Enhanced immunostimulation by novel platinum anticancer agents.

'Poly-plat', SSP and SAP are second generation analogs of cisplatin which have been shown to activate murine peritoneal macrophages in vivo and in vitro. Murine peritoneal macrophages treated with 'poly-plat', SSP or SAP (5 microg/mg) for 2 h are stimulated to form cytoplasmic extensions. Drug-treated macrophages also elicit an increase in the number of lysosomes. In addition, analysis of tissue culture supernatants shows increased levels of interleukin-1alpha and tumor necrosis factor-alpha. These results show that 'poly-plat', SSP and SAP enhance the immune system with greater efficacy and potency than cisplatin.

Specific and effective interaction of a guanine nucleotide analogue with small G proteins.

G proteins are molecular switches that use a cycle of GTP binding and hydrolysis to regulate a wide variety of cellular biochemical processes. Because the functional state of these proteins is allosterically determined by bound guanine nucleotides, a nucleotide analogue with protein specificity might have pharmacological or biochemical value. The binding of [alpha-32P]GTP to four small G proteins immobilized on nitrocellulose was competed by a series of analogues with modifications at multiple sites. One analogue, N2-(p-n-butylphenyl)guanosine 5'-(beta,gamma-difluoromethylene)triphosphate, had a approximately 40-fold higher affinity for one small G protein than for two of the others. Systematic analysis of each modification in the synthetic nucleotide revealed that specificity was conferred by the carbon substitution in the beta,gamma-phosphoanhydride bond. These observations were then extended to purified proteins of known sequence in solution by filtration binding studies with H-ras and rab5. Ras was 9-fold more discriminant between guanosine-5'-(beta,gamma-difluoromethylene)triphosphate and guanosine-5'-O-(3-thiotriphosphate) than was rab5, and the Q79L GTPase-defective mutant of rab5 was 6-fold more discriminant than wild-type rab5. Guanosine-5'-(beta,gamma-difluoromethylene)triphosphate protected a 20-kDa fragment of rab5 from tryptic proteolysis with greater efficacy than guanosine-5'-O-(3-thiotriphosphate) or guanosine-5'-(beta,gamma-imido)triphosphate despite its lower affinity, and GMP stabilized a conformation indistinguishable from apo-rab5. These results identify a synthetic guanine nucleotide analogue with differential affinity for closely related G proteins, determine the atomic substitution in the analogue that confers specificity, demonstrate discrimination by the analogue between wild-type and a point-mutant G protein, and establish efficacy of the analogue in inducing conformational change of a target protein disproportionate to the affinity of the interaction.

Chemical and enzymatic incorporation of N2-(p-n-butylphenyl)-2'-deoxyguanosine into an oligodeoxyribonucleotide.

An 18mer oligodeoxyribonucleotide containing a N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPdG) residue at the 3' end has been synthesized by both chemical and enzymatic methods. Chemical synthesis involved attachment of 5'-DMT-BuPdG as the 3'-H-phosphonate to uridine-controlled pore glass (CPG), followed by extension via H-phosphonate chemistry. After oxidation of the backbone, deprotection of bases, and removal from CPG, the uridine residue was removed by periodate cleavage and beta-elimination. The resulting oligomer 3'-phosphate was digested with alkaline phosphatase to give the free BuPdG-18mer. E.coli DNA polymerase I (Klenow) incorporated BuPdGTP at the 3' end of the corresponding 17mer primer annealed to a complementary 29mer template, and the properties of this product were identical to those of chemically synthesized BuPdG-18mer. E.coli DNA polymerase I (Klenow) was unable to extend the BuPdG-18mer, and the 3' to 5' exonuclease activity of the enzyme was unable to remove the modified nucleotide.

The in vitro evaluation of nucleoside analogues as probes for use in the noninvasive diagnosis of herpes simplex encephalitis.

1-(2-Deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) and the 5-iodo (FIRU), 5-bromo (FBrRU), 5-chloro (FClRU) and 5-fluoro (FFRU) analogues of 1-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)uracil were examined in four in vitro tests designed to evaluate their potential as radiopharmaceuticals in a non-invasive diagnostic test for herpes simplex encephalitis (HSE). The tests involved, (a) cellular uptake studies--which showed that all five nucleosides are selectively trapped in rabbit kidney cells infected with herpes simplex virus, type I, which codes for its own thymidine kinase; (b) incubation studies--which showed that, in human platelets, FIAU, FIRU and FFRU are not degraded by thymidine phosphorylase (an enzyme which catalyzes the glycosidic bond cleavage of physiological nucleosides); (c) a transport study, using mouse erythrocytes--which indicated that all five nucleosides have good affinity for a NBMPR-sensitive nucleoside transporter, with inhibition constants (Ki values in the range of 0.177-0.41; and (d) determination of octanol/water partition coefficients--which (log P = -0.14 to -1.67) indicated that FIAU is the most lipophilic of the five compounds studied, and may therefore cross the blood-brain barrier most readily. We conclude from the results that FIAU is the most promising compound for further evaluation in HSE animal models. The combination of tests described in this study provide a useful screening protocol for evaluation of other nucleoside analogues of potential use in a diagnostic test for HSE.

Effect of acyclovir on the uptake of 131I-labelled 1-(2'fluoro-2'-deoxy-beta-D-arabinofuranosyl)-5-iodouracil in herpes infected cells.

Selective uptake of nucleoside analogues by herpes simplex virus infected cells may serve as the basis for a specific non-invasive diagnostic test for herpes simplex encephalitis. We have examined the effect of acyclovir on the selective uptake of [131I] 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-5-iodouracil in herpes simplex virus infected primary rabbit kidney cells. Infected cells treated with acyclovir continued to concentrate [131I] 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-5-iodouracil for up to 24 h after the addition of the antiviral agent. These results indicated that therapy with acyclovir for as long as 24 h would not prevent the selective trapping of nucleoside analogues. This has important implications for the use of nucleoside analogues in diagnostic brain scans to detect herpes simplex encephalitis.

A proposed mechanism for the selective inhibition of human cytomegalovirus replication by 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-fluorouracil.

The biological activities of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-fluorouracil (2'F-ara-FU), 1-(3'-deoxy-3'-fluoro-beta-D-arabinofuranosyl)-5-fluorouracil (3'F-ara-FU) and 1-beta-D-arabinofuranosylthymine (ara-T) were compared in human cytomegalovirus (HCMV)-infected and noninfected human fibroblasts. 2'F-ara-FU inhibited HCMV plaque formation (ED50, 16 microM for AD 169 strain) at lower concentrations than 3'F-ara-FU (ED50, 100 microM for AD 169). These nucleoside analogues are expected to be phosphorylated to their 5'-phosphate forms by cellular thymidine kinase in HCMV-infected cells. The thymidine kinase activities in the virus-infected and noninfected cells were compared. Cellular thymidine kinase was increased in the virus-infected cells and showed better phosphorylation of 2'F-ara-FU than did 3'F-ara-FU. HCMV DNA polymerase was purified using affinity column chromatography, and the inhibitory effect of the 5'-triphosphate derivatives of 2'F-ara-FU (2'F-ara-FUTP) and 3'F-ara-FU (3'F-ara-FUTP) against viral and host DNA polymerase alpha was examined. No significant difference in the effectiveness of inhibition was observed between viral DNA polymerase and host polymerase alpha. However, viral polymerase incorporated 2'F-ara-FUTP into newly synthesized DNA, whereas polymerase alpha did not utilize 2'F-ara-FUTP as a substrate. Thus, viral polymerase differs from host polymerase alpha in its recognition and utilization of 2'F-ara-FUTP. This difference may be important to the design of selective antiviral agents for HCMV.

Structural modifications at the 2'- and 3'-positions of some pyrimidine nucleosides as determinants of their interaction with the mouse erythrocyte nucleoside transporter.

Modifications in the sugar moiety of pyrimidine nucleosides may affect their ability to function as permeants of the mouse erythrocyte nucleoside transporter. In this investigation, a number of synthetic uracil and thymine nucleosides which differ from the physiological nucleosides, uridine, deoxyuridine and thymidine, through structural changes at the 2'- and 3'-positions were studied. Interaction of the analogs with the transporter has been assessed in terms of their affinities for an external site on the transporter as well as their abilities to effect trans-acceleration of thymidine efflux. 1-(beta-D-Arabinofuranosyl) uracil (araU) and 1-(beta-D-arabinofuranosyl)thymine (araT) were comparable to thymidine as permeants while nucleosides in which the 3'-hydroxyl was replaced with hydrogen or a halogen had a decreased affinity for the transporter. 3'-Fluoro-3'-deoxy-araU weakly accelerated thymidine efflux while its ribo-isomer and the other 3'-halogeno-3' deoxy-arabino analogs as well as dideoxythymidine inhibited efflux. The absence of 2'- and 3'-carbons in acyclothymidine and acyclouridine strongly decreased the affinities of these nucleosides for the transporter; efflux of thymidine was not accelerated in the presence of these compounds. The conformationally constrained cyclic nucleoside 2,2'-anhydro-araU had a very low affinity for the transporter, and influx of the radiolabeled compound could not be demonstrated. The results suggest that modification at the 3'-position, loss of a portion of the sugar ring, and lack of conformational flexibility are factors which decrease the abilities of some pyrimidine nucleosides to function as permeants. It is suggested that combined effects of substituents which play a role in determining nucleoside conformation should be considered in assessing structural requirements for permeants of the transporter.

Synthesis and evaluation of substituted 4(3H)-quinazolone derivatives for antimicrobial and antiacetylcholinesterase activities. Part 3.

The synthesis of some new 6,8-dibromo-2-N-(N-arylcarbamoyl) carbamoylmethylthio-3-aryl-4(3H)-quinazolone derivatives is described. The synthesized derivatives were screened for their antibacterial, antifungal and antiacetylcholinesterase activities in vitro. Most of the compounds exhibited interesting antimicrobial activities. The relation between this and their chemical structure has been studied.

Synthesis and evaluation of substituted quinazolone derivatives for antibacterial, antifungal, and antiacetylcholinesterase activities.

The synthesis of new 6-bromo- and 6,8-dibromo-2-[N-(2'-alkyl-1',3',4'-thiadizol-5'-yl)carbamoylmethylthio] -3-aryl/cyclohexyl-4-(3H)-quinazolones is described. The synthesis derivatives were screened for antibacterial, antifungal, and antiacetylcholinesterase activities in vitro. Most of the compounds exhibited significant biological activity. The relation between their biological activity and chemical structure was studied.