John Lazo
University of Virginia, Pharmacology, Faculty Member
Etoposide (VP-16) is one of several DNA-damaging agents that induce subcellular structural changes associated with apoptosis. VP-16 exerts its DNA-damaging and cytotoxic effects subsequent to interference with DNA topoisomerase II... more
Etoposide (VP-16) is one of several DNA-damaging agents that induce subcellular structural changes associated with apoptosis. VP-16 exerts its DNA-damaging and cytotoxic effects subsequent to interference with DNA topoisomerase II activity. VP-16 also stimulates c-jun and c-fos mRNA expression in some cell lines, including human leukemia K562 and HL-60 cells. To compare the temporal relationship between drug-induced c-jun expression and apoptosis, we examined cell morphology, cell viability, DNA integrity, and c-jun induction during VP-16 treatment of K562 and HL-60 cells. VP-16 (10 microM)-induced internucleosomal DNA damage and nuclear fragmentation were readily apparent within 6 hr in HL-60 cells but were absent in K562 cells treated for up to 24 hr. Some internucleosomal DNA damage was observed in K562 cells but only after treatment with 100 microM VP-16 for 24 hr. In contrast, VP-16-induced DNA single-strand breaks, VP-16-induced topoisomerase II/DNA covalent complex formation,...
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Overexpression of metallothioneins (MTs) protects some cells against heavy metals, mutagens, anticancer agents, and reactive oxygen species. We have examined the effect of the loss of MT expression on the cytotoxicity of anticancer agents... more
Overexpression of metallothioneins (MTs) protects some cells against heavy metals, mutagens, anticancer agents, and reactive oxygen species. We have examined the effect of the loss of MT expression on the cytotoxicity of anticancer agents and mutagens using embryonic fibroblast cells from transgenic mice with targeted disruptions of MT I and II genes (MT -/-). MT -/- cells expressed no detectable MT. Compared to wild type cells, MT -/- cells showed enhanced sensitivity to a 2-h exposure to cisplatin, melphalan, bleomycin, cytarabine, or N-methyl-N'-nitro-N-nitrosoguanidine but were equally sensitive to doxorubicin and neocarzinostatin. Basal expression of the DNA damage-response genes, gadd 45 and gadd 153, were elevated in MT -/- cells compared to MT +/+ cells. Anticancer drug treatment, however, did not produce a greater increase in gadd 45 or gadd 153 expression in MT null cells compared to MT +/+ cells. These results support the hypothesis that endogenous MT levels affect th...
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Research Interests: Organic Chemistry, Kinetics, Enzyme Inhibitors, Humans, Enzyme Inhibition, and 9 morePhosphorylation, Mitogen Activated Protein Kinase, Protein tyrosine phosphatase, Substrate Specificity, Bioorganic and medicinal Chemistry, Imidazoles, Structure activity Relationship, Cell Cycle Proteins, and Protein Tyrosine Phosphatases
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... more
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.
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Research Interests: Organic Chemistry, Medicinal Chemistry, Kinetics, Enzyme Inhibitors, Magnetic Resonance Spectroscopy, and 19 moreMacromolecular X-Ray Crystallography, Cell Division, Lymphoma, Humans, Hungary, Mice, Multidrug Resistance, Animals, P-glycoprotein, Quinones, Terpenes, Quinolones, Diterpenes, Medicinal, Euphorbia, Quinolines, Structure activity Relationship, Recombinant Proteins, and Antineoplastic Agents
The unicellular eukaryote Trypanosoma brucei (T. brucei) is the causative agent of human African trypanosomiasis (HAT), a disease that annually infects ~500,000 people in sub-Saharan Africa resulting in 50,000 – 70,000 deaths per year.... more
The unicellular eukaryote Trypanosoma brucei (T. brucei) is the causative agent of human African trypanosomiasis (HAT), a disease that annually infects ~500,000 people in sub-Saharan Africa resulting in 50,000 – 70,000 deaths per year. Without treatment, HAT is fatal. Unfortunately, current treatments are limited in availability, have toxic side effects, are difficult to administer and are not well characterized in terms of their mechanism of action. Thus, the lack of affordable, safe, and effective therapies for those with African trypanosomiasis makes the identification of molecular target-specific chemotypes a priority in our effort to understand the mechanisms involved with parasite growth and viability, as well as for future therapeutic development. The probe identified from this effort, ML205, is a stable, small molecule possessing submicromolar activity (IC50 = 0.98 μM) against a defined T. brucei hexokinase 1 (rTbHK1) target. The probe was not toxic to mammalian cells (IMR-9...
1. Mol Interv. 2010 Apr;10(2):72-5. Anti-leishmanial drug discovery: rising to the challenges of a highly neglected disease. Sharlow ER, Grögl M, Johnson J, Lazo JS. Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA... more
1. Mol Interv. 2010 Apr;10(2):72-5. Anti-leishmanial drug discovery: rising to the challenges of a highly neglected disease. Sharlow ER, Grögl M, Johnson J, Lazo JS. Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA. ...
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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... more
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.
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Bioassay-directed fractionation of an extract of the marine species Spongia sp. led to the discovery of the new sesquiterpenoid derivative 17-O-isoprenyldictyoceratin-C (1), the known sesquiterpenoid derivative dictyoceratin-C (2), and... more
Bioassay-directed fractionation of an extract of the marine species Spongia sp. led to the discovery of the new sesquiterpenoid derivative 17-O-isoprenyldictyoceratin-C (1), the known sesquiterpenoid derivative dictyoceratin-C (2), and the sesquiterpenoid quinone ilimaquinone (3), in addition to the nucleoside 2'-deoxyuridine. The structure of the new compound 1 was determined on the basis of spectroscopic methods and by conversion of dictyoceratin-C (2) to 1.
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Research Interests: Organic Chemistry, Medicinal Chemistry, Protein-Protein Interaction, Cell line, Humans, and 13 moreCircular Dichroism, Case Study, Animals, Drug Design, Rats, Pyridines, Three Dimensional, S100 protein family, Cell Proliferation, Biological activity, Thiazoles, Human Fibroblasts, and Protein Binding
We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP-dependent protein kinase, three calcium/calmodulin-dependent protein kinases,... more
We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP-dependent protein kinase, three calcium/calmodulin-dependent protein kinases, protein kinase C, and at least one tyrosine kinase. No cyclic GMP-dependent protein kinase activity was found. The cells also contained numerous substrates for cyclic AMP-dependent protein kinase and protein kinase C. Fewer substrates were found for the calcium/calmodulin-dependent protein kinases. There was little difference between either protein kinase activities or substrates when pulmonary artery endothelium was compared to aortic endothelium grown under similar culture conditions. It is likely that these various protein kinases and their respective substrate proteins are involved in mediating several of the actions of the hormones and drugs which affect the vascular endothelium.
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The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently... more
The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with Kd of approximately 3.4 microm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The approximately 80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway.
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Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by the second messenger diacylglycerol. It has been implicated in many important cellular processes and pathological conditions. However, further analysis of... more
Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by the second messenger diacylglycerol. It has been implicated in many important cellular processes and pathological conditions. However, further analysis of PKD in these processes is severely hampered by the lack of a PKD-specific inhibitor that can be readily applied to cells and in animal models. We now report the discovery of the first potent and selective cell-active small molecule inhibitor for PKD, benzoxoloazepinolone (CID755673). This inhibitor was identified from the National Institutes of Health small molecule repository library of 196,173 compounds using a human PKD1 (PKCmu)-based fluorescence polarization high throughput screening assay. CID755673 suppressed half of the PKD1 enzyme activity at 182 nm and exhibited selective PKD1 inhibition when compared with AKT, polo-like kinase 1 (PLK1), CDK activating kinase (CAK), CAMKIIalpha, and three different PKC isoforms. Moreover, it was not competitive with ATP for enzyme inhibition. In cell-based assays, CID755673 blocked phorbol ester-induced endogenous PKD1 activation in LNCaP cells in a concentration-dependent manner. Functionally, CID755673 inhibited the known biological actions of PKD1 including phorbol ester-induced class IIa histone deacetylase 5 nuclear exclusion, vesicular stomatitis virus glycoprotein transport from the Golgi to the plasma membrane, and the ilimaquinone-induced Golgi fragmentation. Moreover, CID755673 inhibited prostate cancer cell proliferation, cell migration, and invasion. In summary, our findings indicate that CID755673 is a potent and selective PKD1 inhibitor with valuable pharmacological and cell biological potential.
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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... more
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.
Research Interests: Fluorescence Microscopy, Enzyme Inhibitors, Biological Chemistry, DNA damage, Biological Sciences, and 17 moreHumans, Natural Product, Biological, Alkaloids, Phosphorylation, Mitogen Activated Protein Kinase, CHEMICAL SCIENCES, Plant extracts, Time Factors, HeLa cells, Neoplasms, Kolmogorov-Smirnov test, Transfection, Two-Dimensional Gel Electrophoresis, Antitumor Activity, Cell Cycle Proteins, and Protein Tyrosine Phosphatases
Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial... more
Malaria caused by Plasmodium falciparum is a disease that is responsible for 880,000 deaths per year worldwide. Vaccine development has proved difficult and resistance has emerged for most antimalarial drugs. To discover new antimalarial chemotypes, we have used a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library-many of which showed potent in vitro activity against drug-resistant P. falciparum strains-and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in several organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Our findings provide the scientific community with...
Research Interests: Genetics, Drug Discovery, Multidisciplinary, Nature, Toxoplasma gondii, and 16 morePhylogeny, Cell line, Humans, Mice, Animals, Scientific Communication, Drug Resistance, Phenotype, Plasmodium falciparum, Antimalarials, Reproducibility of Results, Combination drug therapy, Biological activity, Murine Model, Erythrocytes, and Drug Targeting
The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the... more
The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the gamma-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay.