Chemical library screens targeting an HIV-1 accessory factor/host cell kinase complex identify novel antiretroviral compounds.

Nef is an HIV-1 accessory protein essential for AIDS progression and an attractive target for drug discovery. Lack of a catalytic function makes Nef difficult to assay in chemical library screens. We developed a high-throughput screening assay for inhibitors of Nef function by coupling it to one of its host cell binding partners, the Src-family kinase Hck. Hck activation is dependent upon Nef in this assay, providing a direct readout of Nef activity in vitro. Using this screen, a unique diphenylfuropyrimidine was identified as a strong inhibitor of Nef-dependent Hck activation. This compound also exhibited remarkable antiretroviral effects, blocking Nef-dependent HIV replication in cell culture. Structurally related analogs were synthesized and shown to exhibit similar Nef-dependent antiviral activity, identifying the diphenylfuropyrimidine substructure as a new lead for antiretroviral drug development. This study demonstrates that coupling noncatalytic HIV accessory factors with host cell target proteins addressable by high-throughput assays may afford new avenues for the discovery of anti-HIV agents.

Cdc25B dual-specificity phosphatase inhibitors identified in a high-throughput screen of the NIH compound library.

The University of Pittsburgh Molecular Library Screening Center (Pittsburgh, PA) conducted a screen with the National Institutes of Health compound library for inhibitors of in vitro cell division cycle 25 protein (Cdc25) B activity during the pilot phase of the Molecular Library Screening Center Network. Seventy-nine (0.12%) of the 65,239 compounds screened at 10 muM met the active criterion of > or =50% inhibition of Cdc25B activity, and 25 (31.6%) of these were confirmed as Cdc25B inhibitors with 50% inhibitory concentration (IC(50)) values <50 microM. Thirteen of the Cdc25B inhibitors were represented by singleton chemical structures, and 12 were divided among four clusters of related structures. Thirteen (52%) of the Cdc25B inhibitor hits were quinone-based structures. The Cdc25B inhibitors were further characterized in a series of in vitro secondary assays to confirm their activity, to determine their phosphatase selectivity against two other dual-specificity phosphatases, mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-3, and to examine if the mechanism of Cdc25B inhibition involved oxidation and inactivation. Nine Cdc25B inhibitors did not appear to affect Cdc25B through a mechanism involving oxidation because they did not generate detectable amounts of H(2)O(2) in the presence of dithiothreitol, and their Cdc25B IC(50) values were not significantly affected by exchanging the dithiothreitol for beta-mercaptoethanol or reduced glutathione or by adding catalase to the assay. Six of the nonoxidative hits were selective for Cdc25B inhibition versus MKP-1 and MKP-3, but only the two bisfuran-containing hits, PubChem substance identifiers 4258795 and 4260465, significantly inhibited the growth of human MBA-MD-435 breast and PC-3 prostate cancer cell lines. To confirm the structure and biological activity of 4260465, the compound was resynthesized along with two analogs. Neither of the substitutions to the two analogs was tolerated, and only the resynthesized hit 26683752 inhibited Cdc25B activity in vitro (IC(50) = 13.83 +/- 1.0 microM) and significantly inhibited the growth of the MBA-MD-435 breast and PC-3 prostate cancer cell lines (IC(50) = 20.16 +/- 2.0 microM and 24.87 +/- 2.25 microM, respectively). The two bis-furan-containing hits identified in the screen represent novel nonoxidative Cdc25B inhibitor chemotypes that block tumor cell proliferation. The availability of non-redox active Cdc25B inhibitors should provide valuable tools to explore the inhibition of the Cdc25 phosphatases as potential mono- or combination therapies for cancer.

Bioactivities of simplified adociaquinone B and naphthoquinone derivatives against Cdc25B, MKP-1, and MKP-3 phosphatases.

Some simplified adociaquinone B analogs and a series of 1,4-naphthoquinone derivatives were synthesized and tested against the three enzymes Cdc25B, MKP-1, and MKP-3. Cdc25B and MKP-1 in particular are enzymes overexpressed in human cancer cells, and they represent potential molecular targets for novel cancer chemotherapeutic treatments. A number of analogs exhibited significant inhibitory activity against these enzymes, and the bioassay data in addition to structure-activity relationships of these compounds will be discussed.

Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents.

We report here the development and optimization of a simple 384-well colorimetric assay to measure H(2)O(2) generated by the redox cycling of compounds incubated with reducing agents in high-throughput screening (HTS) assay buffers. The phenol red-horseradish peroxidase (HRP) assay readily detected H(2)O(2) either added exogenously or generated by the redox cycling of compounds in dithiothreitol (DTT). The generation of H(2)O(2) was dependent on the concentration of both the compound and DTT and was abolished by catalase. Although both DTT and tris(2-carboxyethyl) phosphine sustain the redox cycling generation of H(2)O(2) by a model quinolinedione, 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione (NSC 663284; DA3003-1), other reducing agents such as beta-mercaptoethanol, glutathione, and cysteine do not. The assay is compatible with HTS. Once terminated, the assay signal was stable for at least 5 h, allowing for a reasonable throughput. The assay tolerated up to 20% dimethyl sulfoxide, allowing a wide range of compound concentrations to be tested. The assay signal window was robust and reproducible with average Z-factors of > or =0.8, and the redox cycling generation of H(2)O(2) by DA3003-1 in DTT exhibited an average 50% effective concentration of 0.830 +/- 0.068 microM. Five of the mitogen-activated protein kinase phosphatase (MKP) 1 inhibitors identified in an HTS were shown to generate H(2)O(2) in the presence of DTT, and their inhibition of MKP-1 activity was shown to be time dependent and was abolished or significantly reduced by either 100 U of catalase or by higher DTT levels. A cross-target query of the PubChem database with three structurally related pyrimidotriazinediones revealed active flags in 36-39% of the primary screening assays. Activity was confirmed against a number of targets containing active site cysteines, including protein tyrosine phosphatases, cathepsins, and caspases, as well as a number of cellular cytotoxicity assays. Rather than utilize resources to conduct a hit characterization effort involving several secondary assays, the phenol red-HRP assay provides a simple, rapid, sensitive, and inexpensive method to identify compounds that redox cycle in DTT or tris(2-carboxyethyl)phosphine to produce H(2)O(2) that may indirectly modulate target activity and represent promiscuous false-positives from a primary screen.

Development and implementation of a 384-well homogeneous fluorescence intensity high-throughput screening assay to identify mitogen-activated protein kinase phosphatase-1 dual-specificity protein phosphatase inhibitors.

We report here the miniaturization, development, and implementation of a homogeneous 384-well fluorescence intensity high-throughput screening (HTS) assay for identifying mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) dual-specificity phosphatase inhibitors. As part of the National Institutes of Health (NIH) Molecular Libraries Screening Center Network (MLSCN), the MKP-1 assay was utilized to screen an NIH diversity library of 65,239 compounds for inhibitors of MKP-1 activity at 10 microM and was also used to confirm the concentration dependence of active agents identified in the primary screen. We observed 100 (0.15%) compounds that inhibited MKP-1 in vitro by > or =50% at 10 microM in the primary assay, and 46 of the 100 compounds were confirmed as concentration-dependent inhibitors of MKP-1 with 50% inhibitory concentration (IC(50)) values of <50 microM; four exhibited IC(50) values <1.0 microM, six produced IC(50) values in the 1-10 microM range, and 36 produced IC(50) values in the 10-50 microM range. A clustering and classification analysis of the compound structures of the 46 confirmed MKP-1 inhibitors produced 29 singleton structures and seven clusters of related structures. Some MKP-1 inhibitors were members of structural classes or contained substructure pharmacophores that previously were reported to inhibit either MKP-1 or other protein tyrosine phosphatases, validating the HTS assay. Importantly, we have identified several attractive and novel MKP-1 inhibitor structures that warrant further investigation as potential probes to study the biology of MKP-1 and its role in controlling the amplitude and/or duration of MAPK signaling, cell survival, and tumor progression.

Development and optimization of high-throughput in vitro protein phosphatase screening assays.

We describe here detailed protocols to design, optimize and validate in vitro phosphatase assays that we have utilized to conduct high-throughput screens for inhibitors of dual-specificity phosphatases: CDC25B, mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-3. We provide details of the critical steps that are needed to effectively miniaturize the assay into a 384-well, high-throughput format that is both reproducible and cost effective. In vitro phosphatase assays that are optimized according to these protocols should satisfy the assay performance criteria required for a robust high-throughput assay with Z-factors >0.5, and with low intra-plate, inter-plate and day-to-day variability (CV <20%). Assuming the availability of sufficient active phosphatase enzyme and access to appropriate liquid handling automation and detection instruments, a single investigator should be able to develop a 384-well format high-throughput assay in a period of 3-4 weeks.

Synthesis and biological activity of a focused library of mitogen-activated protein kinase phosphatase inhibitors.

Mitogen-activated protein kinase phosphatase 1 is a tyrosine phosphatase superfamily member that dephosphorylates and inactivates mitogen-activated protein kinase substrates, such as p38, c-Jun-N-terminal kinase, and extracellular signal-related kinase. These mitogen-activated protein kinase substrates regulate many cellular processes associated with human diseases. In spite of this potential as a molecular target for chemotherapy, however, pharmacologically tractable inhibitors of mitogen-activated protein kinase phosphatase-1 have yet to be developed. Based on the results from a high-throughput screen for small molecule inhibitors of mitogen-activated protein kinase phosphatase-1, we designed, synthesized, and evaluated a focused library in an effort to further understand the structural requirements for mitogen-activated protein kinase phosphatase-1 inhibitory activity.

Independent mechanistic inhibition of cdc25 phosphatases by a natural product caulibugulone.

Caulibugulones are novel but poorly characterized cytotoxic isoquinoline quinones and iminoquinones identified in extracts from the marine bryozoan Caulibugula intermis. We now report that the caulibugulones are selective in vitro inhibitors of the Cdc25 family of cell cycle-controlling protein phosphatases compared with either human vaccinia H1-related phosphatase (VHR) or tyrosine phosphatase 1B (PTP1B). The in vitro inhibition of Cdc25B by caulibugulone A was irreversible and attenuated by reducing agents or catalase, consistent with direct oxidation of the enzyme by reactive oxygen species. Mechanistically, caulibugulone A directly inhibited cellular Cdc25B activity, generated intracellular reactive oxygen species and arrested cells in both G1 and G2/M phases of the cell cycle. Caulibugulone A also caused the selective degradation of Cdc25A protein by a process that was independent of reactive oxygen species production, proteasome activity, and the Chk1 signaling pathway. Instead, caulibugulone A stimulated the phosphorylation and subsequent activation of p38 stress kinase, leading to Cdc25A degradation. Thus, caulibugulone inhibition of cellular Cdc25A and B phosphatases occurred through at least two different mechanisms, leading to pronounced cell cycle arrest.

Redox regulation of Cdc25B by cell-active quinolinediones.

Intracellular reduction and oxidation pathways regulate protein functionality through both reversible and irreversible mechanisms. The Cdc25 phosphatases, which control cell cycle progression, are potential subjects of oxidative regulation. Many of the more potent Cdc25 phosphatase inhibitors reported to date are quinones, which are capable of redox cycling. Therefore, we used the previously characterized quinolinedione Cdc25 inhibitor DA3003-1 [NSC 663284 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] and a newly synthesized congener JUN1111 [7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] to test the hypothesis that quinone inhibitors of Cdc25 regulate phosphatase activity through redox mechanisms. Like DA3003-1, JUN1111 selectively inhibited Cdc25 phosphatases in vitro in an irreversible, time-dependent manner and arrested cells in the G1 and G2/M phases of the cell cycle. It is noteworthy that both DA3003-1 and JUN1111 directly inhibited Cdc25B activity in cells. Depletion of glutathione increased cellular sensitivity to DA3003-1 and JUN1111, and in vitro Cdc25B inhibition by these compounds was sensitive to pH, catalase, and reductants (dithiothreitol and glutathione), consistent with oxidative inactivation. In addition, both DA3003-1 and JUN1111 rapidly generated intracellular reactive oxygen species. Analysis of Cdc25B by mass spectrometry revealed sulfonic acid formation on the catalytic cysteine of Cdc25B after in vitro treatment with DA3003-1. These results indicate that irreversible oxidation of the catalytic cysteine of Cdc25B is indeed a mechanism by which these quinolinediones inactivate this protein phosphatase.

Four diterpenoid inhibitors of Cdc25B phosphatase from a marine anemone.

Three new diterpenoids and one known diterpenoid have been isolated from a sea anemone of the order Actiniara, and the structures of the new compounds, actiniarins A-C (1-3) were established on the basis of extensive 1D and 2D NMR spectroscopic data interpretation. Compound 1 has a six-membered ring hemiacetal ring, and the equilibrium of this ring is discussed. All the isolates were evaluated for their inhibition of Cdc25B and for cytotoxicity against the A2780 ovarian cancer cell line.

Sesterterpenoids and an alkaloid from a Thorectandra sp. as inhibitors of the phosphatase Cdc25B.

Bioassay-directed separation of an extract of a Thorectandra sp. sponge led to the isolation of three new sesterterpenoids, 16-oxoluffariellolide (1), 16-hydroxyluffariellolide (2) and (2E,6E,10E)-3-formyl-7,11-dimethyl-13-(2,6,6-trimethylcyclohex-1-enyl)trideca-2,6,10-trienoic acid (3); two known sesterterpenoids, luffariellolide (4) and dehydroluffariellolide diacid (5); and one known alkaloid, fascaplysin (6). The structures of the new compounds 1-3 were established on the basis of extensive 1D and 2D NMR spectroscopic data interpretation. Compound 6 showed inhibitory activity in the Cdc25B assay, with an IC50 value of 1.0 microg/mL.

Halenaquinone and xestoquinone derivatives, inhibitors of Cdc25B phosphatase from a Xestospongia sp.

Separation of an extract of a Xestospongia sp., guided by bioassay against Cdc25B, led to the isolation of nine compounds, halenaquinone (1), xestoquinone (2), adociaquinones A (3) and B (4), 3-ketoadociaquinones A (5) and B (6), tetrahydrohalenaquinones A (7) and B (8), and 13-O-methyl xestoquinol sulfate (9). The structures of the new natural products 6 and 9 were established on the basis of extensive one- and two-dimensional NMR studies. Compounds 1, 4, and 6 inhibited recombinant human Cdc25B in vitro with IC50 values of 0.7, 0.07, and 0.2 microM, respectively, and were 19- to 150-fold less active against two related protein phosphatases. Compound 4 blocked cell cycle progression through mitosis.