Phenotypic protein profiling of different B cell sub-populations using antibody CD-microarrays.

Antibody microarrays enable extensive protein expression profiling, and provide a valuable complement to DNA microarray-based gene expression profiling. In this study, we used DotScan antibody microarrays that contain antibodies against 82 different cell surface antigens, to determine phenotypic protein expression profiles for human B cell sub-populations. We then demonstrated that the B cell protein profile can be used to delineate the relationship between normal B cells and malignant counterparts. Principle component analysis showed that the lymphomas did not cluster with the normal memory B cells or germinal centre B cells, but they did cluster with germinal centre founder cells and naïve B cells.

Immunophenotyping of leukemias using a cluster of differentiation antibody microarray.

Different leukemias express on their plasma membranes particular subsets of the 247 defined cluster of differentiation (CD) antigens, which may resemble those of precursor cells along the lineages of differentiation to mature myeloid and lymphoid leukocytes. The extent of use of CD antigen expression (immunophenotyping) for identification of leukemias has been constrained by the technique used, flow cytometry, which commonly specifies only three CD antigens in any one assay. Currently, leukemias and lymphomas are diagnosed using a combination of morphology, immunophenotype, cytochemistry, and karyotype. We have developed a rapid, simple procedure, which enables concurrent determination of 50 or more CD antigens on leukocytes or leukemia cells in a single analysis using a microarray of antibodies. A suspension of cells is applied to the array, and cells only bind to antibody dots for which they express the corresponding CD antigen. For patients with significantly raised leukocyte counts, the resulting dot pattern then represents the immunophenotype of those cells. For patients at earlier stages of disease, the diagnosis depends on recognition of dot patterns distinct from the background of normal leukocytes. Distinctive and reproducible dot patterns have been obtained for normal peripheral blood leukocytes, chronic lymphocytic leukemia (CLL), hairy cell leukemia, mantle cell lymphoma, acute myeloid leukemia, and T-cell acute lymphoblastic leukemia. The consensus pattern for CD antigen expression found on CLL cells taken from 20 patients in descending order of cells bound was CD44, HLA-DR, CD37, CD19, CD20, CD5, CD52, CD45RA, CD22, CD24, CD45, CD23, CD21, CD71, CD11c, and CD9. The antigens that provided the best discrimination between CLL and normal peripheral blood leukocytes were CD19, CD20, CD21, CD22, CD23, CD24, CD25, and CD37. Results obtained for the expression of 48 CD antigens from the microarray compared well with flow cytometry. The microarray enables extensive immunophenotyping, and the intact cells captured on antibody dots can be further characterized using soluble, fluorescently labeled antibodies.

Dual effects of pyrazofurin and 3-deazauridine upon pyrimidine and purine biosynthesis in mouse L1210 leukemia.

Pyrazofurin (NSC 143095) as the monophosphate derivative is a potent inhibitor of orotidine 5'-monophosphate (OMP) decarboxylase of the pyrimidine pathway and has been proposed to inhibit 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (EC 2.1.2.3) of the purine pathway (J. F. Worzalla, and M. J. Sweeney, Pyrazofurin inhibition of purine biosynthesis via 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate formyltransferase. Cancer Res., 40: 1482-1485, 1980). Measurement of levels of pyrimidine and purine intermediates in cultured mouse L1210 leukemia cells has shown that 25 microM pyrazofurin induces an 8-fold accumulation of OMP and large accumulations of intermediates proximal to the blockade with abrupt decreases in uridine and cytidine nucleotides. Considerable increases in the cellular concentrations of N-succino-AICAR (SAICAR), AICAR, 5-formamidoimidazole-4-carboxamide ribotide (FAICAR), IMP, XMP, and GMP at later times indicate that AICAR transformylase is not significantly inhibited in cultured cells; rather the purine pathway and the GMP branch are stimulated. However, addition of 25 microM 3-deazauridine (NSC 126849) to leukemia cells did result in inhibition of AICAR transformylase: AICAR and SAICAR accumulated, IMP disappeared and there was a large accumulation of guanosine nucleotides. Blockade of pyrimidine biosynthesis by derivatives of pyrazofurin or 3-deazauridine spares 5-phosphoribosyl-1-pyrophosphate and L-glutamine, elevated concentrations of which may stimulate initial reactions of purine biosynthesis and the reaction XMP----GMP.

Cytotoxic effects of dihydroorotase inhibitors upon human CCRF-CEM leukemia.

6-L-Thiodihydroorotate (TDHO) and 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylate (HDDP) are potent inhibitors of mammalian dihydroorotase in vitro (R. I. Christopherson, K. J. Schmalzl, E. Szabados, R. J. Goodridge, M. C. Harsanyi, M. E. Sant, E. M. Algar, J. E. Anderson, A. Armstrong, S. C. Sharma, W. A. Bubb, and S. D. Lyons, Biochemistry, 28: 463-470, 1989). Using human CCRF-CEM leukemia cells growing in culture, TDHO and HDDP as the free acids have 50% inhibitory concentration (IC50) values of 32 microM and greater than 1000 microM, respectively, whereas for TDHO methyl ester, the IC50 value is 25 microM, and for HDDP dimethyl ester, the IC50 value is 21 microM. These IC50 values were not affected by addition of dihydroorotate, uridine, or deoxycytidine to the culture medium. TDHO methyl ester (25 microM) had only slight inhibitory effects upon the dihydroorotase reaction of de novo pyrimidine biosynthesis in growing leukemia cells, cells arrested in G2 + M phases of the cell cycle. At 250 microM TDHO methyl ester, analysis of cell extracts by high-performance liquid chromatography showed that after 4 h carbamyl aspartate had accumulated from undetectable levels to 760 microM, whereas UTP decreased from 580 to 110 microM and CTP from 350 to 86 microM, indicating inhibition of dihydroorotase in growing leukemia cells. IMP accumulated from 63 to 350 microM, total guanylates increased while adenylates decreased, and the adenylate energy charge decreased from 0.91 to 0.69 after 4 h. The cellular concentration of 5-phosphoribosyl 1-pyrophosphate increased from 180 to 290 microM due to sparing from pyrimidine nucleotide biosynthesis resulting in complementary stimulation of the de novo purine pathway. HDDP dimethyl ester at concentrations of up to 250 microM had no discernable effect upon pyrimidine or purine nucleotide biosynthesis. At 25 microM HDDP-dimethyl ester, cells arrested in G2 + M phases initially, with accumulation of cells in G1/G0 at later times. These data suggest that the primary mechanisms of growth inhibition for TDHO and HDDP involve inhibition of cell cycle progression from late G2 or M phase to G1 phase and that blockade of the pyrimidine pathway by TDHO is a secondary effect found at higher concentrations.

Regulation of aspartate carbamoyltransferase of Escherichia coli by the interrelationship of magnesium and nucleotides.

Purified aspartate carbamoyltransferase from Escherichia coli K12 (carbamoylphosphate: L-aspartate carbamyltransferase, EC 2.1.3.2) shows greater activity with nucleotide effectors as the magnesium nucleotide complex than with similar amounts of the sodium nucleotide. Regulation of aspartate carbamoyltransferase activity in vivo may occur by changes in the total concentration of regulatory nucleotides or, under conditions of magnesium-limited growth, by variation of the saturation of the nucleotides with magnesium.

Nucleotide and nucleic acid metabolism in rat thymocytes during cell cycle progression.

A complete cell cycle of mature, concanavalin A (Con A) stimulated rat thymocytes was documented by analyzing the cell number as well as the content and synthesis of DNA and RNA. Cell cycle progression is accompanied by an elevation of class I, II and III RNA polymerase activities (about 10-fold) in the S phase maximum, 48 h after stimulation. Moreover, maximal cellular contents of DNA, ATP, ADP and AMP were observed at this culture period, whereas the RNA level peaked at 60 h. The synthesis of purine and pyrimidine nucleotides de novo was detected by use of [14C]HCO3-. Maximal incorporation rates of [14C]HCO3- into nucleotides (de novo synthesis) and of [3H]adenine into adenylates ('salvage pathway') occur during the S phase. However, the de novo synthesis rates were markedly lower than those of the 'salvage pathway'. The highest cellular level of the nucleotide precursor 5-phosphoribosyl-1-pyrophosphate (8.4-fold increase) also coincided with the S phase.

Thiol-bearing compounds selectively inhibit protein kinase C-dependent oxidative events and proliferation in human T cells.

The aminothiol cysteamine at 10(-5) to 10(-4) M concentrations inhibited both the proliferation of mitogenically stimulated human peripheral mononuclear cells and the phorbol myristate acetate-mediated oxidation of 2',7'-dichlorofluorescein within these cells. Both 2',7'-dichlorofluorescein oxidation and the proliferative response were maximally sensitive to cysteamine-induced inhibition during the first 2 h of mitogenic stimulation. This period of sensitivity indicates that cysteamine preferentially arrests cells transiting from G0 to G1 and is the first such demonstration, of an early cell cycle site of arrest for this compound. 2,3-Dimercapto-1-propane-sulfonic acid and WR 1065 were found to be more effective than cysteamine in attenuating T cell replication but not N-acetylcysteine. Aminothiols preferentially inhibited the intracellular oxidation of 2',7'-dichlorofluorescein, rather than the activity of protein kinase C, which initiates the oxidation, indicating that oxidative events are one of a number of crucial and independent events required for the successful transition through G0-G1. Since aminothiols affect both lectin and PMA/ionomycin-directed proliferation, these aminothiol-sensitive events may serve to integrate and regulate common pathways in T cell activation.

Identification of the replication terminator protein binding sites in the terminus region of the Bacillus subtilis chromosome and stoichiometry of the binding.

DNase I footprinting of the interaction between the replication terminator protein (RTP) of Bacillus subtilis and the inverted repeat region (IRR) at the chromosome terminus, to which it binds to block the clockwise replication fork, showed that two major regions of 41 base pairs (bp) were protected from cleavage. These regions corresponded approximately to the imperfect inverted repeats (IRI and IRII) identified previously. Band retardation analyses of the interaction between RTP and portions of the IRR established that each inverted repeat (IRI or IRII) contained two RTP binding sites. By sedimentation equilibrium in the ultracentrifuge, RTP was found to exist as a dimer of 29 kDa at neutral pH and concentrations above 0.2 g/l. Quantitative studies of the RTP-IRR interaction using [3H]RTP and [32P]IRR showed that the fully saturated complex contained eight RTP monomers per IRR. It is concluded that a dimer of RTP binds to each of the four sites in IRR. The apparent dissociation constant for the interaction was estimated (in the presence of 50% glycerol) to be 1.2 x 10(-11) M (dimer of RTP). Glycerol was found to have a marked effect on the affinity of RTP for the IRR and on the relative amounts of the interaction complexes formed; in the absence of glycerol the dissociation constant was approximately 50-fold higher and there was pronounced co-operative binding of RTP dimers to adjacent sites in each inverted repeat. Examination of the DNA sequence in IRI and IRII identified two 8 bp direct repeats in each. The regions protected from DNase I cleavage in each inverted repeat and the protection afforded by a core sequence spanning just one of the 8 bp direct repeats were consistent with each 8 bp repeat representing a recognition sequence for the RTP dimer. A model describing the binding of RTP to the IRR is presented.

Partial inactivation of chorismate mutase-prephenate dehydrogenase from Escherichia coli in the presence of analogues of chorismate.

Chorismate-5,6-epoxide, chorismate-5,6-diol, various adamantane derivatives and 2-hydroxy-phenyl acetate are structural analogues of chorismate that act as competitive inhibitors of both the chorismate mutase and prephenate dehydrogenase activities of the bifunctional enzyme, hydroxyphenylpyruvate synthase. The interactions of these chorismate analogues with both activities of the synthase are investigated further. Chorismate mutase and prephenate dehydrogenase activities were assayed spectrophotometrically at 290 and 340 nm, respectively. Data were fit by non-linear regression to appropriate equations describing the time-dependent formation of product or decay of enzymic activity. In the presence of these chorismate analogues, both the mutase and dehydrogenase activities undergo a time-dependent partial inactivation. Progress curves for synthesis of product by the mutase or dehydrogenase in the presence of chorismate-5,6-epoxide, chorismate-5,6-diol or adamantane-1,3-diacetate resemble time-courses characteristic of slow-binding inhibition. However, if the bifunctional enzyme was preincubated with a chorismate analogue prior to addition of substrate, only a minor proportion of enzymic activity was recovered, excluding the possibility of reversible, slow-binding inhibition. When hydroxyphenylpyruvate synthase binds certain chorismate analogues to form an EI complex, there is a slow conformational transition to an ET complex, which may be susceptible to oxidation leading to partial inactivation. Some protection against this inactivation is provided by high concentrations of dithiothreitol (20 mM), suggesting that the inactivation may be due to chemical oxidation.

Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates.

Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2-14C]orotate (OA) is quantitatively converted to [2-14C]OMP and then [2-14C]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose (PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102+/-11 microM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N(alpha)(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2-d]pyrimidine (PY873) and multitargeted antifolate, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (MTA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3-12-fold. These data indicate that LTX induces the most potent inhibition of APRT.

Relationship between the catalytic sites of human bifunctional IMP synthase.

BACKGROUND AND AIMS: The bifunctional enzyme, IMP synthase, contains 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase and IMP cyclohydrolase activities and catalyses the ninth and tenth reactions of the pathway for de novo biosynthesis of purine nucleotides (AICAR-->FAICAR-->IMP). The spatial relationship between the two active sites on IMP synthase has been investigated along with the possibility that the intermediate, FAICAR, may be channelled between the two sites. METHODS: The two catalytic activities and the overall reaction (AICAR-->FAICAR-->IMP) were assayed using 3H-labelled AICAR or FAICAR with isolation of the reaction products by thin-layer chromatography. RESULTS: Inhibition constants for the interactions of six purine nucleoside 5'-monophosphate derivatives with AICAR transformylase and IMP cyclohydrolase were 24- to 820-fold higher for the transformylase. N-ethylmaleimide inactivated IMP cyclohydrolase but not AICAR transformylase. The rate of IMP synthesis from AICAR was consistent with a high local concentration of FAICAR at the cyclohydrolase site but addition of exogenous unlabelled FAICAR reduced the amount of [3H]AICAR formed from [3H]AICAR indicating that the channelling of FAICAR was not absolute. CONCLUSION: The AICAR transformylase and IMP cyclohydrolase active sites of IMP synthase are distinct but sufficiently close for the FAICAR produced by a transformylase site to be preferentially utilized as a substrate by a cyclohydrolase site on the same molecule if dimeric, bifunctional IMP synthase.

Metabolism of adenosine and deoxyadenosine by human erythrocytes and CCRF-CEM leukemia cells.

Human lymphocytes lacking adenosine deaminase die and T-cell leukemias are killed by deoxycoformycin (dCf), an inhibitor of adenosine deaminase, due to impaired metabolism of dAdo. The initial metabolism of exogenous adenosine (Ado) and deoxyadenosine (dAdo) has been compared in human erythrocytes and CCRF-CEM leukemia cells and the data obtained have been simulated using kinetic constants obtained in vitro for the enzymes involved. Cells were mixed with 3H-labelled Ado and dAdo, samples were taken at 3 sec intervals and progress curves for the 3H-labelled metabolites formed were determined by quantitative two-dimensional thin layer chromatography. Erythrocytes rapidly take up Ado and the predominant metabolite after 60 sec is hypoxanthine (Hyp), while for dAdo, deoxyinosine (dIno) predominates. By contrast, leukemia cells convert to Ado predominantly to AMP, while dAdo is converted first to Hyp and the to AMP. The presence of dCf had little effect upon Ado metabolism by induced accumulation of dAdo. Erythrocytes rapidly degrade Ado and dAdo to Hyp, although the phosphorolysis of dIno is relatively slow. Human CCRF-CEM leukemia cells convert most of the Ado or dAdo to AMP after 60 sec. For dAdo, the sequence of reactions would be dAdo-->dIno-->Hyp-->IMP-->sAMP-->AMP. dCf does not significantly affect the conversion of Ado-->AMP, but dCf blocks AMP accumulation from dAdo, consistent with the reaction sequence shown above. A computer model has been developed for the metabolism of Ado and dAdo, but some of the kinetic constants determined in vitro for this model do not pertain to intact cells.

Metabolic effects of thiopurine derivatives against human CCRF-CEM leukaemia cells.

UNLABELLED: BACKGROUND and aims. To compare the metabolic effects induced by the anticancer drugs, 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and 6-methylmercaptopurine riboside (MMPR), which may inhibit the de novo biosynthesis of purine nucleotides or be mis-incorporated into DNA or RNA. METHODS: Leukaemia cells were grown in culture, exposed to a thiopurine and cell extracts were analyzed for NTPs, dNTPs, drug metabolites and P-Rib-PP. RESULTS: In leukaemia cells, 6-MP was converted to MPR-MP, thio-XMP, thio-GMP, thio-GDP and thio-GTP. Metabolites of 6-TG included thio-XMP, thio-GMP, thio-GDP and thio-GTP, while MMPR-MP was the only major metabolite of MMPR, MMPR (25 microM, 4 h) induced a 16-fold increase in P-Rib-PP and 6-MP (25 microM, 4 h) induced a delayed 5.2-fold increase. MPR-MP, thio-GMP and MMPR-MP are inhibitors of amido phosphoribosyltransferase from leukaemia cells with Ki values of 114 +/- 7.10 microM, 6.20 +/- 2.10 microM and 3.09 +/- 0.30 microM, respectively. CONCLUSION: The nucleoside-5'-monophosphate derivatives of the 3 thiopurines inhibit amido phosphoribosyltransferase in growing leukaemia cells but there is also an initial inhibition of the further conversion of IMP in the pathway. In growing cells, MMPR acts solely as an inhibitor of de novo purine biosynthesis while 6-TG and to a lesser extent, 6-MP, are converted to significant concentrations of di- and tri-phosphate derivatives which may have other mechanisms of cytotoxicity.

Comparative effects of cladribine, fludarabine and pentostatin on nucleotide metabolism in T- and B-cell lines.

BACKGROUND AND AIMS: the purine nucleoside analogues cladribine (CdA), fludarabine (F-Ara-AMP) and pentostatin (dCf), are effective therapy for a range of T- and B-cell lymphoid malignancies. The effects upon nucleotide metabolism in human CCRF-CEM T-cell leukaemia and Raji B-cell lymphoma cell lines of these drugs have been compared to assess possible mechanisms of cytotoxicity. METHODS: Leukaemia cells were exposed to a purine nucleoside analogue and perchloric acid extracts were analysed by HPLC for 2'-deoxynucleoside-5'-triphosphates (dNTPs), nucleoside-5'-triphosphates (NTPs) and drug metabolites. RESULTS: After addition of a purine nucleoside analogue, CdA-TP and F-Ara-ATP accumulate in cells while the levels of dCf-TP formed were not detectable by ultra-violet absorbance. In response to accumulating concentrations of drug triphosphate, the cellular levels of dNTPs initially decrease (0-4 h), then accumulate above their initial levels (4-10 h) before slowly declining beyond 10 h. NTPs also accumulate during the period 4-10 h before declining at later times. CONCLUSION: The temporal effects on the levels of dNTPs and NTPs of the 3 purine nucleoside analogues are similar against CCRF-CEM and Raji cells. However, CdA induces major depletions of dTTP, dGTP and dATP in CCRF-CEM cells and F-Ara-A induces a major accumulation of dATP in Raji cells.

Effects of cytosine arabinoside on human leukemia cells.

Cytosine arabinoside (Ara-C) is used to treat leukemias, with complete remission induced by combination chemotherapy in approximately 70% of cases of acute myelogenous leukemia (AML). Ara-CTP acts as a competitive inhibitor of DNA polymerase and may also be incorporated into DNA. Accumulation of deoxyribonucleoside triphosphates (dNTPs) induced by Ara-C may indicate disruption of DNA synthesis in susceptible leukemia cells. A procedure has been developed for the quantification of Ara-CTP and dNTPs from small samples of leukaemia cells from patients (4 x 10(7) cells) activated with concanavalin A (10 micrograms/ml, 48 hr) and grown in the presence of [32P]orthophosphate (1.1 microM, 9 x 10(6) Ci/mol, 16 hr). The susceptibilities to Ara-C of the human leukemia cell lines CCRF-CEM (IC50 = 6.30 nM), CCRF-HSB-2 (IC50 = 10.4 nM) and MOLT-4 (IC50 = 10.0 nM) may be correlated with their abilities to accumulate high concentrations of Ara-CTP (> 1000 amol/cell) with increases of between 1.3- and 3.4-fold in dATP, dGTP and dTTP for the four cell lines, while dCTP decreased between 0.23- and 0.78-fold. By contrast, an Ara-C-resistant derivative of HL-60 cells (IC50 = 400 nM) accumulated only low concentrations of Ara-CTP (71 amol/cell) without significant changes in dNTPs. High concentrations of Ara-CTP in leukemia cells induce accumulations of dATP, dGTP and dTTP due to inhibition of DNA synthesis, and depletion of dCTP. This imbalance in the pools of the four dNTPs could lead to genetic miscoding and cell death.

Location of the dihydroorotase domain within trifunctional hamster dihydroorotate synthetase.

Mammalian dihydroorotase (DHOase, EC 3.5.2.3) is part of a trifunctional protein, dihydroorotate synthetase which catalyzes the first three reactions of de novo pyrimidine biosynthesis. We have subcloned a portion of the cDNA from the plasmid pCAD142 and obtained a nucleotide sequence which extends 2.1 kb in the 5' direction from the sequence encoding the aspartate transcarbamoylase (ATCase) domain at the 3'-end of the cDNA. The DHOase and ATCase domains have been purified from an elastase digest of the trifunctional protein and subjected to amino acid (aa) sequencing from their N termini. The sequence of the N-terminal 24 aa of the DHOase domain has been obtained and aligned with the cDNA sequence. The C-terminal residues of the DHOase domain have been identified as Leu followed by Val which, when taken with partial sequences of the CNBr fragments of this domain, defines the coding sequence of the active, globular DHOase domain released by proteolysis. Prediction of protein secondary structure from the deduced aa sequence showed that the DHOase domain (Mr 37,751) is separated from the C-terminal ATCase domain (Mr 34,323) by a bridging sequence (Mr 12,532) consisting of multiple beta-turns.

Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites.

The infection of human erythrocytes by two strains of the human malarial parasite, Plasmodium falciparum (FCQ-27 or the multi-drug-resistant strain K-1), markedly changed the transport characteristics of the nucleosides, adenosine and tubercidin, compared to uninfected erythrocytes. A component of the transport of these nucleosides was insensitive to the classical mammalian nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In vitro studies with tubercidin demonstrated ID50 values of 0.43 and 0.51 microM for FCQ-27 and K-1, respectively. In addition, the nucleoside transport inhibitors NBMPR, nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole also independently exhibited antimalarial activity in vitro. The combination of tubercidin and NBMPR or NBTGR in vitro demonstrated synergistic activity, whilst tubercidin together with dilazep or dipyridamole showed subadditive activity. Analysis by HPLC indicated that NBMPR could permeate the infected cell membrane and provided evidence for the catabolism of NBMPR in vitro, with subsequent alteration of the purine pool in the infected erythrocyte. These observations further indicated the possibility of the utilization of cytotoxic nucleosides against P. falciparum infection in conjunction with a nucleoside transport inhibitor to protect the host tissue.

Synthesis and enzymic evaluation of 4-mercapto-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide as an inhibitor of mammalian dihydroorotase.

The design, synthesis, and enzymic evaluation of cis- and trans-4-mercapto-6-oxo-1,4-azaphosphinane-2-carboxylic acid 4-oxide 5 against mammalian dihydroorotase is presented. The design strategy for 5 was based on the strong affinity of phosphinothioic acids for zinc and that 5 also resembles the postulated tetrahedral transition state for the enzyme-catalyzed reaction. The synthesis of 5 utilized a novel protection/deprotection sequence upon 4-hydroxy-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide 4, followed by incorporation of alpha-phenyl benzenemethanethiol and exhaustive deprotection to afford 5 in 40% overall yield from 4. The activities of both isomers of 5 as inhibitors of mammalian dihydroorotase were marginally greater than that of the parent phosphinic acid 4, indicating a weak binding enhancement due to the phosphinothioic acid moiety.

Effects of dual combinations of antifolates with atovaquone or dapsone on nucleotide levels in Plasmodium falciparum.

The triazine antifolates, cycloguanil and 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-[(2,4,5-trichlorophenoxy)propy loxy]-1,3,5-triazine hydrobromide (WR99210), and their parent biguanide compounds, proguanil and N-[3-(2,4,5-trichlorophenoxy)propyloxy]-n-(1-methylethyl)-imido dicarbonimidic-diamine hydrochloride (PS-15), were tested in combination with a series of antimalarial drugs for synergism against Plasmodium falciparum growing in erythrocytic culture. Four synergistic combinations were found: cycloguanil dapsone, WR99210-dapsone, proguanil-atovaquone, and PS-15-atovaquone. Cycloguanil-dapsone or WR99210-dapsone had a profound suppressive effect on the concentration of dTTP in parasites while that of dATP increased. Depletion of dTTP is consistent with cycloguanil or WR99210 inhibiting dihydrofolate reductase and dapsone inhibiting dihydropteroate synthase. For the combinations proguanil-atovaquone and PS-15-atovaquone, the levels of nucleoside triphosphates (NTPs) and dNTPs were generally suppressed, suggesting that inhibition is not through nucleotide pathways but probably through another metabolic mechanism(s). Combinations of two synergistic pairs of antimalarial drugs, (proguanil-atovaquone)-(cycloguanil-dapsone) and (PS-15-atovaquone)-(WR99210-dapsone), were tested, and it was found that NTPs and dNTPs decreased much more than for a single synergistic combination. Dual synergistic combinations could play an important role in the therapy of multidrug-resistant malaria, just as combination chemotherapy is used to treat cancer.

Flavin analogs with antimalarial activity as glutathione reductase inhibitors.

10-(4'-Chlorophenyl)-3-methylflavin has antimalarial activity in vitro and in vivo (Cowden et al., J Med Chem 31: 799, 1988). This flavin analog and two of its derivatives were found to inhibit the antioxidant flavoenzyme glutathione reductase from human erythrocytes in its isolated form as well as in hemolysates. The mixed-type inhibition was completely reversible, the Ki-values being of the order of 1 microM. Surprisingly, the drugs were not competitive with FAD, but with GSSG, one of the enzyme's substrates. Malaria parasite glutathione reductase, extracted from Plasmodium falciparum, could also be inhibited by the compounds. Studies on the effects of the substances on P. falciparum in vitro, which were demonstrated morphologically and by growth inhibition, confirmed previous observations with 10-(4'-chlorophenyl)-3-methylflavin and showed similar parasiticidal characteristics for the two new derivatives. The activities of five other erythrocytic enzymes tested were not impaired by the drugs, nor was the nucleotide metabolism of erythrocytes and/or parasites significantly changed. Permeation into red blood cells was demonstrated for one compound by 19F-NMR-spectroscopy. Inhibition of glutathione reductase might contribute to, or account for, the antimalarial activity of this group of flavin analogs.