Query String: NH4Cl
- ChEMBL_2444012 Inhibition of human NHE5 transfected in chinese hamster PS120 cells measured after 9 mins by BCECF-AM staining based NH4Cl pre-pulse method
- ChEMBL_2444013 Inhibition of rat NHE1 expressed in Chinese hamster ovary cells (AP-1) measured after 9 mins by BCECF-AM staining based NH4Cl pre-pulse method
- ChEMBL_1985815 (CHEMBL4619221) Inhibition of CPS1 in human pooled hepatocytes assessed as reduction in urea production measured after 16 hrs in presence of NH4Cl by tecan-plate reader analysis
- MGAT2 Human Assay A solution of 1 M LiHMDS in THF (51.7 ml, 51.7 mmol) was added dropwise to a mixture of Intermediate 3 (7.5 g, 17.2 mmol) and 5-(tert-butyl)-4-methylisoxazol-3-amine (5.32 g, 34.5 mmol) in anhydrous THF (200 ml) that had been cooled to −78° C. and placed under a N2 atmosphere. The acetone bath was removed and the solution aged at room temperature for 3 hrs. The reaction was quenched with saturated aqueous NH4Cl solution and diluted with EtOAc. The two layers were separated and the aqueous phase extracted with EtOAc×2. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting crude was purified by MPLC (KP-Sil 340 g SNAP column, BIOTAGE system) eluting with a gradient of 20-50% EtOAc/Hexane to afford the title compound.
- Cell-Based Assay of NHE-3 Activity Rat NHE-3-mediated Na+-dependent H+ antiport was measured using a modification of the pH sensitive dye method originally reported by Tsien (Proc. Natl. Acad. Sci. USA. (1984) 81(23): 7436-7440). Opossum kidney (OK) cells were obtained from the ATCC and propagated per their instructions. The rat NHE-3 gene was introduced into OK cells via electroporation, seeded into 96 well plates and grown overnight. Medium was aspirated from the wells, cells were washed twice with NaCl-HEPES buffer (100 mM NaCl, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4), then incubated for 30 min at room temperature with NH4Cl-HEPES buffer (20 mM NH4Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) containing 5 uM BCECF-AM (Invitrogen). Cells were washed twice with Ammonium free, Na+-free HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) and incubated in the same buffer for 10 minutes at room temperature to lower intracellular pH. NHE-3-mediated recovery of neutral intracellular pH was initiated by addition of Na-HEPES buffer containing 5 uM ethyl isopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity that does not inhibit NHE-3) and 0-30 uM test compound, and monitoring the pH sensitive changes in BCECF fluorescence (ex 505 nm, λem 538 nm) normalized to the pH insensitive BCECF fluorescence (λex 439 nm, λem 538 nm). Initial rates were were plotted as the average 3-6 replicates, and pIC50 values were estimated using GraphPad Prism.
- PDE5 assay Experimental protocol: The test compound, i.e the compound of the present invention, reference compound or water (control) are added to a buffer containing 40 mM Tris/HCl (pH 7.8), 3 mM MgCl2, 1.4 mM DTT, 0.21% BSA, 200 mM NH4Cl, 1 μM cGMP and 0.1 μCi [3H]cGMP. Thereafter, the reaction is initiated by addition of the enzyme and the mixture is incubated for 60 min at 22° C. For basal control measurements, the enzyme is omitted from the reaction mixture. Following incubation SPA beads are added. After 20 min at 22° C. under shaking, the amount of [3H]5′GMP is quantified with a scintillation counter (Topcount, Packard).The results shown in Table 1 are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is dipyridamole, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC50 value is calculated.
- Cell-Based Assay of NHE-3 Activity (Pre-Incubation Inhibition) Rat and human NHE-3-mediated Na+-dependent H+ antiport was measured using a modification of the pH sensitive dye method originally reported by Paradiso (Proc. Natl. Acad. Sci. USA. (1984) 81(23): 7436-7440). PS120 fibroblasts stably expressing human NHE3 and NHERF2 were obtained from Mark Donowitz (Baltimore, Md.). Opossum kidney (OK) cells were obtained from the ATCC and propagated per their instructions. The rat NHE-3 gene (GenBank M85300) was introduced into OK cells via electroporation, and cells were seeded into 96 well plates and grown overnight. Medium was aspirated from the wells then incubated for 30 min at 37° C. with NH4Cl-HEPES buffer (20 mM NH4Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) containing 5 μM BCECF-AM. Cells were washed once with Ammonium free, Na+-free HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) and incubated in the same buffer for 10 minutes at room temperature to lower intracellular pH with 0-30 μM test compound. After incubation, NHE-3-mediated recovery of neutral intracellular pH was initiated by addition of Na-HEPES buffer containing 0.4 μM ethyl isopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity that does not inhibit NHE-3). Changes in intracellular pH were monitored using a FLIPR Tetra (Molecular Devices, Sunnyvale, Calif.) by excitation at λex 439 to 505 nm, and measuring BCECF fluorescence at λem 538 nm. The initial rate of the fluorescence ratio change was used as a measure of NHE-mediated Na+/H+ activity, and reported as the change in fluorescence ratio per minute. Initial rates were plotted as the average of 2 or more replicates, and pIC50 values were estimated using GraphPad Prism.
- Cell-Based Assay of NHE3 Activity under Persistent Conditions The ability of compounds to inhibit Rat NHE3-mediated Na+-dependent H+ antiport after application and washout was measured using a modification of the pH sensitive dye method described above. Opossum kidney (OK) cells were obtained from the ATCC and propagated per their instructions. The rat NHE3 gene was introduced into OK cells via electroporation, and cells were seeded into 96 well plates and grown overnight. Medium was aspirated from the wells, cells were washed twice with NaCl-HEPES buffer (100 mM NaCl, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4), then overlayed with NaCl-HEPES buffer containing 0-30 μM test compound.After a 60 min incubation, the test drug containing buffer was aspirated from the cells, cells were washed twice with NaCl-HEPES buffer without drug, then incubated for 30 min at room temperature with NH4Cl-HEPES buffer (20 mM NH4Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) containing 5 uM BCECF-AM. Cells were washed twice with Ammonium free, Natfree HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) and incubated in the same buffer for 10 minutes at room temperature to lower intracellular pH. NHE3-mediated recovery of neutral intracellular pH was initiated (40 min after compound washout) by addition of Na-HEPES buffer containing 0.4 uM ethyl isopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity that does not inhibit NHE3), and monitoring the pH sensitive changes in BCECF fluorescence (λex 505 nm, λem, 538 nm) normalized to the pH insensitive BCECF fluorescence (λex 439 nm, λem, 538 nm). Initial rates were plotted as the average 2 or more replicates, and pIC50 values w
- Cell-based activity under Prompt Conditions Cell-based activity under Prompt Conditions. Rat or human NHE3-mediated Na+-dependent H+ antiport was measured using a modification of the pH sensitive dye method originally reported by Paradiso (PNAS USA. 81:7436-7440, 1984). Opossum kidney (OK) cells were obtained from the ATCC and propagated per their instructions. The rat NHE3 gene (GenBank M85300) or the human NHE3 gene (GenBank NM_004174.1) was introduced into OK cells via electroporation, and cells were seeded into 96 well plates and grown overnight. Medium was aspirated from the wells, cells were washed twice with NaCl-HEPES buffer (100 mM NaCl, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4), then incubated for 30 min at room temperature with NH4Cl-HEPES buffer (20 mM NH4Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) containing 5 μM bis(acetoxymethyl) 3,3′-(3′,6′-bis(acetoxymethoxy)-5-((acetoxymethoxy)carbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-2′,7′-diyl)dipropanoate (BCECF-AM).Cells were washed twice with Ammonium free, Na+-free HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) and incubated in the same buffer for 10 minutes at room temperature to lower intracellular pH. NHE3-mediated recovery of neutral intracellular pH was initiated by addition of Na-HEPES buffer containing 0.4 μM ethyl isopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity that does not inhibit NHE3) and 0-30 μM test compound, or a pharmaceutically acceptable salt thereof, and monitoring the pH sensitive changes in BCECF fluorescence (λex 505 nm, λem, 538 nm) normalized to the pH insensitive BCECF fluorescence (λex 439 nm, λem, 538 nm). Initial rates were plotted as the average 2 or more replicates, and pIC50 values were estimated using GraphPad Prism.
- PDE5 Assay Purpose: Evaluation of the effects of compounds of the present invention on the activity of the human phosphodiesterase-5 quantified by measuring the formation of 5′GMP from cGMP using PDE5 enzyme isolated from human platelets. The latter was effected in accordance with the method as described by Masaaki I, Nishikawa M, Fujioka M, Miyahara M, Isaka N, Shiku H, Nakano T, Cell Signal (1996), 8(8):575-581.Experimental protocol: The test compound, i.e. the compound of the present invention, reference compound or water (control) are added to a buffer containing 40 mM Tris/HCl (pH 7.8), 3 mM MgCl2, 1.4 mM DTT 0.21% BSA, 200 mM NH4Cl, 1 μM cGMP and 0.1 μCi [3H]cGMP. Thereafter, the reaction is initiated by addition of the enzyme and the mixture is incubated for 60 min at 22° C.For basal control measurements, the enzyme is omitted from the reaction mixture. Following incubation SPA beads are added. After 20 min at 22° C. under shaking, the amount of [3H]5′GMP is quantified with a scintillation counter (Topcount, Packard).
- Urease Inhibition Assay The inhibition studies of soybean urease were initiated with boric acid and boronic acids (butylboronic acid, 4-bromophenylboronic acid, and phenylboronic acid). Also, heavy metal ions (HgCl2, AgCl, and Cu(CH3COO)2) and sodium salts of mineral acids (NaF, NaCl, NaNO3, and Na2SO4) were investigated for their inhibitory effects. Stock solutions of inhibitors, except for 4-bromophenylboronicacid, were prepared in 0.05 M Tris-acetate buffer, pH 7.0, and were suitably diluted for experiments, whereas a stock solution of 4-bromophenylboronic acid was prepared in absolute ethanol and subsequently diluted in respective buffer. The activity assay was carried out at standard conditions as described earlier in the presence of varying concentrations of inhibitors. The yellow-orange colored solution was measured at 405 nm on a Unicam UV-2 spectrophotometer. The amount of NH3 liberated in the reaction mixture was estimated by calibrating Nessler's reagent with standard NH4Cl solution. One enzyme unit is defined as the amount of urease required to liberate 1 μmol of ammonia per minute under our test conditions (0.1 M urea, 0.05 M Trisacetatebuffer, pH 7.0, 37°C).
- Cell-Based Assay of NHE-3 Activity (Persistent Inhibition) The ability of compounds to inhibit human and rat NHE-3-mediated Na+ dependent H+ antiport after application and washout was measured using a modification of the pH sensitive dye method described above. PS120 fibroblasts stably expressing human NHE3 and NHERF2 were obtained from Mark Donowitz (Baltimore, Md.). Opossum kidney (OK) cells were obtained from the ATCC and propagated per their instructions. The rat NHE-3 gene was introduced into OK cells via electroporation, and cells were seeded into 96 well plates and grown overnight. Medium was aspirated from the wells, cells were washed once with NaCl-HEPES buffer (100 mM NaCl, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4), then overlayed with NaCl-HEPES buffer containing 0-30 μM test compound. After a 60 min incubation at room temperature, the test drug containing buffer was aspirated from the cells. Following aspiration, cells were washed once with NaCl-HEPES buffer without drug, then incubated for 30 min at 37° C. with NH4Cl-HEPES buffer (20 mM NH4Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) containing 5 μM BCECF-AM. Cells were washed once with Ammonium free, Na+-free HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) and incubated in the same buffer for 10 minutes at room temperature to lower intracellular pH. NHE-3-mediated recovery of neutral intracellular pH was initiated (10 min after compound washout) by addition of Na-HEPES buffer. For the rat NHE3 assay, the Na-HEPES buffer contained 0.4 ethyl isopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity that does not inhibit NHE-3). Changes in intracellular pH were monitored using a FLIPR Tetra (Molecular Devices, Sunnyvale, Calif.) by excitation at λex 439 to 505 nm, and measuring BCECF fluorescence at λem 538 nm. The initial rate of the fluorescence ratio change was used as a measure of NHE-mediated Na+/H+ activity, and reported as the change in fluorescence ratio per minute. Initial rates were plotted as the average of 2 or more replicates, and pIC50 values were estimated using GraphPad Prism.
- Aminoacylation Assay Compounds were solubilized in DMSO. Serial 2-fold dilutions covering two concentration ranges, 10 mM to 19.5 μM and 100 μM to 195 nM, were prepared. 0.6 μl/well of the diluted compound solutions (50× the final assay concentration) were added to white 384-well polystyrene assay plates (Thermo Fisher Scientific/Matrix Technology Corp., Hudson, NH). Uninhibited control wells (MAX)received 2 μl of 30% (v/v) DMSO. Baseline wells (MIN) received 2 μl of a solution containing 30% (v/v)DMSO and 15 mM L-phenylalanine (Sigma-Aldrich). Assays were performed in a buffer consisting of 50 mM Tris-HCl (pH 8.0), 50 mM NH4Cl, 10 mM MgCl2, 2 mM DTT, 0.005% Tween 20 (Surfact-Amps-20, Thermo Fisher Scientific/Pierce Protein Research products, Suwanee, GA), and 0.1 mM EDTA-NaOH (pH 8.0). Compounds were preincubated with 15 μl/well of either 2 nM E. coli PheRS, 2 nM H. influenzae PheRS, or 0.8 nM P. aeruginosa PheRS in buffer for 30 min at 2× final assay concentrations. The reactions were initiated with 15 μl/well of a 2× substrate solution in buffer containing 2 μM E. coli phenylalanine tRNA (Sigma-Aldrich), 100 μM ATP, and 2 μM [3H]Phe with a specific radioactivity of 6.3 Ci/mmol (PerkinElmer Life Sciences). The reactions were quenched after 30 min with 15 μl/well of a solution containing 4 mg/ml PVT/PEI/WGA type A SPA beads (PerkinElmer Life Sciences), 262 mM sodium citrate (pH 2.0), and 150 mM NaCl. The plates were sealed with transparent film (PerkinElmer Life Sciences). The beads were allowed to settle for a minimum of 2 h before scintillation counting with a Top-Count plate reader (PerkinElmer Life Sciences). [3H] counts (CPM) for aminoacylated tRNA were measured for 1 min/well.
- MATE2-K IC50 Assay MATE2-K (multidrug and toxin extrusion protein 2) is expressed in the apical membrane in the kidney and mediates the elimination of compounds to urine. MDCK-II cells were maintained in DMEM with low glucose and 10% FBS. Cells passages up to 40 were seeded at 60K±10K cells/well on 96-well, transwell membrane plates approximately 24 hours before transfection. Transport assays were carried out approximately 48 hours after transfection. On assay day, the DMEM was removed, and cells were washed with HBSS. After washing, the cells in each well were pre-incubated with HBSS containing 30 mM NH4Cl and either vehicle, the compound being tested at 6 concentrations ranging from 0.127 μM to 40 AM, or 100 μM cimetidine as a reference inhibitor. The assay plate was then placed in a 37° C. incubator with orbital shaking at approximately 60 RPM for the pre-incubation time of 15 minutes. The pre-incubation solutions were then removed, and cells washed with HBSS once. 100 μL of incubation buffer was added to each well containing HBSS with 10 μM 14[C]-metformin as the probe substrate and either vehicle control, the compound being tested (at 6 concentrations ranging from 0.127 μM to 40 μM) or reference inhibitors. The assay plate was incubated at 37° C. with orbital shaking at approximately 60 RPM for the incubation time of 5 minutes. At end of the 5-minute incubation, 15 μL of dosing solution was removed from each well containing the compound being tested and measured using LC/MS/MS for dose recovery assessment. The assay wells were then washed four times with ice cold PBS. 60 μL cell extraction solution was added to each well and the plate was incubated at 37° C. with orbital shaking at approximately 60 RPM for the 15 minutes. After this incubation, 30 μL was removed from each well, added to 200 μL scintillation fluid, and counted on a 1450 Microbeta (Perkin-Elmer) to measure the probe substrate uptake. Inhibition potential of the compound being tested was calculated by dividing the transporter-mediated uptake rate in presence of the compound being tested or the reference inhibitor by the transporter-mediated uptake rate in presence of vehicle control and fitted to a sigmoidal function to determine the IC50 values.
- BCA Protein Assay Escherichia coli BL21*DE3 pET30a-Ec yeaWX #1 (Ec YeaWX) strain was generated as described below. The contiguous Escherichia coli coding sequence yeaW (equivalent to uniprot ID P0ABR7.1 (YeaW) (SEQ ID NO: 2)) and yeaX (equivalent to uniprot ID P76254.1 (YeaX) (SEQ ID NO: 3)) were PCR amplified from Escherichia coli strain K-12 substr. BW25113 genomic DNA. PCR primers (YeaW_Nde I_fwd2-SEQ ID NO: 4; YeaX_rev2-SEQ ID NO: 5) were designed to create a 5′ NdeI restriction site including the ATG start codon of yeaW and create a PstI restriction site just 3′ of the yeaX TAG stop codon.The bacteria were grown aerobically in 50 mL LB broth (Difco #244620; 10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 50 μg/mL kanamycin), in a 500 mL Erlenmeyer flask. The cultures were inoculated from glycerol stock of BL21*DE3 pET30a-Ec yeaWX #1 strain. Strains were cultured all day at 37° C. with 250 rpm shaking. Two 300 mL Minimal M9 Medium (6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 0.1 mM CaCl2, 1 mM MgSO4, 0.2% Dextrose, 1 mg/L Thiamine, 50 μg/mL kanamycin), in 1 L Erlenmeyer flasks, were inoculated with 5 mL of the LB broth day culture and cultured overnight at 37° C. with 250 rpm shaking. The overnight cultures were used to inoculate twelve 1 L cultures of Minimal M9 media in 2.8 L fluted Erlenmeyer flasks to an OD 600 nm of 0.05 (typically approximately 28 mLs), which were grown at 37° C. with 250 rpm shaking until an OD600 of approximately 0.4 was reached. Expression of YeaWX was induced with 1 mM IPTG and the induced cultures were further grown overnight at 37° C. with 250 rpm shaking. The biomass was pelleted by centrifugation at 6000×g for 12 minutes at 4° C. The cell pellet was suspended in 240 mL of ice-cold 1× Phosphate Buffered Saline (Ca2+ and Mg2+ free). Ninety micrograms of Lysozyme (Sigma #L6876 Lot #SLBG8654V; Sigma-Aldrich Corp., St. Louis, Mo.) was added and incubated with 320 rpm shaking for 30 minutes at 4° C. Lysis was achieved via French press with a 4° C. prechilled 1″ diameter chamber at 1000 psi (high ratio; internal PSI equivalent 16000). The lysate was centrifuged at 6,000×g for 12 minutes at 4° C. to pellet extra debris. Glycerol was added to the centrifuged lysate supernatant at a final concentration of 15% A protein concentration of the centrifuged lysate supernatant was determined by a BCA Protein Assay Kit (Pierce #23225), typically in the 2.5 to 4.5 mg/ml range. The centrifuged Ec YeaWX lysate supernatant was aliquoted into 20 mL volumes and stored frozen at −80° C.Ec YeaWX lysate was diluted to 2.0 mg/mL protein with 1× Dulbecco's phosphate buffered saline (DPBS) plus 15% glycerol. Nicotinamide adenine dinucleotide phosphate (NADPH) was added to 250 μM. One hundred and fifty microliters of Ec YeaWX lysate was dispensed into a deep-well plate (polypropylene, 2 mL volume, Corning Axygen catalogue #P-DW-20-C). Candidate IC50 compounds from TABLE 1 and vehicle control (respective vehicle control of DMSO or water), or control compounds (IC50 control, 8-Quinolinol hemisulfate salt (Sigma Catalog #55100)) were added at a 1:100 dilution (e.g., 1.5 μL per well). The plates were agitated on a plate shaker for 1 minute. d9-carnitine chloride (1.5 μL of 5 mM) was added to all wells to reach a final d9-carnitine chloride concentration of 50 μM.