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 Intraperitoneal LD50 in mice >100 mg/kg, Journal of Antibiotics, Series A, 15(231), 1962
5282071 Intraperitoneal LD50 in mice >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 antibiotic produced by the fermentation of Streptomyces hygroscopicus and possesses potent immunosuppressive properties. It can be used as an immunosuppressant, antifungal agent, and bacterial metabolite. It is a macrolide antibiotic, ether antibiotic, lactone antibiotic, and secondary alcohol antibiotic. Ascomycin has been reported to exist in Streptomyces ascomycinicus, Streptomyces hygroscopicus, and Streptomyces clavuligerus, and relevant data are available. See also: ... See more ...

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Polyketide macrolide FK506 inhibits the growth of Plasmodium falciparum in culture and inhibits the enzymatic activity (peptidyl prolyl cis-trans isomerase [PPIase]) and molecular chaperone activity of the recently discovered Plasmodium falciparum FK506-binding protein (PfFKBP35). However, the potent immunosuppressive properties of FK506 make it unsuitable as an antimalarial drug. We describe the antimalarial effects of related compound FK520 and some of its non-immunosuppressive analogues. All compounds exhibited potent inhibitory effects on parasite growth, regardless of their immunosuppressive potency. Although some compounds inhibited the PPIase activity of recombinant PfFKBP35, they all inhibited the molecular chaperone activity of this bifunctional protein. These findings suggest that the antimalarial effects of such drugs may be achieved by inhibiting molecular chaperone activity rather than the enzymatic activity of PfFKBP35. Elucidating the precise intracellular function of PfFKBP35 may help in designing more effective inhibitors that are specific to parasite target proteins. [3] However, the effects of these compounds may not be achieved directly by inhibiting the molecular chaperone activity of PfFKBP35, but rather indirectly by inhibiting the activity of a specific essential parasite protein that is dependent on PfFKBP35. For example, hFKBP52 is involved in the targeted transport of steroid receptors to their sites of action in the nucleus. The C-terminal region of hFKBP52 contains three TPR motifs that guide its binding to a heterologous steroid receptor complex, while its N-terminal PPIase domain guides its interaction with dynein (a microtubule-associated motility protein involved in retrograde transport). Interestingly, the interaction between the hFKBP52 PPIase domain and dynein is independent of the hFKBP52's own PPIase activity. This arrangement of the N-terminal PPIase domain and the C-terminal ternary TPR domain is remarkably similar to the domain architecture of PfFKBP35. Our lab's current research focus is on identifying the intracellular binding chaperones of PfFKBP35, as this may be key to elucidating the protein's role in parasites and the precise mechanisms of action of these drugs. By further elucidating the modes of action of these drugs (especially 18-ene-20-oxa-FK520 and 13-dM(Me)-18-ene-20-oxa-FK520), more effective derivatives can be designed while maintaining their specificity for parasite proteins. [3]

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This study reports the in vivo effects of asscomycin on cucurbitacin-induced seizures, an effect previously unexplored. We found that asscomycin had an anticonvulsant effect on cucurbitacin-induced seizures when perfused into the hippocampus of rats at concentrations of 50 and 100 μM. No effect was observed at a dose of 10 μM. Previous studies have aimed to determine the proconvulsant or anticonvulsant effects of several calcineurin inhibitors (Moia et al., 1994; Suzuki et al., 2001; Sanchez et al., 2005), but these studies have been limited by the complexities of systemic administration, such as blood-brain barrier transport and brain tissue distribution. This study is the first to use direct administration to the hippocampus of conscious rats, thereby maximizing the inhibitor concentration at the epileptic focus rather than distributing it over a larger brain region. Continuous microperfusion also maintained a stable extracellular asscomycin concentration during microdialysis, minimizing the impact of individual differences in enzyme inhibitor uptake and clearance. However, further investigation is needed to rule out the possibility of chemical interactions between asscomycin and cucurbitacin. [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.)