Antibiotic A23187 Research Articles

Therapeutic Synergy from Combined Inhibition of the SERCA Channel and MAPK Signaling Pathway in MAPK-Dependent Leukemia

BackgroundThe generation and maintenance of electrochemical gradients by ion transporters are vital in almost all cellular processes. Ion transporters are often dysregulated in cancer cells and play important roles in cancer cell function. For example, aberrant expression of voltage gated potassium channels Kv10.1 (Eag1) and Kv11.1, (hERG) have been associated with increased proliferation and worse prognosis in hematological malignancies. Targeting ion transporters may thus offer a novel therapeutic strategy against cancer. To this end, we identified ion transporters that when inhibited, result in selective toxicity in hematological malignancies with a focus on acute myeloid leukaemia (AML).Method and ResultsIon transporter inhibitors exhibiting selective activity against leukaemia/lymphoma cells were identified from screening compound libraries targeting ion transporters (total 126 inhibitors) against 7 leukaemia/lymphoma cell lines (HL-60, Kasumi-1, HuT-78, BC-2, U266B1, K562 and Raji). Eight unique compounds were selected (Antibiotic A-23187; Niguldipine; Ouabain; Penitrem A; SKF-96365; Tetrandrine; Thapsigargin; Triamterene). The most potent compound was thapsigargin, exhibiting toxicity at nano-molar concentrations against leukemia cells (IC50 1.8 nM - 8.4 nM) whilst sparing human umbilical cord blood cells (IC50 20 nM). In AML cell lines dependent on activated MAPK signaling pathway (HL-60, Kasumi-1), the potency of thapsigargin was enhanced when combined with MEK inhibitor trametinib (thapsigargin IC50 0.7 nM - 2.6 nM in presence of trametinib), with significantly increased apoptosis as measured by both caspase 3 activation and Annexin-V surface binding. The therapeutic activity of thapsigargin likely acted through the inhibition of sarco-endoreticulum calcium ATPase (SERCA) channel as similar efficacy against the AML cells could be reproduced by treatment with trametinib combined with other SERCA inhibitors (CPA and BHQ).SERCA is ubiquitously expressed on the endoplasmic reticulum (ER) and functions to sequester cytoplasmic calcium into the ER. SERCA inhibition by thapsigargin is known to trigger ER stress and to activate the unfolded protein response (UPR), which we postulated might underlie the mechanism of activity from combined trametinib/thapsigargin treatment. Compared to human umbilical cord blood cells, inhibition of the MAPK pathway in AML cell lines by trametinib reduced the expression of GRP-78, a major chaperone protein acting both as an initiator and effector of UPR. When trametinib was combined with thapsigargin, the downregulation of GRP-78 was associated with increased level of CHOP, a pro-apoptotic effector downstream of UPR activation. Taken together, these data suggested the downregulation of GRP-78 by trametinib in the AML cells potentially reduces cellular capacity to adapt to the ER stress induced by thapsigargin, thus triggering UPR mediated apoptosis pathway. Indeed, the addition of the chemical chaperone tauro-ursodeoxycholic acid (TUDCA) rescued for the loss of GRP-78 and the toxicity from combined trametinib and thapsigargin treatment.ConclusionInhibition of SERCA channel either alone or in combination with MAPK pathway inhibition was selectively toxic in MAPK-dependent AML cell lines. The combination-induced toxicity resulted from a deficiency in GRP-78 chaperone protein that sensitized the cells to ER stress mediated apoptosis. The combined inhibition of SERCA channel and MAPK signaling may thus offer a novel therapeutic opportunity in MAPK-dependent leukemia. DisclosuresNo relevant conflicts of interest to declare.

ChemInform Abstract: Total Synthesis of Antibiotic A23187 (Calcimycin) from D‐Glucose.

AbstractThe pyranoside (III) serves as a common intermediate for the preparation of the synthons (IV) and (V) (syntheses described).

The role of calcium in the rapid adaptation of an insect mechanoreceptor

The femoral tactile spine of the cockroach is a rapidly adapting mechanoreceptor in which the response to a step displacement is a burst of action potentials lasting about 1 sec. This adaptation seems to occur during the encoding of action potentials from receptor current, since there is no evidence of adaptation in the receptor potential, and the adaptation can be demonstrated by direct electrical stimulation of the single sensory neuron in the spine. We have recently presented evidence for a calcium-activated potassium conductance in the tactile spine receptor neuron, although it was not directly linked to adaptation. Since this conductance could be involved in the rapid adaptation of the tactile spine neuron, we sought an association between adaptation and the entry of calcium ions into the cell. Adaptation in the tactile spine neuron was examined by direct electrical stimulation using a randomly fluctuating current followed by frequency-response analysis. The dynamic behavior was studied as a function of the concentration of calcium ions in the bathing solution, the presence of calcium channel blocking agents, or the divalent cation ionophore antibiotic A23187. No evidence could be found that entry of calcium ions into the cell has a major role in the sensory adaptation.

Total synthesis of antibiotic A23187 (calcimycin) from D-glucose

The fully stereocontrolled synthesis of A23187 by using the chirons derived from D-glucose is described.

INFLUENCE OF OCTOPINE, CALCIUM AND COMPOUNDS THAT AFFECT CALCIUM TRANSPORT ON ZEATIN‐INDUCED BUD FORMATION BY PYLAISIELLA SELWYNII

Decreasing the calcium chloride content of the medium for the moss, Pylaisiella selwynii (Kindb.) Steere and Anderson, reduces the number of developed buds obtained in the presence of zeatin (1 μm), whereas similar changes in magnesium sulfate concentration have no effect. Cobalt (10 μm), lanthanum (10 μm), zinc (10 μm) and cadmium (0.01 μm) chlorides inhibit zeatin‐induced bud formation even in the presence of 680 μm calcium, the normal concentration in the medium. Chlorpromazine (0.1 μm), cyclic adenosine 3′,5′‐monophosphate (100 μm), and glutamate (100 μm) also inhibit zeatin‐induced bud initiation. Ethylene glycol‐bis(β3‐aminoethyl ether)N,N,N′,N′‐tetraacetic acid (1 μm), the antibiotic A23187 (100 μm) and octopine (100 μm) each act synergistically with zeatin in promoting bud development and are generally more effective at calcium concentrations of 6.8 μm or less. The combination of either ethylene glycol‐bis(β‐aminoethyl ether)N,N,N′,N′‐tetraacetic acid, A23817 or octopine with zeatin plus any of the inhibitory metals or compounds results in more buds than are obtained with zeatin alone and these combinations are more effective relative to zeatin response at calcium concentrations lower than 6.8 μm. Experiments with Funaria hygrometrica (L.) Sibth. indicate a similar response to calcium, octopine and glutamate. These results appear to be related through changes in calcium availability at or within moss protonemal cells.

Total Synthesis of Antibiotic A23187 (Calcimycin) from D-Glucose: Communication

Abstract Antibiotic A23187 1 is a cation ionophore highly specific for Ca2+. Because of its unique dioxaspiro ring structure as well as its biological activities,2 several synthetic approaches to the compound have been reported.3 We report here the first example of the total synthesis of based on the use of a carbohydrate template.4 The target structure was synthesized from the chiral synthons or “chirons”5 and via a key intermediate , as shown in Scheme I. A thermodynamically controlled, acid catalyzed ring closure of was expected to provide a correct configuration at the spiro center C-14 according to the anomeric effect.6 The two chirons and were prepared from -glucose via the same intermediate -as follows (Scheme II).

Active Potassium Transport by the Isolated Lepidopteran Larval Midgut: Stimulation of Net Potassium Flux and Elimination of the Slower Phase Decline of the Short-Circuit Current

ABSTRACT Adenosine 3’:5’-cyclic monophosphate (cyclic AMP), Antibiotic A23187, caffeine, cholera toxin, dibutyryladenosine 3’ :5’-cyclic monophosphate (dbcAMP), 5-hydroxytryptamine (5-HT), larval blood, and theophylline were tested for their effects on active potassium transport, as measured by the short-circuit current (SCC), in isolated Manduca sexta larval midgut. None of the tested materials affected the short-circuit current during the rapid phase of its usual exponential decline, and only dbcAMP and larval blood affected the short-circuit current during the slower phase of its exponential decline. Dibutyryl-cyclic AMP stimulated an increase in short-circuit current and net potassium flux (measured using 42K). Both unidirectional potassium fluxes increased but there was no effect on the flux ratio or on the rate at which net potassium transport declined with time. A dialysable extract of fifth instar larval blood effected potassium transport in a manner similar to that of dbcAMP, indicating that larval blood contains natural transport-stimulating factors. A dialysable extract of fourth instar larval blood not only stimulated potassium transport but also prevented the slow phase of decline in the short-circuit current. Therefore, in addition to transport-stimulating factors present in fifth instar blood, fourth instar blood appears to contain substances which prevent degeneration of at least the transport related functions of midgut in vitro. It is argued that the action of the transport-stimulating factor in larval blood on the electrogenic cation pump in larval midgut cells is mediated by cyclic AMP without involving a change in intracellular calcium ion concentration.

The biosynthesis of antibiotic A23187

The biosynthesis of antibiotic A23187 (I) by S. chartreusis was examined using 14C and 13C labeled substrates in conjunction with chemical degradation and 13C NMR. It has been demonstrated that the three district structural units of the antibiotic are derived from proline (the α-ketopyrrole unit), propionate/acetate (diox-aspiro(5,5)undecane ring), glucose metabolism via a shikimate-type pathway (C 7N 2 or diaminobenzyl unit), and the S-methyl group of methionine (methyl amino of the C 7N 2 unit). Unlike antibiotics that contain a C 7N 1 unit, shikimic acid [ 14C] was incorporated efficiently into the antibiotic and apparently specifically into the C 7N 2 unit. The incorporation of D-glucose[1- 13C], [6- 13C] and [U- 13C 6] is also consistent with a shikimate-type pathway to this unit. None of the aromatic compounds tested were incorporated into the C 7N 2 unit, suggesting that the introduction of the nitrogen functionalities in this moiety occurs before or concommitently with aromization of the six membered carbocyclic ring. These results are in accord with the involvement of a unique biosynthetic pathway in the formation of A23187.

Stereocontrolled total synthesis of Antibiotic A-23187 (calcimycin)

Der ausgehend vom Keton (I) zugangliche Alkohol (Va) wird durch Oxidation und Grignardreaktion zu einem trennbaren Gemisch der Vinylderivate (Vb) + (Vc) umgesetzt, die sich beide in mehreren Stufen in den Ester (VIa) bzw. dessen Folgeprodukt (VIb) uberfuh; ren lassen.

Synthetic studies on antibiotic A23187 I. Chiral synthons for C9-C13 and C14-C20

Chiral synthons 3 and 4 for the synthesis of antibiotic A23187 1 were prepared from methyl α- D-glucopyranoside.

Biosynthesis of antibiotic A23187. Incorporation of precursors into A23187.

Biosynthesis of antibiotic A23187. Incorporation of precursors into A23187.

Ionomycin stimulates mast cell histamine secretion by forming a lipid-soluble calcium complex.

The activation of cellular function following the direct introduction of Ca2+ into the cytosol by the use of a Ca2+-transporting ionophore has served to confirm the widely held idea that Ca2+ has the status of a second messenger in many cell types. However, this evidence has been obtained largely from the use of a single ionophore, the antibiotic A23187; experiments with X537A, which is another ionophorous antibiotic capable of transporting Ca2+ (ref. 3), have failed to show the expected characteristics. For example, histamine release from rat mast cells mediated by X537A is neither dependent on extracellular Ca2+ nor prevented by metabolic inhibitors. Ionomycin is a recently described polyether antibiotic produced by Streptomyces conglobatus ATCC 31005, and is active against Gram-positive bacteria. The antibiotic action is presumably due to its ionophorous properties, as it extracts Ca2+ from an aqueous phase into an organic phase with a stoichiometry of 1:1 (ref. 6). The ionophore is also capable of transporting 45Ca2+ across biological membranes (our unpublished results). Here we report the application of ionomycin to rat mast cells. We show that ionomycin stimulates mast cell secretion solely through its ability to form a lipid-soluble calcium complex, and thus to convey Ca2+ across the hydrocarbon region of the cell membrane.

Morphologic Characteristics of the Chemically Induced Acrosome Reaction in Human Spermatozoa

The morphologic changes accompanying the acrosome reaction in human spermatozoa, as it is induced by the antibiotic A23187 and calcium ions, are described. The reaction is shown to be similar to that observed in other species when the reaction occurs spontaneously or is induced by physiologic fluids. The reaction in human spermatozoa differs from the chemically induced reaction in other species in that plasma membrane microfilaments, prominent in the boar, and tubular-like elements prominent in boar, rabbit, and monkey sperm, are not observed. Motility remains high when human spermatozoa are treated with A23187 and calcium and it is possible that these agents may be useful in the study of certain causes of infertility.

ChemInform Abstract: STUDIES DIRECTED TOWARDS THE TOTAL SYNTHESIS OF THE IONOPHORE ANTIBIOTIC A‐23187

Chemischer InformationsdienstVolume 9, Issue 21 Natural Products ChemInform Abstract: STUDIES DIRECTED TOWARDS THE TOTAL SYNTHESIS OF THE IONOPHORE ANTIBIOTIC A-23187 D. A. EVANS, D. A. EVANSSearch for more papers by this authorC. E. SACKS, C. E. SACKSSearch for more papers by this authorR. A. WHITNEY, R. A. WHITNEYSearch for more papers by this authorN. G. MANDEL, N. G. MANDELSearch for more papers by this author D. A. EVANS, D. A. EVANSSearch for more papers by this authorC. E. SACKS, C. E. SACKSSearch for more papers by this authorR. A. WHITNEY, R. A. WHITNEYSearch for more papers by this authorN. G. MANDEL, N. G. MANDELSearch for more papers by this author First published: May 23, 1978 https://doi.org/10.1002/chin.197821346AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume9, Issue21May 23, 1978 RelatedInformation

Studies directed towards the total synthesis of the ionophore antibiotic A-23187

Studies directed towards the total synthesis of the ionophore antibiotic A-23187

Granule release by polymorphonuclear leukocytes treated with the lonophore A23187

Polymorphonuclear leukocytes (PMN's) incubate three to eight minutes at 37 degrees C in medium containing 1 X 10(-6) M of the ionophore antibiotic A23187 released their cytoplasmic granules into the extracellular medium. Transmission electron microscopy of treated cells showed microfilament bundles extending between adjacent granules within the cytoplasm and between granules and the plasma membrane. Tiny dense projections (beads) 8-12 nm in diameter were observed along segments of the cytoplasmic surface of the plasma membrane with a periodicity of 20-30 nm. These beads were observed on the plasma membrane only in the vicinity of intra- or extracytoplasmic granules. The structural relationships of the beads with the plasma membrane microfilaments suggest they play a role in the process of ionophore-induced granule release from polymorphonuclear leukocytes.

The effect of calcium on the respiratory responses of mung bean mitochondria

Purified mung bean hypocotyl mitochondria were examined for their capacity to carry out respiration-dependent accumulation of calcium. The addition of 0.1–1.0 mM calcium to mung bean mitochondria supplemented with succinate gave no stimulation of state 4 respiration even in the presence of inorganic phosphate and the ionophoretic antibiotic A-23187. Even at high calcium concentrations, no transient changes in the respiratory activity occurred and subsequent addition of ADP initiated a further state 3 response. Although the additions of calcium resulted in a rapid H + ejection, it was insensitive to lanthanum and uncoupling agents. Similarly, additions of calcium failed to initiate any transient changes in the oxidation-reduction states of either pyridine nucleotides or cytochrome b. Direct spectrophotometric recordings of absorbance changes of murexide revealed no respiration-linked calcium transport. It is proposed that although mung bean mitochondria possess a respiration-linked electrochemical potential gradient it would appear that this potential cannot be expressed as calcium transport even at high ion concentrations, probably due to a low calcium membrane permeability.

Effects of prostaglandins and oxytocin on calcium release from a uterine microsomal fraction.

A microsomal fraction resembling striated muscle sarcoplasmic reticulum was isolated from uterine smooth muscle. ATP induces calcium accumulation in this fraction. Increased temperature enhances calcium accumulation and calcium-activated ATPase. In the absence of ATP, approximately 35% of the intrinsic calcium exchanges with the 45Ca in the incubation medium. In the presence of ATP, exchange of intrinsic calcium with 45Ca increases by an amount which equals the ATP-dependent calcium binding. In preparations partially preloaded with calcium, a steady state of bound calcium is reached when the ATP is exhausted. Calcium is released under these conditions by prostaglandins E2 and F2alpha, but not by PGF1beta. The antibiotic ionophores X537A and A23187, as well as oxytocin, also release calcium previously accumulated under ATP stimulation. None of these agents, with the exception of oxytocin, release intrinsic calcium. Thus, the effect of prostaglandins resembles that of the ionophores, suggesting an ionophoretic action of these prostaglandins. The release of calcium conforms with the in vivo smooth muscle contracting action of these agents.

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187

The antibiotic A23187 carries Ca 2+ across Müller-Rudin membranes made from 1,2- dierucoyl-sn- glycero-3- phosphocholine and n- decane . The conductance of the membranes is not increased by the Ca 2+-transport. The flux depends linearly on Ca 2+ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4–5 between pH 6 and pH 8. Maximal Ca 2+-fluxes of about 10 −10 mol · cm −2 · s −1 were found. A counter transport of H + could not be detected. The complex formation between A23187 and Ca 2+ in egg phosphatidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2 : 1 complex. Optical absorption measurements on single phosphatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.

Proliferation of human peripheral blood lymphocytes induced by A23187, a streptomyces antibiotic.

Incubation of human peripheral blood lymphocyte cultures with streptomyces antibiotic A23187, a divalent cation ionophore, resulted in an increased rate of calcium uptake, enhanced rates of RNA and DNA synthesis, and lymphoblastic transformation. An optimal response was obtained with an initial ionophore concentration of 3–5 μM. The highest rate of thymidine incorporation was detected when the cells were labelled from the 3rd to 4th day of culture. In long-term culture the ionophore was highly toxic to the lymphocytes and optimal response was detected only if the cells were transferred to fresh medium after incubating for some hours with A23187. Both RNA and DNA synthesis, as well as calcium uptake induced by A23187 were completely inhibited if ethyleneglycol- bis -(aminoethylether)tetraacetic acid (EGTA) was present in the culture during the first 6 h of incubation. These findings support the hypothesis that calcium ion has a critical role in the mitogenic response of lymphocytes, and that calcium influx may be an important event in the initiation of proliferation. Possible mechanisms of the effects of A23187 on lymphocytes are discussed.