Structural basis for inhibition of mycobacterial and human adenosine kinase by 7-substituted 7-(Het)aryl-7-deazaadenine ribonucleosides.

Adenosine kinase (ADK) from Mycobacterium tuberculosis (Mtb) was selected as a target for design of antimycobacterial nucleosides. Screening of 7-(het)aryl-7-deazaadenine ribonucleosides with Mtb and human (h) ADKs and testing with wild-type and drug-resistant Mtb strains identified specific inhibitors of Mtb ADK with micromolar antimycobacterial activity and low cytotoxicity. X-ray structures of complexes of Mtb and hADKs with 7-ethynyl-7-deazaadenosine showed differences in inhibitor interactions in the adenosine binding sites. 1D (1)H STD NMR experiments revealed that these inhibitors are readily accommodated into the ATP and adenosine binding sites of Mtb ADK, whereas they bind preferentially into the adenosine site of hADK. Occupation of the Mtb ADK ATP site with inhibitors and formation of catalytically less competent semiopen conformation of MtbADK after inhibitor binding in the adenosine site explain the lack of phosphorylation of 7-substituted-7-deazaadenosines. Semiempirical quantum mechanical analysis confirmed different affinity of nucleosides for the Mtb ADK adenosine and ATP sites.

9-Norbornyl-6-chloropurine is a novel antileukemic compound interacting with cellular GSH.

AIM: 6-Chloropurines substituted at position 9 with bicyclic skeletons represent promising chemotherapeutic agents. We explored the metabolism and membrane transport of 9-norbornyl-6-chloropurine (NCP) aiming to understand its mechanism of action. MATERIALS AND METHODS: The metabolism of NCP was studied in vitro in whole cells (CCRF-CEM), cellular extracts, subcellular fractions and purified enzymes. Transport experiments were conducted in Caco-2 cell monolayers. RESULTS: Three metabolites were identified, a glutathione conjugate (NCP-GS), NCP-cysteinylglycine and NCP-cysteine. Both glutathione-S-transferase inhibition and glutathione (GSH) depletion prevented metabolite formation and increased the cytotoxicity of NCP. Transepithelial transport (Caco-2) indicated good permeability, with Papp (12.6±0.3) ×10(-5) cm/s. Importantly, the drug induced glutathione depletion in treated cells and affected the activity of several GSH-dependent enzymes. CONCLUSION: The novel nucleoside analog NCP represents a promising orally available antileukemic agent, acting through lowering of GSH levels in tumor cells.

Mutations in adenosine deaminase-like (ADAL) protein confer resistance to the antiproliferative agents N6-cyclopropyl-PMEDAP and GS-9219.

BACKGROUND/AIM: GS 9219 is a double prodrug of antiproliferative nucleotide analog 9-(2-Phosphonylmethoxyethyl)guanine (PMEG), with potent in vivo efficacy against various hematological malignancies. This study investigates the role of adenosine deaminase-like (ADAL) protein in the intracellular activation of GS-9219. MATERIALS AND METHODS: A cell line resistant to 9-(2-Phosphonylmethoxyethyl)-N(6)-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), an intermediate metabolite of GS-9219, was generated and characterized. RESULTS: The resistant cell line was cross-resistant to cPrPMEDAP and GS-9219, due to a defect in the deamination of cPrPMEDAP to PMEG. Mutations in the ADAL gene (H286R and S180N) were identified in the resistant cells that adversely-affected its enzymatic activity. Introduction of the wild-type ADAL gene re-sensitized resistant cells to both cPrPMEDAP and GS-9219. CONCLUSION: The ADAL protein plays an essential role in the intracellular activation of GS-9219 by catalyzing the deamination of cPrPMEDAP metabolite to PMEG. Mutations affecting the activity of ADAL confer resistance to both GS-9219 and its metabolite cPrPMEDAP.

Sugar-modified derivatives of cytostatic 7-(het)aryl-7-deazaadenosines: 2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides.

A series of novel sugar-modified derivatives of cytostatic 7-hetaryl-7-deazaadenosines (2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides) was prepared and screened for biological activity. The synthesis consisted of preparation of the corresponding sugar-modified 7-iodo-7-deazaadenine nucleosides and their aqueous-phase Suzuki-Miyaura cross-coupling reactions with (het)arylboronic acids or Stille couplings with hetarylstannanes in DMF. The synthesis of 7-iodo-7-deazaadenine nucleosides was based on a glycosidation of 6-chloro-7-iodo-7-deazapurine with a suitable sugar synthon or on an interconversion of 2'-OH stereocenter (for arabinonucleosides). Several examples of 2'-C-Me-ribonucleosides showed moderate anti-HCV activities in a replicon assay accompanied by cytotoxicity. Several 7-hetaryl-7-deazaadenine fluoroarabino- and arabinonucleosides exerted moderate micromolar cytostatic effects. The most active was 7-ethynyl-7-deazaadenine fluoroarabinonucleoside which showed submicromolar antiproliferative activity. However, all the sugar-modified derivatives were less active than the parent ribonucleosides.

Involvement of MAP kinases in the cytotoxicity of acyclic nucleoside phosphonates.

BACKGROUND: 9-[2-(phosphonomethoxy)ethyl] guanine (PMEG) is a nucleotide analogue with anticancer activity. Here we investigate the role of ERK, p38, JNK and AKT kinases in PMEG-induced apoptosis. MATERIALS AND METHODS: CCRF-CEM and HL-60 leukemia cells were used to assess MAPK mRNA and protein expression in PMEG-treated cells. MAPK activation was measured using phospho-specific antibodies. Apoptosis was evaluated by caspase-3 and PARP cleavage. RESULTS: Up-regulation of p38ß, γ and δ mRNA were observed following PMEG treatment of CCRF-CEM cells, however, the total protein expression remained unchanged. Neither PMEG nor its analogue 9-[2-(phosphonomethoxy) ethyl]-2,6-diaminopurine (PMEDAP) induced p38 kinase phosphorylation in CCRF-CEM cells, whereas increased p38 phosphorylation was observed in HL-60 cells. The ERK pathway was also activated by these compounds. Pretreatment of the cells with the p38 inhibitor SB203580 diminished drug-induced apoptosis whereas inhibition of ERK, JNK or AKT pathways did not. [corrected]. CONCLUSION: PMEG- and PMEDAP-induced. [corrected].

Synthesis and biological activity of desmethoxy analogues of coruscanone A.

A series of simple desmethoxy analogues of coruscanone A was prepared via a novel version of Ti(iPrO)(4)-mediated Knoevenagel condensation of cyclopentenedione with substituted benzaldehydes and cinnamic aldehydes, and the compounds were evaluated for antifungal activity and cytotoxicity. The most potent 2-benzylidenecyclopent-4-ene-1,3-dione possessed antifungal effect comparable to coruscanone A and a somewhat broader spectrum of activity against Candida species. The compound was also superior to fluconazole against several non-albicans Candida sp. Evaluation of the ability of the compound to influence cell proliferation using two different assays showed that 2-benzylidenecyclopent-4-ene-1,3-dione has lower cytotoxicity compared to the natural product.

Antiviral activity of tenofovir against Cauliflower mosaic virus and its metabolism in Brassica pekinensis plants.

The antiviral effect of the acyclic nucleoside phosphonate tenofovir (R)-PMPA on double-stranded DNA Cauliflower mosaic virus (CaMV) in Brassica pekinensis plants grown in vitro on liquid medium was evaluated. Double antibody sandwich ELISA and PCR were used for relative quantification of viral protein and detecting nucleic acid in plants. (R)-PMPA at concentrations of 25 and 50 mg/l significantly reduced CaMV titers in plants within 6-9 weeks to levels detectable neither by ELISA nor by PCR. Virus-free plants were obtained after 3-month cultivation of meristem tips on semisolid medium containing 50 mg/l (R)-PMPA and their regeneration to whole plants in the greenhouse. Studying the metabolism of (R)-PMPA in B. pekinensis revealed that mono- and diphosphate, structural analogs of NDP and/or NTP, are the only metabolites formed. The data indicate very low substrate activity of the enzymes toward (R)-PMPA as substrate. The extent of phosphorylation in the plant's leaves represents only 4.5% of applied labeled (R)-PMPA. In roots, we detected no radioactive peaks of phosphorylated metabolites of (R)-PMPAp or (R)-PMPApp.

A novel and efficient one-pot synthesis of symmetrical diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates and evaluation of their biological activities.

A novel and efficient method for the one-pot synthesis of diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates, starting from free phosphonic acids or phosphonate diesters is reported. The approach from phosphonate diesters via their bis(trimethylsilyl) esters is highly convenient, eliminates isolation and tedious purification of the phosphonic acids, and affords the corresponding bis-amidates in excellent yields (83-98%) and purity. The methodology has been applied to the synthesis of the potent anticancer agent GS-9219, and symmetrical bis-amidates of other biologically active phosphonic acids. Anti-HIV, antiproliferative, and immunomodulatory activities of the compounds are discussed including the bis-amidate prodrugs 14 and 17 that exhibited anti-HIV activity at submicromolar concentrations with minimal cytotoxicity.

Synthesis and significant cytostatic activity of 7-hetaryl-7-deazaadenosines.

A series of 7-aryl- and 7-hetaryl-7-deazaadenosines was prepared by the cross-coupling reactions of unprotected or protected 7-iodo-7-deazaadenosines with (het)arylboronic acids, stannanes, or zinc halides. Nucleosides bearing 5-membered heterocycles at the position 7 exerted potent in vitro antiproliferative effects against a broad panel of hematological and solid tumor cell lines. Cell cycle analysis indicated profound inhibition of RNA synthesis and induction of apoptosis in treated cells. Intracellular conversion to triphosphates has been detected with active compounds. The triphosphate metabolites showed only a weak inhibitory effect on human RNA polymerase II, suggesting potentially other mechanisms for the inhibition of RNA synthesis and quick onset of apoptosis. Initial in vivo evaluation demonstrated an effect of 7-(2-thienyl)-7-deazaadenine ribonucleoside on the survival rate in syngeneic P388D1 mouse leukemia model.

2´-deoxy-5,6-dihydro-5-azacytidine - a less toxic alternative of 2´-deoxy-5-azacytidine: a comparative study of hypomethylating potential.

Restoration of transcriptionally silenced genes by means of methyltransferases inhibitors plays a crucial role in the current therapy of myelodysplastic syndromes and certain types of leukemias. A comparative study of hypomethylating activities of a series of 5-azacytidine nucleosides: 5-azacytidine (AC), 2'-deoxy-5-azacytidine (DAC) and its α-anomer (α-DAC), 5,6-dihydro-5-azacytidine (DHAC), 2'-deoxy-5,6-dihydro-5-azacytidine (DHDAC, KP-1212) and its α-anomer (α-DHDAC), and of a 2-pyrimidone ribonucleoside (zebularine) was conducted. Methylation-specific PCR was employed to detect the efficiency of individual agents on cyclin-dependent kinase inhibitor 2B and thrombospondin-1 hypermethylated gene loci. Overall changes in DNA methylation level were quantified by direct estimation of 5-methyl-2'-deoxycytidine-5'-monophosphate by HPLC using digested genomic DNA. Flow cytometric analysis of cell cycle progression and apoptotic markers was used to determine cytotoxicity of the compounds. mRNA expression was measured using qRT-PCR. 2'-deoxy-5,6-dihydro-5-azacytidine was found to be less cytotoxic and more stable than 2'-deoxy-5-azacytidine at the doses that induce comparable DNA hypomethylation and gene reactivation. This makes it a valuable tool for epigenetic research and worth further investigations to elucidate its possible therapeutic potential.

Point mutations in human guanylate kinase account for acquired resistance to anticancer nucleotide analogue PMEG.

Acyclic nucleotide analogue PMEG represents promising drug candidate against lymphomas. In the present work we describe the ability of PMEG to induce resistance and we elucidate the mechanisms involved in this process. CCRF-CEM T-lymphoblastic cells resistant to either PMEG or its 6-amino congener PMEDAP were prepared and assayed for the expression of membrane transporters, PMEG and PMEDAP uptake and intracellular metabolism. Genes for guanylate kinase (GUK) and adenylate kinase (AK) isolated from PMEG- and PMEDAP-resistant cells were sequenced and cloned into mammalian expression vectors. PMEG-resistant cells were transfected with GUK vectors and catalytic activities of GUKs isolated from PMEG-sensitive and resistant cells were compared. PMEG phosphorylation to PMEG mono- and diphosphate was completely impaired in resistant cells. GUK obtained from PMEG-resistant cells revealed two point mutations S(35)N V(168)F that significantly suppressed its catalytic activity. Transfection of resistant cells with wtGUK led to the recovery of phosphorylating activity as well as sensitivity towards PMEG cytotoxicity. No differences in PMEG uptake have been found between sensitive and resistant cells. In contrast to GUK no changes in primary sequence of AK isolated from PMEDAP resistant cells were identified. Therefore, resistance induced by PMEDAP appears to be conferred by other mechanisms. In conclusion, we have identified GUK as the sole molecular target for the development of acquired resistance to the cytotoxic nucleotide PMEG. Therefore, PMEG is unlikely to cause cross-resistance in combination therapeutic protocols with most other commonly used anticancer drugs.

Synthesis and antiviral activity of N9-[3-fluoro-2-(phosphonomethoxy)propyl] analogues derived from N6-substituted adenines and 2,6-diaminopurines.

An efficient method for the synthesis of N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases has been developed. Both (R)- and (S)-enantiomers of the N(6)-substituted FPMP derivatives of adenine and 2,6-diaminopurine were prepared and their anti-human immunodeficiency virus (HIV) and anti-Moloney murine sarcoma virus (MSV) activity was evaluated. Whereas none of the 6-substituted FPMPA derivatives showed any antiviral activity, several FPMPDAP derivatives had a moderate antiretroviral activity. Moreover, the data obtained from the study of the substrate activity of the active derivatives towards N(6)-methyl-AMP aminohydrolase support the notion that the studied N(6)-substituted FPMPDAP derivatives act as prodrugs of the antiretroviral FPMPG analogues.

Inhibition of SAH-hydrolase activity during seed germination leads to deregulation of flowering genes and altered flower morphology in tobacco.

Developmental processes are closely connected to certain states of epigenetic information which, among others, rely on methylation of chromatin. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are key cofactors of enzymes catalyzing DNA and histone methylation. To study the consequences of altered SAH/SAM levels on plant development we applied 9-(S)-(2,3-dihydroxypropyl)-adenine (DHPA), an inhibitor of SAH-hydrolase, on tobacco seeds during a short phase of germination period (6 days). The transient drug treatment induced: (1) dosage-dependent global DNA hypomethylation mitotically transmitted to adult plants; (2) pleiotropic developmental defects including decreased apical dominance, altered leaf and flower symmetry, flower whorl malformations and reduced fertility; (3) dramatic upregulation of floral organ identity genes NTDEF, NTGLO and NAG1 in leaves. We conclude that temporal SAH-hydrolase inhibition deregulated floral genes expression probably via chromatin methylation changes. The data further show that plants might be particularly sensitive to accurate setting of SAH/SAM levels during critical developmental periods.

8-Aza-7,9-dideazaxanthine acyclic nucleoside phosphonate inhibitors of thymidine phosphorylase.

3- and 8-(8-phosphonooctyl)-8-aza-7,9-dideazaxanthine, and 1,8-bis(8-aza-7,9-dideazaxanthin-8-yl)octane were prepared and found to inhibit thymidine phosphorylase from Escherichia coli, human recombinant TP expressed in V79, and TP purified from human placenta. The IC(50) values ranged from 3.5 to 27µM.

Phosphoramidate pronucleotides of cytostatic 6-aryl-7-deazapurine ribonucleosides.

A series of O-phenyl methyl-, ethyl- and benzylalanyl phosphoramidate pronucleotides derived from cytostatic 6-aryl-7-deazapurine ribonucleosides were prepared by the cross-coupling reactions of the 2',3'-isopropylidene protected 6-chloro-7-deazapurine ribonucleoside phosphoramidates with (het)arylboronic acids or -stannanes followed by deprotection. Most of the prepared prodrugs exerted in vitro cytostatic effects against both solid tumor and lymphoid cancer cells within low micromolar range of concentrations. These activities were in general weaker or comparable to the activities of the parent nucleosides. Additional testing of selected prodrugs suggests that the lack of activity improvement over parent nucleosides is not due to the lack of permeability or inefficient catabolism of alanyl-ester by intracellular hydrolases. More likely, active efflux of prodrugs may play a role in their weak cytotoxic activity.

3,5-disubstituted pyranone analogues of highly antifungally active furanones: conversion of biological effect from antifungal to cytostatic.

A series of 3-aryl-5-acyloxymethyl-5,6-dihydro-2H-pyran-2-ones, related to highly antifungally active butenolides, was synthesized via cyclization of substituted δ-hydroxy acids as the key step, and evaluated for their in vitro antifungal activity and cytostatic activity. While the extension of the furanone ring to pyranone led to a complete loss of the antifungal effect, some of the compounds displayed promising effect against several cell lines, including the resistant colorectal carcinoma cells.

Role of caspases and CD95/Fas in the apoptotic effects of a nucleotide analog PMEG in CCRF-CEM cells.

BACKGROUND/AIM: 9-[2-(phosphonomethoxy)ethyl] guanine (PMEG) is a guanine acyclic nucleotide analog whose targeted prodrugs are being investigated for chemotherapy of lymphomas. Its antiproliferative effects have been attributed to cell cycle arrest and induction of apoptosis, however, the underlying mechanisms remain poorly understood. The objective of this study was to determine the requirements for caspase and CD95/Fas activation in PMEG-induced apoptosis. Additionally, the influence of PMEG on cell cycle regulatory proteins was explored. MATERIALS AND METHODS: CCRF-CEM cells were exposed to PMEG with/without caspase inhibitor or anti-Fas blocking antibody and assayed for phosphatidyl serine externalization, mitochondrial depolarization and the cleavage of procaspase 3 and the nuclear protein poly (ADP-ribose) polymerase (PARP). RESULTS: Despite an observed increase of caspase 3, 8 and 9 proteolytic activity, neither pretreatment of the cells with cell-permeable caspase inhibitors nor blocking the death receptor with anti-Fas antibody did prevent apoptosis induced by PMEG. CONCLUSION: PMEG-induced apoptosis is caspase- and CD95/Fas-independent.

Distinct modulation of telomere length in two T-lymphoblastic leukemia cell lines by cytotoxic nucleoside phosphonates PMEG and PMEDAP.

We have previously shown that PMEG diphosphate (PMEGpp) and PMEDAP diphosphate (PMEDAPpp) inhibit the enzymatic activity of human telomerase in a cell-free assay. Here, we investigated the ability of PMEG and PMEDAP to induce telomere shortening and telomerase inhibition at both transcriptional and activity level in T-lymphoblastic leukemia cells CCRF-CEM and MOLT-4. At defined time points (3days and 9weeks), the telomerase activity and relative levels of hTERT and c-myc mRNA were determined using real-time RT-PCR. Telomere length was measured by the flow-FISH method. Both PMEDAP and PMEG induced telomere shortening in CCRF-CEM cells after 9weeks of exposure by 50% and 20%, respectively, without major impairment of telomerase activity. The effect of the tested compounds on telomere length in MOLT-4 cells was the opposite, with telomere elongation by 50% and 40% after 9-week treatment with PMEDAP and PMEG, respectively. At this time point, telomerase activity in MOLT-4 cells appeared to be slightly higher than that of CCRF-CEM cells, nevertheless no correlation between telomerase activity and telomere length was found. Both compounds down-regulated the expression of hTERT and c-myc mRNA in CCRF-CEM and MOLT-4 cells at 72h in a concentration-dependent manner while prolonged exposure to PMEG or PMEDAP for 9weeks had weaker effects. In conclusion, PMEDAP and PMEG are able to modulate telomere length in leukemic cells and this effect is cell-type specific. It is neither due to direct telomerase inhibition nor impairment of hTERT expression and it is likely to be telomerase-independent.

CycloSal-phosphate pronucleotides of cytostatic 6-(Het)aryl-7-deazapurine ribonucleosides: Synthesis, cytostatic activity, and inhibition of adenosine kinases.

A series of cycloSal-phosphate prodrugs of a recently described new class of nucleoside cytostatics (6-hetaryl-7-deazapurine ribonucleosides) was prepared. The corresponding 2',3'-isopropylidene 6-chloro-7-deazapurine nucleosides were converted into 5-O'-cycloSal-phosphates. These underwent a series of Stille or Suzuki cross-couplings with diverse (het)arylstannanes or -boronic acids to yield the protected 6-(het)aryl-7-deazapurine pronucleotides that were subsequently deprotected to give 12 derivatives of free pronucleotides. The in vitro cytostatic effect of the pronucleotides was compared with parent nucleoside analogues. In most cases, the activity of the pronucleotide was similar to or somewhat lower than that of the corresponding parent nucleosides, with the exception of 7-fluoro pronucleotides 13 a, 13 b, and 13 d, which had exhibited GIC(50) values that were improved by one order of magnitude (to the low nanomolar range). The presence of a cycloSal-phosphate group also influenced selectivity toward various cell lines. Several pronucleotides were found which strongly inhibit human adenosine kinase but only weakly inhibit the MTB adenosine kinase.

The acyclic 2,4-diaminopyrimidine nucleoside phosphonate acts as a purine mimetic in HIV-1 reverse transcriptase DNA polymerization.

The acyclic pyrimidine nucleoside phosphonate (ANP) phosphonylmethoxyethoxydiaminopyrimidine (PMEO-DAPym) differs from other ANPs in that the aliphatic alkyloxy linker is bound to the C-6 of the 2,4-diaminopyrimidine base through an ether bond, instead of the traditional alkyl linkage to the N-1 or N-9 of the pyrimidine or purine base. In this study, we have analyzed the molecular interactions between PMEO-DAPym-diphosphate (PMEO-DAPym-pp) and the active sites of wild-type (WT) and drug-resistant HIV-1 reverse transcriptase (RT). Pre-steady-state kinetic analyses revealed that PMEO-DAPym-pp is a good substrate for WT HIV-1 RT: its catalytic efficiency of incorporation (k(pol)/K(d)) is only 2- to 3-fold less than that of the corresponding prototype purine nucleotide analogs PMEA-pp or (R)PMPA-pp. HIV-1 RT recognizes PMEO-DAPym-pp as a purine base instead of a pyrimidine base and incorporates it opposite to thymine (in DNA) or uracil (in RNA). Molecular modeling demonstrates that PMEO-DAPym-pp fits into the active site of HIV-1 RT without significant perturbation of key amino acid residues and mimics an open incomplete purine ring that allows the canonical Watson-Crick base pairing to be maintained. PMEO-DAPym-pp is incorporated more efficiently than (R)PMPA-pp by mutant K65R HIV-1 RT and is not as efficiently excised as (R)PMPA by HIV-1 RT containing thymidine analog mutations. Overall, the data revealed that PMEO- DAPym represents the prototype compound of a novel class of pyrimidine acyclic nucleoside phosphonates that are recognized as a purine nucleotide and should form the rational basis for the design and development of novel purine nucleo(s)(t)ide mimetics as potential antiviral or antimetabolic agents.