72124-77-7

In a P2RX7-dependent way, calcimycin (A-23187) hemimagnesium induces intracellular calcium-regulated autophagy, which kills mycobacteria[4].Phenotypic screening led to the identification of Calcimycin as a potent inhibitor of Mycobacterium bovis BCG (M. bovis BCG) growth in vitro and in THP-1 cells. In the present study, we aim to decipher the mechanism of antimycobacterial activity of calcimycin. We noticed that treatment with calcimycin led to up-regulation of different autophagy markers like Beclin-1, autophagy-related gene (Atg) 7, Atg 3 and enhanced microtubule-associated protein 1A/1B-light chain 3-I (LC3-I) to LC3-II conversion in macrophages. This calcimycin-mediated killing of intracellular M. smegmatis and M. bovis BCG was abrogated in the presence of 3-methyladenine (3-MA). We also demonstrate that calcimycin binding with purinergic receptor P2X7 (P2RX7) led to increase in intracellular calcium level that regulates the extracellular release of ATP. ATP was able to regulate calcimycin-induced autophagy through P2RX7 in an autocrine fashion. Blocking of either P2RX7 expression by 1-[N,O-bis(5-Isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) or reducing intracellular calcium levels by 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy-methyl) ester (BAPTA-AM) abrogated the antimycobacterial activity of calcimycin. Taken together, these results showed that calcimycin exerts its antimycobacterial effect by regulating intracellular calcium-dependent ATP release that induces autophagy in a P2RX7 dependent manner. The killing of branching E is mediated by calcimycin (A-23187). coli by using P2RX7 activation to trigger intracellular calcium-regulated autophagy [4].

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Calcimycin above 0.4 μM is cytotoxic to THP-1 cells [4]
Our unpublished in vitro screening experiment using the small molecular library of pharmacologically active compounds led to the identification of calcimycin as an antimycobacterial agent. Calcimycin displayed MIC99 value of 1.25 μM against in vitro growing culture of M. bovis BCG. To determine the mechanism of action of calcimycin against intracellular bacteria, present work was undertaken. Initially, to measure the cytotoxicity of calcimycin on THP-1 cells, cell viability assays were performed with varying concentration of calcimycin at different time points. We observed a reduction in cell viability in a dose-dependent (0.001–1 μM) and time- dependent (24–72 h) manner (Fig. 1A). After 72 h of calcimycin treatment, cell viability was reduced to 83.6 ± 3.5%, 76.4 ± 3.6% and 60 ± 2.2% at 0.6, 0.8 and 1 μM calcimycin respectively (Fig. 1A). MTT cell viability results were further validated using Trypan blue dye exclusion assay in THP-1 cells after 24, 48 and 72 h of calcimycin treatment. After 72 h of calcimycin treatment, doses above 0.4 μM were found to be cytotoxic (Fig. 1B). Hence, 0.4 μM calcimycin was chosen for further experiments in THP-1 cells.

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Calcimycin upregulates autophagy in THP-1 cells [4]
Since calcimycin is known to increase intracellular calcium level and this level affects mycobacterial viability by regulating autophagy so we further checked the effect of calcimycin on autophagy induction in THP-1 cells. Time-dependent kinetic experiment (Figs. S1A, B, 1C and D) was performed to measure protein expression of Beclin-1, Atg 7, Atg 3 and conversion of LC3-I to LC3-II. We found optimum expression of Beclin-1, Atg 7 and Atg 3 after 12 h of calcimycin treatment in THP-1 cells (Figs. 1C, D and 2A) clearly suggesting that calcimycin is able to induce autophagy in THP-1 macrophages. In concordance, we also found more LC3 puncta formation in calcimycin treated cells compared to control samples substantiating increased autophagy in treated cells (Fig. 2B). As expected, this induction in expression levels of Beclin-1, Atg 7 and Atg 3 by calcimycin was abrogated in the presence of 3-MA, a selective PI3K inhibitor that inhibits autophagy (Fig. 2, C and D). In parallel, experiment was also done in calcimycin treated THP-1 cells where vacuolar ATPase inhibitor, Bafilomycin A1 (Baf-A1, 50 nM) was added 3 h before the end of the treatment. Baf-A1 addition increased LC3 puncta formation in calcimycin treated cells compared to only treated cells (Fig. 2E and F) suggesting that calcimycin treatment affects autophagic flux in THP-1 cells. These observations confirmed that treatment with calcimycin resulted in induction of autophagy in macrophages.

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Calcimycin induced autophagy in infected cells affects intracellular survival of mycobacteria [4]
Next, we studied the effect of calcimycin in intracellular survival of M. smegmatis. Firstly, THP-1 cells were infected with M. smegmatis for 3 h and subsequently treated with calcimycin for 12 h. As shown in Fig. 3(A and B), we interestingly observed 1.7, 2.3 and 2.1 fold expressions of Beclin-1, Atg 7 and Atg 3 respectively, in infected and treated cells compared to only infected cells. The addition of 3-MA abolished autophagy induction potential of calcimycin thus suggesting specificity of calcimycin in inducing autophagy in infected THP-1 cells. Induction of autophagy has been demonstrated to mediate clearance of intracellular bacteria. Likewise, we observed a 4 fold reduction in the intracellular growth of M. smegmatis in calcimycin treated sample in comparison to control cells (Fig. 3C). As expected, 3-MA pretreatment abrogated the killing activity of calcimycin in macrophages (Fig. 3C). These observations confirmed that induction of autophagy is the mechanism by which calcimycin inhibited the growth of intracellular M. smegmatis.

Highly pathogenic mycobacterial species are mostly slow growers, so next, we evaluated the ability of calcimycin to induce autophagy in THP-1 macrophages infected with M. bovis BCG, one of the slow growing mycobacteria. Similarly to M. smegmatis infected cells, we also observed 1.3, 4.4 and 1.7 fold upregulation of Beclin-1, Atg 7 and Atg 3 expression in infected and calcimycin treated cells compared to infected cells (Fig. 4, A and B). As shown in Fig. 4(A and B), the ability ofCalcimycin to induce autophagy was reduced in the presence of 3-MA. As expected, we observed increased conversion of LC3-I to LC3-II in infected and treated samples as compared to only infected cells (Fig. 4C). Calcimycin addition also increased LC3 puncta formation in M. bovis BCG infected cells compared to either treated or infected cells (Fig. 4D and E). We also noticed that calcimycin increased autophagy had a detrimental effect on the intracellular M. bovis BCG viability starting day 4 to day 6 post-infection. For three independent experiments, we observed that treatment with calcimycin significantly reduced the numbers of intracellular M. bovis BCG by 4.4 fold at 6 day post-infection (Fig. 4F). As shown in Fig. 4F, this reduction of bacterial counts in calcimycin treated macrophages was not observed in the presence of 3-MA. Since we found results in M. bovis BCG infected cells similar to M. smegmatis so further experiments involving bacteria were done with M. smegmatis.

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Calcimycin treatment increases intracellular calcium level through ATP-mediated P2RX7 dependent pathway in THP-1 cells [4]
Further experiments were performed to understand the mechanism by which calcimycin induces autophagy in THP-1 macrophages. Previous studies have shown that ATP plays an important role in increasing autophagy and alleviating intracellular mycobacterial growth, so we first checked the expression of ATP receptor, P2RX7 in THP-1 cells by qRT-PCR using gene-specific primers. We observed ≈ 14 fold (p = 0.04) increase in mRNA expression upon calcimycin treatment in THP-1 cells. Similarly, we noticed ≈ 8 (p = 0.048) or ≈ 12 (p = 0.001) fold increase in P2RX7 mRNA expression in M. bovis BCG or M. smegmatis infected cells respectively upon calcimycin treatment, indicating that treatment with calcimycin results in enhancement of P2RX7 expression (Fig. 5A). This observation led us to speculate that calcimycin treatment may increase the extracellular release of ATP because P2RX7 is the known receptor of ATP. We observed 2.5 (p = 0.001) and 2 (p = 0.004; M. bovis BCG) or 2.2 (p = 0.01; M. smegmatis) fold increase in extracellular ATP levels in only calcimycin treated and infected and treated macrophages in comparison to non-treated cells (Fig. 5B). Since ATP release has been shown to be regulated by intracellular calcium levels so we also studied the calcium level in calcimycin treated samples. As expected, we observed 2 fold increase in intracellular calcium level in calcimycin-treated samples compared to untreated cells (Fig. 5C). Confocal microscopy was also performed to see the percentage of fluorescence positive cells for calcium signal. We observed ≈ 3 fold (p = 0.003) increase in fluorescence positive cells upon calcimycin treatment compared to untreated cells (Fig. 5D and E) indicative of increased intracellular calcium in treated cells.

Based on these results, we next asked the question that how Calcimycin is regulating intracellular calcium level through P2RX7. To address this question, we performed molecular docking experiments. As described in methods, for each docking 10 models were calculated and the model with lowest binding energy was chosen for a detailed analysis. As shown in Fig. 6(A and B), calcimycin is able to form H-bonds with LYS311 and LYS64 of human P2RX7. The binding free energy observed was − 5.05 kcal/mol for the best-docked structure that had grid center placed at (x, y, z): 169.816, 145.646, 159.337. Since, docking was done using one ligand molecule and 3 chains of protein, every time the ligand binds to only one chain. Two more reliable docked structures of calcimycin and P2RX7 showed significant docking scores of − 4.40 kcal/mol (Fig. S2A) and − 5.46 kcal/mol (Fig. S2B). These observations were then verified using pre-treatment with P2RX7 inhibitor KN-62. We observed that pre-treatment with KN-62 led to 1.6 fold reduction of intracellular calcium levels in calcimycin treated cells (Fig. 6C). These results suggest the role of calcimycin binding with P2RX7 in upregulation of intracellular calcium level. Interestingly, chelation of intracellular calcium by BAPTA-AM in calcimycin treated cells led to 1.9 fold decrease in the extracellular release of ATP (Fig. 6D). These results clearly suggest that initial binding of calcimycin with P2RX7 results in enhanced P2RX7 expression and more intracellular calcium level that regulates the extracellular release of ATP.

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Inhibition of P2RX7 and intracellular calcium level affect autophagy and intracellular mycobacterial viability in Calcimycin treated cells [4]
In continuation to the previous result, we further checked the effect of calcium chelator (BAPTA-AM) or P2RX7 inhibitor (KN-62) on autophagy induction and mycobacterial viability in calcimycin treated cells. As expected, we observed a significant reduction in the expression of Beclin-1, Atg 7 and Atg 3 proteins through western blotting and reduced LC3 puncta formation by confocal microscopy in THP-1 cells pretreated with either BAPTA-AM or KN-62 before calcimycin treatment. These observations suggested the regulatory role of P2RX7 in the induction of autophagy in calcimycin treated cells (Fig. 7A–D).

To rule out the possibility that increase in intracellular calcium upon Calcimycin treatment is responsible for induction of autophagy in an ATP-independent pathway, we added KN-62, 2 h after the calcimycin treatment. We did not find any significant increase in autophagy in those samples where KN-62 was added after the calcimycin treatment clearly negating the possibility of ATP-independent autophagy (Fig. S3A and B). Abrogation of the calcimycin-induced autophagy by these inhibitors also provided a permissive environment for the intracellular growth of M. smegmatis. As expected, 7 fold reduction was observed in M. smegmatis growth upon calcimycin treatment but BAPTA-AM and KN-62 treatment reversed the antimycobacterial effect of calcimycin by 4.7 and 6 fold respectively (Fig. 7E). These results clearly suggest the host defensive role of calcimycin-induced autophagy on mycobacteria that is regulated by intracellular calcium level through ATP-dependent P2RX7 pathway. So, overall our results clearly show that binding of calcimycin to P2RX7 leads to enhancement of intracellular calcium level that regulates ATP release. This eventually enhances autophagy that inhibits the growth of intracellular mycobacteria (Fig. 8).

Protein leakage is caused by calcimycin (A-23187) hemimagnesium (2.5 or 7.5 nM; intrapleurally)[5].

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A23187-induced pleurisy in the mouse was demonstrated in this study. The protein leakage, leukocyte accumulation, LTB4 and PGE2 production in the pleural cavity of mice were increased by A23187 in a dose-dependent manner. At 7.5 nmole A23187 intrapleural injection, the protein level peaked at 0.5-2 h, PMN leukocytes accumulation peaked at 3-4 h, and LTB4 and PGE2 production peaked at 0.5-1 h. In this in vivo model we investigated the anti-inflammatory effect of norathyriol, isolated from Tripterospermum lanceolatum. A23187-induced protein leakage was reduced by norathyriol (ID50 was about 30.6 mg/kg i.p.), indomethacin and BW755C. A23187-induced PMN leukocytes accumulation was suppressed by norathyriol (ID50 was about 16.8 mg/kg, i.p.) and BW755C, while enhanced by indomethacin. Like BW755C, norathyriol reduced both LTB4 and PGE2 production (ID50 was about 18.6 and 29.1 mg/kg i.p., respectively), while indomethacin reduced PGE2 but not LTB4 generation. We also demonstrated the analgesic effect of norathyriol on the acetic acid-induced writhing response. Acetic acid-induced writhing response was depressed by norathyriol (ID50 was about 27.9 mg/kg i.p.), indomethacin and ibuprofen. These results suggest that norathyriol, like BW755C, might be a dual, yet weak, cyclooxygenase and lipoxygenase pathway blocker. The inhibitory effect of norathyriol on the A23187-induced pleurisy and acetic acid-induced writhing response in mice is proposed to be dependent on the reduction of eicosanoids mediators formation in the inflammatory site.[5]

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Calcimycin/A 23 187-induced protein leakage [5]
When A23 187 was injected into the pleural cavity, the level of leakaged protein of 7.5 nmole A23 187-treated group was higher than that of 2.5 nmole A23 187-treated animals (the values of area under curve were 8.8 + 0.4 vs. 6.1+0.4, P<0.01, and the peak values were 2.67+0.18 mg/mouse vs. 2.21+0.18 mg/mouse) (Fig. 1). In this respect, however, the profile of the response was similar at both concentrations of A23 187. The significant increase in the level of protein was observed at 0.25-0.5 h after A23 187 injection. The level of protein peaked at 0.5-1 h for 2.5 nmole A23 187, and 0.5-2 h for 7.5 nmole A23 187, intrapleural injection, then progressively declined. Two hours after 2.5 nmole A23 187, or 3 h after 7.5 nmole A23 187, challenge the protein levels in the pleural cavity were equivalent to about a half of their corresponding peak values. The resting protein level was 1.16 + 0.06 mg/mouse. Indomethacin 3 and 10mg/kg i.p. (Fig. 2a, b), BW755C, a dual cyclooxygenase and lipoxygenase inhibitor (Higgs et al. 1979), 3 and 30mg/kg i.p. (Fig. 2c, d), and norathyriol 30 mg/kg i.p. (Fig. 2g) was administrated to mice 30 min prior to 7.5 nmole A23 187 challenge, showed significant reduction of protein levels in pleural cavity. Norathyriol reduced the protein leakage in A23187-induced pleurisy with an IDs0 value was about of 30.6 mg/kg.

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PMN leukocytes infiltration [5]
Figure 3 shows the time course of the PMN leukocytes infiltration in the pleural cavity after intrapleural injection of sterile saline, 2.5 or 7.5 nmole Calcimycin/A23 187. Two hours after A23 187 injection the percentage of PMN leukocyte was increased significantly (P < 0.05). These values peaked at 3h (51.3+6.8% for 2.5nmole A23187 and 80.5+3.5% for 7.5nmole A23187 challenge) then declined. Three hours after intrapleural injection of sterile saline in normal mice, the pleural washes consisted of 25% of PMN leukocytes. The number of total cells and PMN leukocytes in resting state was 1.58_+0.18 and 0.39_+ 0.02 × 106 cells/mouse, respectively. Mice pretreated with indomethacin 3 mg/kg i.p. significantly increased (P<0.05) the number of PMN leukocytes infiltration in the 7.5 nmole A23 187-induced pleurisy (Table 1). Furthermore, both total cell and PMN leukocyte counts in pleural cavity was increased (P <0.01) in indomethacin (10 mg/kg i.p.)-treated mice. However, BW755C 3 and 30mg/kg i.p. abrogated the leukocytes accumulation. Norathyriol at a dose of 10 mg/kg i.p. little affect on leukocyte infiltration, while at a dose of 20 and 30 mg/kg i.p. significantly reduced the number of total cells and PMN leukocytes. Norathyriol reduced the PMN leukocytes infiltration in A23 187-induced pleurisy with an ID50 value was about 16.8 mg/kg.

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PGE 2 and LTB 4 levels in the pleural cavity [5]
Intrapleural injection of 2.5 or 7.5 nmole Calcimycin/A23 187 significantly increased (P <0.01) the LTB 4 and PGE 2 levels in comparison with that of control mice which were injected with sterile saline (Table 2). The PGE 2 and LTB 4 levels were markedly increased at 0.5 h. The peak level of LTB 4 was sustained for at least 1 h. The LTB 4 level was still significantly increased (P <0.05) at 3 h after challenge with 7.5 nmole A23 187. The levels of LTB4 and PGE 2 declined quickly at a lower concentration of A23 187 (2.5 nmole) was used. Indomethacin 3 and 10 mg/kg i.p. reduced (P < 0.01) the PGE z level, but left the LTB4 level unaffected (Table 3). BW755C significantly reduced (P <0.01) both the PGE 2 and LTB 4 levels. Norathyriol 10 mg/kg i.p. not affected on LTB4 and PGE 2 levels, while a significant reduction of LTB 4 and PGE 2 levels in comparison with the control values was shown at a dose of 20 and 30 mg/kg intraperitoneal administration. Norathyriol reduced the LTB4 and PGE 2 formation in A23 187 pleurisy with IDs0 values were about 18.6 and 29.1 mg/kg, respectively. In order to evaluate the possible action mechanism of indomethacin increased the number of PMN leukocyte infiltration in the pleural cavity, the LTB 4 level in the pleural exudate collected at 3 h after 7.5 nmole A23 187 challenge was also determined. At 3 h after A23 187 challenge, the LTB 4 levels in the pleural fluid was greatly reduced in normal mice (Table 1). Mice pretreated with indomethacin 3 mg/kg i.p.

The pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis.[1]

Phenotypic screening led to the identification of calcimycin as a potent inhibitor of Mycobacterium bovis BCG (M. bovis BCG) growth in vitro and in THP-1 cells. In the present study, we aim to decipher the mechanism of antimycobacterial activity of calcimycin. We noticed that treatment with calcimycin led to up-regulation of different autophagy markers like Beclin-1, autophagy-related gene (Atg) 7, Atg 3 and enhanced microtubule-associated protein 1A/1B-light chain 3-I (LC3-I) to LC3-II conversion in macrophages. This calcimycin-mediated killing of intracellular M. smegmatis and M. bovis BCG was abrogated in the presence of 3-methyladenine (3-MA). We also demonstrate that calcimycin binding with purinergic receptor P2X7 (P2RX7) led to increase in intracellular calcium level that regulates the extracellular release of ATP. ATP was able to regulate calcimycin-induced autophagy through P2RX7 in an autocrine fashion. Blocking of either P2RX7 expression by 1-[N,O-bis(5-Isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) or reducing intracellular calcium levels by 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy-methyl) ester (BAPTA-AM) abrogated the antimycobacterial activity of calcimycin. Taken together, these results showed that calcimycin exerts its antimycobacterial effect by regulating intracellular calcium-dependent ATP release that induces autophagy in a P2RX7 dependent manner.[4]

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [4]
2.5 × 104 cells were seeded per well and PMA differentiated overnight for performing cell viability experiments. Next day after PMA wash, cells were treated with different concentration of Calcimycin (0.001–1 μM) for 24, 48 and 72 h. At the end of the treatment, 10 μl of MTT (5 mg/ml) was added to each well and incubated at 37 °C. After 2–3 h of incubation, 100 μl of lysis solution was added to each well and OD was measured at 562 nm using microplate reader (PerkinElmer, USA). The percentage of viable cells was calculated according to the following formula:

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Trypan blue dye exclusion assay [4]
PMA differentiated THP-1 cells were seeded at a density of 1 × 106 cells/well in 24 well plate and treated with varying concentration of Calcimycin (0.2–1 μM) at different time points. At indicated time points after treatment, cells were harvested, diluted 1:1 with 0.4% Trypan blue dye and 10 μl was loaded onto hemocytometer chamber. Total no. of cells with and without the dye was counted and cell viability was calculated according to the following formula:

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Western blot analysis [4]
Western blot analysis was done by seeding 1 × 106 cells/well in 12 well plate and then treated with Calcimycin (0.4 μM) at varying time points. In some of the experiments, cells were infected with either M. smegmatis or M. bovis BCG before adding calcimycin. Briefly, after the calcimycin treatment, cells were lysed in RIPA (Radioimmunoprecipitation Assay) buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% Sodium deoxycholate and 0.1% SDS with 1 × protease inhibitor cocktail and amount of protein in these lysates was determined using BCA protein kit. For western blot analysis, 40 μg of protein was resolved on 15% SDS-PAGE and subjected to immunoblot analysis. Briefly, after electrophoresis, proteins were transferred to nitrocellulose membranes and blocked with PBST (PBS containing 0.5% Tween-20) containing 5% skimmed milk at room temperature for 1 h. Membranes were subsequently incubated with primary antibodies at a dilution of 1:1000 in PBST followed by incubation with HRP-conjugated secondary antibody before visualizing the proteins using ECL kit. Respective band intensities from the blots were quantified using the ImageJ software.

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Confocal microscopy [4]
Differentiated THP-1 cells were grown overnight on glass cover slips at a density of 105 cells/ml in 35-mm dishes. Next day, cells were washed, treated with Calcimycin for 12 h and then fixed in 1% p-formaldehyde. In some of the experiments, cells were infected with M. bovis BCG before adding calcimycin. The fixed cells were permeabilized, blocked with 2 mg/ml BSA, incubated with LC3 primary antibody at 4 °C for overnight and stained with respective Alexa 568 coupled secondary antibody as per manufacturer's recommendations. These stained cells were mounted with ProLong Gold anti-fade reagent with DAPI. Images of Fig. 2B were acquired on Olympus FV1000 confocal microscope and Figs. 2E and 7C on confocal scanning laser microscope (CSLM). The scanned images were exported and processed using Adobe Photoshop version 7 software.

Animal/Disease Models: Mice (ICR, 25-30 g )[5]
Doses: 2.5 or 7.5 nM
Route of Administration: Intrapleurally
Experimental Results: Two hrs (hours) after 2.5 nM, or three hrs (hours) after 7.5 nM, challenge the protein levels in the pleural cavity were equivalent to about a half of their corresponding peak values.

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Calcimycin/A 23187-induced pleurisy. [5]
Mice (ICR, 25 - 30 g) were anesthetized with sodium pentobarbital (60 mg/kg). Twenty-five ~tl of sterile saline, 2.5 or 7.5 nmole Calcimycin/A23187 (A23 187 was prepared as 10 mM stock solution in DMSO and diluted with sterile saline) was injected intrapleurally through a 27-gauge needle. At a given intervals, mice were sacrificed by exsanguination. The pleural cavity was opened, and then washed once with 0.5 ml of 0.2% (w/v) EDTA in phosphate-buffered saline. The fluid in pleural cavity was harvested and immediately kept in ice bath. 91 Protein assay. The harvested fluid was centrifuged at 600 xg 4 °C for 5 min. An aliquot of supernatant was used to determine the protein content (Bradford 1976). Briefly, standard or sample was mixed with Coomassie Brilliant Blue G 250. The color change was detected by spectrophotometry at 595 nm. Protein content was estimated from the standard curve. The data were also analyzed to compare the area under the time-protein level curve based on the Trapezoidal rule.

[1]. Characterization of the biosynthesis gene cluster for the pyrrole polyether antibiotic calcimycin(A23187) in Streptomyces chartreusis NRRL 3882. Antimicrob Agents Chemother. 2011 Mar;55(3):974-82.

[2]. IKCa1 activity is required for cell shrinkage, phosphatidylserine translocation and death in Tlymphocyte apoptosis. EMBO Rep. 2003 Feb;4(2):189-94.

[3]. Modulation of intracellular calcium homeostasis blocks autophagosome formation. Autophagy. 2013 Oct;9(10):1475-90.

[4]. Calcimycin mediates mycobacterial killing by inducing intracellular calcium-regulated autophagy in a P2RX7 dependent manner. Biochim Biophys Acta Gen Subj. 2017 Dec;1861(12):3190-3200.

[5]. Effect of norathyriol, isolated from Tripterospermum lanceolatum, on A23187-induced pleurisy and analgesia in mice. Naunyn Schmiedebergs Arch Pharmacol. 1994 Jul;350(1):90-5.

[1]. Characterization of the biosynthesis gene cluster for the pyrrole polyether antibiotic calcimycin(A23187) in Streptomyces chartreusis NRRL 3882. Antimicrob Agents Chemother. 2011 Mar;55(3):974-82.

[2]. IKCa1 activity is required for cell shrinkage, phosphatidylserine translocation and death in Tlymphocyte apoptosis. EMBO Rep. 2003 Feb;4(2):189-94.

[3]. Modulation of intracellular calcium homeostasis blocks autophagosome formation. Autophagy. 2013 Oct;9(10):1475-90.

[4]. Calcimycin mediates mycobacterial killing by inducing intracellular calcium-regulated autophagy in a P2RX7 dependent manner. Biochim Biophys Acta Gen Subj. 2017 Dec;1861(12):3190-3200.

[5]. Effect of norathyriol, isolated from Tripterospermum lanceolatum, on A23187-induced pleurisy and analgesia in mice. Naunyn Schmiedebergs Arch Pharmacol. 1994 Jul;350(1):90-5.

An ionophorous, polyether antibiotic from Streptomyces chartreusensis. It binds and transports CALCIUM and other divalent cations across membranes and uncouples oxidative phosphorylation while inhibiting ATPase of rat liver mitochondria. The substance is used mostly as a biochemical tool to study the role of divalent cations in various biological systems.

Apoptotic cell volume decrease (AVD) and exposure of phosphatidylserine (PtdSer) at the cell surface are early events in apoptosis. However, the ion channels responsible for AVD, and their relationship to PtdSer translocation and cell death are poorly understood. Real-time analysis of calcium-induced apoptosis in lymphocytes and thymocytes showed that AVD occurs rapidly, and precedes PtdSer translocation. Blockers of the K(+) channel IKCa1 completely inhibited AVD. Blockade of IKCa1, and hence AVD, also completely prevented PtdSer translocation and cell death. Thus, IKCa1-mediated AVD is the earliest-defined essential step in calcium-induced apoptosis, required for both PtdSer translocation and cell death.[2]

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.[3]

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Taken together, in the present study, we unravel the mechanism by which Calcimycin induces autophagy and kills intracellular mycobacteria in THP-1 cells. We show that treatment of THP-1 macrophages with Calcimycin results in increasing calcium that augments ATP release, which in turn regulates autophagy through P2RX7. Thus, understanding the mechanism of autophagy by calcium ionophores may provide an attractive target for the control of mycobacterial infection, which will help in developing better therapeutic interventions against TB.[4]

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Calcimycin/A23187-induced pleurisy in the mouse was demonstrated in this study. The protein leakage, leukocyte accumulation, LTB4 and PGE2 production in the pleural cavity of mice were increased by A23187 in a dose-dependent manner. At 7.5 nmole A23187 intrapleural injection, the protein level peaked at 0.5-2 h, PMN leukocytes accumulation peaked at 3-4 h, and LTB4 and PGE2 production peaked at 0.5-1 h. In this in vivo model we investigated the anti-inflammatory effect of norathyriol, isolated from Tripterospermum lanceolatum. A23187-induced protein leakage was reduced by norathyriol (ID50 was about 30.6 mg/kg i.p.), indomethacin and BW755C. A23187-induced PMN leukocytes accumulation was suppressed by norathyriol (ID50 was about 16.8 mg/kg, i.p.) and BW755C, while enhanced by indomethacin. Like BW755C, norathyriol reduced both LTB4 and PGE2 production (ID50 was about 18.6 and 29.1 mg/kg i.p., respectively), while indomethacin reduced PGE2 but not LTB4 generation. We also demonstrated the analgesic effect of norathyriol on the acetic acid-induced writhing response. Acetic acid-induced writhing response was depressed by norathyriol (ID50 was about 27.9 mg/kg i.p.), indomethacin and ibuprofen. These results suggest that norathyriol, like BW755C, might be a dual, yet weak, cyclooxygenase and lipoxygenase pathway blocker. The inhibitory effect of norathyriol on the A23187-induced pleurisy and acetic acid-induced writhing response in mice is proposed to be dependent on the reduction of eicosanoids mediators formation in the inflammatory site.[5]

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Recently, we have found that norathyriol reduced the intracellular calcium concentration in compound 48/80-activated rat peritoneal mast cells and inhibited the purified 5-1ipoxygenase activity of potato (data not shown). Whether or not norathyriol also act as a calcium blocker in PMN leukocytes and/or redox inhibitor to 5-1ipoxygenase which contribute to its inhibitory effect on leukotriene formation needs further study. In conclusion, we have demonstrated that Calcimycin/A23 187-induced pleurisy in mice is a useful in vivo model for studying drugs effect on prostaglandins and leukotrienes production. The results suggest that norathyriol, like BW755C, inhibited both the cyclooxygenase and lipoxygenase pathway, and that this action probably accounts for its anti-inflammatory and analgesic effects. [5]

C58H72MGN6O12

1069.5303

1068.51

72124-77-7

Calcimycin;52665-69-7;Calcimycin hemicalcium salt;59450-89-4; Calcimycin hemimagnesium;72124-77-7; 76455-48-6 (bromo)

17749232

Typically exists as solid at room temperature

8.536

4

16

12

77

868

14

CC1CCC2(C(CC(C(O2)C(C)C(=O)C3=CC=CN3)C)C)OC1CC4=NC5=C(O4)C=CC(=C5C(=O)[O-])NC.CC1CCC2(C(CC(C(O2)C(C)C(=O)C3=CC=CN3)C)C)OC1CC4=NC5=C(O4)C=CC(=C5C(=O)[O-])NC.[Mg+2]

XBWKBTZDBYFEMH-UIOMRPQBSA-L

InChI=1S/2C29H37N3O6.Mg/c2*1-15-10-11-29(17(3)13-16(2)27(38-29)18(4)26(33)20-7-6-12-31-20)37-22(15)14-23-32-25-21(36-23)9-8-19(30-5)24(25)28(34)35;/h2*6-9,12,15-18,22,27,30-31H,10-11,13-14H2,1-5H3,(H,34,35);/q;;+2/p-2/t2*15-,16-,17-,18-,22-,27+,29+;/m11./s1

magnesium;5-(methylamino)-2-[[(2S,3R,5R,6S,8R,9R)-3,5,9-trimethyl-2-[(2S)-1-oxo-1-(1H-pyrrol-2-yl)propan-2-yl]-1,7-dioxaspiro[5.5]undecan-8-yl]methyl]-1,3-benzoxazole-4-carboxylate

Magnesium;5-(methylamino)-2-[[(2S,3R,5R,6S,8R,9R)-3,5,9-trimethyl-2-[(2S)-1-oxo-1-(1H-pyrrol-2-yl)propan-2-yl]-1,7-dioxaspiro[5.5]undecan-8-yl]methyl]-1,3-benzoxazole-4-carboxylate; Calcimycin hemimagnesium; DTXSID50585115

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)