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.

A series of 29 madurahydroxylactone derivatives was evaluated for dual inhibition of human immunodeficiency virus type 1 (HIV-1) integrase and RNase H. While most of the compounds exhibited similar potencies for both enzymes, two of the derivatives showed 10-to 100-fold-higher selectivity for each enzyme, suggesting that distinct pharmacophore models could be generated. This study exemplifies the common and divergent structural requirements for the inhibition of two structurally related HIV-1 enzymes and demonstrates the importance of systematically screening for both integrase and RNase H when developing novel inhibitors.

A new dimeric lactone, ardimerin digallate (1), was isolated from the whole plants of Ardisia japonica, along with six known constituents. The structure of 1 was established on the basis of spectroscopic analysis including 1D- and 2D-NMR techniques. Compound 1 inhibited HIV-1 and HIV-2 RNase H in vitro with IC50 values of 1.5 and 1.1 microM, respectively.

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A new compound, 1,3,4,5-tetragalloylapiitol ( 1), was isolated from the aqueous extract of the plant Hylodendron gabunensis and was found to be a potent inhibitor of RNase H enzymatic activity. The structure of 1 was elucidated by NMR analyses to be an apiitol ( 2) sugar moiety substituted with four gallic acid residues. Optical rotation measurements of the free sugar following basic hydrolysis indicated that the 3 S absolute configuration was the same as that of d-apiitol. Compound 1 inhibited HIV-1, HIV-2, and human RNase H with IC 50 values of 0.24, 0.13, and 1.5 microM, respectively, but it did not show inhibition of E. coli RNase H at 10 microM.

Three new galloyl arbutins, hyemalosides A-C (1-3), along with nine known compounds were isolated from the evergreen tree Eugenia hyemalis. The structures of compounds 1-3 were determined by analysis of NMR and MS data. Compounds 1-3 inhibited HIV-1 RNase H in vitro with IC 50 values of 1.46, >18, and 1.19 μM, respectively. However, in a XTT-based cell viability assay using the human T-cell line CEM-SS infected with HIV-1 RT , none of the compounds inhibited the cytopathic effect of HIV-1 infection at the highest dose tested (20 μg/mL).As part of a molecularly targeted screening program to discover drug leads from natural products, we investigated the chemistry of the evergreen tree Eugenia hyemalis L. Cambess (Myrtaceae). The organic extract of the plant showed inhibition of ribonuclease H (RNase H) enzymatic activity in a high-throughput screening assay. 1 RNase H is a second, distinct enzymatic activity of the HIV-1 reverse transcriptase protein. It is a nonspecific nuclease that is responsible for hydrolyzing the RNA strand of a RNA/DNA heteroduplex and allowing the incorporation of viral genetic information into the host cell genome. Because RNase H activity is required for viral infectivity, it is a viable target for screening for potential anti-HIV drugs, and a high-throughput fluorescence resonance energy transfer (FRET) assay 1 was developed for this purpose.Bioassay-guided fractionation of the extract led to the isolation of three new compounds, hyemalosides A-C (1-3), along with nine known compounds. The structures of the nine known compounds were determined on the basis of spectroscopic evidence and comparison with literature values. These were characterized as 2-O-galloylarbutin, 2 4-O-galloylarbutin, 2,6-di-O-galloylarbutin, 3 2,4,6-tri-O-galloylarbutin, 4 1,2,6-tri-O-galloyl-β-D-glucose, 5 kaempferol-3-O-(6-O-galloyl-β-D-glucopyranoside), 6 afzelin 2″-O-gallate, 7 afzelin 3″-Ogallate, 7 and quercitrin. 7 HREIMS of compound 1 showed a peak at m/z 727.1191 [M -H] − consistent with a molecular formula of C 33 H 28 O 19 . The 1 H NMR spectrum showed a proton doublet at δ 5.13 (J = 7.9 Hz) along with six resonances from δ 3.88 to 5.49, which were linked by COSY data and indicated a hexose moiety. Large coupling constants for the sugar ring protons (J = 7.9-9.5 Hz) indicated a series of trans diaxial couplings characteristic of glucose and also revealed that the anomeric oxygen was β-oriented.

The recent demonstration that adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) plays an oncogenic role in a number of important cancers has led to a renaissance in drug development interest targeting this kinase. We therefore have established a suite of biochemical, cell-based, and structural biology assays for identifying and evaluating new pharmacophores for PKA inhibition. This discovery process started with a 384-well high-throughput screen of more than 200,000 substances, including fractionated natural product extracts. Identified active compounds were further prioritized in biochemical, biophysical, and cell-based assays. Priority lead compounds were assessed in detail to fully characterize several previously unrecognized PKA pharmacophores including the generation of new X-ray crystallography structures demonstrating unique interactions between PKA and bound inhibitor molecules.