In Silico Exploration of Potential Natural Inhibitors against SARS-Cov-2 nsp10.

In continuation of our previous effort, different in silico selection methods were applied to 310 naturally isolated metabolites that exhibited antiviral potentialities before. The applied selection methods aimed to pick the most relevant inhibitor of SARS-CoV-2 nsp10. At first, a structural similarity study against the co-crystallized ligand, S-Adenosyl Methionine (SAM), of SARS-CoV-2 nonstructural protein (nsp10) (PDB ID: 6W4H) was carried out. The similarity analysis culled 30 candidates. Secondly, a fingerprint study against SAM preferred compounds 44, 48, 85, 102, 105, 182, 220, 221, 282, 284, 285, 301, and 302. The docking studies picked 48, 182, 220, 221, and 284. While the ADMET analysis expected the likeness of the five candidates to be drugs, the toxicity study preferred compounds 48 and 182. Finally, a density-functional theory (DFT) study suggested vidarabine (182) to be the most relevant SARS-Cov-2 nsp10 inhibitor.

Structural Basis for Inhibition of Human Primase by Arabinofuranosyl Nucleoside Analogues Fludarabine and Vidarabine.

Nucleoside analogues are widely used in clinical practice as chemotherapy drugs. Arabinose nucleoside derivatives such as fludarabine are effective in the treatment of patients with acute and chronic leukemias and non-Hodgkin's lymphomas. Although nucleoside analogues are generally known to function by inhibiting DNA synthesis in rapidly proliferating cells, the identity of their in vivo targets and mechanism of action are often not known in molecular detail. Here we provide a structural basis for arabinose nucleotide-mediated inhibition of human primase, the DNA-dependent RNA polymerase responsible for initiation of DNA synthesis in DNA replication. Our data suggest ways in which the chemical structure of fludarabine could be modified to improve its specificity and affinity toward primase, possibly leading to less toxic and more effective therapeutic agents.

Determination and quantification of intracellular fludarabine triphosphate, cladribine triphosphate and clofarabine triphosphate by LC-MS/MS in human cancer cells.

Purine nucleoside analogues are widely used in the treatment of haematological malignancies, and their biological activity is dependent on the intracellular accumulation of their triphosphorylated metabolites. In this context, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to study the formation of 5'-triphosphorylated derivatives of cladribine, fludarabine, clofarabine and 2'-deoxyadenosine in human cancer cells. Br-ATP was used as internal standard. Separation was achieved on a hypercarb column. Analytes were eluted with a mixture of hexylamine (5 mM), DEA (0.4%, v/v, pH 10.5) and acetonitrile, in a gradient mode at a flow rate of 0.3mLmin-1. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI-) were used for detection. The application of this method to the quantification of these phosphorylated cytotoxic compounds in a human follicular lymphoma cell line, showed that it was suitable for the study of relevant biological samples.

Comparative Analysis between [(18)F]Fludarabine-PET and [(18)F]FDG-PET in a Murine Model of Inflammation.

Lymphoma research has advanced thanks to introduction of [(18)F]fludarabine, a positron-emitting tool. This novel radiotracer has been shown to display a great specificity for lymphoid tissues. However, in a benign process such as inflammation, the uptake of this tracer has not been questioned. Indeed, in inflammatory zones, elevated glucose metabolism rate may result in false-positives with [(18)F]FDG-PET Imaging. In the present investigation, it has been argued that cells, involved in inflammation, might be less avid of [(18)F]fludarabine. To generate inflammation, Swiss mice were intramuscularly injected with 0.1 mL of turpentine oil into the right front paw. Imaging sessions with (18)F-labeled tracers named above were conducted on days 5 and 25 after inoculation. For each animal, volumes of interest (VOI), delineating the muscle of the inflamed (IP) and normal paws (NP), were determined on PET scans. For characterization of inflammation, muscle samples from IP and NP were stained with hematoxylin and eosin (H&E). In early (day 5) inflammation, [(18)F]FDG accumulation was 4.00 ± 1.65 times greater in the IP than in the contralateral NP; for [(18)F]fludarabine, this IP/NP ratio was 1.31 ± 0.28, resulting in a significant difference between radiotracer groups (p < 0.01). In late (day 25) inflammation, the IP/NP ratios were 2.07 ± 0.49 and 1.03 ± 0.07, for [(18)F]FDG and [(18)F]fludarabine, respectively (p < 0.001). [(18)F]Fludarabine showed significantly weaker uptake in inflammation when compared with [(18)F]FDG. This encouraging finding suggests that [(18)F]fludarabine-PET might well be a robust approach for distinguishing tumor from inflammatory tissue, avoiding false-positive PET results and thus enabling an accurate imaging of lymphoma.

Efficient Fludarabine-Activating PNP From Archaea as a Guidance for Redesign the Active Site of E. Coli PNP.

The combination of the gene of purine nucleoside phosphorylase (PNP) from Escherichia coli and fludarabine represents one of the most promising systems in the gene therapy of solid tumors. The use of fludarabine in gene therapy is limited by the lack of an enzyme that is able to efficiently activate this prodrug which, consequently, has to be administered in high doses that cause serious side effects. In an attempt to identify enzymes with a better catalytic efficiency than E. coli PNP towards fludarabine to be used as a guidance on how to improve the activity of the bacterial enzyme, we have selected 5'-deoxy-5'-methylthioadenosine phosphorylase (SsMTAP) and 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII), two PNPs isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. Substrate specificity and catalytic efficiency of SsMTAP and SsMTAPII for fludarabine were analyzed by kinetic studies and compared with E. coli PNP. SsMTAP and SsMTAPII share with E. coli PNP a comparable low affinity for the arabinonucleoside but are better catalysts of fludarabine cleavage with k(cat)/K(m) values that are 12.8-fold and 6-fold higher, respectively, than those reported for the bacterial enzyme. A computational analysis of the interactions of fludarabine in the active sites of E. coli PNP, SsMTAP, and SsMTAPII allowed to identify the crucial residues involved in the binding with this substrate, and provided structural information to improve the catalytic efficiency of E. coli PNP by enzyme redesign.

Annexin V-Directed Enzyme Prodrug Therapy Plus Docetaxel for the Targeted Treatment of Pancreatic Cancer.

OBJECTIVES: The bleak prognosis associated with pancreatic cancer (PDAC) drives the need for the development of novel treatment methodologies. Here, we evaluate the applicability of 3 enzyme prodrug therapies for PDAC, which are simultaneously targeted to the tumor, tumor vasculature, and metastases via annexin V. In these therapies, annexin V is fused to an enzyme, creating a fusion protein that converts nontoxic drug precursors, prodrugs, into anticancer compounds while bound to the tumor, therefore mitigating the risk of side effects. METHODS: The binding strength of fusion proteins to the human PDAC cell lines Panc-1 and Capan-1 was measured via streptavidin-horseradish peroxidase binding to biotinylated fusion proteins. Cytotoxic efficacy was evaluated by treatment with saturating concentrations of fusion protein followed by varying concentrations of the corresponding prodrug plus docetaxel. RESULTS: All fusion proteins exhibited strong binding to PDAC cells, with dissociation constants between 0.02 and 1.15 nM. Cytotoxic efficacy was determined to be very good for 2 of the systems, both of which achieved complete cell death on at least 1 cell line at physiologically attainable prodrug concentrations. CONCLUSIONS: Strong binding of fusion proteins to PDAC cells and effective cytotoxicity demonstrate the potential applicability of enzyme prodrug therapy to the treatment of PDAC.

IMP-GMP specific cytosolic 5'-nucleotidase regulates nucleotide pool and prodrug metabolism.

BACKGROUND: Type II cytosolic 5'-nucleotidase (cN-II) catalyzes the hydrolysis of purine and, to some extent, of pyrimidine monophosphates. Recently, a number of papers demonstrated the involvement of cN-II in the mechanisms of resistance to antitumor drugs such as cytarabine, gemcitabine and fludarabine. Furthermore, cN-II is involved in drug resistance in patients affected by hematological malignancies influencing the clinical outcome. Although the implication of cN-II expression and/or activity appears to be correlated with drug resistance and poor prognosis, the molecular mechanism by which cN-II mediates drug resistance is still unknown. METHODS: HEK 293 cells carrying an expression vector coding for cN-II linked to green fluorescent protein (GFP) and a control vector without cN-II were utilized. A highly sensitive capillary electrophoresis method was applied for nucleotide pool determination and cytotoxicity exerted by drugs was determined with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. RESULTS: Over-expression of cN-II causes a drop of nucleoside triphosphate concentration and a general disturbance of nucleotide pool. Over-expressing cells were resistant to fludarabine, gemcitabine and cytarabine independently of cN-II ability to hydrolyze their monophosphates. CONCLUSIONS: An increase of cN-II expression is sufficient to cause both a general disturbance of nucleotide pool and an increase of half maximal inhibitory concentration (IC50) of the drugs. Since the monophosphates of cytarabine and gemcitabine are not substrates of cN-II, the protection observed cannot be directly ascribed to drug inactivation. GENERAL SIGNIFICANCE: Our results indicate that cN-II exerts a relevant role in nucleotide and drug metabolism through not only enzyme activity but also a mechanism involving a protein-protein interaction, thus playing a general regulatory role in cell survival. SENTENCE: Resistance to fludarabine, gemcitabine and cytarabine can be determined by an increase of cN-II both through dephosphorylation of active drugs and perturbation of nucleotide pool.

Attenuation of nucleoside and anti-cancer nucleoside analog drug uptake in prostate cancer cells by Cimicifuga racemosa extract BNO-1055.

This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport.

Phosphorylation of deoxycytidine kinase on Ser-74: impact on kinetic properties and nucleoside analog activation in cancer cells.

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A.

Cytotoxic purine nucleoside analogues bind to A1, A2A, and A3 adenosine receptors.

Fludarabine, clofarabine, and cladribine are anticancer agents which are analogues of the purine nucleoside adenosine. These agents have been associated with cardiac and neurological toxicities. Because these agents are analogues of adenosine, they may act through adenosine receptors to elicit their toxic effects. The objective of this study was to evaluate the ability of cytotoxic nucleoside analogues to bind and activate adenosine receptor subtypes (A(1), A(2A), A(2B), and A(3)). Radioligand binding studies utilizing Chinese hamster ovary cells, stably transfected with adenosine A(1), A(2A), or A(3) receptor subtype, were used to assess the binding affinities of these compounds, whereas adenylyl cyclase activity was used to assess the binding to A(2B) receptors. Clofarabine and cladribine both bound to the A(2A) receptor with a K (i) of 17 and 15 µM, respectively. Clofarabine was the only adenosine analogue to bind to the A(3) receptor with a K (i) of 10 µM, and none of these compounds bound to the A(2B) receptor. Results show that clofarabine, cladribine, and fludarabine bind to the A(1) receptor. In addition, clofarabine, cladribine, and fludarabine were A(1) agonists (IC(50) 3.1, 30, and 30 µM, respectively). Neither pyrimidine nucleoside analogues gemcitabine nor cytarabine associated with any of the adenosine receptor subtypes (K (i) > 100µM). This is the first report of an interaction between all adenosine receptor subtypes and chemotherapeutic nucleoside analogues commonly used in the treatment of cancer. Therefore, activation of these receptors may be at least one mechanism through which fludarabine-associated toxicity occurs.

ROS-mediated upregulation of Noxa overcomes chemoresistance in chronic lymphocytic leukemia.

In recent years considerable progress has been made in treatment strategies for chronic lymphocytic leukemia (CLL). However, the disease remains incurable because of the development of chemoresistance. Strategies to overcome resistance mechanisms are therefore highly needed. At least two mechanisms contribute to the development of resistance to drugs; acquired mutations resulting in a dysfunctional p53 response and shifts in the balance between apoptosis-regulating proteins. Platinum-based compounds have been successfully applied in relapsed lymphoma and recently also in high-risk CLL. In this study we investigated the efficacy and mechanism of action of cisplatinum (CDDP) in chemorefractory CLL. Independent of p53-functional status, CDDP acted synergistically with fludarabine (F-ara-A). The response involved generation of reactive oxygen species (ROS), which led to specific upregulation of the proapoptotic BH3-only protein Noxa. Induction of Noxa resulted in cell death by apoptosis as inhibition of caspase activation completely abrogated cell death. Furthermore, drug-resistance upon CD40-ligand stimulation, a model for the protective stimuli provided in lymph nodes, could also be overcome by CDDP/F-ara-A. ROS accumulation resulted in Noxa upregulation mainly at the transcriptional level and this was, at least in part, mediated by the mitogen-activated protein kinase p38. Finally, Noxa RNA-interference markedly decreased sensitivity to CDDP/F-ara-A, supporting a key role for Noxa as mediator between ROS signaling and apoptosis induction. Our data indicate that interference in the cellular redox balance can be exploited to overcome chemoresistance in CLL.

A novel phenotypic method to determine fludarabine triphosphate accumulation in T-lymphocytes from hematopoietic cell transplantation patients.

PURPOSE: Fludarabine is an integral anticancer agent for patients with chronic lymphocytic leukemia (CLL) and those receiving conditioning regimens prior to allogeneic hematopoietic cell transplantation (HCT). An individual's response to fludarabine may be influenced by the amount of CD4(+) and CD8(+) T-lymphocyte suppression. Fludarabine undergoes cellular uptake and activation to form the cytotoxic metabolite, fludarabine triphosphate (F-ara-ATP). METHODS: We have previously developed a highly sensitive LC-MS method to quantitate intracellular F-ara-ATP concentrations in a leukemic cell line. However, quantitation of F-ara-ATP concentrations within CD4(+) and CD8(+) T-lymphocytes from pharmacokinetic blood samples obtained from patients receiving fludarabine therapy is not feasible because of the limited number of T-lymphocytes that can be isolated from each blood sample. Thus, we sought to determine F-ara-ATP accumulation after ex vivo exposure of freshly isolated human CD4(+) or CD8(+) T-lymphocytes to fludarabine. The method was optimized in T-lymphocytes obtained from healthy volunteers, and proved to be a feasible method to determine F-ara-ATP accumulation in patients undergoing HCT. RESULTS: Considerable variability was observed in F-ara-ATP accumulation in HCT patients (10.5- and 12.5-fold in CD4(+) and CD8(+) cells, respectively), compared to healthy volunteers (1.6- and 1.9-fold in CD4(+) and CD8(+) cells, respectively). Larger variability was also observed in gene expression of transporters and enzymes involved in F-ara-ATP accumulation in HCT patients; however, F-ara-ATP accumulation was not correlated with gene expression, which is in agreement with previous studies. CONCLUSIONS: The quantitation of F-ara-ATP accumulation in T-lymphocytes provides a novel tool to evaluate patient sensitivity to fludarabine. This tool can be used in future studies to evaluate whether intracellular F-ara-ATP accumulation is associated with efficacy and/or toxicity in patients receiving fludarabine.

Collision induced dissociation studies of alkali metal adducts of tetracyclines and antiviral agents by electrospray ionization, hydrogen/deuterium exchange and multiple stage mass spectrometry.

The collision induced dissociation (CID) mass spectra were obtained for the X(+)-adducts (X=Na(+) or Li(+)) of five tetracyclines, four pyrimidine and three purine derivatives and their fully D-exchanged species in which the labile hydrogens were replaced by deuterium by either gas phase or liquid phase exchange. The CID spectra were obtained for [M + Na](+) and [M + Li](+) and the exchanged analogs, [M(D) + Na](+) and [M(D) + Li](+), and compositions of product ions and mechanisms of decomposition were determined by comparison of the MS(n) spectra of the undeuterated and deuterated species. Metal ions are bound to the base of purine and pyrimidine antiviral agents and dissociate primarily to give the metal complexes of the base [B + X](+). For vidarabine monophosphate, however, the metal ions are bound to the phosphate group, resulting in unique and characteristic cleavage reactions not observed in the uncomplexed system, and dissociate through the loss of phosphate and/or phosphate metal ion complex. The [B + X](+) of these antiviral agents are relatively stable and show no or little fragmentation compared to [B + H](+). The CID of [B + X](+) of guanine derivative occurs mainly through elimination of NH(3) and that of trifluoromethyl uracil dissociates primarily through the loss of HF. For tetracyclines, metal ions are bound to ring A at the tricarbonylmethyl group and dissociate initially by the loss of NH(3)/ND(3) from [M(H) + X](+) and [M(D) + X](+). The CID spectra of [M + X](+) of tetracyclines are somewhat similar to those of [M + H](+). The dominant fragments from the metal complexes of these compounds are charge remote decompositions involving molecular rearrangements and the loss of small stable molecules. Additionally, tetracyclines and the antiviral agents show more selectivity towards Li+ ion than the corresponding complexes with Na(+) or K(+).

Fludarabine induces differential expression of proteins in human leukemia and lymphoma cells.

The purine analog fludarabine (FdAMP) is widely used for chemotherapy of B-lymphoid malignancies, and multiple mechanisms of action leading to apoptosis have been proposed. We examined changes at the protein level induced in the Raji cell line (Burkitt's lymphoma) by fludarabine nucleoside (FdA). Raji cells are sensitive to FdA. Raji cells treated with FdA (3 micro M, 24 hours), accumulate multiple phosphorylated forms of p53 in the nucleus that in turn degrade to phosphorylated forms of p40. Using CD antibody microarrays to determine surface expression profiles for Raji cells treated with FdA, we found up-regulation of the following CD antigens: CD20, CD54, CD80, CD86, and CD95. FdA thus induces changes in the genetic program of the cells that might be exploited to obtain synergy with therapeutic antibodies.

Imatinib inhibition of fludarabine uptake in T-lymphocytes.

PURPOSE: We investigated the potential drug-drug interaction between imatinib and fludarabine, which may be concomitantly administered in chronic myeloid leukemia (CML) patients receiving fludarabine-based conditioning for allogeneic hematopoietic cell transplantation (HCT). Imatinib is an inhibitor of human equilibrative transporters (hENTs), which are responsible for the intracellular uptake of fludarabine. METHODS: Intracellular accumulation of fludarabine triphosphate (F-ara-ATP), the active metabolite of fludarabine, was measured in CD4(+) and CD8(+) T-lymphocytes isolated from healthy volunteers, which were treated in vitro with fludarabine alone, and in the presence of either imatinib or NBMPR, a known hENT inhibitor. RESULTS: Imatinib significantly inhibited F-ara-ATP accumulation in CD4(+) and CD8(+) T-lymphocytes in a concentration-dependent manner. The observed imatinib inhibition was comparable to inhibition observed with NBMPR. The inhibition of F-ara-ATP by imatinib is likely due to inhibition of nucleoside transporters hENT1 and hENT2. CONCLUSIONS: There is significant in vitro drug interaction between imatinib and fludarabine. This effect may be of important consideration in patients receiving fludarabine-based conditioning prior to HCT.

A comparison of the transportability, and its role in cytotoxicity, of clofarabine, cladribine, and fludarabine by recombinant human nucleoside transporters produced in three model expression systems.

2-Chloro-9-(2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl)adenine (Cl-F-ara-A, clofarabine), a purine nucleoside analog with structural similarity to 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine) and 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A, fludarabine), has activity in adult and pediatric leukemias. Mediated transport of the purine nucleoside analogs is believed to occur through the action of two structurally unrelated protein families, the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). The current work assessed the transportability of Cl-F-ara-A, Cl-dAdo, and F-ara-A in cultured human leukemic CEM cells that were either nucleoside transport-defective or possessed individual human nucleoside transporter types and in Xenopus laevis oocytes and Saccharomyces cerevisiae yeast that produced individual recombinant human nucleoside transporter types. Cells producing hENT1 or hCNT3 exhibited the highest uptake of Cl-F-ara-A, whereas nucleoside transport-deficient cells and cells producing hCNT1 lacked uptake altogether. When Cl-F-ara-A transport rates by hENT1 were compared with those of Cl-dAdo and F-ara-A, Cl-dAdo had the highest efficiency of transport, although Cl-F-ara-A showed the greatest accumulation during 5-min exposures. In cytotoxicity studies with the CEM lines, Cl-F-ara-A was more cytotoxic to cells producing hENT1 than to the nucleoside transport-deficient cells. The efficiency of Cl-F-ara-A transport by oocytes with recombinant transporters was hCNT3 > hENT2 > hENT1 > hCNT2; no transport was observed with hCNT1. Affinity studies with recombinant transporters produced in yeast showed that hENT1, hENT2, and hCNT3 all had higher affinities for Cl-F-ara-A than for either Cl-dAdo or F-ara-A. These results suggest that the nature and activity of the plasma membrane proteins capable of inward transport of nucleosides are important determinants of Cl-F-ara-A activity in human cells.

[Metabolism, mechanism of action and resistance to cytotoxic nucleoside analogues]. / Métabolisme, mécanisme d'action et résistance aux analogues de nucléosides cytotoxiques.

Cytotoxic nucleoside analogues are widely used in treatment of patients with hematological malignancies as well as for some solid tumors. Resistance developed against these molecules limit their clinical use. Many studies on cell models and clinical samples have identified cellular mechanisms involved in this phenomenon. Here, we describe the available data concerning the proteins involved in the metabolism and the mechanism of action of nucleoside analogues, as well as the clinical studies showing their implication in the resistance to these drugs.

Functional analysis of genetic variants in the human concentrative nucleoside transporter 3 (CNT3; SLC28A3).

The human concentrative nucleoside transporter, CNT3 (SLC28A3), plays an important role in mediating the cellular entry of a broad array of physiological nucleosides and synthetic anticancer nucleoside analog drugs. As a first step toward understanding the genetic basis for interindividual differences in the disposition and response to antileukemic nucleoside analogs, we examined the genetic and functional diversity of CNT3. In all, 56 variable sites in the exons and flanking intronic region of SLC28A3 were identified in a collection of 270 DNA samples from US populations (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). Of the 16 coding region variants, 12 had not been previously reported. Also, 10 resulted in amino-acid changes and three of these had total allele frequencies of >/=1%. Nucleotide diversity (pi) at nonsynonymous and synonymous sites was estimated to be 1.81 x 10(4) and 18.13 x 10(4), respectively, suggesting that SLC28A3 is under negative selection. All nonsynonymous variants, constructed by site-directed mutagenesis and expressed in Xenopus laevis oocytes, transported purine and pyrimidine model substrates, except for c. 1099G>A (p. Gly367Arg). This rare variant alters an evolutionarily conserved site in the putative substrate recognition domain of CNT3. The presence of three additional evolutionarily conserved glycine residues in the vicinity of p. Gly367Arg that are also conserved in human paralogs suggest that these glycine residues are critical in the function of the concentrative nucleoside transporter family. The genetic analysis and functional characterization of CNT3 variants suggest that this transporter does not tolerate nonsynonymous changes and is important for human fitness.

Design, synthesis, and characterization of a series of cytochrome P(450) 3A-activated prodrugs (HepDirect prodrugs) useful for targeting phosph(on)ate-based drugs to the liver.

A new class of phosphate and phosphonate prodrugs, called HepDirect prodrugs, is described that combines properties of rapid liver cleavage with high plasma and tissue stability to achieve increased drug levels in the liver. The prodrugs are substituted cyclic 1,3-propanyl esters designed to undergo an oxidative cleavage reaction catalyzed by a cytochrome P(450) (CYP) expressed predominantly in the liver. Reported herein is the discovery of a prodrug series containing an aryl substituent at C4 and its use for the delivery of nucleoside-based drugs to the liver. Prodrugs of 5'-monophosphates of vidarabine, lamivudine (3TC), and cytarabine as well as the phosphonic acid adefovir were shown to cleave following exposure to liver homogenates and exhibit good stability in blood and other tissues. Prodrug cleavage required the presence of the aryl group in the cis-configuration, but was relatively independent of the nucleoside and absolute stereochemistry at C4. Mechanistic studies suggested that prodrug cleavage proceeded via an initial CYP3A-catalyzed oxidation to an intermediate ring-opened monoacid, which subsequently was converted to the phosph(on)ate and an aryl vinyl ketone by a beta-elimination reaction. Studies in primary rat hepatocytes and normal rats comparing 3TC and the corresponding HepDirect prodrug demonstrated the ability of these prodrugs to effectively bypass the rate-limiting nucleoside kinase step and produce higher levels of the biologically active nucleoside triphosphate.

In vitro susceptibility of CD4+ and CD8+ T cell subsets to fludarabine.

Administration of the adenosine analogue fludarabine (FLU) in vivo induces a profound and prolonged T lymphopenia which mainly affects CD4(+) cells. To better understand the mechanistic basis underlying this preferential depletion, we analyzed the in vitro susceptibility of T cell subsets to FLU-induced apoptosis. Contrasting with observations in vivo, our results showed that treatment of peripheral blood mononuclear cells with FLU induced a higher level of apoptosis in CD8(+) than in CD4(+) T lymphocytes. This increased sensitivity of CD8(+) T cells to FLU was observed in samples from both, healthy donors and B cell chronic lymphocytic leukemia patients, and resulted in higher CD4:CD8 ratios in FLU-treated than in untreated cultures (P<0.01). Expression of factors involved in FLU transport and metabolism was then evaluated by quantitative real time-PCR in normal T cell subsets. It was found that mRNA levels of human equilibrative nucleoside transporter-1 nucleoside transporter were higher whereas deoxycytidine kinase and IMP/GMP selective 5'-nucleotidase mRNA levels were lower in CD4(+) cells. However the dCK/cN-II ratio was 2-fold greater in CD8(+) than in CD4(+) T lymphocytes, which could account for the higher apoptosis levels observed in the CD8(+) subset. These results favor the view that decreased CD4:CD8 ratios in FLU-treated patients should be attributed to differences in cell recovery and/or homing between T cell subsets.