Eckart Matthes

Inhibition of HIV-associated reverse transcriptase by sugar-modified derivatives of thymidine 5′-triphosphate in comparison to cellular DNA polymerases α and β☆

Abstract The sugar-modified dTTP analogues 2′,3′-didehydro-2′,3′-dideoxythymidine 5′-triphosphate (ddeTTP), 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP), 3′-fluorothymidine 5′-triphosphate (FdTTP), and 3′-azidothymidine 5′-triphosphate (N3dTTP) are demonstrated to be very effective and selective inhibitors of the HIV-associated reverse transcriptase (HIV-RT). This conclusion is based on a comparison of the IDso values of the compounds for the HIV-RT (ranging from 0.03 μM for ddeTTP to 0.1 μM for ddTTP) and the cellular DNA polymerase α (> 200 μM). DNA polymerase β is partially affected by N3dTTP (IDso=31 μM) and by the other analogues (IDso=1–2.2 μM). FdTTP has proved as effective as N3dTTP (IDso=0.05 μM) in suppressing the HIV-RT activity. Kinetic analysis revealed for both dTTP analogues a competitive type of inhibition and the same K1 values (about 0.05 μM).

Detection of activity of telomerase in tumor cells using fiber optical biosensors.

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.

Avarol restores the altered prostaglandin and leukotriene metabolism in monocytes infected with human immunodeficiency virus type 1

Infection of monocytes with human immunodeficiency virus type 1 (HIV-1) (strain Ada-M) caused increased levels of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) in vitro. These two products result from the activities of the two enzymes cyclooxygenase and 5-lipoxygenase. The addition of the sesquiterpenoid hydroquinone Avarol, an HIV inhibitor, strongly reduced the levels of LTB4 and PGE2 via inhibition of both cyclooxygenase and lipoxygenase in monocytes. The 50% inhibition concentrations (IC50) for the enzymes were determined to be 2.26 microM (cyclooxygenase) and 1.97 microM (lipoxygenase). A 50% reduction of the extent of PGE2 and LTB4 production in HIV-infected monocytes was measured at a concentration of 0.9 microM Avarol, a dose which caused an 80% anti-HIV effect in vitro (50% inhibition of virus release from infected cells: 0.3 microM). We conclude that Avarol inhibits the enzymes cyclooxygenase and lipoxygenase and suggest that, in general, inhibitors of these enzymes are promising anti-HIV compounds.

Alteration of DNA topoisomerase II activity during infection of H9 cells by human immunodeficiency virus type 1 in vitro: A target for potential therapeutic agents

Infection of H9 cells with human immunodeficiency virus type 1 (HIV-1) was found to decrease the phosphorylation of DNA topoisomerase II during the initial phase of infection. Simultaneously, with a later overshoot of phosphorylation and the subsequent activation of DNA topoisomerase II, the production of HIV-1 started. Applying three new protein kinase C inhibitors from the class of O-alkylglycerophospholipids we demonstrated that inhibition of protein kinase C-mediated phosphorylation of DNA topoisomerase II resulted in an inhibition of HIV-1 production. Based on the differential effect of the two protein kinase C activators, phorbol ester and bryostatin, we conclude that phosphorylation of DNA topoisomerase II is mediated by the form alpha and gamma of protein kinase C. These data suggest that agents which inhibit these two forms of protein kinase C are also potential candidates for an anti-HIV therapy.

Inhibition of adenovirus DNA polymerase by modified nucleoside triphosphate analogs correlate with their antiviral effects on cellular level

Abstract Adenovirus (Ad) infection results in significant morbidity and mortality in both immunocompetent and immunosuppressed hosts. There is currently no licensed chemotherapy effective in dealing with this virus infection. In this study the anti-adenoviral activity of a group of modified nucleoside analogs was investigated. The most efficient 3-fluorosubstituted nucleoside triphosphate inhibitors of Ad DNA polymerase were 3′-fluorothymidine triphosphate (IC50 0.63 μM), 2′,3′-dideoxy-3′-fluoroguanosine triphosphate (IC50 0.71 μM) and 2′,3′-dideoxy-3′-fluorouridine triphosphate (IC50 2.96 μM). The most efficient 2′,3′-dideoxynucleoside triphosphates were 2′,3′-dideoxycytidine triphosphate (ddCTP; IC50 1.0 μM), 2′,3′-dideoxyadenosine triphosphate (IC50 1.6 μM) and 2′,3′-dideoxythymidine triphosphate (IC50 1.82 μM). Kinetic studies indicate competitive inhibition of adenovirus DNA polymerase by ddCTP. These data confirm results previously obtained at the cellular level using a focus reduction assay involving Ad2-infected FL cells. Whereas the D-enantiomers 3′-fluorothymidine and 2′,3′-dideoxycytidine are potent inhibitors of adenoviral replication, the corresponding L-enantiomers exhibited no inhibitory activity.

Inhibition of human immunodeficiency virus-1 infection by human conglutinin-like protein : in vitro studies

The lectin‐like protein analogous to bovine conglutinin was purified from human serum. The carbohydrate‐binding ability of conglutinin‐like protein was inhibited by D‐mannose, N‐acetylglucosamine and L‐fucose as well as by mannan‐containing oligosaccharides. By applying a lectin‐based ELISA system it was demonstrated that conglutinin‐like protein binds to human immunodeficiency virus‐1 (HIV‐1) glycoprotein 120 (gpl20) via its carbohydrate binding site. In vitro experiments with T‐lymphoblastoid CEM cells revealed that conglutinin‐like protein abolishes infection by HIV‐1; a 50% cytoprotective concentration of 23.9 μg/ml was measured. These findings demonstrate that human conglutinin‐like protein binds to HIV‐gp120 and inhibits, under the described in vitro conditions, CEM cell infection.

Chemo-enzymatic synthesis of a new type of enantiomerically pure carbocyclic nucleoside analogues with strong inhibitory effects on terminal deoxynucleotidyl transferase

The synthesis of enantiomerically pure carbocyclic adenosine derivatives which have been prepared based on the kinetic resolution of a trans-2-(hydroxymethyl)cyclopentanol derivative is described. Their corresponding triphosphates were evaluated as inhibitors of DNA polymerase beta, terminal deoxynucleotidyl transferase (TdT), telomerase, Escherichia coli DNA polymerase I and reverse transcriptase of human immunodeficiency virus. Surprisingly, the triphosphate of (1S,2R)-1-(6-aminopurin-9-yl)-2-(hydroxymethyl)cyclopentane [(1S,2R)-6] and its enantiomer (1R,2S)-6 emerged as strong inhibitors of TdT (Ki = 0.5 and 1.9 mM, Kmapp dATP = 40 mM), whereas the activities of all other enzymes tested proved to be unaffected.

Strong and Selective Inhibitors of Hepatitis B Virus Replication among Novel N4-Hydroxy- and 5-Methyl-β-l-Deoxycytidine Analogues

ABSTRACT Novel N4-hydroxy- and 5-methyl-modified β-l-deoxycytidine analogues were synthesized and evaluated as anti-hepatitis B virus (HBV) agents. Their in vitro efficiencies were investigated in HepG2.2.15 cells stably transfected with HBV. β-l-2′,3′-Didehydro-2′,3′-dideoxy-N4-hydroxycytidine (β-l-Hyd4C) was most effective in reducing secreted HBV DNA (50% effective concentration [EC50], 0.03 μM), followed by β-l-2′,3′-dideoxy-3′-thia-N4-hydroxycytidine (EC50, 0.51 μM), β-l-2′,3′-dideoxy-N4-hydroxycytidine (EC50, 0.55 μM), and β-l-5-methyl-2′-deoxycytidine (EC50, 0.9 μM). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the β-l-cytidine derivatives was also assessed. In accordance with the cell culture data, β-l-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 μM. The cytotoxicities of some of the 4-NHOH-modified β-l-nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH2 group. The 50% cytotoxic concentrations for β-l-Hyd4C in HepG2 and HL-60 cells were 2,500 μM and 3,500 μM, respectively. In summary, our results demonstrate that at least β-l-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.