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PubMed1984

Characterization of the estrogen receptor and its dynamics in MCF-7 human breast cancer cells using a covalently attaching antiestrogen.

We have used a covalently attaching antiestrogen, tamoxifen aziridine [TA; (Z)-(1-[4-(2-[N-aziridinyl] ethoxy)phenyl])1,2-diphenyl-1-butene], to analyze the structure and dynamics of the estrogen receptor in MCF-7 human breast cancer cells. The labeling of receptor with [3H]TA is specific, being blocked only by estrogens and antiestrogens, and the labeling is very efficient in that TA labels covalently the same number of receptors that are labeled reversibly by estradiol. In cells exposed to [3H]TA for 1 h, most of the covalently associated radioactivity is found in the 0.6 M KCl extract of the nuclear fraction; this receptor has an apparent mol wt of 63,000 +/- 2000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.7 by gel isoelectric focusing in the presence of 8 M urea. The mol wt and pI of cytosol receptor labeled with [3H] TA are identical. In cells labeled with [3H]TA (20 nM) for 1 h and then exposed to a chase of 10(-6) M estradiol, [3H]TA-labeled nuclear receptor disappears with a half-life of 4 h. Analysis of nuclear receptor by sodium dodecyl sulfate-gels during the chase period reveals that this loss reflects a decrease in the 63,000 mol wt species; no significant quantities of lower mol wt TA-labeled fragments are observed in the nuclear, cytosol, or membrane fractions. Affinity labeled receptor interacts with several monoclonal antibodies to MCF-7 estrogen receptor, and it can be purified extensively by immunoadsorbent chromatography. TA has a low affinity (8% that of tamoxifen) for microsomal antiestrogen-binding sites that are distinct from the estrogen receptor, but TA reacts reversibly, rather than covalently, with these sites. The findings of similar mol wt and isoelectric points for soluble cytosol and nuclear extracted receptors under strongly denaturing and disaggregating conditions reveal that nuclear localization of receptor after ligand binding is not associated with major structural alterations in the receptor component labeled by TA. In addition, the receptor, even when occupied by a covalently attached ligand, is rapidly turned over in these cells.

Affinity Labels↗
PubMed1985

Selection and characterization of a breast cancer cell line resistant to the antiestrogen LY 117018.

We have selected and cloned a stable variant of the MCF-7 human breast cancer cell line (LY 2) that is resistant to LY 117018 (LY), a potent antiestrogen that inhibits cell growth at concentrations as low as 10(-10) M. The cell line was selected by increasing the concentration of LY in the growth medium in a stepwise manner from 10(-8) to 10(-6) M as the cells become resistant. LY2 has been cloned in soft agar and carried for over 50 passages with no change in resistance. Other antiestrogens, such as tamoxifen and 40-hydroxytamoxifen no longer inhibit cell proliferation of LY 2. The cell line is still responsive to estrogen in a cell proliferation assay, but contains somewhat less estrogen receptors than MCF-7. The cytosolic estrogen receptor sediments to a 4S position on high salt sucrose density gradient centrifugation and is completely shifted to a denser gradient region when the receptor is incubated with a monoclonal antiestrophilin. The nuclear estrogen receptor when covalently labeled with [3H]tamoxifen aziridine has the same mol wt (62,000) in both MCF-7 and LY2 cells, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a competitive binding assay, LY 117018 competes for [3H]estradiol binding to its cytosol receptor with the same Ki in both MCF-7 and LY2 cells. When the induction of estrogen-specific proteins was examined, no detectable progesterone receptor could be detected in either estrogen-induced or control LY2 cells, in contrast to MCF-7 cells. However, both 52,000- and 160,000-dalton proteins were estrogen inducible in the medium of LY2 and MCF-7 cells, as measured by labeling with [35S]methionine. The phenotypic stability of the antiestrogen resistance in LY2 cells coupled with the cross-resistance the antiestrogens of widely different structures make this cell line an ideal model system for the study of hormone resistance in human breast cancer. In addition, while the mechanism of resistance is currently not elucidated, the selective loss of estrogen-inducible functions in this cell line may provide powerful clues for future study.

Binding, Competitive↗
PubMed1986

Dynamics of estrogen receptor turnover in uterine cells in vitro and in uteri in vivo.

The rate at which the estrogen receptor (ER) is synthesized and degraded (turns over) in target cells is a major factor regulating the levels of ER and cell sensitivity to estrogen. We have used the ability of tamoxifen aziridine [(TAZ)(Z)-1-[4-(2-[N-aziridinyl]ethoxy)phenyl]1,2-diphenyl-1-buten e] to affinity label the ER in intact cells to study the dynamics of ER turnover in uterine cells in vitro and in intact rat uteri in vivo. In primary cultures of rat uterine cells (from 21-day-old rats), ER is degraded with a half-life of 3-4 h, as determined by pulse-chase experiments in which ER in cells are covalently labeled with [3H]TAZ (20 nM) and then exposed to a chase of 10(-6) M estradiol. Density shift experiments, in which uterine cells are exposed for different time periods to medium containing dense (15N,13C,2H) amino acids and the shift of receptor from a normal density to a more dense species is analyzed on sucrose gradients, also confirm this rapid turnover for the uterine ER. A similar half-life is obtained for ER in rat uteri in vivo, after intralumenal installation of [3H]TAZ to label effectively ER covalently. Analysis of nuclear ER on sodium dodecyl sulfate-polyacrylamide gels during the chase period reveals the loss of the 65,000 mol wt [3H]TAZ-labeled receptor species; no lower mol wt [3H]TAZ-labeled fragments are observed. Likewise, immunoblot analyses of receptor with the ER monoclonal antibody H222Sp gamma reveal only the 65,000 mol wt receptor species, with no evidence of any smaller receptor forms in cells. Interestingly, incubation of uterine cells with 10(-5) M cycloheximide (which inhibits 98% of protein synthesis) completely arrests ER turnover, suggesting that ER turnover, which is rapid, is probably dependent upon the synthesis of other rapidly turning over proteins. Hence, we conclude that ER turnover is rapid in uterine cells, that turnover of uterine ER occurs at a rate similar to that previously reported for ER turnover in MCF-7 human breast cancer cells, and that ER turnover is similar in uterine cells under in vitro and in vivo conditions. These findings indicate that the receptor is a dynamic and rapidly turning over protein whose level could be adjusted quickly by alterations in the rate of synthesis and/or degradation.

Affinity Labels↗
PubMed1987

Extensive in situ activation of nuclear estrogen receptors after exposure of murine uteri to [3H]estradiol or [3H]4-hydroxytamoxifen.

Estrogen receptor activation has been examined in murine uteri by characterizing binding to ATP-Sepharose. Determinations were performed under conditions in which specific binding to estrogen receptors was demonstrated by both agonist and antagonist without participation by nonreceptor antiestrogen-binding components. Cell-free activation of estrogen receptors in cytosol was more effectively promoted by [3H]estradiol estradiol than by [3H]4-hydroxytamoxifen or [3H]tamoxifen aziridine. However, when in situ activation was examined after intact uteri were exposed to [3H]estradiol or [3H]4-hydroxytamoxifen, virtually all extracted nuclear receptors demonstrated activated binding to ATP-Sepharose within 20 min of hormone exposure. Profiles of nuclear receptor activation were remarkably similar after exposure to either agonist or antagonist. Estrogen receptors in cytosol prepared after exposing intact uteri to 3H-labeled ligands were characterized by much less ability to bind to ATP-Sepharose than nuclear receptors. After uteri were exposed to [3H]estradiol, the activated receptor fraction in the cytosol progressively increased in contrast to preparations obtained after uteri were exposed to [3H]4-hydroxytamoxifen, which demonstrated a constant level of activation. Thus, even when activation has occurred within the intact uterus, agonists and antagonists may be characterized by different apparent levels of receptor activation in the cytosol fraction. These differences in the cytosol, however, are considerably overshadowed by the extensive activation occurring within the nuclear fraction, which we have observed to be similar with agonist and antagonist. Since estrogen receptors appear to act within chromatin, and cytosol receptors may be produced by receptor redistribution during preparation, we interpret these observations to indicate that in situ receptor activation is very similar after exposure to either agonist or antagonist. Consequently, antagonism does not appear to be associated with antiestrogens that impede receptor activation within intact murine uteri.

Animals↗
PubMed1987

Purification and characterization of mouse uterine estrogen receptor under conditions of varying hormonal status.

Mouse uterine estrogen receptor (ER) was purified about 11,000-fold from normal mouse uteri by affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified ER demonstrated a major component of 65,000 mol wt with minor fragments of the 54,000- and 37,000-dalton species, as judged by affinity labeling with [3H]tamoxifen aziridine and immunodetection with an ER monoclonal antibody (H-222). The minor fragments were not detected with additional monoclonals (H-226 or D-547), which recognize different domains on the ER molecule. Two-dimensional gel electrophoresis revealed that the major component with a mol wt of 65,000 had a pI of about 6.5. The 54,000- and 37,000-dalton components had similar pI values. Saturation binding and Scatchard plot analysis of purified ER yielded one class of binding sites with an apparent dissociation constant of about 1.4 nM. Changes in the hormonal status resulted in changes in the size of the ER even in the presence of molybdate and leupeptin.

Animals↗
PubMed1991

Estrogen-like activity of a subpopulation of natural antiestrogen receptor autoantibodies in man.

We recently reported that a subpopulation of immunoglobulin G (IgG) in man interacts with the hormone-binding site of estrogen receptors (ER), competes with [3H]estradiol (E2) uptake, and decreases effective ER concentrations in cell cultures. The present work further characterizes the immunological properties of these antibodies and defines their biological activity. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques, enriched preparations of the natural anti-ER IgG subpopulation (IgGs) were found to specifically immunoprecipitate ER extracted from MCF-7 mammary carcinoma cells and to compete with [3H]tamoxifen-aziridine for ER binding. During 18-h incubations IgGs decreased [3H]E2 binding capacity of MCF-7 cells in a dose-dependent manner similar to E2. Like E2 but unlike antiestrogens, this biological effect corresponded to down-regulation of the receptor protein and depended on a mechanism specifically inhibited by actinomycin D. Moreover, IgGs antagonized the decrease of [3H]E2 binding capacity produced by the strong antiestrogen methyl-hydroxytamoxifen; this antagonism was additive to that of E2. On the other hand, IgGs like estrogens increased progesterone receptor concentrations and cathepsin D secretion. The biological activity of IgGs was neutralized by anti-IgG antibodies and by ICI 164,384, a "pure" steroid antagonist of E2, confirming that immunoglobulins G were responsible for this activity and acted at the E2-binding site. These observations indicate that some natural antibodies in man can function like potent estrogens on ER and mammary cells.

Autoantibodies↗
PubMed1992

Multiple forms of affinity-labeled estrogen receptors in rat distinct pituitary cells.

The presence of multiple monomeric forms has been described for estrogen receptor (ER) in different target tissues. Using [3H]tamoxifen aziridine ([3H]TA) to covalently label ER and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze labeled products, ER forms were investigated in pituitary cytosol and purified nuclei from male rats. ER forms were also compared in cellular extracts from gonadotrope-enriched populations (GP), prepared using the fast method of centrifugal elutriation, and from lactotrope-somatotrope fractions (LSP), obtained by sequential use of both elutriation and Percoll gradient sedimentation. A major labeled protein of 60,000-65,000 mol wt (M(r)) and a minor species of 50,000-55,000 M(r) were found in the pituitary cytosol and nuclear extracts covalently labeled with [3H]TA. The same results were obtained after ER covalent labeling from cellular extracts or intact dispersed cells. In gonadotrope-enriched cell population (greater than or equal to 50% LH-immunoreactive cells), the 65,000 M(r) species is the single unique ER form; in the LSP (80% PRL- and GH-immunoreactive cells), the major TA-labeled species is the 50,000 M(r) form, while the 65,000 M(r) ER is hardly detectable. Thus, the prevalence of 65,000 M(r) protein in the initial cell population can be explained by the higher number of binding sites per gonadotrope than per lactotrope cell. In conclusion, ER heterogeneity is demonstrated in pituitary cell populations. The source of this heterogeneity could be due to 1) different ER mRNAs according to cell type, or 2) a specific posttranslational processing, such as proteolytic activity within lactotrope cells.

Animals↗
PubMed1992

Identification of two cysteines closely positioned in the ligand-binding pocket of the human estrogen receptor: roles in ligand binding and transcriptional activation.

We have previously identified cysteine 530 in the human estrogen receptor (ER) as the major site of attachment for covalently binding affinity ligands and have shown that when this cysteine is mutated to alanine (C530A mutant), the affinity ligand [tamoxifen aziridine (TAZ)] can still bind covalently to the ER, presumably by interaction with a different cysteine(s) in the hormone-binding domain (HBD). Using site-directed mutagenesis, we have determined the alternative ligand attachment site and the functional importance of the cysteines (residues 381, 417, 447, and 530) in the HBD of the ER to the hormone-binding and transcriptional responses to estrogens and antiestrogens. Cysteine 530 plus one or more of these other cysteines were mutated to alanines. Analysis of these mutant ERs expressed in Chinese hamster ovary cells provides strong evidence that cysteine 381 is the residue that is preferentially covalently labeled by TAZ in the C530A mutant. Hence, portions of the HBD that are far apart in the linear receptor sequence, namely regions near C381 and C530, are probably closely positioned in the ligand-binding pocket, with the cysteine thiols being 1.1 nm or less apart. The affinity of estradiol binding to receptors was reduced only 2- and 5-fold, respectively, in the double and quadruple Cys to Ala mutants, and estradiol was an effective stimulator of transcription from an estrogen-responsive reporter gene [(ERE)2-TATA-CAT].(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Molecular characterization by mass spectrometry of the human estrogen receptor ligand-binding domain expressed in Escherichia coli.

The ligand binding domain of the human estrogen receptor (hER-LBD), encompassing the sequence MDPS282AG...V595, has been expressed at high levels in Escherichia coli from a pET-23d vector, and a purified preparation has been characterized both by mass spectrometry and biochemical methods. Inclusion bodies from the bacterial expression were solubilized by sonication and the hER-LBD was purified to near homogeneity by affinity chromatography over an estradiol-Sepharose column in urea-containing buffer. This material ran as a single peak on reversed-phase HPLC, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band with apparent molecular mass of 31-32 kilodaltons (kDa), somewhat smaller than that expected from the construct (35.6 kDa). Edman degradation revealed a single sequence of MDPSAGDMRA, consistent with an intact N terminus. Further characterization of this material using low resolution matrix-assisted laser desorption ionization mass spectrometry indicated an apparent single protein species of average mass 33,143 daltons (Da). Detailed molecular analysis by electrospray ionization mass spectrometry, however, revealed that this purified hER-LBD preparation was actually composed of a number of both modified and unmodified LBD protein fragments between 32,900-33,400 Da. The bulk of this 33-kDa protein mixture consisted of three LBD protein fragments with C termini at Ala571 (70%), Ala569 (23%), and Ser575 (4%), with only a trace amount (3%) of the full length expressed LBD (MDPS282...V595; 35, 602 Da). These four protein species also appear to be partially chemically modified by carbamylation. The functional integrity of this hER-LBD preparation was investigated by a ligand-exchange assay, which showed that the hER-LBD retained full estradiol-binding capacity; specific, covalent labeling was also observed using either the electrophilic affinity-labeling ligand tamoxifen aziridine (TAZ) or the photoaffinity-labeling ligand hexestrol diazirine. Thus, this expressed hER-LBD preparation, while appearing nominally pure by conventional biochemical techniques and having the expected ligand-binding capacity, was shown by sensitive high resolution electrospray ionization mass spectrometry techniques to be largely truncated 20-26 amino acids from the expected C terminus and to have a substantial level of covalent modification arising from the urea.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2002

Syntheses of 1beta-methylcarbapenems bearing 5-methyl-4-hydroxypyrrolidinone.

A new series of 1beta-methylcarbapenems 1a-d bearing 5-methyl-4-mercaptopyrrolidinone rings has been prepared and evaluated for in vitro antibacterial activity and pharmacokinetic parameters. Most compounds showed excellent antibacterial activity and high stability to dehydropeptidase-1. We have synthesized optically active 5-methyl-4-hydroxypyrrolidinones from enantiomerically pure aziridine esters.

Anti-Infective Agents↗
PubMed2005

Induction of apoptosis and cell cycle arrest by imexon in human pancreatic cancer cell lines.

Imexon is an aziridine-containing small molecule currently in Phase I clinical trials. This agent has been shown to bind to thiols and increase intracellular oxidants, inducing apoptosis in hematologic cancer cells. Pancreatic cancers are known to be sensitive to oxidation, suggesting this disease may be an appropriate target for this agent. The current report examines the activity of imexon in pancreatic cells. Imexon induced concentration-dependent and time-dependent apoptosis in a panel of six human pancreatic carcinoma cell (PCC) lines. The mean IC50 (SD) for growth inhibition by the SRB assay was 200 (101) microM for a 48 h exposure with a range of 64-358 microM. Cell killing was schedule-dependent, favoring exposure times > or =48 h. Imexon-treated MiaPaCa-2 cells underwent non-lethal growth arrest following exposure to concentrations < or =200 microM for 48 h. When concentrations were increased to 300 microM for > or =48 h, the MiaPaCa-2 cells arrested in G2 phase and activated caspases 3, 8, and 9 were detected. After a 72 h exposure to the IC80 concentration of imexon, cells exhibited a loss of mitochondrial membrane potential detected by CMXRos staining. However, there was no loss of reduced cellular thiols unless very high concentrations of > or =400 microM were used. In contrast, reactive oxygen species (ROS) were elevated in a dose-dependent fashion, starting at very low imexon concentrations. Imexon also significantly inhibited MiaPaCa-2 tumor growth in SCID mice at 100 mg/kg/d for 9 d. The tumor growth inhibition (% T/C) was 27% of control, and the tumor growth delay was 21 d, indicating an active agent by NCI standards. The levels of imexon that are cytotoxic in human PCC's are achievable based on the preliminary results of the ongoing Phase I trial. Imexon appears to be active against PCCs in vitro and has an entirely novel mechanism of action involving G2 arrest, accumulation of ROS, and the induction of apoptosis.

Apoptosis↗
PubMed1989

Synthesis of thiol-functionalized N-acetylmuramyl peptide congeners suitable for their conjugation to target molecules.

N-Acetylmuramyl-dipeptide and tripeptide derivatives containing at the C-terminus a masked thiol function, i.e. the S-tert-butylthiocysteamine residue, were synthesized via direct condensation of N-acetylmuramic acid with the peptide moiety using the dicyclohexylcarbodiimide/N-hydroxysuccinimide procedure. Reduction with tributylphosphine in aqueous organic media generates the free thiol function for a selective conjugation of these immunomodulants with target molecules via unsymmetrical disulfide bridging with a second thiol group by the sulfenohydrazide procedure or via thio-ether linkage by the addition to maleimido--or aziridine-derivatives.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1993

The nature and significance of multiple isoforms of the oestrogen receptor in breast tumours.

Oestrogen receptors (ERs) in breast tumours are highly heterogeneous. In previous studies we have shown that at least four isoforms may exist. These migrate in isoelectric focusing (IEF) gels to isoelectric points (pI values) 6.1, 6.3, 6.6 and 6.8. Of these the first (pI 6.1) corresponds to the 8S isoform as detected by sucrose gradient fractionation, while the others all sediment at 4S. In a series of 66 breast tumours it was found that those at pI 6.3 and pI 6.8 were significantly correlated with the presence of progesterone receptors. To characterize the isoforms more fully, ER isoforms labelled by [3H]oestradiol binding were fractionated by IEF. The results were compared with those obtained after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the H222 anti-ER monoclonal antibody. In other experiments, tumour ER isoforms were covalently labelled with [ring-3H] tamoxifen aziridine and separated by IEF. The individual isoforms were electroeluted from the IEF gel and further analysed by SDS-PAGE and non-denaturing PAGE. In summary, the evidence shows that the isoforms of pI values 6.3, 6.8 and 6.6 have molecular masses of 50, 65 and 70 kDa respectively. In addition, all three of these isoforms, i.e. the pI 6.3, 6.8 and 6.6 isoforms, could form dimers. We conclude that the three isoforms sedimenting at 4S have the capacity to form dimers and thus may have the potential for binding to oestrogen response elements in the genome.

Blotting, Western↗
PubMed1992

Modulatory effect of nonesterified fatty acids on structure and binding characteristics of estrogen receptor from MCF-7 human breast cancer cells.

Arachidonic, docosahexaenoic and oleic acids were found to produce a dose-dependent cytotoxic effect on MCF-7 cells; palmitic and stearic acids were totally ineffective in this regard suggesting that solely unsaturated fatty acids were able to arrest mammary tumor cell growth. Similarly, only former acids were able to decrease the binding capacity and affinity (increase Kd value) of the cells for 3H-E2 in a dose-dependent manner. In the case of arachidonic acid (the reference fatty acid), this decrease was associated with a slight cleavage of the native 67 KDa estrogen receptor (ER) into 50 and 25-30 KDa peptides as demonstrated by sequential labeling of high-salt cell extracts with 3H-tamoxifen aziridine, specific immunoadsorption with H-222 anti-ER monoclonal antibody, SDS-PAGE and fluorography. Both, modifications in binding characteristics of ER and cleavage of the native 67 KDa receptor were found to be extremely marked when unsaturated fatty acids were directly added to the high-salt cell extracts. This clear influence on the ER structure was reflected on enzyme immunological assay (EIA) by a reduction of ER immunoreactivity of approximately 50% in presence of arachidonic acid. Our observations are discussed in terms of possible interference of unsaturated fatty acids either through transmembrane modulation of phosphokinases and/or phospholipases implicated in ER mechanism of action, or through an intracellular interaction between ER and these acids acting as second messengers in regulation of cellular functions.

Breast Neoplasms↗
PubMed1997

Decrease of hormone binding capacity of estrogen receptor by calcium.

We have addressed the question as to whether calcium may modify the [3H]estradiol ([3H]E2) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues. Ca++ (0.1-10 mM) always produced an important loss of [3H]E2 binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [125I]Z-17 alpha-(2-iodovinyl)-11 beta-chloromethyl estradiol-17 beta (an compound with very high selectivity for ER) as well as [125I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca(++)-induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [3H]E2 binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca++ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca++ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.

Animals↗
PubMed1980

Synthesis of demethyl derivatives of istamycin A.

4-N,6'-N,3-O-Tridemethylistamycin A0 (9) and 6'N,3-O-didemethylistamycin A0 (15) were synthesized from 3,2',6'-tri-N-benzyloxycarbonyl-3',4'-dideoxyneamine 1,6-carbamate (1) through an aziridine derivative 6 by an analogous procedure employed in the total synthesis of istamycin A0 (19). Acylation of 15 with glycine at the 4-methylamino group gave 6'-N,3-O-didemethylistamycin A (18) having interesting activities especially against pseudomonas, but 4-N,6'-N,3-O-tridemethylistamycin A (12) derived from 9 showed only weak activity. Therefore, the 4-N-methyl group of istamycin A (20) is essential for the antimicrobial activity.

Aminoglycosides↗
PubMed1986

New mitomycin analogs produced by directed biosynthesis.

When the normal fermentation medium for the production of mitomycin C with Streptomyces caespitosus is supplemented with a number of primary amines, two new types of mitomycin analogs described as Type I and Type II are produced. Type I analogs are related to mitomycin C with the amine substitution at position C7 on the mitosane ring. Type II analogs also contain the same substitutions at C7 but the conformation of the mitosane ring is related to mitomycin B having an OH at positions C9a and a methyl substituted aziridine. The products obtained from the supplementation of the medium with methylamine, ethylamine, propylamine, propargylamine and 2-methylallylamine were isolated and characterized. In all cases the Type I analogs are more active in a prophage induction test and against L1210 lymphatic leukemia in mice. A number of other amines have been tested and shown to yield new products that have not yet been isolated. No secondary amines are incorporated.

Animals↗
PubMed1990

The derivation of 1a-demethylmitomycin G from mitomycin C.

Mitomycin G (2) was derived from porfiromycin (10b) in 3 steps via the methanesulfonate (14b) in an overall yield of 39%. On the basis of the established method for the introduction of an exomethylene group in mitomycins with a 9a-methoxy group, the preparation of biologically more important 1a-demethylmitomycin G (5) from mitomycin C (1) was accomplished by the use of a protective acetyl group on the aziridine in an overall yield of 57%. 1a-Demethylmitomycin K (6) was obtained from 5 in a yield of 42%. In a preliminary evaluation of their antitumor activity, compound 5 showed superior activity against sarcoma 180 (sc-ip) to its 1a-methyl congener, i.e., mitomycin G (2).

Animals↗