8-thioguanosine and 8-bromoguanosine

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates the feedback inhibition of GTP cyclohydrolase I activity by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) through protein complex formation. Since guanine and BH4 have a common pyrimidine ring structure, we examined the inhibitory effect of guanine and its analogs on the enzyme activity. Guanine, 8-hydroxyguanine, 8-methylguanine, and 8-bromoguanine inhibited the enzyme activity in a GFRP-dependent and pH-dependent manner and induced complex formation between GTP cyclohydrolase I and GFRP. The type of inhibition by this group is a mixed type. All these properties were shared with BH4. In striking contrast, inhibition by 8-azaguanine and 8-mercaptoguanine was GFRP-independent and pH-independent. The type of inhibition by 8-azaguanine and 8-mercaptoguanine was a competitive type. The two compounds did not induce complex formation between the enzyme and GFRP. These results demonstrate that guanine compounds of the first group bind to the BH4-binding site of the GTP cyclohydrolase I/GFRP complex, whereas 8-azaguanine and 8-mercaptoguanine bind to the active site of the enzyme. Finally, the possible implications in Lesch-Nyhan syndrome and Parkinson diseases of the inhibition of GTP cyclohydrolase I by guanine and 8-hydroxyguanine are discussed.

Topics: Adjuvants, Immunologic; Animals; Antimetabolites, Antineoplastic; Azaguanine; Binding Sites; Binding, Competitive; Chromatography, Gel; Dose-Response Relationship, Drug; GTP Cyclohydrolase; Guanine; Guanosine; Guanosine Triphosphate; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Kinetics; Lesch-Nyhan Syndrome; Models, Chemical; Parkinson Disease; Rats; Thionucleosides

Topics: Adjuvants, Immunologic; Animals; Cells, Cultured; Guanosine; Leishmania mexicana; Macrophage Activation; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Thionucleosides

C8-substituted guanine ribonucleosides activate B cells by a novel pathway that apparently is independent of GTP-binding proteins and protein kinase C. B lymphocytes from SJL mice are hyporesponsive to antigen-independent inductive signals transmitted by these nucleosides. In the current studies, the basis for this observation was explored. Responses of normal murine strains to these agents have been dissociated into antigen-independent (inductive) and antigen-dependent (differentiative) types by use of the 7,8-disubstituted guanine ribonucleosides. Dose-response profiles for inductive responses appear to correlate with apparent Kd values for low-affinity nucleoside binding sites; dose-response curves for antigen-dependent differentiative responses correlate with apparent Kd values for high-affinity binding sites. It was found that the SJL low-affinity site exhibits an apparent Kd that is approximately 10- to 20-fold lower in affinity for 8BrGuo than that of normal CBA mice. Although the low-affinity site in normal murine strains displays nearly equivalent affinity toward C8-substituted and 7,8-disubstituted nucleosides, the low-affinity site of SJL mice binds 7,8-disubstituted compounds with approximately 5-fold higher affinity than it does monosubstituted compounds. The dissociation constant for high-affinity nucleoside binding sites of SJL mice was only slightly different from that of CBA mice, consistent with the observation of essentially normal antigen-dependent nucleoside-mediated activity in SJL mice. The current observations support (a) a role for low-affinity binding sites in antigen-independent inductive events, (b) a role for high-affinity binding sites in antigen-dependent differentiative events mediated by substituted guanine nucleosides, and (c) the existence of aberrant low-affinity binding sites in B cells from SJL mice.

Topics: Animals; B-Lymphocytes; Binding Sites; Disease Models, Animal; Guanosine; Immunologic Deficiency Syndromes; Kinetics; Mice; Mice, Inbred CBA; Mice, Inbred Strains; Thionucleosides

The 8-substituted guanosine derivatives (8-sGs), mercaptoguanosine and bromoguanosine, are shown to induce immunoglobulin production and proliferation in a subset of normal human B lymphocytes. Limiting dilution analysis indicated that the 8-sG-responsive B cell pool is approximately 10 times larger than that activated by pokeweed mitogen (PWM), 10 times smaller than that activated by Epstein-Barr virus (EBV), and contains approximately equal proportions of cells committed to the expression of IgG, IgA or IgM isotypes. Although some B cells seem able to respond to 8-sGs in the absence of T helper cells, maximal immunoglobulin production is only observed in the presence of T cells. The 8-sG response pattern of human B lymphocytes appeared similar but not identical to that reported for rodent cells. The mitogenic 8-sGs are unique activators as they bypass surface membrane interactions obligatory for other agents including anti-IgM, Staphylococcus aureus particles, PWM, and EBV.

Topics: B-Lymphocytes; Guanosine; Herpesvirus 4, Human; Humans; Immunoglobulin Isotypes; In Vitro Techniques; Lymphocyte Activation; Pokeweed Mitogens; T-Lymphocytes; Thionucleosides

5-Halo-6-phenyl pyrimidinones, represented by 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP) and 2-amino-5-iodo-6-phenyl-4(3H)-pyrimidinone (AIPP), and 8-substituted guanosines, represented by 8-bromoguanosine (8-BrGuo) and 8-mercaptoguanosine (8-MGuo), are well-documented biological response modifiers. We have found that these substituted pyrimidinones and guanosines are very similar in their abilities to activate B cells. ABPP, AIPP, 8-BrGuo, and 8-MGuo induced murine B cells to polyclonally proliferate and differentiate in vitro. The maximal B-cell response levels and the kinetics of the responses elicited with both classes of compounds were comparable; however, ABPP and AIPP were approximately 10-fold more potent than 8-BrGuo and 8-MGuo. An additional similarity observed between the two classes was that polyclonal activation of B cells by ABPP, AIPP, 8-BrGuo, and 8-MGuo was limited to large B cells which had probably been activated previously in vivo. This is in contrast to lipopolysaccharide which is capable of inducing both large, activated B cells and small, resting B cells to proliferate and differentiate. Although substituted pyrimidinones and guanosines were not able to induce new DNA synthesis or antibody production in small B cells, both classes of compounds increased the expression of Ia antigens on the surface of both small and large B cells. These data, together with the recent observations that 8-BrGuo, like ABPP and AIPP, can stimulate NK and cytotoxic macrophage activity via the induction of interferon, strongly suggest that 5-halo-6-phenyl pyrimidinones and 8-substituted guanosines belong to the same structural class of biological response modifiers. Thus, the residues held in common by these two classes of stimulators may interact with the same cellular constituent in the target cells.

Topics: Animals; B-Lymphocytes; Cell Differentiation; Cytosine; Female; Guanosine; Histocompatibility Antigens Class II; Interphase; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Thionucleosides

Production of an IL-1-like activity in cultures of irradiated splenic adherent cells can be elicited by the C8-substituted guanine ribonucleosides 8-bromoguanosine (8BrGuo) and 8-mercaptoguanosine (8MGuo). This report constitutes the first evidence for activation of a non-lymphocytic cell type by these agents to secrete an immunologically-active mediator. The secreted activity is mitogenic for murine thymocytes, co-stimulates these cells synergistically in the presence of concanavalin A, and co-stimulates low cell density cultures of purified B-cells in the presence of anti-mu antibodies. Production of this activity increases in a dose-dependent manner as the concentration of nucleoside is increased, both in cultures of splenic adherent cells and of the macrophage cell line P388D1. The P388D1 results indicate that this effect of the nucleoside is not mediated by another cell type, but can proceed by direct nucleoside-cell interaction. Optimal amounts of IL-1-like activity are produced after about 24 h of culture. Anti-IL-1 antibodies that neutralize the biologic activity of an IL-1 standard also eliminate the IL-1-like activity induced by 8BrGuo. These antibodies, however, fail to alter the magnitude of the primary humoral immune response to sheep erythrocytes amplified by 8BrGuo. These data indicate that C8-substituted guanosines, known intracellular stimuli for B-lymphocytes, can also induce non-lymphocytic cells (including a macrophage-like cell line) to elaborate an active principle which exhibits IL-1-like activity. These nucleosides thus are apparently able to elicit secretion of monokines, lymphokines and immunoglobulin from macrophages, T-cells and B-cells, respectively.

Topics: Adjuvants, Immunologic; Animals; B-Lymphocytes; Guanosine; In Vitro Techniques; Interleukin-1; Lymphocyte Activation; Macrophages; Male; Mice; Mice, Inbred CBA; Thionucleosides

Previous studies have documented the ability of 8-bromoguanosine (8-BrGuo) and 8-mercaptoguanosine (8-MGuo) to induce polyclonal proliferation and differentiation of B cells from a variety of mouse strains. In the present study, we have defined the cellular target of this mitogenic activity. Using B cells fractionated according to size, we have found that large B cells are responsive to 8-BrGuo- and 8-MGuo-induced proliferation and differentiation whereas small, resting B cells are relatively unresponsive to these compounds. Addition of splenic adherent cells to the small B-cell fraction partially restored the proliferative but not the differentiative responses to 8-BrGuo and 8-MGuo. Although small B cells alone did not proliferate or differentiate in response to 8-BrGuo and 8-MGuo, cell surface expression of Ia antigens increased following incubation with these compounds. Thus, the biological activity of 8-BrGuo and 8-MGuo appears to be dictated by the cell type upon which it is acting. Small B cells are activated as evidenced by increased levels of surface Ia whereas large B cells are not only activated but are also induced to proliferate and differentiate.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibody Formation; Antigens, Surface; B-Lymphocytes; Cell Differentiation; Cell Division; Guanosine; Histocompatibility Antigens Class II; Lymphocyte Activation; Macrophages; Mice; Muramidase; Receptors, Antigen, B-Cell; Spleen; Thionucleosides

The current studies compare the inductive and differentiative properties of 8-mercaptoguanosine with those of 7-methyl-8-oxoguanosine. 7-Methyl-8-oxoguanosine (7m8oGuo) is a new member of the family of C8-substituted guanine ribonucleosides, the first such biologically active compound described that differs from guanosine other than by the specific substituent at the 8 position. Like 8MGuo, 7m8oGuo stimulates proliferation selectively in B lymphocytes. However, 7m8oGuo possesses greater activity than 8MGuo as a mitogen and greater potency as an adjuvant for humoral immune responses. Thus, as a B-lymphocyte mitogen, 7m8oGuo induces quantitatively greater [3H]TdR uptake than does 8MGuo, but with the same concentration optimum. As an adjuvant for in vitro antibody responses, however, 7m8oGuo achieves the same degree of immunoenhancement as 8MGuo but at approximately 10-fold lower concentrations, that is, the dose-response profile has been shifted to the left. Moreover, whereas the mitogenic responses to 8MGuo and 7m8oGuo exhibit parallel kinetic profiles, the adjuvant activity of 7m8oGuo arises earlier and persists later than does that of 8MGuo. These results are interpreted in terms of two distinct intracellular pathways: one mediating mitogenesis and the other adjuvanticity.

Topics: Adjuvants, Immunologic; Animals; Cells, Cultured; Guanosine; Kinetics; Lymphocyte Activation; Mice; Spleen; Structure-Activity Relationship; Thionucleosides

A variety of 8-substituted guanosine and 2'-deoxyguanosine derivatives were synthesized and tested as inducers of the differentiation of Friend murine erythroleukemia cells in culture. The most active agents in the guanosine series were 8-substituted-N(CH3)2, -NHCH3, -NH2, -OH, and -SO2CH3, which caused 68, 42, 34, 33, and 30% of erythroleukemia cells to attain benzidine positivity, a functional measure of maturation, at concentrations of 5, 1, 0.4, 5, and 5 mM, respectively. The 8-OH derivative of the 2'-deoxyguanosine series produced comparable activity, causing 62% benzidine-positive cells at a level of 0.2 mM. These findings indicate that 8-substituted analogues of guanosine and 2'-deoxyguanosine have the potential to terminate leukemia cell proliferation through conversion to end-stage differentiated cells.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Chemical Phenomena; Chemistry; Deoxyguanosine; Friend murine leukemia virus; Guanosine; Hypoxanthine Phosphoribosyltransferase; Leukemia, Erythroblastic, Acute; Mice; Structure-Activity Relationship

The observed enhancement of B-lymphocyte activation by 8-bromoguanosine and 8-mercaptoguanosine is hypothesized to occur via a "binding protein" which requires a guanine nucleoside as the syn conformer for productive interaction. In addition, because of the 7-substituent, Q nucleoside also is hypothesized to bind as the syn conformer and, therefore, to be a potential B-lymphocyte activator.

Topics: Animals; Anticodon; B-Lymphocytes; Base Sequence; Carrier Proteins; Guanosine; Humans; Lymphocyte Activation; Nucleoside Q; RNA, Transfer; Structure-Activity Relationship; Thionucleosides

Guanine ribonucleosides, substituted at the C8 position with either a bromine or thiol group, are potent adjuvants when added to culture with antigen. Responses from both naive and antigen-experienced B cells are augmented by the presence of the nucleoside derivatives. Like the induction of B cell proliferation and polyclonal immunoglobulin secretion, enhancement of antibody responses is not attributable to structural analogy between 8BrGuo and the cyclic nucleotide 8Br-cGMP, because the latter compound is unable to augment the antibody response. The capacity of 8MGuo to augment both T-independent and T helper factor-supported antibody responses suggests that the nucleoside acts by directly interacting with B cells and/or antigen-presenting cells. 8MGuo retains its full adjuvant activity even when added 3 days after culture initiation, a time that is too late for freshly added T cells to support a response. Finally, supplementation of cultures of spleen cells from immunodeficient (CBA/N x CBA/CaJ)F1 male mice with the nucleoside effectively restored to normal their capacity to generate an antibody response.

Topics: Adjuvants, Immunologic; Animals; Antigens, T-Independent; Cyclic GMP; Guanosine; Immunoglobulin G; Immunoglobulin M; Immunologic Deficiency Syndromes; Isoantibodies; Male; Mice; Mice, Inbred CBA; T-Lymphocytes; Thionucleosides