High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives β-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). β-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 μM, while the IC50 for Escherichia coli and human RNases H was 50 μM and 5.7 μM, respectively. In contrast, the related tropolone analog β-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC50 = 1.5 μM and 40 μM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both β-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 μM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between β-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted.

RNA ligase type 1 from bacteriophage T4 (Rnl1) is involved in countering a host defense mechanism by repairing 5-PO 4 and 3-OH groups in tRNA Lys . Rnl1 is widely used as a reagent in molecular biology. Although many structures for DNA ligases are available, only fragments of RNA ligases such as Rnl2 are known. We report the first crystal structure of a complete RNA ligase, Rnl1, in complex with adenosine 5-(␣,␤-methylenetriphosphate) (AMPcPP). The N-terminal domain is related to the equivalent region of DNA ligases and Rnl2 and binds AMPcPP but with further interactions from the additional N-terminal 70 amino acids in Rnl1 (via Tyr 37 and Arg 54 ) and the C-terminal domain (Gly 269 and Asp 272 ). The active site contains two metal ions, consistent with the two-magnesium ion catalytic mechanism. The C-terminal domain represents a new all ␣-helical fold and has a charge distribution and architecture for helix-nucleic acid groove interaction compatible with tRNA binding.Bacteriophage T4 RNA ligase 1 (EC 6.5.1.3), the founding member of the RNA ligase family (1), is a very well studied representative of the nucleotidyltransferase superfamily, which includes RNA ligases, DNA ligases, and RNA capping enzymes. All members of this family hydrolyze a pyrophosphate bond of a ribonucleotide triphosphate and make a high energy phosphoramidate linking the nucleotide monophosphate with an essential lysine in the active site. This lysine is identified within a conserved motif KX(D/N)G motif (motif I) and is responsible for the formation of the covalent bond to ATP, NAD, or GTP (2). DNA ligases and RNA capping enzymes share five amino acid sequence motifs: I, III, IIIa, V, and IV (3). Motifs III and IIIa are missing in the Rnl1 sequence (4) but are present in the recently discovered T4 RNA ligase 2 (Rnl2) (5, 6). Rnl2 shares greater sequence homology with DNA ligases and RNA capping enzymes compared with Rnl1. It may suggest that Rnl1 has a more specific role than Rnl2, although the function of Rnl2 still remains unknown.The biological role of Rnl1 involves the countering of a host defense mechanism invoked following phage infection of the bacterial host. The bacterial tRNA Lys -specific anticodon nuclease (ACNase) is kept latent because of the association of its core protein, PrrC, with the endonuclease EcoprrI, which stabilizes PrrC and masks its activity (7). Upon infection, T4 bacteriophage expresses a T4 Stp peptide (8), which inhibits EcoprrI and activates the latent enzyme. Anticodon nuclease is involved in the 5Ј cleavage of the wobble base of tRNA Lys (9). This modification of tRNA Lys acts as a defense mechanism by inhibiting phage protein synthesis and, as a consequence, stops the infection. Bacteriophage T4 has developed a counter-defense mechanism using Rnl1 and polynucleotide kinase (PnK) to repair the break in the tRNA anticodon loop. Thus Rnl1 plays an important in vivo role in the spread of the bacteriophage. It has been shown that if PnK and Rnl1 are not present, viral protein synthesis is blocked by depletio...

High-throughput screening of National Cancer Institute libraries of synthetic and natural compounds identified the vinylogous ureas 2-amino-5, 6,7,8-tetrahydro-4H-cyclohepta[b] thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT).A Yonetani-Theorell analysis demonstrated that NSC727447, and the active-site hydroxytropolone RNase H inhibitor β-thujaplicinol were mutually exclusive in their interaction with the RNase H domain. Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the thumb subdomain of p51 were affected by ligand binding. Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb. Sitedirected mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or nonnatural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity. NSC727447 thus represents a novel class of RNase H antagonists with a mechanism of action differing from active site, diva-lent metal-chelating inhibitors that have been reported.Although an absolute requirement for reverse transcriptase (RT)-associated ribonuclease H (RNase H) activity for human immunodeficiency virus (HIV) replication was documented almost two decades ago (1,2), development of potent and selective RNase H inhibitors has been surprisingly slow compared with the nucleoside and non-nucleoside DNA polymerase inhibitors currently in clinical use. Recently, however,4), diketo acids (5,6), and dihydroxytropolones (7) have shown promise by specifically inhibiting RNase H activity of HIV-1 and HIV-2 RT, and in some instances acting synergistically with clinically approved RT inhibitors. The preliminary crystal structure of an N-hydroxyimide bound to the RNase H domain of HIV-1 RT (4) suggests that it sequesters the divalent metal cofactor, laying the foundation for rational design of improved inhibitors. Increasing the diversity of RNase H © 2008 American Chemical Society * Corresponding author, slegrice@ncifcrf.gov.. Supporting Information Available:This material is free of charge via the Internet. In order to examine the NSC727447 binding site, we performed mass spectrometric protein footprinting based on biotin modification of exposed lysine residues in the free protein and the protein-inhibitor complex (8-10). Cys280 and Lys281, located in helix I of the thumb subdomain, were protected from modification by inhibitor binding. Proximity between the p51 thumb subdomain and the p66 RNase H domain implies that inhibitor binding adjacent to the catalytic center affects either divalent metal coordination or positioning of the nucleic acid substrate in the active site. Although DNA polymerase activity was less sensi...

Carcinoembryonic antigen (CEA) is a widely used tumor marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. As a first step toward analyzing the regulation of expression of CEA at the transcriptional level, we have isolated and characterized a cosmid clone (cosCEAl), which contains the entire coding region of the CEA gene. A close correlation exists between the exon and deduced immunoglobulin-like domain borders. We have determined a cluster of transcriptional starts for CEA and the closely related nonspecffic cross-reacting antigen (NCA) gene and have sequenced their putative promoters. Regions of sequence homology are found as far as approximately 500 nucleotides upstream from the translational starts of these genes, but farther upstream they diverge completely. In both cases we were unable to find classic TATA or CAAT boxes at their expected positions. To characterize the CEA and NCA promoters, we carried out transient transfection assays with promoter-indicator gene constructs in the CEA-producing adenocarcinoma cell line SW403, as well as in nonproducing HeLa cells. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the SW403 than in the HeLa cell line. This indicates that cis-acting sequences which convey cell type-specific expression of the CEA gene are contained within this region.

A key step in proliferation of retroviruses is the conversion of their RNA genome to double-stranded DNA, a process catalysed by multifunctional reverse transcriptases (RTs). Dimeric and monomeric RTs have been described, the latter exemplified by the enzyme of Moloney murine leukaemia virus. However, structural information is lacking that describes the substrate binding mechanism for a monomeric RT. We report here the first crystal structure of a complex between an RNA/DNA hybrid substrate and polymerase-connection fragment of the single-subunit RT from xenotropic murine leukaemia virus-related virus, a close relative of Moloney murine leukaemia virus. A comparison with p66/p51 human immunodeficiency virus-1 RT shows that substrate binding around the polymerase active site is conserved but differs in the thumb and connection subdomains. Small-angle X-ray scattering was used to model full-length xenotropic murine leukaemia virus-related virus RT, demonstrating that its mobile RNase H domain becomes ordered in the presence of a substrate—a key difference between monomeric and dimeric RTs.