Glucuronidation associated with intrinsic resistance to mycophenolic acid in human colorectal carcinoma cells.

The in vivo efficacy of the antitumor, immunosuppressive antibiotic mycophenolic acid is known to be limited by its rapid conversion to the biologically inactive 7-0-glucuronide, catalyzed by UDP-glucuronosyl transferase activity, which is widely distributed among normal tissues, including intestinal epithelium. We have found that mycophenolic acid is also converted to its glucuronide by several lines of human colorectal carcinoma cells, including HT29, Lovo, and Colo-205. In contrast, malignant cell lines not of colorectal origin, including EMT6, HeLa, and SKOV3, showed no ability to metabolize mycophenolic acid. The 7-amino derivative of mycophenolic acid was not metabolized by HT29 cells. This compound was less potent than mycophenolic acid versus EMT6 and HeLa cells but showed inhibitory activity against HT29 cells comparable with the parent antibiotic. The rapid metabolism of mycophenolic acid by HT29 cells was associated with a markedly lower sensitivity to both the antiproliferative activity of the drug and to its ability to inhibit GTP synthesis, compared with cells lacking the capacity for significant glucuronidation. After an initial decline in cellular GTP in HT29 cells induced by mycophenolic acid, there was a progressive recovery in GTP over 48 h, accompanying the metabolism of the antibiotic. This recovery process was not observed in EMT6 cells. It is suggested that glucuronosyl transferase activity may occur widely in colorectal cancer cells and could contribute to resistance to drugs that are susceptible to inactivation by glucuronide conjugation.

Cell cycle specific induction of HL-60 cell differentiation and apoptosis by mycophenolic acid.

HL-60 cells undergo terminal differentiation and apoptosis in response to different types of sub-toxic and toxic perturbations respectively. The mechanism by which cells sense different amounts of perturbation to activate pathways that lead to the engagement of a relevant biological response is not known. The response of HL-60 cells to treatment with the immunosuppressant mycophenolic acid (MPA), a specific inhibitor of dGTP/GTP-synthesis, allowed quantitation of a metabolic perturbation which triggered a cellular response. 1.5 microM MPA induced 38% terminal differentiation to CD14 positive, early monocyte-like cells and 22% cell death by apoptosis, whereas 3 microM MPA induced 70% apoptosis but no differentiation. Despite the difference in biological outcomes, 72 h exposure to both 1.5 microM and 3 microM MPA caused a similar ( approximately 75%) depletion of total GTP levels. Cells synchronized by centrifugal elutriation were treated with MPA. Elutriated cells were overall less sensitive to the effects of MPA but 3 microM MPA induced significantly less apoptosis and more differentiation in an elutriation-enriched G1-population than in a population normally distributed in the cell cycle, suggesting that the effects of MPA in S-phase may subsequently lead to cell death. However, analysis of apoptosis by using a terminal deoxynucleotidyltransferase assay and measurement of bromodeoxyuridine incorporation showed that apoptosis was engaged in G1. Analysis of the phosphorylation status of the retinoblastoma protein demonstrated that Rb was hypophosphorylated prior to apoptosis and that in apoptotic cells, separated by flow cytometry, Rb protein was absent, presumably due to proteolysis. The loss of Rb protein did not appear to permit transit to S-phase, and was not accompanied by an expression of c-Myc. Surprisingly, therefore, an antimetabolite inducing a loss of GTP brought about cell death by apoptosis in the G1 phase of the cell cycle.

Therapeutic approaches to organ fibrosis.

Scarring of superficial tissues and chronic fibroses of major organs present major medical problems ranging from disfigurement to progressive disability and death. Growing understanding of the cellular and molecular events, which are common to these intractable disorders, now provides a favourable basis for the development of effective drug therapies. Much attention is focussed on the roles of the many cytokines and growth factors, which contribute to the fibrogenic process. The transforming growth factor (TGF)-beta 1 and 2 isoforms are among the most significant of these and approaches to control their activity include blocking the activation of latent TGF-beta, preventing the ligand-receptor interactions and the inhibition of down-stream signal transduction. Concerns regarding possible risks of the long-term suppression of TGF-beta function point to connective tissue growth factor (CTGF) as a possible alternative target. CTGF is induced by and appears to mediate at least some of the fibrogenic actions of TGF-beta, although not its important antimitogenic activity on epithelial cells. The fibrogenic effects of endothelins and angiotensin II have aroused considerable interest in the anti-fibrotic potential of antihypertensive agents designed primarily to limit the vasoconstrictive activities of these peptides. Polypeptides including interferons alpha and gamma, relaxin, TGF-beta 3 and hepatocyte growth factor, all show an ability to limit fibrogenesis in either clinical or experimental situations. Finally, inhibitors of the enzymes required for the post-translational processing of collagens, including prolyl 4-hydroxylase, C-proteinase and lysyl oxidase provide a more direct means of reducing the deposition of fibrillar collagens into the extracellular matrix although the potentially adverse effects of sustained manipulation of collagen metabolism remain to be investigated.

The complete cDNA derived sequence of the rat prolyl 4-hydroxylase alpha subunit.

A rat cDNA encoding the prolyl 4-hydroxylase alpha subunit (P4H alpha) was isolated and sequenced. The primary aa sequence deduced from the nucleotide sequence reveals a 534-aa protein that shows extensive aa identity with the human (88%) and chick (77%) P4H alpha.

Novel inhibitors of prolyl 4-hydroxylase. 3. Inhibition by the substrate analogue N-oxaloglycine and its derivatives.

N-Oxaloglycine (3) is an alpha-ketoglutarate (1) analogue that is a competitive inhibitor of prolyl 4-hydroxylase (EC 1.14.11.2). A study of the structure-activity relationships of some other oxalo derivatives shows that substitution on the glycine moiety modulates activity stereoselectively and that if the omega-carboxylate is homologated or replaced by either acylsulfonamides or anilide, then activity is sharply reduced. This sensitivity to these changes is contrasted with the relative insensitivity of another putative alpha-ketoglutarate analogue, pyridine-2,5-dicarboxylic acid (2), and the implication is discussed that compounds of both series are unlikely to bind to prolyl hydroxylase in the same way even though both inhibit the enzyme competitively.

A novel series of non-nucleoside inhibitors of inosine 5'-monophosphate dehydrogenase with immunosuppressive activity.

Inhibitors of inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) are effective immunosuppressive drugs that may also have additional potential applications as antitumour and antimicrobial agents. The clinical value of the most potent and specific inhibitor of IMPDH, mycophenolic acid, is limited by its rapid metabolism in vivo to an inactive glucuronide derivative. There is, therefore, a considerable incentive to develop structurally novel, preferably non-nucleoside, inhibitors with greater metabolic stability than mycophenolic acid. Here, we describe a high throughput screen for inhibitors of IMPDH, which facilitated the discovery of a single novel non-nucleoside inhibitor from a collection of approximately 80,000 compounds. The inhibitor is a pyridazine, which, like mycophenolic acid, exerts uncompetitive inhibition of IMPDH. Analysis of the enzyme kinetics suggests that the inhibitory action of the pyridazine is similar to that of mycophenolic acid, which involves trapping of a covalent intermediate formed during the conversion of IMP to xanthosine monophosphate. Chemical modification of the lead compound resulted in pyridazine derivatives with enhanced potency against IMPDH and guanine nucleotide synthesis in cultured cells in vitro and also against guanine nucleotide synthesis in the mouse spleen in vivo. One of the compounds was available in sufficient quantity to demonstrate highly effective immunosuppressive activity in a model of delayed type hypersensitivity in mice. To our knowledge, the novel pyridazines described in this report represent the first non-nucleoside uncompetitive inhibitors of IMPDH with immunosuppressive activity since the discovery of the inhibitory activity of mycophenolic acid and its derivatives thirty years ago.

Pharmacodynamics of the inhibition of GTP synthesis in vivo by mycophenolic acid.

Mycophenolic acid is effective against a wide range of experimental tumors in rodents when given orally, despite rapid metabolism to the inactive glucuronide derivative and rapid clearance from plasma. In the light of this, the pharmacodynamic action of mycophenolic acid on the radiolabelling of GTP and ATP by [14C]hypoxanthine in spleen and heart has been investigated in vivo in the rat as a preliminary to studies in tumor tissue. The data indicate that inhibition of GTP, and more surprisingly, ATP synthesis in spleen was sustained for at least 24.25 hr after single oral doses of the disodium salt of mycophenolic acid, indicating that the inhibitor is retained in sensitive cells for considerably longer than might be expected from the pharmacokinetic profile in the plasma in this species. GTP and ATP levels became depressed in rat spleen subsequent to the inhibition of nucleotide radiolabelling. The persistence of mycophenolic acid in proliferating cells may account for the effectiveness of once daily dosing against rapidly growing experimental tumors. In contrast with spleen, there was no incorporation of radiolabel from [14C]hypoxanthine into either GTP or ATP in rat heart and mycophenolic acid had no effect on the cardiac content of either nucleotide. The lack of effect of mycophenolic acid on cardiac GTP levels is consistent with the absence of any pharmacological action on cardiac functions associated with receptor-G-protein-GTP interactions. The ability of the morpholinoethyl ester of mycophenolic acid (a clinically effective immunosuppressive agent) to inhibit GTP synthesis and depress GTP levels in rat spleen in vivo was compared with that of mycophenolic free acid and its disodium salt. The ester derivative was clearly more effective than the poorly water-soluble free acid but showed comparable activity with the freely soluble disodium salt.

Glucuronidation by human colorectal adenocarcinoma cells as a mechanism of resistance to mycophenolic acid.

Mycophenolic acid (MPA), a potent and specific inhibitor of IMP dehydrogenase, exerts its anti-mitotic action by a rapid depletion of the cellular content of guanine nucleotides. Although MPA is a potent inhibitor of GTP synthesis in the HT29 line of human colorectal adenocarcinoma cells in short-term culture, its ability to depress the cloning efficiency of these cells was found to be markedly less than against the mouse mammary carcinoma line, EMT6. In vivo, MPA is efficiently converted to the biologically inactive O-glucuronide derivative thereby limiting its effectiveness as an anti-tumor agent. Investigation of the fate of MPA incubated with monolayer cultures of HT29 and EMT6 cells revealed that the compound is rapidly converted to the O-glucuronide derivative by HT29 cells, but not by EMT6 cells. Confirmation of the identity of the glucuronide formed by HT29 cells was obtained by its conversion to MPA after incubation with beta-glucuronidase and by comparison of the mass spectrum of its HPLC peak with that of synthetic MPA O-glucuronide. Cultures of two other lines of human colorectal adenocarcinoma cells, Colo-205 and LoVo, also depleted their culture media of MPA although we have not yet established whether these cells also synthesize the glucuronide. The intrinsic partial resistance of HT29 cells to MPA appears to be associated with the ability of these cells to convert MPA to the biologically inactive glucuronide. These results, in conjunction with other reports of the capacity of colorectal cancer cells for Phase I and II metabolism of xenobiotics, may have implications for the design of drugs intended for the treatment of colorectal cancer.

Energetics of ligand binding to receptors.

The primary events in the biological signals elicited by hormonal ligands are the initial binding of the ligands to their receptors and the consequent changes in the receptors that this induces. Although the effects of chemical modifications of ligands on their affinity for receptors have been intensively studied in several systems, the influence of such changes upon the ability of ligands to induce the conformational changes in receptors that are necessary for the expression of agonism has been largely ignored. This paper considers some theoretical and experimental approaches to the problem of the relationships between ligand structure, binding energy and the induction of conformational change in receptors. The significance of the concept of productive and non-productive ligand binding to proteins previously applied with considerable success by Jencks to the understanding of enzyme catalysis, is highlighted. Given a series of closely related ligands it is possible to estimate the energetic contributions of substituent groups not only to the binding affinity to the receptor but also to the induction of conformational change in the receptor provided that the rate of conformational change, can be monitored.

Resistance of Escherichia coli to tetracyclines. Changes in permeability to tetracyclines in Escherichia coli bearing transferable resistance factors.

1. When strains of Escherichia coli, bearing transferable factors for resistance to the tetracyclines (R-factors), and previously cultured in the absence of the tetracyclines, are grown for 15-30min. in a low, subinhibitory, concentration (10mug./ml.) of oxytetracycline or tetracycline, there is a rapid and striking increase in resistance to oxytetracycline or tetracycline, this being associated with a marked fall in the absorption of the drug by the cells. 2. Very short preincubation (1min.) with oxytetracycline, followed by growth for 15-30min. in drug-free medium, produces a marked fall in the absorption of the drug by the resistant cells. Preincubation for 30min. with very low concentrations (0.05mug./ml.) of oxytetracycline produces a similar effect. 3. beta-Apo-oxytetracycline, which has very little antibacterial activity, also induces a decreased absorption of oxytetracycline. 4. The ability to exclude oxytetracycline is retained by preincubated resistant cells after growth for 2hr. in drug-free medium. However, after growth for 16hr. in drug-free medium, the cells absorb oxytetracycline freely. 5. Chloramphenicol and proflavine inhibit the adaptive decrease in tetracycline absorption. 5-Fluorouracil has only a slight effect. 6. Spheroplasts prepared from resistant cells show an impaired response to preincubation with tetracycline, compared with intact cells. 7. The relevance of these results to the probable mechanism of tetracycline resistance in R-factor-bearing E. coli is discussed.

Uptake of tetracycline by membrane preparations from Escherichia coli.

1. A membrane fraction from Escherichia coli has been prepared essentially free from ribosomes by treatment of the membranes with Triton X-100 at 0 degrees C followed by differential centrifugation. 2. The ribosome-free membrane vesicles absorbed tetracycline by a reversible temperature-dependent process with an apparent K(m) of 0.029mm at pH7.5 and 37 degrees C. 3. The absorption process was negligible below 25 degrees C and had an optimum at 40 degrees C; a pH optimum at 7.5 was observed. 4. The absorption of tetracycline was strongly inhibited by EDTA and ATP; ADP inhibited less strongly and AMP had no effect. 5. There was no significant difference in the rates or extent of uptake of tetracycline by membranes prepared from tetracycline-sensitive and tetracycline-resistant, R-factor-bearing E. coli.

Inhibition of prolyl 4-hydroxylase by hydroxyanthraquinones.

Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. We have found that anthraquinones with at least two hydroxy groups ortho to each other are potent inhibitors of this enzyme. Kinetic studies revealed that 2,7,8-trihydroxyanthraquinone (THA) competitively inhibited the co-substrate, 2-oxoglutarate, but was non-competitive with regard to ascorbate and was tentatively considered to be uncompetitive with regard to protocollagen. The inhibition by THA was greatly enhanced in the absence of added Fe2+ and was partially reversed by the addition of concentrations of Fe2+ in excess of the optimum for the enzymic reaction. Binding studies indicated that THA is an effective chelating agent for Fe2+. Several non-quinoidal compounds bearing the catechol moiety also inhibited the enzyme. The results suggest that THA inhibited prolyl 4-hydroxylase by binding to the enzyme at the site for 2-oxoglutarate possibly involving the Fe2+ atom, rather than by complexing with Fe2+ in free solution. The inhibition of prolyl 4-hydroxylase by THA exhibited strong positive co-operativity and may involve three distinct but non-independent binding sites.

Biochemical studies with a new cytotoxic immunosuppressive agent, 3-acetyl-5-(4-fluorobenzylidene)-2,5-dihydro-4-hydroxy-2-oxothiophen (I.C.I. 47776).

1. A new cytotoxic agent, 3-acetyl-5-(4-fluorobenzylidene)-2,5-dihydro-4-hydroxy-2-oxothiophen (I.C.I. 47776), strongly inhibits protein and nucleic acid synthesis and, to a smaller extent, respiration in lymph-node cells and Landschütz ascites-tumour cells in vitro. 2. The activity of I.C.I. 47776 in vitro declines as the pH of the medium is increased and is inversely proportional to the concentration of serum in the medium. 3. The compound has no effect on the incorporation of leucine by a cell-free preparation from Landschütz ascites cells containing ATP and phosphoenolpyruvate. 4. I.C.I. 47776 stimulates glycolysis in suspensions of Landschütz ascites cells in the presence of excess of glucose but has no effect on glycolysis in suspensions of rat lymph-node cells. 5. I.C.I. 47776 markedly depresses ATP concentration in ascites cells in the absence of glucose but has no effect on the ATP concentration in the presence of glucose. The inhibition of protein synthesis by I.C.I. 47776 in ascites cells is, however, only partially reversed by the addition of glucose. 6. The ATP concentration of rat lymph-node cells incubated with I.C.I. 47776 in the absence of glucose is also markedly depressed but the addition of glucose increases the ATP concentration only slightly. Further, glucose has no effect on the inhibition of protein synthesis in lymph-node cells by I.C.I. 47776. 7. It is suggested that I.C.I. 47776 inhibits protein and nucleic acid synthesis in cell suspensions indirectly by acting as a mitochondrial poison. 8. The relevance of studies on the activity of I.C.I. 47776 in vitro to its cytotoxic and immunosuppressive action in vivo is discussed.