Plasmodium

Ascomycin, isolated from Streptomyces, suppresses immune response in vitro with IC50 of 0.55 nM for mouse mixed lymphocyte. [1] Ascomycin inhibits calcineurin phosphatase with an IC50 of 49 nM by forming an FKBP12-FK520-calcineurin ternary complex. Additionally, FK520 accelerates the rate of nerve regeneration and encourages neurite outgrowth. [2] By blocking this bifunctional protein's chaperone activity, ascomycin exhibits antimalarial effects. [3]

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FR-900520 and FR-900523, novel neutral macrolide immunosuppressants, were isolated from the cultured broth of Streptomyces hygroscopicus subsp. yakushimaensis No. 7238. Their molecular formulae were determined as C43H69NO12 and C42H67NO12, respectively. The compounds suppressed immune response in vitro. IC50 values of FR-900520 and FR-900523 for mouse mixed lymphocyte reaction were 0.55 nM and 1.6 nM, respectively. [1]

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The polyketides FK506 (tacrolimus) and FK520 (ascomycin) are potent immunosuppressants that function by inhibiting calcineurin phosphatase through formation of an FKBP12-FK506/520-calcineurin ternary complex. They also have calcineurin-independent neuroregenerative properties in cell culture and animal models of nervous system disorders. Based on the crystal structure of the FKBP12-FK506-calcineurin complex, we deduced that the 13- and 15-methoxy groups of FK506 or FK520 are important for inhibition of calcineurin phosphatase but not for binding to FKBP12. By genetic modification of the FK520 gene cluster, we generated 13- and 15-desmethoxy analogs of FK520 that contain hydrogen, methyl, or ethyl instead of methoxy at one or both of these positions. These analogs bind FKBP12 tightly, have decreased calcineurin phosphatase inhibition and immunosuppressive properties, and enhance neurite outgrowth in cell cultures. [2]

Ascomycin (3.2 mg/kg, i.m.) clearly prolongs skin allograft survival in rats. [1] Infusion of ascomycin into the rat hippocampus at concentrations of 50 or 100 μM has an anticonvulsant effect against picrotoxin-induced seizures. [4]
The potential in vivo anticonvulsant effect of calcineurin (protein phosphatase 2B) inhibitor ascomycin against seizures induced by intrahippocampal microdialysis of picrotoxin was examined in the present study. After establishing individual picrotoxin seizure thresholds, ascomycin was continually microperfused into the rat hippocampus through microdialysis probes at concentrations 10, 50 and 100 microM. No behavioral or electroencephalographic effects were observed during microperfusion of ascomycin alone. Low concentrations (10 microM) of ascomycin did not prevent picrotoxin seizures, however, 50 and 100 microM ascomycin showed antiepileptic effect, completely suppressing seizures in 41.7% and 75% of the animals studied respectively. Mean seizure duration and mean number of seizures were significantly reduced (P < 0.01) by microperfusion of 100 microM ascomycin. Calcineurin activity might be involved in the biochemical changes leading to picrotoxin-induced epileptic seizures. The present findings provide additional in vivo evidence of the involvement of phosphorylation/dephosphorylation mechanisms in the development of epileptic seizures, suggesting that calcineurin modulation may be a possible strategy in the search for new anticonvulsant drugs[4].

PPIase assay [3]
Recombinant maltose binding protein (MBP)–PfFKBP35-His6 was produced in E. coli and purified to homogeneity by sequential nickel-chelate and ion-exchange chromatographies, as described elsewhere. Its PPIase activity in the absence or presence of drugs was assessed by use of a standard protease-coupled assa. Briefly, the cis-trans conversion of a chromogenic peptide substrate, which is cleaved by chymotrypsin only in its trans conformation, was measured spectrophotometrically. The concentration of enzyme was 0.25 μmol/L, the assay buffer consisted of 50 mmol/L HEPES and 100 mmol/L NaCl (pH 8.0), and the assay temperature was 0°C (to minimize the nonenzymic background isomerization). In assays in which drugs were included, they were added as 1-μL volumes of 1000 times the desired concentration (final concentration, 0.05–5 μmol/L), prepared in DMSO. One microliter of solvent alone served as a control. The IC50 values of PPIase activity were determined graphically from the respective dose-response curves

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Chaperone assays [3]
MBP-FKBP-His6 was produced and purified as described elsewhere. The thermal denaturation of pig heart mitochondrial citrate synthase and bovine liver rhodanese was achieved essentially as described elsewhere. Briefly, citrate synthase (1.5 μmol/L monomer) was incubated at 43°C in 40 mmol/L HEPES (pH 7.5) for 30 min, and aggregation during the denaturation process was measured by monitoring the increase in absorbance at 360 nm in a Shimadzu UV-1601PC spectrophotometer with a thermostatted cuvette holder, by use of a quartz microcuvette. Rhodanese (4.4 μmol/L) was incubated at 44°C in 40 mmol/L sodium phosphate (pH 8.0) for 30 min, and aggregation was monitored as for citrate synthase. The effects of additional components on aggregation were assessed as described in Results.

Growth-inhibition assays [3]
To assess the effects of ascomycin/FK520 and its analogues on cultured P. falciparum asynchronous parasitized human erythrocytes at 0.8% parasitemia and 2% hematocrit were grown, for 72 h, in RPMI 1640 culture medium supplemented with the appropriate compound in 96-well flat-bottom microtiter plates. Drugs were diluted from stock solutions in DMSO into culture medium and then serially diluted 2-fold, in wells of the microtiter plates, down to subinhibitory concentrations. After incubation, the effect of the compounds on parasite growth was determined by use of the parasite lactate dehydrogenase–based assay of Makler et al. Dose-response curves were constructed for each drug. The IC50 values were determined graphically from the respective dose-response curves.

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Metabolism of compounds by cultured parasites [3]
The ability of parasites to metabolize 18-ene-20-oxa-FK520 and 13-dM(Me)-18-ene-20-oxa-FK520 into other forms was assessed by treating P. falciparum cultures with 5 μmol/L of these compounds for 48 h (∼IC30). Cultures were transferred to microfuge tubes and centrifuged, and the pellets were frozen at −70°C in preparation for analysis by high-resolution mass spectrometry, as described elsewhere. Cells treated identically but exposed to ascomycin/FK520 or 13-dM(Me)-FK520 served as controls.

Recipient WKA rats transplanted with F344 skin allografts.
~32 mg/kg 5 days a week.
i.m.

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Ascomycin was dissolved in Ringer and perfused continuously throughout the experiment in all the animals on different days at 10, 50 and 100 μM concentrations in a random order, following the same protocol for Ringer's solution and picrotoxin administration in the control experiments (Table 1). Each dose was administered once in each animal with resting periods between experiments of at least one week during a total period of 2–3 months.
Threshold control experiments were performed on all animals to ensure that no permanent modification had been induced in the duration or number of seizures using the same picrotoxin dose. After finishing ascomycin administration, frequent 3 h EEG controls (2–3 times a week,) with simultaneous video recording were performed in all animals without probe introduction, in order to monitor possible long-term effects of ascomycin and the picrotoxin/ascomycin combination.

5282071 mouse LD intraperitoneal >100 mg/kg Journal of Antibiotics, Series A., 15(231), 1962
5282071 mouse LD intraperitoneal >100 mg/kg Journal of Antibiotics., 41(1592), 1988 [PMID:2461926]

[1]. J Antibiot (Tokyo). 1988 Nov;41(11):1592-601.

[2]. J Pharmacol Exp Ther. 2002 Sep;302(3):1278-85.

[3]. J Infect Dis. 2005 Apr 15;191(8):1342-9.

[4]. Pharmacol Biochem Behav. 2006 Jul;84(3):511-6.

Ascomycin is a macrolide that is produced by the fermentation of Streptomyces hygroscopicus and exhibits strong immunosuppressant properties. It has a role as an immunosuppressive agent, an antifungal agent and a bacterial metabolite. It is a macrolide, an ether, a lactol and a secondary alcohol.
Ascomycin has been reported in Streptomyces ascomycinicus, Streptomyces hygroscopicus, and Streptomyces clavuligerus with data available.
See also: ... View More ...

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The polyketide macrolactone FK506 inhibits the growth of Plasmodium falciparum in culture and the enzymatic (peptidyl-prolyl cis-trans isomerase [PPIase]) and chaperone activities of a recently identified P. falciparum FK506-binding protein (PfFKBP35). However, the potent immunosuppressive properties of FK506 exclude it from consideration as an antimalarial drug. We describe the antimalarial actions of the related compound FK520 and a number of its nonimmunosuppressive analogues. All compounds were shown to be strong inhibitors of parasite growth, regardless of their immunosuppressive potency. Although some of the compounds inhibited the PPIase activity of recombinant PfFKBP35, they all inhibited the chaperone activity of this bifunctional protein. These findings suggest that the antimalarial effects of this class of drug may be mediated via inhibition of the chaperone activity rather than via the enzymatic activity of PfFKBP35. Elucidating the precise intracellular functions of PfFKBP35 may facilitate the design of more potent inhibitors that retain their specificity for parasite target protein. [3]

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It is possible, however, that the effects of the compounds are not mediated directly through inhibition of the chaperone activity of PfFKBP35 per se but, rather, through an indirect effect caused by loss of activity of a specific, essential parasite protein whose activity is dependent on PfFKBP35. For example, hFKBP52 has been implicated in the targeted movement of steroid receptors to their sites of action in the nucleus. The C-terminal region of hFKBP52, which contains 3 TPR motifs, directs its association with the steroid receptor heterocomplex, whereas its N-terminal PPIase domain directs its interaction with dynein, the microtubule-associated motor protein involved in retrograde transport. Interestingly, the interaction of the hFKBP52 PPIase domain with dynein is independent of hFKBP52’s intrinsic PPIase activity. This arrangement of an N-terminal PPIase domain followed by a C-terminal tripartite TPR domain is strikingly similar to the domain architecture of PfFKBP35. Work in our laboratory is currently focused on identifying intracellular binding partners for PfFKBP35, because this may well hold the key to elucidating the role of the protein in the parasite and the precise mechanism of action of these drugs. By further dissecting the mode of action of this class of drugs (18-ene-20-oxa-FK520 and 13-dM(Me)-18-ene-20-oxa-FK520 in particular), more potent derivatives could be designed that retain their specificity for the parasite protein. [3]

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This study reports on the previously uninvestigated in vivo effect of ascomycin on picrotoxin-induced seizures. We have found that ascomycin shows anticonvulsant effect against picrotoxin seizures when perfused into the rat hippocampus at 50 and 100 μM concentrations. No effects were observed with a 10 μM dose. Previous studies have been performed in order to determine the pro- or anticonvulsant effect of several calcineurin inhibitors (Moia et al., 1994, Suzuki et al., 2001, Sanchez et al., 2005), however, they have been limited by the complications of systemic administration such as blood–brain barrier transport and brain tissue distribution. The present study is the first to use direct application to the hippocampus of awake rats, thus allowing the maximum inhibitor concentration at the seizure focus rather than distributed over the large brain areas. Continuous microperfusion permits also to keep a steady extracellular ascomycin concentration over the microdialysis period, minimizing the effect of individual differences in absorption and clearance of the enzyme inhibitor. However, the possibility of chemical interaction among ascomycin and picrotoxin will have to be excluded in further research. [4]

C43H69NO12

792.01

791.481

C, 65.21; H, 8.78; N, 1.77; O, 24.24

104987-12-4

104987-11-3 (tacrolimus free base); 109581-93-3 (tacrolimus hydrate);11011-38-4 (Ascomycin)

5282071

White to off-white solid powder

1.2±0.1 g/cm3

868.3±75.0 °C at 760 mmHg

153-157ºC

478.9±37.1 °C

0.0±0.6 mmHg at 25°C

1.546

3.81

3

12

6

56

1430

14

O1[C@]2(C(C(N3C([H])([H])C([H])([H])C([H])([H])C([H])([H])[C@@]3([H])C(=O)O[C@]([H])(/C(/C([H])([H])[H])=C(\[H])/[C@]3([H])C([H])([H])C([H])([H])[C@]([H])([C@@]([H])(C3([H])[H])OC([H])([H])[H])O[H])[C@]([H])(C([H])([H])[H])[C@]([H])(C([H])([H])C([C@]([H])(C([H])([H])C([H])([H])[H])C([H])=C(C([H])([H])[H])C([H])([H])[C@]([H])(C([H])([H])[H])C([H])([H])[C@@]([H])([C@]1([H])[C@]([H])(C([H])([H])[C@@]2([H])C([H])([H])[H])OC([H])([H])[H])OC([H])([H])[H])=O)O[H])=O)=O)O[H] |c:77|

ZDQSOHOQTUFQEM-NURRSENYSA-N

InChI=1S/C43H69NO12/c1-10-30-18-24(2)17-25(3)19-36(53-8)39-37(54-9)21-27(5)43(51,56-39)40(48)41(49)44-16-12-11-13-31(44)42(50)55-38(28(6)33(46)23-34(30)47)26(4)20-29-14-15-32(45)35(22-29)52-7/h18,20,25,27-33,35-39,45-46,51H,10-17,19,21-23H2,1-9H3/b24-18+,26-20+/t25-,27+,28+,29-,30+,31-,32+,33-,35+,36-,37-,38+,39+,43+/m0/s1

(1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-17-ethyl-1,14-dihydroxy-12-[(E)-1-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]prop-1-en-2-yl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetrone

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)

Solubility in Formulation 1: 2.5 mg/mL (3.16 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (2.63 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (2.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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