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Biomedical subjects

John S Lazo

Publications and source records attributed to John S Lazo.

At least 19 recordsLinked to original sources
PubMed2007

Discovery of protein kinase phosphatase inhibitors.

Dynamic protein phosphorylation, a major cellular regulatory system, is tightly controlled by coordinating the reversible action of protein kinases and phosphatases. Recent evidence is consistent with sophisticated mechanisms that regulate both kinases and phosphatases in the cell. Dual specificity phosphatases, which act on phosphorylated serine, threonine, and tyrosine residues in proteins, are valid targets for drug discovery. Chemical complementation combines genetic manipulations with chemical biology and high-content multiparametric analyses and was developed as a screening approach to discover protein phosphatase inhibitors. Using a dual specificity mitogen-activated protein kinase phosphatase as an example, a detailed protocol, discussion of issues relating to data analysis, and high-throughput implementation of the chemical complementation approach to drug discovery is presented.

Cell Cycle Proteins↗
PubMed2006

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.

Animals↗
PubMed2006

Biological evaluation of newly synthesized quinoline-5,8-quinones as Cdc25B inhibitors.

Cdc25B protein phosphatase represents an attractive potential therapeutic target for small molecule intervention because of its central role in positively regulating cyclin dependent kinases and thus cell proliferation, as well as its elevated levels observed in many human tumors. Among the most potent previously identified Cdc25 inhibitors have been quinoline quinones, which have a rich legacy as therapeutic agents but have also been associated with nonspecific interactions. In this study, we have interrogated the structure-activity relationship of a focused series of C2-, C3-, or C4-modified quinoline-5,8-quinones on Cdc25B inhibition in vitro. Substitution at the C3-position in this small chemical series were slightly superior to substitutions at the C3-position. For all compounds, recombinant human Cdc25B was approximately 5-fold more sensitive compared to recombinant human PTP1B. Two compounds inhibited HeLa cell growth with IC50 values of approximately 2 microM. Consistent with other para-quinones, some members of this series generated intracellular reactive oxygen species and the in vitro enzyme inhibition was mitigated by addition of reductants or catalase. These results indicate that chemical modifications on the pyridine core are tolerated, providing additional sites for future structural modification of this biologically active pharmacophore.

Cell Cycle Proteins↗
PubMed2006

Novel benzofuran inhibitors of human mitogen-activated protein kinase phosphatase-1.

Protein tyrosine phosphatases have a central role in the maintenance of normal cellular functionality. For example, PTP1B has been implicated in insulin-resistance, obesity, and neoplasia. Mitogen-activated protein kinase phosphatase-1 (MKP-1 or DUSP1) dephosphorylates and inactivates mitogen-activated protein kinase (MAPK) substrates, such as p38, JNK, and Erk, and has been implicated in neoplasia. The lack of readily available selective small molecule inhibitors of MKP family members has severely limited interrogation of their biological role. Inspired by a previously identified inhibitor (NSC 357756) of MKP-3, we synthesized seven NSC 357756 congeners, which were evaluated for in vitro inhibition against several protein phosphatases. Remarkably, none displayed potent inhibition against MKP-3, including the desamino NSC 357756 analog NU-154. Interestingly, NU-154 inhibited human PTP1B in vitro with an IC(50) value of 24 +/- 1 microM and showed little inhibition against Cdc25B, MKP-1, and VHR phosphatases. NU-126 [2-((E)-2-(5-cyanobenzofuran-2-yl)vinyl)-1H-indole-6-carbonitrile] inhibited MKP-1 and VHR in vitro but was less active against human MKP-3, Cdc25B, and PTP1B. The inhibition of MKP-1 by NU-126 was independent of redox processes. The benzofuran substructure represents a new potential scaffold for further analog development and provides encouragement that more selective and potent inhibitors of MKP family members may be achievable.

Benzofurans↗
PubMed2006

Cellular analysis of disorazole C and structure-activity relationship of analogs of the natural product.

Structure-activity analyses of synthetic disorazole C(1) and eight of its analogs indicate that the presence of a vinyl oxirane moiety or a tetraene sequence is not necessary for potent cytotoxic and antimitotic properties. Using an automated multiparameter fluorescence-based cellular assay to simultaneously probe the effects of disorazole analogs on cellular microtubules, mitotic arrest, and cytotoxicity, we found that disorazole C(1) enhanced the mitotic index and chromatin condensation and arrested cells in the G2/M phase of the cell cycle. All structural analogs and synthesis precursors of disorazole C(1) were at least two orders of magnitude less potent than the parent compound, thus indicating that both the functional group array and the three-dimensional conformation of the parent compound are critical for interaction with the biological target. We conclude that disorazole C(1) is a potent inducer of mitotic arrest and hypothesize that this biological activity may be mediated by microtubule perturbation.

Antimitotic Agents↗
PubMed2006

Airing out an antioxidant role for the tumor suppressor p53.

The tumor suppressor p53 exerts its activity by preventing DNA-damaged cells from dividing until either the chromosomal repair is effected or the cell undergoes apoptosis. Reactive oxygen species (ROS) are enhanced through the action of p53-mediated transcription of apoptosis-promoting genes; however, p53 also can promote the expression of many antioxidant genes that prevent apoptosis. New research indicates that in low cellular stress, low concentrations of p53 induce the expression of antioxidant genes, whereas in severe cellular stress, high concentrations of p53 promote the expression of genes that contribute to ROS formation and p53-mediated apoptosis. p53-depleted cells injected into athymic mice increased significantly in tumor volume, whereas injected p53(+/+) cells did not grow to the same degree. Interestingly, administration of the antioxidant compound N-acetylcysteine (NAC) inhibited the growth of tumor volume in p53-depleted injected cells, and NAC supplementation of p53(-/-) mice from birth greatly decreased the number of karyotype abnormalities and tumors formed in these mice by six months of age. Thus, under normal (or low stress) conditions, p53 appears to have an antioxidant role that protects cells from oxidative DNA damage and although this effect might depend on the concentration of p53, other cellular factors likely participate in a cell's final fate.

Animals↗
PubMed2005

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.

Anemone↗
PubMed2005

Diallyl trisulfide-induced G(2)-M phase cell cycle arrest in human prostate cancer cells is caused by reactive oxygen species-dependent destruction and hyperphosphorylation of Cdc 25 C.

Molecular mechanism of cell cycle arrest caused by diallyl trisulfide (DATS), a garlic-derived cancer chemopreventive agent, has been investigated using PC-3 and DU 145 human prostate cancer cells as a model. Treatment of PC-3 and DU 145 cells, but not a normal prostate epithelial cell line (PrEC), with growth suppressive concentrations of DATS caused enrichment of the G(2)-M fraction. The DATS-induced cell cycle arrest in PC-3 cells was associated with increased Tyr(15) phosphorylation of cyclin-dependent kinase 1 (Cdk 1) and inhibition of Cdk 1/cyclinB 1 kinase activity. The DATS-treated PC-3 and DU 145 cells also exhibited a decrease in the protein level of Cdc 25 C and an increase in its Ser(216) phosphorylation. The DATS-mediated decrease in protein level and Ser(216) phosphorylation of Cdc 25 C as well as G(2)-M phase cell cycle arrest were significantly attenuated in the presence of N-acetylcysteine implicating reactive oxygen species (ROS) in cell cycle arrest caused by DATS. ROS generation was observed in DATS-treated PC-3 and DU 145 cells. DATS treatment also caused an increase in the protein level of Cdk inhibitor p21, but DATS-induced G(2)-M phase arrest was not affected by antisense-mediated suppression of p21 protein level. In conclusion, the results of the present study indicate that DATS-induced G(2)-M phase cell cycle arrest in human prostate cancer cells is caused by ROS-mediated destruction and hyperphosphorylation of Cdc 25 C.

Allyl Compounds↗
PubMed2005

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.

Cell Cycle Proteins↗
PubMed2005

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.

Alkaloids↗
PubMed2005

Live long and prosper.

In this issue of Molecular Pharmacology, Meyn et al. (p. 1320) provide the results of the first comprehensive investigation of the expression pattern of the Src family of nonreceptor tyrosine kinases (SFK) in mouse embryonic stem (ES) cells. They found that self-renewing ES cells express seven of the eight mammalian SFK members and that some undergo distinct expression changes during early differentiation events. One of the most dramatic changes was in Hck transcript levels, which decreased almost 30-fold during the first 3 days of embryoid body formation, a culture system model of early embryogenesis and differentiation. Other SFKs, such as Fyn and Src, remain present and active as ES cells differentiate. Of particular interest was the observation that ES cell self-renewal or differentiation can be manipulated through the selective pharmacological inhibition of SFK members. This information should help in the expanding efforts to exploit ES cells for basic and clinical purposes.

Animals↗
PubMed2005

MKP-8, a novel MAPK phosphatase that inhibits p38 kinase.

Intracellular signaling pathways and their relationship to malignant progression have become a major focus of cancer biology. The dual-specificity phosphatase (DSP) family is a more recently identified family of intracellular signaling modulators. We have identified a novel protein phosphatase with a well-conserved DSP catalytic domain containing the DSP catalytic motif, xHCxxGxSRS, and mitogen-activated protein kinase phosphatase (MKP) motif, AYLM. Because of these unique characteristics, the protein was named mitogen-activated protein kinase phosphatase-8 (MKP-8). This protein is approximately 20kDa in size and mainly localizes to the nuclear compartment of the cell. MKP-8 is expressed in embryonal cancers (retinoblastoma, neuroepithelioma, and neuroblastoma) and has limited expression in normal tissues. MKP-8 displays significant phosphatase activity that is inhibited by a cysteine to serine substitution in the catalytic domain. When co-expressed with activated MAPKs, MKP-8 is able to inhibit p38 kinase phosphorylation and downstream activity.

Amino Acid Sequence↗
PubMed2005

The benzo[c]phenanthridine alkaloid, sanguinarine, is a selective, cell-active inhibitor of mitogen-activated protein kinase phosphatase-1.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase that is overexpressed in many human tumors and can protect cells from apoptosis caused by DNA-damaging agents or cellular stress. Small molecule inhibitors of MKP-1 have not been reported, in part because of the lack of structural guidance for inhibitor design and definitive assays for MKP-1 inhibition in intact cells. Herein we have exploited a high content chemical complementation assay to analyze a diverse collection of pure natural products for cellular MKP-1 inhibition. Using two-dimensional Kolmogorov-Smirnov statistics, we identified sanguinarine, a plant alkaloid with known antibiotic and antitumor activity but no primary cellular target, as a potent and selective inhibitor of MKP-1. Sanguinarine inhibited cellular MKP-1 with an IC50 of 10 microM and showed selectivity for MKP-1 over MKP-3. Sanguinarine also inhibited MKP-1 and the MKP-1 like phosphatase, MKP-L, in vitro with IC50 values of 17.3 and 12.5 microM, respectively, and showed 5-10-fold selectivity for MKP-3 and MKP-1 over VH-1-related phosphatase, Cdc25B2, or protein-tyrosine phosphatase 1B. In a human tumor cell line with high MKP-1 levels, sanguinarine caused enhanced ERK and JNK/SAPK phosphorylation. A close congener of sanguinarine, chelerythrine, also inhibited MKP-1 in vitro and in whole cells, and activated ERK and JNK/SAPK. In contrast, sanguinarine analogs lacking the benzophenanthridine scaffold did not inhibit MKP-1 in vitro or in cells nor did they cause ERK or JNK/SAPK phosphorylation. These data illustrate the utility of a chemical complementation assay linked with multiparameter high content cellular screening.

Alkaloids↗
PubMed2005

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.

Animals↗
PubMed2005

Chemical complementation: a definitive phenotypic strategy for identifying small molecule inhibitors of elusive cellular targets.

Forward Pharmacology seeks to identify small or large molecules that modulate a normal or abnormal biological process in living cells or whole organisms and historically has been responsible for the discovery of many clinically used drugs. Forward Pharmacology approaches have become particularly attractive because advances in combinatorial chemistry and laboratory automation have made it possible to generate and interrogate large compound collections in a short period of time. Because many drug discovery efforts are now directed against specific biochemical targets, however, the utility of Forward Pharmacology is limited by the fact that assays to investigate compounds in biological systems are often phenotypic rather than target specific. We discuss here a novel strategy to discover target-based small molecules in intact cells using contemporary Forward Pharmacology in cells with specific genetic manipulations. The method, which we have termed "chemical complementation", is defined as the ability of small molecules to reverse a genetically induced phenotypic change in intact cells. Chemical complementation represents an extension of the commonly used genetic complementation approach, where cDNA libraries are used to investigate the function of genes based on their ability to rescue a specific genetic defect. We present examples of how chemical complementation has been used to identify and credential cell-active, small molecule inhibitors of 2 dual-specificity phosphatases, Cdc25A and MKP-3, which heretofore have eluded small molecule drug discovery efforts.

Animals↗
PubMed2005

Systems cell biology knowledge created from high content screening.

High content screening (HCS), the large-scale automated analysis of the temporal and spatial changes in cells and cell constituents in arrays of cells, has the potential to create enormous systems cell biology knowledge bases. HCS is being employed along with the continuum of the early drug discovery process, including lead optimization where new knowledge is being used to facilitate the decision-making process. We demonstrate methodology to build new systems cell biology knowledge using a multiplexed HCS assay, designed with the aid of knowledge-mining tools, to measure the phenotypic response of a panel of human tumor cell types to a panel of natural product-derived microtubule-targeted anticancer agents and their synthetic analogs. We show how this new systems cell biology knowledge can be used to design a lead compound optimization strategy for at least two members of the panel, (-)-laulimalide and (+)-discodermolide, that exploits cell killing activity while minimally perturbing the regulation of the cell cycle and the stability of microtubules. Furthermore, this methodology can also be applied to basic biomedical research on cells.

Antineoplastic Agents↗
PubMed2005

Dual specificity protein phosphatases: therapeutic targets for cancer and Alzheimer's disease.

The complete sequencing of the human genome is generating many novel targets for drug discovery. Understanding the pathophysiological roles of these putative targets and assessing their suitability for therapeutic intervention has become the major hurdle for drug discovery efforts. The dual-specificity phosphatases (DSPases), which dephosphorylate serine, threonine, and tyrosine residues in the same protein substrate, have important roles in multiple signaling pathways and appear to be deregulated in cancer and Alzheimer's disease. We examine the potential of DSPases as new molecular therapeutic targets for the treatment of human disease.

Alzheimer Disease↗