Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 31
Filter
1.
J Biol Chem ; 300(6): 107383, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38762182

ABSTRACT

Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. In bacteria, disulfide bonds are catalyzed by DsbA and DsbB/VKOR enzymes. First, DsbA, a periplasmic disulfide oxidoreductase, introduces disulfide bonds into substrate proteins. Then, the membrane enzyme, either DsbB or VKOR, regenerate DsbA's activity by the formation of de novo disulfide bonds which reduce quinone. We have previously performed a high-throughput chemical screen and identified a family of warfarin analogs that target either bacterial DsbB or VKOR. In this work, we expressed functional human VKORc1 in Escherichia coli and performed a structure-activity-relationship analysis to study drug selectivity between bacterial and mammalian enzymes. We found that human VKORc1 can function in E. coli by removing two positive residues, allowing the search for novel anticoagulants using bacteria. We also found one warfarin analog capable of inhibiting both bacterial DsbB and VKOR and a second one antagonized only the mammalian enzymes when expressed in E. coli. The difference in the warfarin structure suggests that substituents at positions three and six in the coumarin ring can provide selectivity between the bacterial and mammalian enzymes. Finally, we identified the two amino acid residues responsible for drug binding. One of these is also essential for de novo disulfide bond formation in both DsbB and VKOR enzymes. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold.


Subject(s)
Bacterial Proteins , Escherichia coli Proteins , Escherichia coli , Vitamin K Epoxide Reductases , Warfarin , Warfarin/metabolism , Warfarin/chemistry , Vitamin K Epoxide Reductases/metabolism , Vitamin K Epoxide Reductases/chemistry , Vitamin K Epoxide Reductases/genetics , Humans , Escherichia coli/metabolism , Escherichia coli/genetics , Escherichia coli/enzymology , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Disulfides/chemistry , Disulfides/metabolism , Coumarins/metabolism , Coumarins/chemistry , Protein Disulfide-Isomerases/metabolism , Protein Disulfide-Isomerases/chemistry , Protein Disulfide-Isomerases/genetics , Anticoagulants/chemistry , Anticoagulants/metabolism , Benzoquinones/metabolism , Benzoquinones/chemistry , Structure-Activity Relationship , Protein Binding , Membrane Proteins
2.
Physiol Rep ; 11(17): e15819, 2023 09.
Article in English | MEDLINE | ID: mdl-37697223

ABSTRACT

Necrotizing enterocolitis (NEC) continues to be a devastating disease in preterm neonates and has a paucity of medical management options. Chondroitin sulfate (CS) is a naturally occurring glycosaminoglycan (GAG) in human breast milk (HM) and has been shown to reduce inflammation. We hypothesized that supplementation with CS in an experimental NEC model would alter microbial diversity, favorably alter the cytokine profile, and (like other sulfur compounds) improve outcomes in experimental NEC via the eNOS pathway. NEC was induced in 5-day-old pups. Six groups were studied (n = 9-15/group): (1) WT breastfed and (2) Formula fed controls, (3) WT NEC, (4) WT NEC + CS, (5) eNOS KO (knockout) NEC, and (6) eNOS KO NEC + CS. Pups were monitored for clinical sickness score and weights. On postnatal day 9, the pups were killed. Stool was collected from rectum and microbiome analysis was done with 16 s rRNA sequencing. Intestinal segments were examined histologically using a well-established injury scoring system and segments were homogenized and analyzed for cytokine profile. Data were analyzed using GraphPad Prism with p < 0.05 considered significant. CS supplementation in formula improved experimental NEC outcomes when compared to NEC alone. CS supplementation resulted in similar improvement in NEC in both the WT and eNOS KO mice. CS supplementation did not result in microbial changes when compared to NEC alone. Our data suggest that although CS supplementation improved outcomes in NEC, this protection is not conferred via the eNOS pathway or alteration of microbial diversity. CS therapy in NEC does improve the intestinal cytokine profile and further experiments will explore the mechanistic role of CS in altering immune pathways in this disease.


Subject(s)
Enterocolitis, Necrotizing , Fetal Diseases , Female , Infant, Newborn , Humans , Animals , Mice , Chondroitin Sulfates/therapeutic use , Enterocolitis, Necrotizing/drug therapy , Disease Models, Animal , Dietary Supplements , Cytokines
3.
J Am Soc Nephrol ; 34(2): 220-240, 2023 02 01.
Article in English | MEDLINE | ID: mdl-36283811

ABSTRACT

BACKGROUND: Translation shutdown is a hallmark of late-phase, sepsis-induced kidney injury. Methods for controlling protein synthesis in the kidney are limited. Reversing translation shutdown requires dephosphorylation of the eukaryotic initiation factor 2 (eIF2) subunit eIF2 α ; this is mediated by a key regulatory molecule, protein phosphatase 1 regulatory subunit 15A (Ppp1r15a), also known as GADD34. METHODS: To study protein synthesis in the kidney in a murine endotoxemia model and investigate the feasibility of translation control in vivo by boosting the protein expression of Ppp1r15a, we combined multiple tools, including ribosome profiling (Ribo-seq), proteomics, polyribosome profiling, and antisense oligonucleotides, and a newly generated Ppp1r15a knock-in mouse model and multiple mutant cell lines. RESULTS: We report that translation shutdown in established sepsis-induced kidney injury is brought about by excessive eIF2 α phosphorylation and sustained by blunted expression of the counter-regulatory phosphatase Ppp1r15a. We determined the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic and antisense oligonucleotide approaches enabled the overexpression of Ppp1r15a, which salvaged translation and improved kidney function in an endotoxemia model. Loss of this uORF also had broad effects on the composition and phosphorylation status of the immunopeptidome-peptides associated with the MHC-that extended beyond the eIF2 α axis. CONCLUSIONS: We found Ppp1r15a is translationally repressed during late-phase sepsis because of the existence of an uORF, which is a prime therapeutic candidate for this strategic rescue of translation in late-phase sepsis. The ability to accurately control translation dynamics during sepsis may offer new paths for the development of therapies at codon-level precision. PODCAST: This article contains a podcast at.


Subject(s)
Acute Kidney Injury , Endotoxemia , Animals , Mice , Protein Biosynthesis , Open Reading Frames , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Endotoxemia/complications , Disease Models, Animal , Acute Kidney Injury/genetics , Protein Phosphatase 1
4.
Mol Ther Methods Clin Dev ; 27: 185-194, 2022 Dec 08.
Article in English | MEDLINE | ID: mdl-36284765

ABSTRACT

Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With the skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays a vital role in serotype confirmation and quality control. Further, peptide mapping the capsid proteins might inevitably be a future requirement by regulatory agencies since it is a critical step in good manufacturing practice (GMP) for biotherapeutic characterization. To overcome many challenges that traditional methods like SDS-PAGE and western blots carry, liquid chromatography and mass spectrometry (LC-MS) allows high resolution and sensitivity with great accuracy in characterizing the AAV capsid proteins. Our optimized LC-MS method provides quick sample preparation, a fast and high-throughput 4-min run, and high sensitivity, which allows for very efficient characterization of wild-type and engineered capsids. This study also reports the usage of LC-MS/MS peptide mapping of AAV capsid proteins to determine the most accessible lysine residues targeted by chemical modifications. Our detailed protocols are anticipated to promote the development and discovery of AAV variants with high accuracy and efficiency.

5.
Eng Life Sci ; 22(5): 376-390, 2022 May.
Article in English | MEDLINE | ID: mdl-35573133

ABSTRACT

There are few reports on the material transformation and dominant microorganisms in the process of greening waste (GW) composting. In this study, the target microbial community succession and material transformation were studied in GW composting by using MiSeq sequencing and PICRUSt tools. The results showed that the composting process could be divided into four phases. Each phase of the composting appeared in turn and was unable to jump. In the calefactive phase, microorganisms decompose small molecular organics such as FA to accelerate the arrival of the thermophilic phase. In the thermophilic phase, thermophilic microorganisms decompose HA and lignocellulose to produce FA. While in the cooling phase, microorganisms degrade HA and FA for growth and reproduction. In the maturation phase, microorganisms synthesize humus using FA, amino acid and lignin nuclei as precursors. In the four phases of the composting, different representative genera of bacteria and fungi were detected. Streptomyces, Myceliophthora and Aspergillus, maintained high abundance in all phases of the compost. Correlation analysis indicated that bacteria, actinomycetes and fungi had synergistic effect on the degradation of lignocellulose. Therefore, it can accelerate the compost process by maintaining the thermophilic phase and adding a certain amount of FA in the maturation phase.

6.
Cell Chem Biol ; 25(12): 1456-1469.e6, 2018 12 20.
Article in English | MEDLINE | ID: mdl-30293938

ABSTRACT

5-Nitrofurans are antibiotic pro-drugs that have potential as cancer therapeutics. Here, we show that 5-nitrofurans can be bio-activated by aldehyde dehydrogenase (ALDH) 1A1/1A3 enzymes that are highly expressed in a subpopulation of cancer-initiating (stem) cells. We discover that the 5-nitrofuran, nifuroxazide, is selective for bio-activation by ALDH1 isoforms over ALDH2, whereby it both oxidizes ALDH1 and is converted to cytotoxic metabolites in a two-hit pro-drug mechanism. We show that ALDH1High melanoma cells are sensitive to nifuroxazide, while ALDH1A3 loss-of-function mutations confer drug resistance. In tumors, nifuroxazide targets ALDH1High melanoma subpopulations with the subsequent loss of melanoma-initiating cell potential. BRAF and MEK inhibitor therapy increases ALDH1 expression in patient melanomas, and effectively combines with nifuroxazide in melanoma cell models. The selective eradication of ALDH1High cells by nifuroxazide-ALDH1 activation goes beyond current strategies based on inhibiting ALDH1 and provides a rational basis for the nifuroxazide mechanism of action in cancer.


Subject(s)
Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Hydroxybenzoates/metabolism , Hydroxybenzoates/pharmacology , Isoenzymes/metabolism , Melanoma/drug therapy , Melanoma/pathology , Neoplastic Stem Cells/drug effects , Nitrofurans/metabolism , Nitrofurans/pharmacology , Retinal Dehydrogenase/metabolism , Aldehyde Dehydrogenase 1 Family , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Hydroxybenzoates/chemistry , Isoenzymes/antagonists & inhibitors , Melanoma/genetics , Melanoma/metabolism , Mice , Molecular Structure , Neoplastic Stem Cells/pathology , Nitrofurans/chemistry , Prodrugs/chemistry , Prodrugs/metabolism , Prodrugs/pharmacology , Retinal Dehydrogenase/antagonists & inhibitors
7.
Mol Biosyst ; 13(12): 2509-2520, 2017 Nov 21.
Article in English | MEDLINE | ID: mdl-29099132

ABSTRACT

The protein arginine methyltransferase (PRMT) family of enzymes comprises nine family members in mammals. They catalyze arginine methylation, either monomethylation or symmetric/asymmetric dimethylation of histone and non-histone proteins. PRMT methylation of its substrate proteins modulates cellular processes such as signal transduction, transcription, and mRNA splicing. Recent studies have linked overexpression of PRMT5, a member of the PRMT superfamily, to oncogenesis, making it a potential target for cancer therapy. In this study, we developed a highly sensitive (Z' score = 0.7) robotic high throughput screening (HTS) platform to discover small molecule inhibitors of PRMT5 by adapting the AlphaLISA™ technology. Using biotinylated histone H4 as a substrate, and S-adenosyl-l-methionine as a methyl donor, PRMT5 symmetrically dimethylated H4 at arginine (R) 3. Highly specific acceptor beads for symmetrically dimethylated H4R3 and streptavidin-coated donor beads bound the substrate, emitting a signal that is proportional to the methyltransferase activity. Using this powerful approach, we identified specific PRMT5 inhibitors P1608K04 and P1618J22, and further validated their efficacy and specificity for inhibiting PRMT5. Importantly, these two compounds exhibited much more potent efficacy than the commercial PRMT5 inhibitor EPZ015666 in both pancreatic and colorectal cancer cells. Overall, our work highlights a novel, powerful, and sensitive approach to identify specific PRMT5 inhibitors. The general principle of this HTS screening method can not only be applied to PRMT5 and the PRMT superfamily, but may also be extended to other epigenetic targets. This approach allows us to identify compounds that inhibit the activity of their respective targets, and screening hits like P1608K04 and P1618J22 may serve as the basis for novel drug development to treat cancer and/or other diseases.


Subject(s)
Enzyme Inhibitors/pharmacology , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/metabolism , Animals , Blotting, Western , Cell Line, Tumor , Humans , Polymerase Chain Reaction , S-Adenosylmethionine/metabolism , Streptavidin/pharmacology
8.
Oncotarget ; 8(25): 39963-39977, 2017 Jun 20.
Article in English | MEDLINE | ID: mdl-28591716

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are notoriously challenging for treatment. Hyperactive nuclear factor κB (NF-κB) is a common culprit in both cancers. Previously, we discovered that protein arginine methyltransferase 5 (PRMT5) methylated and activated NF-κB. Here, we show that PRMT5 is highly expressed in PDAC and CRC. Overexpression of PRMT5 promoted cancer progression, while shRNA knockdown showed an opposite effect. Using an innovative AlphaLISA high throughput screen, we discovered a lead compound, PR5-LL-CM01, which exhibited robust tumor inhibition effects in both cancers. An in silico structure prediction suggested that PR5-LL-CM01 inhibits PRMT5 by binding with its active pocket. Importantly, PR5-LL-CM01 showed higher anti-tumor efficacy than the commercial PRMT5 inhibitor, EPZ015666, in both PDAC and CRC. This study clearly highlights the significant potential of PRMT5 as a therapeutic target in PDAC and CRC, and establishes PR5-LL-CM01 as a promising basis for new drug development in the future.


Subject(s)
Carcinoma, Pancreatic Ductal/drug therapy , Colorectal Neoplasms/drug therapy , Enzyme Inhibitors/pharmacology , Pancreatic Neoplasms/drug therapy , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Amines/chemistry , Amines/pharmacology , Animals , Carcinoma, Pancreatic Ductal/enzymology , Carcinoma, Pancreatic Ductal/pathology , Cell Line , Cell Line, Tumor , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/pathology , Drug Screening Assays, Antitumor/methods , Enzyme Inhibitors/chemistry , HCT116 Cells , HT29 Cells , Humans , Hydrocarbons, Aromatic/chemistry , Hydrocarbons, Aromatic/pharmacology , Male , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/pathology , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Purines/chemistry , Purines/pharmacology , Small Molecule Libraries/chemistry , Treatment Outcome , Xenograft Model Antitumor Assays
9.
Oncotarget ; 7(45): 73817-73829, 2016 Nov 08.
Article in English | MEDLINE | ID: mdl-27650545

ABSTRACT

Melanoma ranks among the most aggressive and deadly human cancers. Although a number of targeted therapies are available, they are effective only in a subset of patients and the emergence of drug resistance often reduces durable responses. Thus there is an urgent need to identify new therapeutic targets and develop more potent pharmacological agents for melanoma treatment. Herein we report that SHP2 levels are frequently elevated in melanoma, and high SHP2 expression is significantly associated with more metastatic phenotype and poorer prognosis. We show that SHP2 promotes melanoma cell viability, motility, and anchorage-independent growth, through activation of both ERK1/2 and AKT signaling pathways. We demonstrate that SHP2 inhibitor 11a-1 effectively blocks SHP2-mediated ERK1/2 and AKT activation and attenuates melanoma cell viability, migration and colony formation. Most importantly, SHP2 inhibitor 11a-1 suppresses xenografted melanoma tumor growth, as a result of reduced tumor cell proliferation and enhanced tumor cell apoptosis. Taken together, our data reveal SHP2 as a novel target for melanoma and suggest SHP2 inhibitors as potential novel therapeutic agents for melanoma treatment.


Subject(s)
Melanoma/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Survival/genetics , Disease Models, Animal , Gene Expression , Humans , MAP Kinase Signaling System , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , Mice , Molecular Targeted Therapy , Phosphatidylinositol 3-Kinases/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Xenograft Model Antitumor Assays
10.
JCI Insight ; 1(7)2016 May 19.
Article in English | MEDLINE | ID: mdl-27275015

ABSTRACT

The PTPN11 gene, encoding the tyrosine phosphatase SHP-2, is overexpressed in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) compared with osteoarthritis (OA) FLS and promotes RA FLS invasiveness. Here, we explored the molecular basis for PTPN11 overexpression in RA FLS and the role of SHP-2 in RA pathogenesis. Using computational methods, we identified a putative enhancer in PTPN11 intron 1, which contained a glucocorticoid receptor- binding (GR-binding) motif. This region displayed enhancer function in RA FLS and contained 2 hypermethylation sites in RA compared with OA FLS. RA FLS stimulation with the glucocorticoid dexamethasone induced GR binding to the enhancer and PTPN11 expression. Glucocorticoid responsiveness of PTPN11 was significantly higher in RA FLS than OA FLS and required the differentially methylated CpGs for full enhancer function. SHP-2 expression was enriched in the RA synovial lining, and heterozygous Ptpn11 deletion in radioresistant or innate immune cells attenuated K/BxN serum transfer arthritis in mice. Treatment with SHP-2 inhibitor 11a-1 reduced RA FLS migration and responsiveness to TNF and IL-1ß stimulation and reduced arthritis severity in mice. Our findings demonstrate how abnormal epigenetic regulation of a pathogenic gene determines FLS behavior and demonstrate that targeting SHP-2 or the SHP-2 pathway could be a therapeutic strategy for RA.

11.
Cancer Res ; 76(16): 4805-15, 2016 08 15.
Article in English | MEDLINE | ID: mdl-27325652

ABSTRACT

Phosphatase of regenerating liver (PRL) oncoproteins are phosphatases overexpressed in numerous types of human cancer. Elevated levels of PRL associate with metastasis and poor clinical outcomes. In principle, PRL phosphatases offer appealing therapeutic targets, but they remain underexplored due to the lack of specific chemical probes. In this study, we address this issue by exploiting a unique property of PRL phosphatases, namely, that they may function as homotrimers. Starting from a sequential structure-based virtual screening and medicinal chemistry strategy, we identified Cmpd-43 and several analogs that disrupt PRL1 trimerization. Biochemical and structural analyses demonstrate that Cmpd-43 and its close analogs directly bind the PRL1 trimer interface and obstruct PRL1 trimerization. Cmpd-43 also specifically blocks the PRL1-induced cell proliferation and migration through attenuation of both ERK1/2 and Akt activity. Importantly, Cmpd-43 exerted potent anticancer activity both in vitro and in vivo in a murine xenograft model of melanoma. Our results validate a trimerization-dependent signaling mechanism for PRL and offer proof of concept for trimerization inhibitors as candidate therapeutics to treat PRL-driven cancers. Cancer Res; 76(16); 4805-15. ©2016 AACR.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor , Melanoma, Experimental/drug therapy , Protein Tyrosine Phosphatases/antagonists & inhibitors , Animals , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Humans , Immunohistochemistry , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Xenograft Model Antitumor Assays
12.
J Clin Invest ; 126(6): 2077-92, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27183387

ABSTRACT

Systemic lupus erythematosus (SLE) is a devastating multisystemic autoimmune disorder. However, the molecular mechanisms underlying its pathogenesis remain elusive. Some patients with Noonan syndrome, a congenital disorder predominantly caused by gain-of-function mutations in the protein tyrosine phosphatase SH2 domain-containing PTP (SHP2), have been shown to develop SLE, suggesting a functional correlation between phosphatase activity and systemic autoimmunity. To test this directly, we measured SHP2 activity in spleen lysates isolated from lupus-prone MRL/lpr mice and found it was markedly increased compared with that in control mice. Similar increases in SHP2 activity were seen in peripheral blood mononuclear cells isolated from lupus patients relative to healthy patients. To determine whether SHP2 alters autoimmunity and related immunopathology, we treated MRL/lpr mice with an SHP2 inhibitor and found increased life span, suppressed crescentic glomerulonephritis, reduced spleen size, and diminished skin lesions. SHP2 inhibition also reduced numbers of double-negative T cells, normalized ERK/MAPK signaling, and decreased production of IFN-γ and IL-17A/F, 2 cytokines involved in SLE-associated organ damage. Moreover, in cultured human lupus T cells, SHP2 inhibition reduced proliferation and decreased production of IFN-γ and IL-17A/F, further implicating SHP2 in lupus-associated immunopathology. Taken together, these data identify SHP2 as a critical regulator of SLE pathogenesis and suggest targeting of its activity as a potent treatment for lupus patients.


Subject(s)
Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Animals , Autoantibodies/biosynthesis , Case-Control Studies , Cell Proliferation , Cytokines/biosynthesis , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Female , Humans , Lupus Erythematosus, Systemic/etiology , MAP Kinase Signaling System , Mice , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/immunology , T-Lymphocyte Subsets/enzymology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/pathology
13.
Nat Commun ; 6: 8859, 2015 Nov 30.
Article in English | MEDLINE | ID: mdl-26617336

ABSTRACT

Ras is phosphorylated on a conserved tyrosine at position 32 within the switch I region via Src kinase. This phosphorylation inhibits the binding of effector Raf while promoting the engagement of GTPase-activating protein (GAP) and GTP hydrolysis. Here we identify SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signalling. In comparison to normal astrocytes, SHP2 activity is elevated in astrocytes isolated from glioblastoma multiforme (GBM)-prone H-Ras(12V) knock-in mice as well as in glioma cell lines and patient-derived GBM specimens exhibiting hyperactive Ras. Pharmacologic inhibition of SHP2 activity attenuates cell proliferation, soft-agar colony formation and orthotopic GBM growth in NOD/SCID mice and decelerates the progression of low-grade astrocytoma to GBM in a spontaneous transgenic glioma mouse model. These results identify SHP2 as a direct activator of Ras and a potential therapeutic target for cancers driven by a previously 'undruggable' oncogenic or hyperactive Ras.


Subject(s)
Glioblastoma/enzymology , Oncogene Protein p21(ras)/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Animals , Carcinogenesis , Cell Line , Female , Glioblastoma/genetics , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Male , Mice , Mice, Inbred ICR , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Oncogene Protein p21(ras)/genetics , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics
14.
Proc Natl Acad Sci U S A ; 112(43): 13342-7, 2015 Oct 27.
Article in English | MEDLINE | ID: mdl-26460004

ABSTRACT

Previous data suggested a negative role of phosphatase and tensin homolog (Pten) and a positive function of SH2-containing tyrosine phosphatase (Shp2)/Ptpn11 in myelopoiesis and leukemogenesis. Herein we demonstrate that ablating Shp2 indeed suppressed the myeloproliferative effect of Pten loss, indicating directly opposing functions between pathways regulated by these two enzymes. Surprisingly, the Shp2 and Pten double-knockout mice suffered lethal anemia, a phenotype that reveals previously unappreciated cooperative roles of Pten and Shp2 in erythropoiesis. The lethal anemia was caused collectively by skewed progenitor differentiation and shortened erythrocyte lifespan. Consistently, treatment of Pten-deficient mice with a specific Shp2 inhibitor suppressed myeloproliferative neoplasm while causing anemia. These results identify concerted actions of Pten and Shp2 in promoting erythropoiesis, while acting antagonistically in myeloproliferative neoplasm development. This study illustrates cell type-specific signal cross-talk in blood cell lineages, and will guide better design of pharmaceuticals for leukemia and other types of cancer in the era of precision medicine.


Subject(s)
Anemia/genetics , Erythropoiesis/physiology , Myelopoiesis/physiology , PTEN Phosphohydrolase/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Anemia/etiology , Animals , Cell Differentiation/genetics , DNA Primers/genetics , Erythrocytes/physiology , Genotype , Histological Techniques , Mice , Mice, Knockout , Mutagenesis , PTEN Phosphohydrolase/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Survival Analysis
15.
ACS Med Chem Lett ; 6(7): 782-6, 2015 Jul 09.
Article in English | MEDLINE | ID: mdl-26191366

ABSTRACT

Protein tyrosine phosphatases (PTPs) are potential therapeutic targets for many diseases. Unfortunately, despite considerable drug discovery efforts devoted to PTPs, obtaining selective and cell permeable PTP inhibitors remains highly challenging. We describe a strategy to explore the existing drug space for previously unknown PTP inhibitory activities. This led to the discovery of cefsulodin as an inhibitor of SHP2, an oncogenic phosphatase in the PTP family. Crystal structure analysis of SHP2 interaction with cefsulodin identified sulfophenyl acetic amide (SPAA) as a novel phosphotyrosine (pTyr) mimetic. A structure-guided and SPAA fragment-based focused library approach produced several potent and selective SHP2 inhibitors. Notably, these inhibitors blocked SHP2-mediated signaling events and proliferation in several cancer cell lines. Thus, SPAA may serve as a new platform for developing chemical probes for other PTPs.

16.
Oncotarget ; 5(15): 6130-41, 2014 Aug 15.
Article in English | MEDLINE | ID: mdl-25026279

ABSTRACT

Acquired mutations in KIT are driver mutations in systemic mastocytosis (SM). Here, we tested the role of SHP2/PTPN11 phosphatase in oncogenic KIT signaling using an aggressive SM mouse model. Stable knock-down (KD) of SHP2 led to impaired growth, colony formation, and increased rates of apoptosis in P815 cells. This correlated with defects in signaling to ERK/Bim, Btk, Lyn, and Stat5 pathways in P815-KD cells compared to non-targeting (NT). Retro-orbital injections of P815 NT cells in syngeneic DBA/2 mice resulted in rapid development of aggressive SM within 13-16 days characterized by splenomegaly, extramedullary hematopoiesis, and multifocal liver tumors. In contrast, mice injected with P815 SHP2 KD cells showed less disease burden, including normal spleen weight and cellularity, and significant reductions in mastocytoma cells in spleen, bone marrow, peripheral blood and liver compared to NT controls. Treatment of human mast cell leukemia HMC-1 cells or P815 cells with SHP2 inhibitor II-B08, resulted in reduced colony formation and cell viability. Combining II-B08 with multi-kinase inhibitor Dasatinib showed enhanced efficacy than either inhibitor alone in blocking cell growth pathways and cell viability. Taken together, these results identify SHP2 as a key effector of oncogenic KIT and a therapeutic target in aggressive SM.


Subject(s)
Mastocytosis, Systemic/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/physiology , Cell Proliferation/physiology , Dasatinib/pharmacology , Disease Progression , Drug Synergism , Humans , Indoles/pharmacology , Mastocytosis, Systemic/drug therapy , Mastocytosis, Systemic/pathology , Mice , Mice, Transgenic , Protein Kinase Inhibitors/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Signal Transduction , Triazoles/pharmacology
17.
J Med Chem ; 57(15): 6594-609, 2014 Aug 14.
Article in English | MEDLINE | ID: mdl-25003231

ABSTRACT

The Src homology 2 domain containing protein tyrosine phosphatase-2 (SHP2) is an oncogenic phosphatase associated with various kinds of leukemia and solid tumors. Thus, there is substantial interest in developing SHP2 inhibitors as potential anticancer and antileukemia agents. Using a structure-guided and fragment-based library approach, we identified a novel hydroxyindole carboxylic acid-based SHP2 inhibitor 11a-1, with an IC50 value of 200 nM and greater than 5-fold selectivity against 20 mammalian PTPs. Structural and modeling studies reveal that the hydroxyindole carboxylic acid anchors the inhibitor to the SHP2 active site, while interactions of the oxalamide linker and the phenylthiophene tail with residues in the ß5-ß6 loop contribute to 11a-1's binding potency and selectivity. Evidence suggests that 11a-1 specifically attenuates the SHP2-dependent signaling inside the cell. Moreover, 11a-1 blocks growth factor mediated Erk1/2 and Akt activation and exhibits excellent antiproliferative activity in lung cancer and breast cancer as well as leukemia cell lines.


Subject(s)
Antineoplastic Agents/chemistry , Indoles/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Enzyme Activation , Humans , Indoles/chemical synthesis , Indoles/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Molecular Docking Simulation , Molecular Targeted Therapy , Protein Conformation , Protein Tyrosine Phosphatase, Non-Receptor Type 11/chemistry , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Small Molecule Libraries , Structure-Activity Relationship
18.
Antioxid Redox Signal ; 20(14): 2130-40, 2014 May 10.
Article in English | MEDLINE | ID: mdl-24180557

ABSTRACT

AIMS: Protein tyrosine phosphatases (PTPs) play an important role in regulating a wide range of cellular processes. Understanding the role of PTPs within these processes has been hampered by a lack of potent and selective PTP inhibitors. Generating potent and selective probes for PTPs remains a significant challenge because of the highly conserved and positively charged PTP active site that also harbors a redox-sensitive Cys residue. RESULTS: We describe a facile method that uses an appropriate hydroxyindole carboxylic acid to anchor the inhibitor to the PTP active site and relies on the secondary binding elements introduced through an amide-focused library to enhance binding affinity for the target PTP and to impart selectivity against off-target phosphatases. Here, we disclose a novel series of hydroxyindole carboxylic acid-based inhibitors for receptor-type tyrosine protein phosphatase beta (RPTPß), a potential target that is implicated in blood vessel development. The representative RPTPß inhibitor 8b-1 (L87B44) has an IC50 of 0.38 µM and at least 14-fold selectivity for RPTPß over a large panel of PTPs. Moreover, 8b-1 also exhibits excellent cellular activity and augments growth factor signaling in HEK293, MDA-MB-468, and human umbilical vein endothelial cells. INNOVATION: The bicyclic salicylic acid pharmacophore-based focused library approach may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. CONCLUSION: A novel method is described for the development of bioavailable PTP inhibitors that utilizes bicyclic salicylic acid to anchor the inhibitors to the active site and peripheral site interactions to enhance binding affinity and selectivity.


Subject(s)
Carboxylic Acids/pharmacology , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Carboxylic Acids/chemical synthesis , Carboxylic Acids/chemistry , Cell Line, Tumor , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , HEK293 Cells , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/enzymology , Humans , Indoles/chemical synthesis , Indoles/chemistry , Molecular Structure , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Structure-Activity Relationship
19.
Biochem Biophys Res Commun ; 439(4): 586-90, 2013 Oct 04.
Article in English | MEDLINE | ID: mdl-24041688

ABSTRACT

Targeted therapy with inhibitors of epidermal growth factor receptor (EGFR) has produced a noticeable benefit to non-small cell lung cancer (NSCLC) patients whose tumors carry activating mutations (e.g. L858R) in EGFR. Unfortunately, these patients develop drug resistance after treatment, due to acquired secondary gatekeeper mutations in EGFR (e.g. T790M). Given the critical role of SHP2 in growth factor receptor signaling, we sought to determine whether targeting SHP2 could have therapeutic value for EGFR inhibitor resistant NSCLC. We show that SHP2 is required for EGF-stimulated ERK1/2 phosphorylation and proliferation in EGFR inhibitor resistant NSCLC cell line H1975, which harbors the EGFR T790M/L858R double-mutant. We demonstrate that treatment of H1975 cells with II-B08, a specific SHP2 inhibitor, phenocopies the observed growth inhibition and reduced ERK1/2 activation seen in cells treated with SHP2 siRNA. Importantly, we also find that II-B08 exhibits marked anti-tumor activity in H1975 xenograft mice. Finally, we observe that combined inhibition of SHP2 and PI3K impairs both the ERK1/2 and PI3K/AKT signaling axes and produces significantly greater effects on repressing H1975 cell growth than inhibition of either protein individually. Collectively, these results suggest that targeting SHP2 may represent an effective strategy for treatment of EGFR inhibitor resistant NSCLCs.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Resistance, Neoplasm , ErbB Receptors/antagonists & inhibitors , Lung Neoplasms/drug therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , ErbB Receptors/metabolism , Erlotinib Hydrochloride , Gefitinib , Humans , Lung Neoplasms/pathology , Mice , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Quinazolines/pharmacology , Quinazolines/therapeutic use , Xenograft Model Antitumor Assays
20.
ChemMedChem ; 8(6): 904-8, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23568546

ABSTRACT

Focused on Mtb: A facile hydroxyindole carboxylic acid based focused amide library was designed to target both the PTP active site and a unique nearby pocket for enhanced affinity and selectivity. HTS of the library led to the identification of a highly potent and selective inhibitor, 11 a, of mPTPB, an essential virulence factor for Mycobacterium tuberculosis. Compound 11 a shows high cellular activity and is capable of reversing the altered immune responses induced by mPTPB in macrophages.


Subject(s)
Carboxylic Acids/pharmacology , Indoles/pharmacology , Mycobacterium tuberculosis/enzymology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Carboxylic Acids/chemical synthesis , Carboxylic Acids/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Indoles/chemical synthesis , Indoles/chemistry , Molecular Structure , Protein Tyrosine Phosphatases/metabolism , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship
SELECTION OF CITATIONS
SEARCH DETAIL
...