Mapping the aspartic acid binding site of Escherichia coli asparagine synthetase B using substrate analogs.

Novel inhibitors of asparagine synthetase, that will lower circulating levels of blood asparagine, have considerable potential in developing new protocols for the treatment of acute lymphoblastic leukemia. We now report the indirect characterization of the aspartate binding site of Escherichia coli asparagine synthetase B (AS-B) using a number of stereochemically, and conformationally, defined aspartic acid analogs. Two compounds, prepared using novel reaction conditions for the stereospecific beta-functionalization of aspartic acid diesters, have been found to be competitive inhibitors with respect to aspartate in kinetic studies on AS-B. Chemical modification experiments employing [(fluorosulfonyl)benzoyl]adenosine (FSBA), an ATP analog, demonstrate that both inhibitors bind to the aspartate binding site of AS-B. Our results reveal that large steric alterations in the substrate are not tolerated by the enzyme, consistent with the failure of previous efforts to develop AS inhibitors using random screening approaches, and that all of the ionizable groups are placed in close proximity in the bound conformation of aspartate.

Rationally designed analogues of tamoxifen with improved calmodulin antagonism.

Computerized molecular modeling studies on the interactions of the antiestrogen tamoxifen (1) and its analogues bound to the calcium-binding protein calmodulin have guided the rational design of more potent antagonists. Compounds with either three or four methylene units in the basic side chain or slim lipophilic 4-substituents were expected to be more potent. All compounds were tested for antagonism of the calmodulin-dependent activity of cAMP phosphodiesterase and for binding affinity to the estrogen receptor from rat uteri. Some compounds were assayed for cytotoxicity against MCF-7 breast tumor cells in vitro. Introduction of lipophilic 4-substituents was accomplished by using palladium(0)-catalyzed coupling reactions with a 4-iodinated precursor. Both the 4-ethynyl (16 and 17) and 4-butyl (18 and 19) compounds were more potent calmodulin antagonists than tamoxifen. Extension of the basic aminoethoxy side chain of 4-iodotamoxifen (3) and idoxifene (2) ((E)-1-[4-[2-(N-pyrrolidino)ethoxy]phenyl]-1-(4-iodophenyl)-2-phen yl-1- butene) by one or two methylene units resulted in modest gains in calmodulin antagonism (10-13). All the compounds assayed retained estrogen receptor binding characteristics. The compound possessing the optimal combination of calmodulin antagonism and estrogen receptor binding was 12 ((E)-1-[4-[3-(N-pyrrolidino)propoxy]phenyl]-1-(4-iodophenyl)-2-phe nyl-1 - butene) (IC50 = 1.1 microM, RBA = 23). Correlation between calmodulin antagonism and cytotoxicity was demonstrated for selected compounds.

Metabolism of the 4-iodo derivative of tamoxifen by isolated rat hepatocytes. Demonstration that the iodine atom reduces metabolic conversion and identification of four metabolites.

The 4-iodo derivative of tamoxifen, which has been reported to possess improved oestrogen receptor affinity and effectiveness as an inhibitor of breast tumour cell growth in vitro, was metabolized by hepatocytes isolated from rats pretreated with phenobarbital four times more slowly than tamoxifen and there was very little formation of glucuronide conjugates. Four principal metabolites were isolated. Examination of mass spectra revealed desmethyl-4-iodotamoxifen, 4-iodotamoxifen N-oxide, and alpha-hydroxydesmethyl-4-iodotamoxifen (4-[4-[2-(methylamino)ethoxy]phenyl]-4-(4-iodophenyl)-3-phenyl-but-3- (Z)-en-2-ol). Their identification was confirmed by comparison with synthesized samples. The structure of the fourth metabolite, 4'-hydroxy-4-iodotamoxifen was revealed by 1H NMR spectroscopy. The iodophenyl moiety is thus retained in all the metabolites. The iodine atom not only blocks metabolism in its vicinity but also reduced the rate of side-chain demethylation and N-oxidation by three-fold. It can be predicted from this study that the presence of the iodine atom should give the compound a greater duration of action in vivo.

Variation of the inhibition of calmodulin dependent cyclic AMP phosphodiesterase amongst analogues of tamoxifen; correlations with cytotoxicity.

The ability of a variety of analogues of tamoxifen to inhibit calmodulin dependent cyclic AMP phosphodiesterase has been determined. Effective inhibition requires that the aminoethoxy side chain bears a positive charge at physiological pH and is not too bulky. Amongst 4-substituents, inhibitory potency increases with lipophilicity. The stereochemistry about the olefinic linkage is not important. The most potent agent found (IC50 1.4 microM, compare tamoxifen = 6.75 microM) has a 4-iodine substituent and pyrrolidino in place of dimethylamino. This analogue is also more cytotoxic than tamoxifen against MCF-7 human breast cancer cells as determined in a 24-hr assay, but there was no correlation found between calmodulin inhibition and cytotoxicity against the L1210 murine leukaemia or Walker rat carcinosarcoma cells in culture. The results are consistent with the possibility that calmodulin is important to the functioning of oestrogen receptor mediated growth in MCF-7 cells.

Metabolism of tamoxifen by isolated rat hepatocytes. Identification of the glucuronide of 4-hydroxytamoxifen.

Metabolism of 4-hydroxytamoxifen by hepatocytes isolated from rats administered with phenobarbital and examination by TLC of the components not extractable into ethyl acetate revealed 4-hydroxytamoxifen beta-glucuronide; its identity was confirmed by comparison of its 1H NMR spectrum with that of synthetic material. This conjugate was also formed on metabolism of tamoxifen. It bound to cytosolic oestrogen receptors with only one thousandth the affinity of 4-hydroxytamoxifen and gave a correspondingly very weak inhibition of growth of the MCF-7 human breast cancer cell line. Therefore, in contrast to reported observations on the 3-glucuronide of oestradiol, the MCF-7 cells were unable to hydrolyse 4-hydroxytamoxifen glucuronide and on this evidence, formation of this metabolite is solely a deactivation pathway.

Metabolism of tamoxifen by isolated rat hepatocytes. Identification of 1-[4-(2-hydroxyethoxy)phenyl]-1-(4-hydroxyphenyl)-2-phenyl-1-butene and the dependence of N-oxidation on oxygen availability.

Metabolism of tamoxifen by rat hepatocytes and hydrolysis of the resulting polar metabolites corresponding to conjugates with beta-glucuronidase gave a major component which was identified as 1-[4-(2-hydroxyethoxy)phenyl]-1-(4-hydroxyphenyl)-2-phenyl-1-butene by comparison of mass spectral properties with those of synthetic material. This compound, which was not observed as a phase I metabolite, is believed to have been found previously in rat bile and in human faeces (metabolite F) but its structure had been incorrectly assigned. Its binding affinity for the estrogen receptor was greater than that of tamoxifen but less than that of 4-hydroxytamoxifen, and it possessed a corresponding degree of antitumour activity against the MCF-7 breast cancer cell line. By carrying out the hepatocyte incubation separately under oxygen and air, it has been shown that the N-oxidation of tamoxifen is favoured by a high concentration of oxygen during in vitro metabolism but that the rate of 4-hydroxylation is not dependent on oxygen availability.

Metabolism of 4-hydroxyandrost-4-ene-3,17-dione by rat hepatocytes.

4-[14C]HAD was rapidly metabolized (99% after 5 min) by hepatocytes from phenobarbital-treated rats. An array of phase I metabolites was formed, variously involving one and two reductions, hydroxylation, hydration and hydroxylation plus one or two reductions. Some of the metabolites were identified by synthesis and others tentatively by mass spectrometry. After 10 min, approximately 30% of the original radioactivity was present in HAD glucuronide and, after 15 min, approximately 60% was present in the total glucuronide fraction which contained several components. Only one of the phase I metabolites (2-hydroxy-HAD) exhibited significant aromatase inhibitory activity (45% of that of HAD).

Role of "lymphotoxin" in the local anti-tumour action associated with inflammation caused by delayed hypersensitivity responses or intralesional BCG. I. Variations in response of different syngeneic mouse tumours.

The anti-tumour effect induced by a delayed hypersensitivity response (DHSR) unrelated to the tumour or by intra-tumoural inoculation of BCG was studied with 6 syngeneic mouse tumours. The growth of the tumours was followed i.p. or s.c. in suitably sensitized animals either in the presence or absence of the specific antigen required to elicit a DHSR. In a Winn-type assay the growth of tumour cells admixed with sensitized lymphocytes was also determined with and without the eliciting antigens. In addition, the effect of admixing different amounts of BCG with the tumour cells was studied on the growth of the tumours in vivo. The different tumours varied widely in their susceptibility to growth inhibition by a DHSR reaction and by BCG but their order of sensitivity was the same in all of the tests. Analysis of the effector population in the Winn test coupled with the inability to observe an anti-tumour action in mice with defective T-cell function showed that the effector mechanism involved allergized T-cells or more probably products released when these were confronted with the specific antigen. In vitro the relative susceptibility of the different tumour cells to killing by activated macrophages and by NK cells was quite different to that found for in vivo growth inhibition but the in vitro response to lymphotoxin of the different tumours paralleled that produced by inflammation in vivo.

Mechanism of the local antitumor action of inflammation.

Some but not all tumor cells are killed in the environment of a delayed hypersensitivity reaction (DHSR) induced by antigens unrelated to the tumor. Of the several cytotoxic components, cellular and humoral, which are present at the site of a DHSR, a lymphokine released by the interaction of specific antigen with immune T cells-presumptively lymphotoxin-was shown to be responsible for the antitumor action of DHSR. The same mechanism may account for the failure of some tumor cells to grow when inoculated in admixture with BCG.

Selective mobilization of specifically cytotoxic T-lymphocytes at sites of inflammation in relation to BCG-induced resistance to implants of syngeneic sarcoma in mice.

The heightened and long-persisting resistance of BCG--immunized C57BL/6 mice (10-week-old males and females) to challenge with syngeneic sarcoma cells was largely restricted to the site of inoculation of the BCG. The specific cytotoxicity of peritoneal T-cells and the total number of T-cells that could be recovered from the peritoneal cavity were more than ten times greater in mice that had received BCG ip 2-4 weeks prior to inoculation of tumor than in non-BCG-treated mice. The specific T-cell-mediated cytotoxic potential of the peritoneal exudate of mice immunized with tumor was therefore at least 100 times greater in mice that had received BCG ip. This effect was detectable by 3 days after inoculation of BCG and reached a maximum 2-4 weeks later. The protection against tumor offered by pretreatment with BCG could be explained by the selective recruitment of committed T-lymphocytes to sites of chronic inflammation. The induction of nonspecifically cytotoxic macrophages and systemic changes such as generalized stimulation of the reticuloendothelial system were not contributing factors.

Rejection of sarcoma cells at the site of an inflammatory reaction: macrophages are not the only effector cells.

Purified protein derivative (PPD) (a soluble protein from tubercle bacilli), when injected together with sarcoma cells into syngeneic mice that had been immunized previously with Bacillus Calmitte-Guérin (BCG), prevents tumour toxic to sarcoma cells [4]. However, non-adherent mononuclear peritoneal exudate cells from BCG-treated mice were also found on addition of PPD to become cytotoxic to sarcoma cells. In vivo assays indicate that such cells may play a major role in the in vivo destruction of tumours at the site of a delayed hypersensitivity reaction.