i and suppression of PTH secretion. NPS R-467 and NPS R-568 define binding sites on the Ca ²⁺ receptor that are not previously recognized. Binding to this site occurs only when the extracellular Ca ²⁺ binding site is partially dominated and / or results in full activation of the Ca ²⁺ receptor. Ligands acting at either site are effective in suppressing serum Ca ²⁺ levels in vivo.

Calcium-like compounds that activate bovine parathyroid cell calcium receptors, such as allosteric sites of parathyroid calcium receptors, NPS R-467 and NPS R-568, are intracellular in the absence of extracellular Ca2 +. It does not cause Ca ²⁺ migration. Rather, they increase the sensitivity of the Ca ²⁺ receptor by extracellular Ca ²⁺ and thus cause a shift to the left of the concentration response curve for extracellular Ca ²⁺ . Because of this, they are unlikely to work at the same site on the receptor where extracellular Ca ²⁺ works. In contrast, organic and inorganic polycations cause intracellular Ca ²⁺ migration in the absence of extracellular Ca ²⁺ , and thus probably work at the same site as extracellular Ca ²⁺ . Compounds such as NPS R-568 work in an allosteric manner and their activity may depend on some minimal extracellular Ca ²⁺ concentration. This suggests that partial occupancy of the extracellular Ca ^{2+ -binding} site of the receptor is necessary for compounds such as NPS R-568 to be effective. This model is consistent with the observations described in Example 20.

Other details of the mechanism of NPS R-568 activity at the parathyroid Ca ²⁺ receptor, however, assess the specific binding of NPS R-568 (eg using ³ H). Has been investigated more accurately. There are several molecular mechanisms that can explain NPS R-568 activity on parathyroid cell Ca ²⁺ receptors. In one mechanism (Model 1), NPS R-568, when occupied, can bind to the Ca ²⁺ receptor where it is not sufficient to functionally activate the receptor. Activation occurs only when a certain level of occupancy of the extracellular Ca ^{2+ -binding} site (s) is achieved. In another mechanism (model 2), occupation of the extracellular Ca ²⁺ -binding site may expose potential binding sites for compounds such as NPS R-568. The occupation of this potential site by NPS R-568 in turn increases the affinity and / or binding effects of the extracellular Ca ²⁺ site. Both mechanisms involve a form of allosteric activation of the Ca ²⁺ receptor by compounds such as NPS R-568. These are not the only mechanisms that can explain the effects of compounds like NPS R-568 on the Ca ²⁺ receptor on parathyroid cells. Other mechanisms of action can be suggested by the binding studies described below.

To further study the mechanism of action of compounds such as NPS R-568 at the parathyroid cell Ca ²⁺ receptor, studies with ³ H-NPS R-568 can be conducted. Specific binding of ³ H-NPS R-568 to intact parathyroid cells or membranes prepared from parathyroid cells is first studied by techniques known in the art. In pair, kinetic parameters of binding are measured as a function of extracellular Ca ²⁺ concentration. In particular, Scatchard analysis of the data will reveal the number of binding sites and the apparent affinity of the receptor site for ³ H-NPS R-568. These parameters are then determined as a function of changes in the level of extracellular Ca ²⁺ in the buffer used for the measurement. If mechanism 1 is correct, then specific binding will occur at a significant level in the absence of extracellular Ca ²⁺ . Large changes in binding kinetic parameters as a function of extracellular Ca ²⁺ levels would support mechanism 2. It is anticipated that various other inorganic and organic polycations described in other examples above will cause changes in ³ H-NPS R-568 binding parameters similar to extracellular Ca ^2+. Is done. This supports the view that these polycations act at a different extracellular Ca ^{2+ -binding} site than the site to which compounds such as NPS R-568 bind.

Example 21
Preparation of NPS467 In a 250 ml round bottom flask, 10.0 g (100 mmol) of 3'-methoxyacetophenone and 13.5 g (100 mmol) of 3-phenyl-propylamine were mixed and titanium (IV) isopropoxide 125 Treat with millimoles (35.5 g). The reaction mixture is stirred for 30 minutes at room temperature under a nitrogen atmosphere. Thereafter, 6.3 g (100 mmol) of sodium cyanoporohydrin in 100 ml of ethanol is added dropwise over 2 minutes. The reaction is stirred at room temperature under nitrogen for 16 hours. The reaction mixture is then transferred to a 2 liter separatory funnel containing 1.5 liters of ethyl ether and 0.5 liters of water. Allow the phases to equilibrate and remove the ether phase. The remaining aqueous phase is thoroughly extracted by treating 4 times with 1 liter of ether. Combine the washings and dry over anhydrous potassium carbonate to obtain a clear, light amber oil.

  TLC analysis of this material (on silica, using chloroform-methanol-isopropylamine = 100: 5: 1) showed the product of Rf 0.65 and Rf 0.99 (3′-methoxy acetophenone) and Rf 0.0 ( Two starting materials with traces of (3-phenylpropylamine) are shown.

  The reaction mixture was purified by chromatography on silica (48 × 4.6 cm) using a gradient elution of chloroform-methanol-isopropylamine (99: 1: 0.1-90: 10: 0.1). NPS 467 (13.66 g) is obtained. This material is dissolved in hexane-isopropanol (99: 1) containing 0.1% diethylamine to give a solution with a concentration of 50 mg / ml. Chiral resolution was chromatographed on 4 ml of this solution (200 mg, maximum to achieve separation) using Chiral Cell OD (25 × 2 cm) using 0.7% isopropylamine and 0.07% diethylamine in hexane. At 100 ml / min while monitoring the optical density at 260 nm. Under these conditions (100 mg injection of material), the earlier eluting isomer (NPS467R) begins to emerge from the column at -26 minutes and the later eluting isomer (NPS467S) begins to emerge at -34 minutes. Baseline decomposition is achieved at these conditions. Each optical isomer (free base) is converted to the corresponding hydrochloride salt by dissolving 3 g of the free base in 100 ml of ethanol and treating with 100 ml of 10 molar equivalents of HCl in water. A white solid is obtained by lyophilization of this solution.

NPS568
NPS568 is a structural analog of NPS467 that is a more effective cause of the increase in [Ca ²⁺ ] _i in bovine and human parathyroid cells. Like NPS467, the effect of NPS568 is stereospecific and the more effective enantiomer is the R-isomer (see Table 6, supra). NPS R-568 is a lead calcium-like compound that currently has selective activity on the parathyroid Ca2 ⁺ receptor. It behaves similarly to NPS R-467 described in the above example, even with great effectiveness. Thus, NPS R-568 induces an increase in [Ca ²⁺ ] _i in bovine parathyroid cells in a stereospecific manner (see Table 6, supra). NPS R-568 is not an induced increase in [Ca ^2+] _i in the absence of extracellular Ca ^2+, it enhances the response of the extracellular Ca ^2+. NPS R-568 shifts the concentration response curve for extracellular Ca ²⁺ to the left.

Oral administration of NPS R-568 to rats causes a dose-dependent decrease in serum Ca ²⁺ concentration (ED ₅₀ = 7 mg / kg). The hypocalcium response elicited by oral administration of NPS R-568 is rapid at onset and parallels the decrease in serum PTH levels. The low calcium response elicited by NPS R-568 is minimally affected only by prior complete nephrectomy. However, NPS R-568 does not induce a hypocalcium response in parathyroidectomized rats. NPS R-568 therefore selectively targets the in vivo parathyroid cell Ca ²⁺ receptor and is responsible for inhibiting PTH secretion. Decrease in serum PTH levels and the resulting hypocalcosis is a desirable therapeutic effect in the case of hyperparathyroidism. The synthesis of NPS R-568 is shown below.

Example 22
Production of NPS 568 NPS 568 is produced by the method described in Example 21. However, instead of 3-phenylpropylamine, an equivalent amount of 3- (2-chlorophenyl) propylamine is used. Stirring a mixture of 3'-methoxy acetophenone, 3- (2-chlorophenyl) propylamine and titanium (IV) isopropoxide for 5 hours and then treating with NaCNBH ₃ / EtOH results in significantly higher yields Was found (98%).

Example 23
Rats (male, Sprague Dawley, 250-300 g) whose serum ionized calcium is reduced by NPS467 when administered orally are fasted overnight prior to the experiment on a standard rat diet. NPS467 is suspended in corn oil and administered as a single oral dose using a nutritional needle. After 3 hours, a blood sample is taken from the tail vein and the ionized Ca ²⁺ level is measured. FIG. 71 shows NPS467 causing a dose-dependent decrease in serum levels of ionized Ca ²⁺ when administered orally.

Example 24
BOPCAR1 cloning method 1
Xenopus laevis (Xenopus laevis) oocytes were used to identify bovine parathyroid calcium receptor-encoding cDNA. Although these techniques are only briefly described herein, this complete and detailed method is in the section describing the techniques preceding this section, which can be used for other cell types Ca 2 ⁺ -. Additional forms of the receptor can be used to clone. Poly (A ⁺ ) -rich RNA was first prepared from bovine epithelial bodies by extraction with guanidinium thiocyanate, centrifugation through CsCl, and oligo (dT) cellulose chromatography. Injection of the resulting poly (A ⁺ ) -rich RNA into the oocyte (25-50 ng / oocyte) was performed using Ca ²⁺ and trivalent cations (1-100 μM) as described herein. It provides sensitivity to increased extracellular concentrations of Gd ³⁺ , and two cations induce a calcium activated chlorine current. Such currents were not induced in control eggs injected with water. Xenopus laevis is the collected oocyte: (i) high level of maturation (ie, stages V, VI); (ii) high activity of Cl ⁻ currents activated by Ca ²⁺ ionophores such as A23187; iii) Basically, when injected with 25 ng / oocyte of total poly (A) ⁺ RNA isolated from bovine parathyroid bodies, it shows a high level of functional expression of Gd ³⁺ -induced Cl ⁻ current To select. The mRNA is then size-fractionated using preliminary continuous flow agarose gel electrophoresis (Hediger M.A., Analytical Biochemistry, 159: 280-286 (1986)), and the Ca ²⁺ -receptor A fraction further enriched in poly (A ⁺ ) -RNA is obtained in the transcript encoding Peaks of activity, such as oocytes injected with mRNA from these fractions, show enhanced expression of Gd ³⁺ -activated Cl ⁻ currents obtained from a size range of 4-5.5 kb. This RNA was used to produce a size-selected, directed cDNA library in plasmid pSPORT1, which is abundant in full length transcripts. Sense complementary RNA (cRNA) is then synthesized from DNA inserts collected from separate 350-500 clones from this library and injected into oocytes. Gd ³⁺ - activated Cl ^- currents were observed after injection of a single filter derived from RNA containing 350 colonies. Production and injection of CRNA from a serially reduced pool of clones is a single with a 5.3 kb cDNA insert that exhibits highly enhanced Ca ²⁺ -receptor activity after injection of cRNA into oocytes. A clone (BoPCaR 1) was isolated. A plasmid containing the BoPCaR 1cRNA (plasmid, FIG. 72; restriction map, FIG. 73; and partial nucleotide sequence, see FIG. 74) has been deposited with ATCC under accession number ATCC75416.

BoPCaR 1 cDNA is neomycin-induced Cl in Xenopus oocytes ^- is out of the range of sizes of the fractionated RNA - size are known to exhibit a channel activity. This is consistent with the possibility that different isoforms or multiple genes mask many other Ca ²⁺ receptor genes.

Several pharmacological and biochemical criteria were used to identify clones encoding authentic bovine parathyroid Ca2 ⁺ -receptors. Oocytes expressing this cloned receptor, not water-injected oocytes, increased poly (A ⁺ ) with an increase in extracellular Ca ²⁺ (1.5-5 mM) or Gd ³⁺ (20-600 μM). ) —Reacted with an increase in Cl ⁻ (at least 1.8 microamperes) several times greater than that observed in injected oocytes. These responses increase markedly over one (1) to four (4) days after injection of cRNA prepared from BoPCaR 1 cDNA into eggs. Furthermore, the range of concentrations of the two cations that elicit this response is known to be very similar to the effect of Ca ²⁺ on parathyroid cells and is caused by Ca ²⁺ and Gd ³⁺ . provoking Cl ^- Cl in current changes substantially the same oocyte ^- causes a change in current, which occurs over the same concentration range as the antibiotic modulates vitro parathyroid function, neomycin It is very similar to the concentration range (20-100 μM) already shown to act on bovine parathyroid cells in vitro. Finally, the in vitro RNA translation made from the clone is a single protein with a molecular weight of about 100 kd on a polyacrylamide gel, and this synthesis is 10-15% of the apparent molecular weight with the addition of canine pancreatic microsomes. The increase was accompanied by an increase. The latter indicates that the cloned receptor interacts strongly with the required membrane protein receptor and the expected membrane and is glycosylated in its native form. Studies of lectin concanavalin A show that the Ca ²⁺ receptor resembles glycoproteins. Thus, the pharmacological properties of the cloned receptor expressed at high levels in oocytes and the biochemical studies performed to date have shown that its bovine parathyroid Ca ²⁺ kinetics (Ca in oocytes). ²⁺ activated Cl ^- not at all consistent with the nature of such) as is illustrated by the change in current.

Use in diagnosis NPS R-568 or other calcium receptor active compounds can be used as a diagnostic tool. In particular, pharmacological preparations of such compounds are useful as diagnostic tools. As one example, a pharmacological formulation comprising a parathyroid calcium-like compound such as NPS R-568 can be administered orally or by other routes of administration to hypercalcemic patients who exhibit symptoms of psychological depression. If these symptoms arise from a potential hyperparathyroidism such as hyperparathyroidism, administration of NPS R-568 or a similarly acting compound will alleviate these symptoms. If symptoms are not relieved, psychosuppression is due to some pathological condition other than hyperparathyroidism. Thus, parathyroid cell calcium-like compounds can be used for differential diagnosis of mental depression.

Symptoms and signs common to hyperparathyroidism and other diseases can also be differentially diagnosed using the methods described above. Such common signs and symptoms include, but are not limited to, high blood pressure, muscle weakness and general discomfort. Alleviation of these symptoms after treatment with parathyroid cell Ca ²⁺ receptor calcium-like compound indicates that the problem arises from potential hyperparathyroidism.

As another example, compounds that act as antagonists (calcium antagonists) at the C-cell Ca ²⁺ receptor can be administered as described above for the diagnosis of thyroid cancer of the bone marrow. In this case, administration of the C-cell Ca ²⁺ receptor calcium antagonist compound will reduce serum levels of calcitonin that can be readily measured by radioimmunoassay. Certain symptoms, such as diarrhea associated with bone marrow thyroid cancer, can be observed and diagnosed as to whether they alleviate or decrease after administration of calcium antagonist compounds.

As a third example, a compound that acts as a calcium antagonist in the glomerular adjacent cell Ca ²⁺ receptor can be used for differential diagnosis of hypertension. In this case, administration of the glomerular adjacent cell Ca ²⁺ receptor calcium-like compound is administered as described above. If hypertension is largely or completely due to increased levels of renin rather than another pathological condition, a decrease in blood pressure to normal levels occurs.

As another example, compounds that act calcium-like specifically at the osteoclast Ca ²⁺ receptor can be used for differential diagnosis of high- or low-level turnovers that form osteoporosis. In this case, such compounds can be administered in suitable pharmacological preparations, serum alkaline phosphatase, osteocalcin, pyridinoline and / or deoxypyridinoline crosslinks and / or other predictions of bone resorption and / or formation The level of the target marker is measured by methods known in the art. A significant decrease in one or more of these parameters is predictive of high turnover osteoporosis, while a slight decrease or no decrease is predictive of low-turnover osteoporosis. Such information is necessary for a suitable procedure. Anti-resorptive agents are not the preferred monotherapy for low-turnover osteoporosis.

Although these examples are not complete, specific Ca ²⁺ receptors are directed to pharmacological formulations, and the observed effects of such formulations on physical function and / or chemical composition can be used in diagnosis Useful as an explanation. In general, the calcium-like and calcium antagonist compounds acting at the Ca ²⁺ receptors of various cells described above can be used for diagnosis of various diseases associated with individual cell types. These diseases include bone and mineral-related diseases (as described in Koe et al., Disorders of Bone and Mineral Metabolism, Raven Publishing, 1990), kidney disease, endocrine disease, cancer, cardiovascular disease and pregnancy. Including but not limited to related diseases. Human diseases or disorders for which such molecules are therapeutically effective are as follows: (1) Calcium-like substances are expected to ameliorate psoriasis by reducing abnormal skin cell proliferation. (2) Since Ca ²⁺ inhibits the vasopressin effect on MTAL and cortex-recovered cells, calcium-like substances are expected to reduce water retention in vasopressin-excess conditions such as inappropriate vasopressin (ADH) secretion syndrome. Is done. Conversely, calcium receptor antagonists used in the ADH deficient state are expected to enhance the activity of any ADH present as in partially central diabetic insipidis. (3) Calcium-like substances are used to treat hypertension by (a) suppressing renin secretion, (b) stimulating the production of vasodilators for vascular smooth muscle such as PTHrP (PTH-related peptide). obtain. (4) Calcium-like substances are expected to increase platelet aggregation ability, which is useful when the platelet count is low. Conversely, calcium antagonists inhibit platelet function when they are highly aggregating. (5) Calcium promotes colon and breast cell differentiation. Calcium-like substances reduce the risk of colon or breast cancer. (6) Calcium promotes urinary calcium excretion in MTAL. Calcium-like substances are expected to have low calcium activity useful in the treatment of hypercalcemia diseases. The inhibitory effect of calcium-like substances in osteoclasts and the stimulation of low calcium peptide calcitonin secretion are expected to be useful in the treatment of hypercalcemia and its symptoms. Calcium-like substances also ameliorate low calcium symptoms by activating calcium receptors. Conversely, calcium antagonists are expected to reduce urinary calcium excretion and are useful in the treatment of kidney stones. In addition, calcium inhibits the synthesis of 1,25-dihydroxyvitamin D in the proximal tubules, and this vitamin D metabolite is often over-accumulated in kidney stone patients and contributes to hypercalciuria. Inhibition of 1,25-dihydroxyvitamin D formation by calcium-like substances is expected to be useful in the treatment of renal calcium stone disease. (7) Endogenous amines can reproduce the symptoms of uremic patients by calcium-like or cytolytic activity. Calcium-like and / or calcium antagonists are expected to ameliorate these symptoms. (8) Certain nephrotoxicities of aminoglycoside antibiotics can be mediated by the interaction of these drugs with renal calcium receptors. With calcium receptors, it is expected that it will be possible to easily screen for minimal nephrotoxicity when designing a class of drugs. In addition, renal calcium receptor antagonists, when associated with this mechanism, prevent or treat this nephrotoxicity. (9) Some of the genetic factors of calcium-related diseases, such as osteoporosis, kidney stones and hypertension, are expected to be related to certain forms of genetic problems in the receptor. These are currently being studied, and genetic screening / testing using receptor-based reagents can be performed. Human disease, family-specific hypocalciuria-like hypercalcemia may be due to calcium receptor defects. Diagnostic separation from eventual primary hyperparathyroidism can be performed using receptor-based techniques. (10) It is expected that calcium receptors are present in the placenta, affect placental function diseases, and transport nutrients to the growing fetus.

  Other embodiments are within the scope of the following claims.

FIG. 1 depicts representative molecules useful in the present invention. FIG. 2 depicts representative molecules useful in the present invention. FIG. 3 depicts representative molecules useful in the present invention. FIG. 4 depicts representative molecules useful in the present invention. FIG. 5 depicts representative molecules useful in the present invention. FIG. 6 depicts representative molecules useful in the present invention. FIG. 7 is a graphical representation showing the increase in [Ca ²⁺ ] _i induced by extracellular Ca ^{2+ in} bovine parathyroid cells given kin-2- or fura-2-. The starting [Ca ²⁺ ] was 0.5 mM (using CaCl ₂ ) and increased with an increase of 0.5 mM in each of the arrows. FIG. 8 is a graphical representation showing the migration of [Ca ²⁺ ] _{i in} bovine epithelial somatic cells. The starting [Ca ²⁺ ] was 0.5 mM and was reduced to <1 μM by the addition of EGTA as indicated. (A) Extracellular Mg ²⁺ (8 mM, final) causes an increase in [Ca ²⁺ ] _i in the absence of extracellular Ca ²⁺ . (B) Pretreatment with inomycin (1 μM) blocks reaction to Mg ²⁺ . (C) Pretreatment with 5 μM molecule 1799 (mitochondrial uncoupler) has no effect on the reaction to Mg ²⁺ . FIG. 9 shows the selective blocking effect of low concentrations of Gd ^{3+ on} the steady state increase of [Ca ²⁺ ] _i , and that high temperature Gd ³⁺ causes a temporary increase of [Ca ²⁺ ] _i. It is a graph expression which shows. Upper panel: control. The starting concentration of extracellular Ca ²⁺ was 0.5 mM, increasing 0.5 mM for each arrowhead. Middle panel: Gd ³⁺ (5 μM) blocks in steady state but does not block the one hour increase in [Ca ²⁺ ] _i caused by extracellular Ca ²⁺ . Lower panel: Gd ³⁺ (50 μM) elicits a transient increase in [Ca ²⁺ ] _i , ending the transient and sustained response to extracellular Ca ²⁺ . In the middle and lower panels, just enough EGTA was added to selectively chelate Gd ³⁺ . Except for the block of Ca ²⁺ influx, [Ca ²⁺ ] _i rises rapidly. FIG. 10 is a graphical representation showing that the effect of PMA on [Ca ²⁺ ] _i , IP ₃ formation and PTH secretion is blocked by increasing the concentration of extracellular Ca ²⁺ . For each variable, there is a shift to the right in the concentration-response curve for extracellular Ca ²⁺ . It should also be noted that the concentration-response curve changes to an S shape as [Ca ²⁺ ] increases linearly. FIG. 11 is a graphical representation showing that the increase in [Ca ²⁺ ] _i induced by spermine in bovine parathyroid cells is gradually attenuated by increasing [Ca ²⁺ ]. Spermine (200 μM) was added at the time indicated by the arrowhead shape. In this and the following drawings, the number attached to the figure is [Ca ²⁺ ] _i in nM. FIG. 12 is a graphical representation showing that spermine migrates intracellular Ca ²⁺ in bovine parathyroid cells. As shown (left graphic), [Ca ²⁺ ] was reduced to <1 μM by adding EGTA prior to the addition of spermine (200 μM). Pretreatment with ionomycin (1 μM) blocks the reaction to spermine (right figure). FIG. 13 is a graphical representation showing that spermine increases [Ca ²⁺ ] _i and inhibits PTH secretion in bovine parathyroid cells, similar to extracellular Ca ²⁺ . The data point for the spermine dose-concentration response curve is the average of two experiments. FIG. 14 is a graphical representation showing that spermine increases [Ca ²⁺ ] _i as well as extracellular Ca ²⁺ and inhibits PTH secretion in bovine parathyroid cells. The data point for the spermine dose-concentration response curve is the average of two experiments. FIG. 15 is a graphical representation showing the contrasting effect of PMA on the response to extracellular Ca ²⁺ and on the response of small epithelial cells to ATPγS. Left panel: The concentration-response curve for extracellular Ca ²⁺ -induced inhibition of cyclic AMP formation is shifted to the right by PMA (100 nM). Middle panel: PMA does not affect the ability of ATPγS to increase [Ca ²⁺ ] _i . Note also that the concentration-response curve for ATPγS shows a classical sigmoidal behavior as a function of log concentration, in contrast to extracellular divalent cations. FIG. 16 is a graphical representation showing the movement of intracellular Ca ²⁺ in human parathyroid cells drawn by extracellular Mg ²⁺ . Cells were obtained from adenomas and placed in a buffer containing 0.5 mM extracellular Ca ²⁺ . (A) Temporary and sustained increase in [Ca ²⁺ ] _i induced by extracellular Mg ²⁺ (10 mM, final) is not affected by nimodipine (1 μM), but La ³⁺ (1 μM) Decreased, indicating that La ³⁺ can be rapidly restored by selective chelation with low concentrations of EGTA. (B) La ³⁺ (1 μM) blocks the sustained increase but does not block the temporary increase in [Ca ²⁺ ] _i caused by extracellular Mg ²⁺ . (C) Cytosolic Ca ²⁺ transients induced by extracellular Mg ²⁺ persist in the absence of extracellular Ca ²⁺ . FIG. 17 is a graphical representation showing the movement of intracellular Ca ²⁺ drawn by neomycin or protamine in bovine parathyroid cells. In all figures, the starting [Ca ²⁺ ] and [Mg ²⁺ ] were 0.5 and 1 mM, respectively. In diagrams (a) and (b), the Ca ²⁺ and Mg ²⁺ concentrations increased from 0.5 and 1 mM to 2 and 8 mM, respectively. In other figures, (c) through (i) were added to indicate neomycin B (30 μM) or protamine (1 ug / ml). La ³⁺ (1 μM), EGTA (1 mM) or iomycin (100 nM) was added as indicated. Each graphic is representative of the pattern seen in 5 or more tests using at least 3 different cell preparations. Bar = 1 minute. FIG. 18 is a graphical representation showing that neomycin B blocks transiently in bovine parathyroid cells but does not block the steady state increase of [Ca ²⁺ ] _i caused by extracellular Ca ²⁺ . Left control: [Ca ²⁺ ] started at 0.5 mM and increased in 0.5 mM increments with each arrowhead used before addition of neomycin B (30 μM). Right: Neomycin B (30 μM) was added before [Ca ²⁺ ]. Bar = 1 minute. FIG. 19 is a graphical representation showing that neomycin B or protamine inhibits PTH secretion at concentrations that induce an increase in [Ca ²⁺ ] _i . Cells were incubated for 30 minutes with the indicated concentrations of organic polycations. Hollow mark: the control responds to PTH secretion in the presence of 0.5 (circle) or 2 mM (diamond) extracellular Ca ²⁺ . The value of [Ca ²⁺ ] _i is a diamond mark. Bovine cells were used for experiments with protamine, and human (adenoma) parathyroid cells were used for experiments with neomycin B. Each point is the mean ± SEM of 3 experiments. FIG. 20 is a graphical representation showing the selective inhibitory effect of PMA on cytosolic Ca ²⁺ transients induced by spermine in bovine parathyroid cells. The starting [Ca ²⁺ ] was 0.5 mM: Spermine (200 μM) or ATP (50 μM) was added as indicated. Bar = 1 minute. FIG. 21 is a graphical representation showing that PMA shifts the concentration-response curve to the right for extracellular Ca 2 ⁺ -and neomycin B-induced increases in [Ca ²⁺ ] _i in bovine parathyroid cells. Cells were pretreated with PMA for 1 min before [Ca ²⁺ ] increase or before adding neomycin B as indicated. Each point is the mean ± SEM of 3-5 experiments. FIG. 22 is a graphical representation showing that PMA shifts the concentration-response curve to the right for extracellular Ca 2 ⁺ -and spermine-induced inhibition of PTH secretion. Cells were incubated with [Ca ²⁺ ] and spermine indicated for 30 minutes in the presence (black circles) or absence (hollow circles) in the presence of 100 nM PMA. Each point is the mean ± SEM of 3 experiments. FIG. 23 is a graphical representation showing that protamine amplifies the formation of inositol phosphates. Parathyroid cells were incubated overnight in culture medium containing ⁴ uCi / ml ³ H-myo-inositol, washed and incubated at 37 ° C. with the indicated concentration of protamine. After 30 seconds, the reaction was stopped by the addition of CHCl ₃ : MeOH: HCl and IP ₁ (circle) and IP ₃ (triangle) were separated by anion exchange chromatography. FIG. 24 is a graphical representation showing that PMA reduces the formation of IP ₁ drawn by extracellular Ca ²⁺ or spermine in bovine parathyroid cells. ³ H-myo-inositol-labeled cells were exposed to [Ca ²⁺ ] or spermine as indicated for 30 seconds before stopping the reaction and separating IP ₁ by anion exchange chromatography. Hatched column: Cells were pretreated with PMA (100 nM) for 5 min before [Ca ²⁺ ] increase or spermine addition. Each value is the average of two experiments performed in triplicate. FIG. 25 is a graphical representation showing the transient and sustained increase in [Ca ²⁺ ] _i induced by neomycin B in human (adenoma) parathyroid cells. [Ca ²⁺ ] was 0.5 mM. (A) The sustained increase in [Ca ²⁺ ] _i induced by neomycin B (10 μM) was reduced by La ³⁺ . (B) The temporary increase in [Ca ²⁺ ] _i elicited by neomycin B was not affected by La ³⁺ . (C) The temporary increase in [Ca ²⁺ ] _i persisted in the absence of extracellular Ca ²⁺ . FIG. 26 is a graphical representation showing that neomycin B induces vibrationally amplified Cl ⁻ currents in Xenopus cells expressing the Ca ²⁺ receptor. Top view from oocyte, 3 days after injection of human (proliferative) parathyroid cell poly (A) ⁺ -mRNA. Bottom shape from oocyte, water injection. Neomycin B did not elicit responses in 5 water-injected oocytes, and carbachol elicited responses as indicated. In both figures, the holding potential was -76 mV. FIG. 27 is a graphical representation showing that neomycin B does not affect basal or attraction increase in C-cells. Control, left figure: Fuller rMTC6-23 cells gave 2-, before increasing to 3mM the [Ca ^2+], were first immersed in buffer containing 1mMCa ^2+. Right figure: Pretreatment with 5 mM neomycin B. FIG. 28 is a graphical representation showing that extracellular Ca ²⁺ induces an increase in [Ca ²⁺ ] _{i in} rat osteoclasts. Microfluorescence quantification in single rat osteoclasts given India-1 and overmelted for the indicated time (bar) with the indicated buffer containing [Ca ²⁺ ]. Normal buffer overmelted between the bars and contained 1 mM Ca ²⁺ . FIG. 29 is a graphical representation showing that spermine or neomycin B does not induce an increase in [Ca ²⁺ ] _{i in} rat osteoclasts. Osteoclasts given India-1- were overmelted with the indicated concentrations of spermine or neomycin B (hollow bars) alone or with 20 mM Ca ²⁺ (black bars). FIG. 30 shows the specific effects of argiotoxin (shown as algiopine in the figure, the structure is also shown in FIG. 1-6) 659 and argiotoxin 636 on [Ca ²⁺ ] _i in bovine parathyroid cells. It is a graph representation. The starting [Ca ²⁺ ] was 0.5 mM and increased to 1.5 mM where indicated (right graphic). Where indicated, Argiotoxin 659 (3 μM) or Argiotoxin 636 (400 μM) was added. FIG. 31 is a graphical representation showing that extracellular Mg ²⁺ or Gd ³⁺ vibrationally amplifies Cl ⁻ current in Xenopus oocytes injected with bovine parathyroid poly (A) ⁺ -mRNA. . In figure (a), the concentration of extracellular Ca ²⁺ was <1 μM and in figure (b) it was 0.7 mM. Figure (c) shows that extracellular Mg ²⁺ does not elicit a response to substance K receptors in oocytes injected only with mRNA, although overmelting with substance K induces the reaction. The holding potential was -70 to -80 mV. FIG. 32 is a graph showing that extracellular Ca ²⁺ causes vibrational amplification of Cl ⁻ conductance in Xenopus oocytes injected with human (proliferating) parathyroid poly (A) ⁺ -mRNA. Is an expression. Oocytes were tested for reactivity to extracellular Ca ²⁺ 3 days after injection of 50 ng poly (A) ⁺ -mRNA. The holding potential was −80 mV. FIG. 33 is a graphical representation showing the movement of intracellular Ca ²⁺ in bovine parathyroid cells induced by budomunkyamine. Budmunkyamine (300 μM, also showing structure) was added where indicated. FIG. 34 is a graphical representation showing that the ability of parathyroid cells to migrate intracellular Ca ²⁺ is stereospecific. Bovine parathyroid cells fed with hula-2 were first suspended in a buffer containing 0.5 mM extracellular Ca ²⁺ prior to the addition of the indicated concentrations of each molecule. FIG. 35 is a graphical representation showing the effect of La ³⁺ on [Ca ²⁺ ] _{i of} osteoclasts. A representative figure from a single rat osteoclast fed with India-1 is shown. At low concentrations, La ³⁺ partially blocks the increase in [Ca ²⁺ ] _i caused by extracellular Ca ²⁺ . FIG. 36 is a graphical representation showing the movement of intracellular Ca ²⁺ induced by extracellular Mn ²⁺ in rat osteoclasts. Extracellular Mn ²⁺ persists in the absence of extracellular Ca ²⁺ (FIG. 37) and induces a concentration-dependent increase in [Ca ²⁺ ] _i (FIG. 36). FIG. 37 is a graphical representation showing the movement of intracellular Ca ²⁺ induced by extracellular Mn ²⁺ in rat osteoclasts. Extracellular Mn ²⁺ persists in the absence of extracellular Ca ²⁺ (FIG. 37) and induces a concentration-dependent increase in [Ca ²⁺ ] _i (FIG. 36). FIG. 38 is a graphical representation showing the movement of [Ca ²⁺ ] _{i in} rat osteoclasts induced by a molecule named NPS449 (see FIG. 65). Isolated rat osteoclasts fed with Indo-1 overmelted with the indicated concentrations of NPS449 in the presence (FIG. 38) or absence (FIG. 39) of 1 mM extracellular CaCl ₂ . FIG. 39 is a graphical representation showing the movement of [Ca ²⁺ ] _{i in} rat osteoclasts induced by a molecule named NPS449 (see FIG. 65). Isolated rat osteoclasts fed with Indo-1 overmelted with the indicated concentrations of NPS449 in the presence (FIG. 38) or absence (FIG. 39) of 1 mM extracellular CaCl ₂ . FIG. 40 is a graphical representation showing the movement of intracellular Ca ²⁺ in C-cells attracted by NPS019 (see FIGS. 1-6). rMTC6-23 cells were given fura-2 and soaked in a buffer containing 0.5 mM [Ca ²⁺ ]. Where indicated, NPS019 was added to a final concentration of 10 μM. The representative figure shows that the temporary increase of [Ca ²⁺ ] _i induced by NPS019 is resistant to inhibition by La ³⁺ (center figure) and persists in the absence of extracellular Ca ²⁺ (right figure). ) FIG. 41 is a graphical representation showing that NPS456 (FIGS. 44-63) induces vibrational amplification of Cl ⁻ currents in Xenopus oocytes that had been injected with bovine parathyroid poly (A) ⁺ -mRNA. It is. FIG. 42 is a graphical representation showing that extracellular Ca ²⁺ induces vibrational amplification of Cl ⁻ flow in Xenopus oocytes that had been injected with human osteoclast mRNA. The oocytes were tested for extracellular Ca ²⁺ reactivity 3 days after injection of 50 ng of total poly (A) ⁺ mRNA. FIG. 43 is a graphical representation showing that the parathyroid cell Ca ²⁺ receptor is encoded by mRNA in the 2.5-3.5 kb size range. Bovine parathyroid poly (A) ⁺ -mRNA was size fractionated with a denatured glycerol gradient and pooled into 10 fractions. Each fraction was injected separately into Xenopus oocytes (50 ng / fraction). After 3 days, the oocytes, Cl ^- were tested for ability to respond to extracellular Ca ²⁺ by vibration amplifying current. FIG. 44 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing the family of molecules that were produced and screened to find useful molecules of the invention. FIG. 45 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing the family of molecules that were produced and screened to find useful molecules of the invention. FIG. 46 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing the family of molecules produced and screened to find useful molecules of the invention. FIG. 47 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 48 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 49 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that were produced and screened to find useful molecules of the invention. FIG. 50 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine, showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 51 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 52 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 53 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 54 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 55 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing the family of molecules that were produced and screened to find useful molecules of the invention. FIG. 56 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 57 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 58 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 59 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 60 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 61 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that have been produced and screened to find useful molecules of the invention. FIG. 62 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing the family of molecules that were produced and screened to find useful molecules of the invention. FIG. 63 shows the chemical structure of a molecule derived from diphenylpropyl-α-phenethylamine showing a family of molecules that were produced and screened to find useful molecules of the invention. FIG. 64 is a graphical representation showing that NPS021 is a calcium antagonist compound that blocks the effect of extracellular Ca ²⁺ on [Ca ²⁺ ] _{i in} bovine parathyroid cells. Cells were immersed in a buffer containing 0.5 mM CaCl ₂ and [Ca ²⁺ ] was increased to a final 2 mM where indicated (left graphic). Addition of NPS021 (200 μM) did not change [Ca ²⁺ ], but prevented the increase of [Ca ²⁺ ] _i induced by extracellular Ca ²⁺ (right figure). FIG. 65 is a graph showing the in vivo Ca ²⁺ response to NPS [R, S] -467. FIG. 66 is a graph showing the in vivo PTH response to NPS [R, S] -467. FIG. 67 is a graph showing the in vivo Ca ²⁺ response to 25 mg / kg NPS [R, S] -467. FIG. 68 is a graph showing the in vivo response of [Ca ²⁺ ] _i in bull parathyroid cells to different enantiomers of NPS467. FIG. 69 is a graph showing the in vivo response of serum Ca ²⁺ in rats to different enantiomers of NPS467. FIG. 70 depicts a reaction scheme for the preparation of fendiline or fendiline analogs or derivatives described in FIGS. 44-63 and for the synthesis of NPS467. FIG. 71 depicts a dose response curve showing that NPS467 reduces serum ionized calcium when administered orally. FIG. 72 is a restriction map of a plasmid containing BoPCaR 1 deposited with ATCC under accession number ATCC75416. FIG. 73 is a restriction map of BoPCaR 1. FIG. 74 is a nucleotide sequence corresponding to an aligned BoPCaR 1 2.2 Kb fragment (approximately 90% accuracy).

Claims (5)

The following restriction map, which encodes a protein derived from bovine parathyroid bodies and capable of functioning as a calcium receptor when expressed on Xenopus oocytes:

A nucleic acid molecule that hybridizes under high stringency conditions to a cDNA complement having   The nucleic acid molecule according to claim 1, wherein the cDNA derived from bovine epithelium has the nucleic acid sequence of the cDNA insert contained in plasmid pSPORT1 deposited under accession number ATCC75416.   The nucleic acid molecule according to claim 1 or 2, which is a recombinant nucleic acid molecule.   The nucleic acid molecule according to claim 1 or 2, which is genomic DNA.   The nucleic acid molecule according to claim 1 or 2, which is cDNA.