We discovered structurally novel human calcium-sensing receptor (CaSR) allosteric agonists and compared their pharmacology to phenylalkylamine calcimimetics. 1-Benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol (AC-265347) activated CaSR signaling in cellular proliferation and phosphatidylinositol (PI) hydrolysis assays with potencies of 30 and 10 nM, respectively. (S)-1-Benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) [(S)-AC-265347], the S-enantiomer of AC-265347, was approximately 10- to 20-fold more potent than (R)-1-benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) [(R)-AC-265347]. The phenylalkylamines cinacalcet and calindol had activity similar to that of AC-265347 in cellular proliferation assays but less activity in PI assays. All compounds had reduced activity when extracellular Ca2+ was removed, indicating that they cooperate with Ca2+ to activate CaSRs, and all activated CaSR isoforms with the N-terminal extracellular domain deleted, indicating that they interact with the transmembrane domains. In both cases, AC-265347 and therefore (S)-AC-265347 were significantly more efficacious than the phenylalkylamines. Mutations E837A7.39 and I841A7.43 strongly reduced phenylalkylamine-induced signaling, but not AC-265347- or (S)-AC-265347-induced signaling, suggesting different modes of binding. AC-265347 and (S)-AC-265347 stimulated significantly greater responses than cinacalcet or calindol at each of four loss-of-function human polymorphic CaSR variants. AC-265347 did not inhibit the CYP2D6 cytochrome P450 isozyme, unlike cinacalcet, which is a potent CYP2D6 inhibitor. In rats, AC-265347, (S)-AC-265347, and (R)-AC-265347 each reduced serum parathyroid hormone (PTH) with a rank order potency correlated with their in vitro potencies. AC-265347 and (S)-AC-265347 also reduced plasma ionizable calcium ([Ca2+]o). AC-265347 was orally active, and its plasma concentrations correlated well with its effects on serum PTH. Thus, these highly efficacious CaSR allosteric agonists represent leads for developing therapeutic agents with potential advantages over existing therapies.
Extracellular calcium ([Ca2+]o) is able to function as a “first” messenger, affecting a wide array of cellular processes, and therefore blood levels of [Ca2+]o are subject to extremely tight control (Brown and McCleod, 1991). This regulation is mediated primarily by a calcium-sensing receptor (CaSR), first cloned from bovine parathyroid cells (Brown et al., 1993). The CaSR is a member of the G protein-coupled receptor (GPCR) superfamily and belongs to the group C family of GPCRs, which also includes the GABAB receptor and the metabotropic glutamate receptors. These receptors contain a large N-terminal extracellular domain that binds ligand, a seven-transmembrane spanning region, and a C-terminal domain that transduces intracellular signals (Brown and MacLeod, 2001). Besides extracellular calcium, CaSR activity is modulated by other divalent and trivalent cations such as Mg2+, Cd2+, Ba2+, La3+, and Gd3+, amino acids (especially aromatic amino acids), spermine, amyloid β-peptides, and ionic strength (Chang and Shoback, 2004).
The CaSR primarily functions to maintain systemic [Ca2+]o homeostasis mainly by suppressing parathyroid hormone (PTH) secretion by the parathyroid glands and by influencing rates of renal tubular calcium reabsorption and secretion of calcitonin by C cells of the thyroid (Hauache, 2001). Increases in [Ca2+]o also affect secretion of many other hormones including adrenocorticotropin, gastrin, insulin, growth hormone, and PTH-related peptide, although in some cases these changes may occur through Ca2+ ion channels rather than through the CaSR itself (Brown and MacLeod, 2001).
Consistent with its main physiological function, the CaSR is highly expressed in tissues involved in mineral ion homeostasis including the parathyroid, thyroidal C cells, kidney, bone (including osteoclasts, osteoblasts, and osteocytes), chondrocytes (cartilage-forming cells), intestine (including duodenum and ileum), and placenta. However, the CaSR is also found in organs not involved in maintaining [Ca2+]o homeostasis such as brain with the highest levels in the subfornical organ (hypothalamic thirst center); neurons, astrocytes, and microglia; pituitary gland; bone marrow and peripheral blood (including platelets and monocytes); keratinocytes; the gastrointestinal system including the esophagus, stomach, small intestine, and colon; and the pancreas where it may affect insulin and glucagon secretion (Brown and MacLeod, 2001). Thus, besides its well documented role in maintaining [Ca2+]o homeostasis, the CaSR may also function as a nutrient sensor, an osmolarity regulator, and a regulator of hormone secretion, cellular chemotaxis, proliferation, differentiation, apoptosis, and gene expression.
The physiological roles of the CaSR have been further validated through correlation of human CaSR polymorphisms with diseases of [Ca2+]o homeostasis. Inactivating mutations in the human CaSR lead to familial hypocalciuric hypercalcemia (or familial benign hypercalcemia), and neonatal severe hyperparathyroidism, whereas activating mutations in the human CaSR cause autosomal dominant hypocalcemia with hypercalciuria and Bartter syndrome (Thakker, 2004). Autoimmune antibodies to the CaSR cause autoimmune hypocalciuric hypercalcemia and acquired hypoparathyroidism (Thakker, 2004).
Small molecules that modulate the sensitivity of CaSR to Ca2+ have been described, including calcimimetics, which allosterically increase the sensitivity and responsiveness of CaSR to [Ca2+]o (Hammerland et al., 1998; Nemeth et al., 1998; Dauban et al., 2000; Kessler et al., 2004; Goodman, 2005), and calcilytics, which allosterically decrease the sensitivity and responsiveness of CaSR to [Ca2+]o (Nemeth et al., 2001; Arey et al., 2005; Kessler et al., 2006). One calcimimetic called cinacalcet (marketed as Sensipar in the United States and Mimpara in Europe) is approved for the clinical treatment of secondary hyperparathyroidism and for the treatment of parathyroid carcinoma (Dong, 2005). Secondary hyperparathyroidism occurs in patients with chronic kidney disease and end-stage renal disease and is characterized by elevated serum levels of PTH and disturbances in calcium and phosphorus metabolism. By activating the CaSR, cinacalcet lowers serum PTH and normalizes calcium and phosphorus metabolism.
We have identified a structurally novel benzothiazole class of human CaSR allosteric agonists and compared their pharmacology with that of the phenylalkylamine calcimetics cinacalcet and calindol in a variety of in vitro and in vivo functional assays. The structural differences of these benzothiazoles from existing calcimimetics translated into greater activity at CaSRs carrying a variety of artificial and naturally occurring loss-of-function mutations. Thus, these novel CaSR allosteric agonists represent important new leads for the development of drugs with potential advantages over existing therapies.
Materials and Methods
MgCl2 and CaCl2 were from Sigma-Aldrich (St. Louis, MO). Pharmacy-grade cinacalcet hydrochloride (Sensipar) tablets were dissolved in dimethyl sulfoxide stock solutions immediately before use. Calindol hydrochloride was from Toronto Research Chemicals (North York, ON, Canada). 1-Benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) (AC-275347) and its resolved enantiomers (S)-1-benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) [(S)-AC-265347] and (R)-1-benzothiazol-2-yl-1-(2,4-dimethyl-phenyl)-ethanol) [(R)-AC-265347] were synthesized at ACADIA Pharmaceuticals, Inc. (San Diego, CA) (Gustafsson et al., 2010). Compound structure was verified by NMR. Purity was greater than 99% as measured by high-performance liquid chromatography and gas chromatography.
NIH 3T3 cells (American Type Culture Collection, Manassas, VA) were incubated at 37°C in a humidified atmosphere (5% CO2) in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 25 mM glucose, 4 mM l-glutamine, 50 U/ml penicillin G, 50 U/ml streptomycin (Invitrogen), and 10% calf serum (Sigma-Aldrich) or 25% UltraCULTURE synthetic supplement (Lonza Walkersville Inc., Walkersville, MD). HEK 293T (American Type Culture Collection) cells were cultured similarly except that 10% fetal calf serum was substituted for 10% calf serum.
The human parathyroid cell CaSR used in this study was cloned from a human kidney cDNA library by polymerase chain reaction using oligonucleotides derived from the GenBank accession entry U20759 and subcloned into pSI and pCI (Promega, Madison, WI) expression vectors. Polymerase chain reactions were performed using PfuTurbo (Stratagene, La Jolla, CA). The N-terminal truncated CaSR was constructed by amplifying the C-terminal 479 amino acids of the wild-type CaSR with a consensus Kozak sequence and an initiating methionine incorporated in frame and subcloning the truncated receptor into the expression vectors. All point mutations described were generated using the QuikChange mutagenesis protocol (Stratagene). All clones were sequence verified.
Cellular Proliferation Assays.
Receptor Selection and Amplification Technology (R-SAT) assays were performed as described previously (Burstein et al., 2006; Gardell et al., 2008) with the following modifications. In brief, cells were plated 1 day before transfection using 7 × 103 cells in 0.1 ml of media/well of a 96-well plate (Falcon; BD Biosciences Discovery Labware (Bedford, MA). Cells were transiently transfected with 10 ng of receptor DNA and 30 ng of pSI-β-galactosidase (Promega) per well of a 96-well plate using PolyFect (QIAGEN, Valencia, CA) according to the manufacturer's instructions. One day after transfection, medium was changed, and cells were combined with ligands in Dulbecco's modified Eagle's medium supplemented with 25% UltraCULTURE synthetic supplement (Lonza Walkersville, Inc.) instead of calf serum to a final volume of 200 μl/well. After 5 days in culture, β-galactosidase levels were measured essentially as described previously (Burstein et al., 2006; Gardell et al., 2008). Cells were rinsed with phosphate-buffered saline (pH 7.4) before the addition of 200 μl of phosphate-buffered saline supplemented with 3.5 mM O-nitrophenyl-β-d-galactopyranoside and 0.5% Nonidet P-40 (both from Sigma-Aldrich). After incubation (2–4 h), the plates were read at 420 nm on a plate reader (EL 310; BioTek Instruments; Winooski, VT, or Molecular Devices, Sunnyvale, CA).
Phosphatidylinositol Hydrolysis Assays.
Phosphatidylinositol (PI) hydrolysis assays were performed using diluted SPA beads (80 μl; GE Healthcare, Little Chalfont, Buckinghamshire, UK), followed by 30 μl of each cell lysate/well of Pico plates (PerkinElmer Life and Analytical Sciences, Waltham, MA) as described previously (Gardell et al., 2008). The “high Ca2+” buffer was identical to the buffer used for the cellular proliferation assays with the addition of 10 mM LiCl as described previously. The “no Ca2+” buffer was composed of Hanks' buffered salt solution (HyClone, Logan, UT) plus 0.5 mM MgCl2, 0.2% bovine serum albumin, and 10 mM LiCl and did not contain antibiotics.
Concentration-response graphs for all functional assays were plotted, and EC50 values were determined by nonlinear regression analysis using Prism software (version 4.0; GraphPad Software, Inc., San Diego, CA) according to the following equation: where X is the logarithm of concentration and Y is the response; Y starts at Bottom and goes to Top with a sigmoid shape.
Male Sprague-Dawley rats (∼150–200 g) were housed with free access to rat chow and water at two animals per cage for at least 2 days before use. Drugs were dissolved in 5% dimethyl sulfoxide-5% water-90% PEG400 and dosed either subcutaneously or orally. The controls were given the same volume of vehicle as the test groups. Blood samples were obtained at the indicated times, and the plasma fraction was separated and stored at −80°C until further use. PTH levels were analyzed using a rat intact PTH radioimmunoassay kit according to the manufacturer's instructions (Immutopics International, San Clemente, CA).
Analysis of Plasma Phosphate and Ca2+ Concentrations.
Plasma phosphate levels were analyzed using a phosphate assay kit with an improved Malachite Green dye from BioAssay Systems (Hayward, CA) according to the manufacturer's instructions. Plasma levels of ionizable Ca2+ were determined using an ion-selective electrode blood-gas analyzer (IDEXX Laboratories, Inc., West Sacramento, CA).
Analysis of AC-265347 Concentrations.
Plasma levels of AC-265347 were measured in rats dosed orally with 10 or 30 mg/kg. Plasma samples were collected at 0, 0.5, 1, 2, 4, 6, and 24 h and analyzed by LC-tandem mass spectrometry. The LC-tandem mass spectrometry analysis was performed using a 4000 QTRAP (Applied Biosciences, Foster City, CA) hybrid triple quadrupole linear ion trap mass spectrometer equipped with electrospray ionization and operated in multiple reaction monitoring mode. The AC-265347 ion pair was 284.2/266. The mass spectrometer was coupled to a high-performance liquid chromatography system consisting of two LC-20AD high-performance pumps interfaced with a CBM-20A controller (Shimadzu, Columbia, MD) and a CTC HTC PAL (LEAP Technologies, Carrboro, NC) autosampler. Separation was performed using a 50 × 2.1-mm Hypersil GOLD aQ (Thermo Fisher Scientific, Waltham, MA) reverse-phase C18 column equipped with a guard column. LC solvent A was water and solvent B was acetonitrile, each containing 1% formic acid. Data collection and processing were performed using Analyst software (version 1.4.2).
Using a cellular proliferation assay (R-SAT) (Burstein et al., 2006), we screened the human parathyroid calcium-sensing receptor against a diverse chemical library containing more than 250,000 compounds and identified a number of active chemical classes of compounds. On the basis of structurally interesting features compared with known calcimimetics, chemical optimization of a benzothiazole class of compounds was undertaken. We pharmacologically characterized one compound from this chemical series called AC-265347 [compound 13 in Gustafsson et al. (2010)], and its enantiomers (S)-AC-265347 and (R)-AC-265347 in greater detail, and compared them with the phenylalkylamine calcimimetics cinacalcet and calindol (Fig. 1).
AC-265347-activated CaSR signaling in cellular proliferation and PI hydrolysis assays with potencies of 30 and 10 nM, respectively (Fig. 2; Tables 1 and 2). The enantiomers of AC-265347 were each active, with (S)-AC-265347 being approximately 10- to 20-fold more potent than (R)-AC-265347. The phenylalkylamine calcimimetics cinacalcet (Nemeth et al., 2004) and calindol had activity similar to that of AC-265347 in cellular proliferation assays, but less activity in PI hydrolysis assays. Under assay conditions of high ambient Ca2+, all of these compounds stimulated 85 to 100% of the maximal functional response to MgCl2 in cellular proliferation assays and 75 to 85% of the maximal functional response to CaCl2 in PI hydrolysis assays. It was not possible to use CaCl2 as a reference standard in cellular proliferation assays because of its cytotoxicity at high concentrations. No responses to AC-265347, cinacalcet, or MgCl2 were observed in cells transfected only with reporter, confirming the specificity of the assay, and AC-265347 did not activate human GABAB receptors or human type 1 parathyroid hormone receptors, confirming its specificity for the CaSR (data not shown). The phenylalkylamine calcimimetics were derived from fendiline, a voltage-gated Ca2+ channel blocker (Nemeth et al., 1998). As expected, given its structural differences, AC-265347 displayed negligible binding to L-type voltage-gated Ca2+ channels and did not inhibit the CYP2D6 cytochrome P450 isoform. In contrast, cinacalcet is a potent CYP2D6 inhibitor (IC50 87 nM)(Nakashima et al., 2007), and both cinacalcet and calidnol have been shown to block L-type Ca2+ channels (Thakore and Ho, 2010).
All of the calcimimetics described to date including cinacalcet allosterically increase the sensitivity and responsiveness of CaSR to [Ca2+]o (Jensen and Bräuner-Osborne, 2007). Therefore, we retested all of these compounds in PI hydrolysis assays in the absence of extracellular Ca2+ (Fig. 2, E and F; Table 2). The maximum responses and potencies of all of the compounds were greatly reduced in the absence of extracellular Ca2+, indicating that they cooperate with Ca2+ to activate CaSRs. Under these conditions, significant differences in the efficacy of these compounds became apparent, with AC-265347 and (S)-AC-265347 stimulating significantly greater responses than cinacalcet, calindol, or (R)-AC-265347. Besides these two buffer systems, we tested these ligands in PI assays in buffers lacking penicillin and streptomycin but containing a range of Ca2+ concentrations (0.5–2 mM) and observed that removal of these antibiotics did not alter the rank order activity of these compounds (data not shown).
The natural ligands for family C GPCRs bind to the large, extracellular N-terminal domain characteristic of this receptor family, whereas drugs and other artificial compounds targeting family C GPCRs interact primarily with the transmembrane helical domains of these receptors (Bräuner-Osborne et al., 2007). Therefore, we tested all of the compounds on cells expressing a CaSR isoform with the N-terminal domain deleted (Fig. 3; Table 2). All the compounds activated CaSR isoforms with the N-terminal extracellular domain deleted (ΔN); however, AC-265347 and (S)-AC-265347 stimulated significantly greater responses than did cinacalcet, calindol, or (R)-AC-265347. As expected, deletion of the N terminus of the CaSR abolished activation by CaCl2. We retested all the compounds at ΔN CaSRs in the absence of extracellular Ca2+ and observed that they still retained agonist activity. Under these conditions, maximal responses to all compounds were further reduced; however, the potencies were not significantly different from the potencies at ΔN CaSRs in the presence of extracellular Ca2+. These results are in agreement with previous studies suggesting that CaSRs may contain more than one Ca2+-binding site (Ray et al., 2005) and indicate that both classes of compounds interact with the transmembrane domains of CaSRs and both have intrinsic agonist activity, but AC-265347 and (S)-AC-265347 have greater intrinsic activity than the phenylalkylamine calcimimetics.
A large number of mutations have been introduced into the human CaSR to define and differentiate the interactions of calcimimetics and calcilytics with CaSRs (Hu and Spiegel, 2007; Jensen and Bräuner-Osborne, 2007). In particular, residues E8377.39 and to a lesser extent I8417.43 are thought to play crucial roles in mediating binding and activation of CaSRs by positive and negative modulators (Hu et al., 2002, 2005, 2006; Petrel et al., 2004). In agreement with previous studies, the activity of cinacalcet was strongly reduced at E837A7.39, and its efficacy was significantly reduced at I841A7.43 (Fig. 4; Tables 1 and 2). Calindol activity was similarly affected. In contrast, the activities of AC-265437 and (S)-AC-265347 were affected to a much smaller degree at the E837A7.39 mutant and were essentially unaffected at I841A7.43. Very similar results were obtained in cellular proliferation and PI hydrolysis assays.
A large number of both activating and inactivating naturally occurring polymorphisms in the human CaSR have been described previously (Hu and Spiegel, 2007). We constructed four of these naturally occurring CaSR mutants (R66C, T138M, R185Q, and R795W) that have previously been shown to be functionally impaired (Bai et al., 1996) and compared the abilities of the benzothiazols and phenylalkylamines to activate these receptors in cellular proliferation and PI hydrolysis assays (Fig. 5; Tables 1 and 2). In agreement with previous results, all four of these CaSR polymorphic variants displayed impaired responses to MgCl2 or CaCl2 compared with those for wild-type CaSR, with reduced potency, reduced maximum response, or both. AC-265347 and (S)-AC-265347 consistently stimulated significantly greater responses than cinacalcet or calindol at each of these four polymorphic variants.
CaSRs play a crucial role in regulating blood concentrations of PTH, [Ca2+]o, phosphorus, and calcitonin (Brown and McCleod, 2001; Hauache, 2001), and treatment with calcimimetics lowers serum levels of PTH and [Ca2+]o (Nemeth et al., 2004). Treatment of normal male Sprague-Dawley rats with AC-265347, (S)-AC-265347, and (R)-AC-265347 each reduced serum PTH (Fig. 6). The approximate ED50 values for PTH suppression were 0.01 mg/kg for AC-265347 and (S)-AC-265347 and 0.1 mg/kg for (R)-AC-265347, a rank order potency correlated with their in vitro potencies. Cinacalcet also suppressed serum PTH, with a similar maximal effect and an ED50 of approximately 0.1 mg/kg. (S)-AC-265347 also suppressed serum ionizable calcium [Ca2+]o in a dose-dependent manner, although it required much higher doses than it needed to suppress serum PTH (Fig. 7A). A similar difference between the potency required to suppress serum PTH and that required to suppress [Ca2+]o has been observed for phenylalkylamine calcimimetics such as cinacalcet (Fox et al., 1999; Nemeth et al., 2004). AC-265347 also produced a significant suppression of serum [Ca2+]o (data not shown); however, although rats treated with (R)-AC-265347 did have lowered serum [Ca2+]o, the trend was not statistically significant (Fig. 7B).
Oral administration of AC-265347 rapidly suppressed serum PTH levels in rats, with the maximal suppression of plasma PTH occurring 30 min after drug administration (Fig. 8A). Lower levels of plasma PTH were maintained for 6 h at 30 mg/kg AC-265347. Plasma concentrations of AC-265347 after oral administration were determined over a 24-h time course (Fig. 8B). Peak plasma concentrations of AC-265347 were 67 and 311 ng/ml for the 10 and 30 mg/kg groups, respectively, and occurred at 1-h postdose. These levels compare favorably with those reported previously for cinacalcet (73 and 124 ng/ml at 10 and 36 mg/kg p.o., respectively) (Nemeth et al., 2004). The plasma concentration-time curves of AC-265347 correlated very well with its observed effects on serum PTH levels.
We have discovered a structurally novel class of benzothiazol CaSR allosteric agonists and compared them pharmacologically with the phenylalkylamine class of calcimimetics (cinacalcet and calindol). Compared with the phenylalkylamine calcimimetics, the benzothiazols showed greater potency and efficacy at wild-type CaSRs and at a variety of artificial and naturally occurring mutant forms of CaSRs.
The novel CaSR allosteric agonists described herein displayed stereoselectivity in their actions on CaSRs, with the (S)-enantiomer being approximately 10- to 20-fold more active than the (R)-enantiomer in both in vitro and in vivo functional assays. These novel compounds demonstrated potent activity, lowering serum PTH and serum [Ca2+]o in vivo, actions expected for calcimimetics. The in vivo actions of these compounds were well correlated with their plasma concentrations and with their in vitro potencies.
The benzothiazol CaSR allosteric agonists appear to interact with CaSRs differently from the phenylalkylamines calcimimetics. Although studies using CaSRs lacking the N-terminal extracellular domain indicated that both classes of compounds interact with the transmembrane domain spanning regions of the CaSR (Fig. 3), studies using point mutations within transmembrane domain 7 clearly indicate that they use different amino acid residues to bind to CaSRs. We observed that mutations E837A7.39 and I8417.43 strongly reduced phenylalkylamine-induced signaling, in agreement with previous studies on cinacalcet and other structurally related calcimimetics and calcilytics (Petrel et al., 2004). In contrast, these mutations had little effect on AC-265347- or (S)-AC-265347-induced signaling (Fig. 4).
The different interactions of AC-265347 and its analogs may translate into certain clinical advantages over cinacalcet and other phenylalkylamine derivatives. The CaSR is highly polymorphic, and a large number of loss-of-function polymorphic variants have been described previously (see http://www.casrdb.mcgill.ca). We tested four loss-of-function CaSR polymorphic variants and found consistently that AC-265347 and in particular (S)-AC-265347 were able to activate these receptors better than either cinacalcet or calindol (Fig. 5). The specific polymorphic variants we tested are thought to be quite rare in normal healthy people; however, each has been found in people with disorders in calcium management, specifically familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, and are significantly associated with these diseases (Pollak et al., 1993; Chou et al., 1995). The improved activation of these loss-of-function polymorphic variants by the benzothiazol CaSR allosteric agonists could stem from their different modes of binding CaSRs or could simply be due to the fact that they have higher intrinsic activity than the phenylalkylamines. These results suggest that AC-265347 or its analogs might provide greater efficacy than cinacalcet in patients harboring loss-of-function CaSR polymorphisms.
Several studies reported that the calcimimetic 3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]propan-1-amine (NPS R-568) improves the signal transduction characteristics of loss-of-function polymorphic variants of the human CaSR associated with human disease (Rus et al., 2008; Lu et al., 2009; White et al., 2009). NPS R-568 is structurally very similar to cinacalcet, and therefore one would expect cinacalcet to have similar effects on those polymorphic receptors, and, vice versa, one would expect NPS R-568 to have effects similar to those of cinacalcet and calindol on the polymorphic receptors studied here. The polymorphisms studied in those previous reports were different from the ones reported in this study, and thus direct comparisons of this study to those studies are not possible. In addition, there are a large number of other polymorphic variants of the CaSR known to exist that were not tested in this study or any of the studies cited above (Pidasheva et al., 2004). It seems reasonable to speculate that benzothiazol CaSR allosteric agonists such as AC-265347 may stimulate greater responses than cinacalcet at these other variant receptors too.
Cinacalcet is a potent inhibitor of the CYP2D6 cytochrome P450 isoform with a Ki of 87 nM (Nahashima et al., 2007). A wide variety of drugs are metabolized by CYP2D6, and the potential for cinacalcet to cause significant drug-drug interactions has been documented (Harris et al., 2007). In contrast, we observed no significant interaction of AC-265347 with CYP2D6 at concentrations up to 100 μM. Thus, drug-drug interactions should be less of a concern with AC-265347 or its analogs.
In conclusion, the data presented herein suggest that AC-265347 and structural analogs of AC-265347 have the potential to be developed into effective calcimimetics and may provide therapeutic advantages over cinacalcet in certain patient populations.
Participated in research design: Ma, Tabatabaei, Olsson, and Burstein.
Conducted experiments: Ma, Owens, and Schmeltzer.
Contributed new reagents or analytic tools: Gustafsson and Jensen.
Performed data analysis: Owens, Ma, and Burstein.
Wrote or contributed to the writing of the manuscript: Burstein.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- calcium-sensing receptor
- G protein-coupled receptor
- parathyroid hormone
- human embryonic kidney
- Receptor Selection and Amplification Technology
- liquid chromatography
- NPS R-568
- Received December 14, 2010.
- Accepted January 12, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics