Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5′-phosphate (PLP) via Schiff base formation

Abstract D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5′-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC50 = 13 ± 1 μM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine β-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures

CD137 Expression Is Induced by Epstein-Barr Virus Infection through LMP1 in T or NK Cells and Mediates Survival Promoting Signals

<div><p>To clarify the mechanism for development of Epstein-Barr virus (EBV)-positive T- or NK-cell neoplasms, we focused on the costimulatory receptor CD137. We detected high expression of <i>CD137</i> gene and its protein on EBV-positive T- or NK-cell lines as compared with EBV-negative cell lines. EBV-positive cells from EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs) patients also had significantly higher <i>CD137</i> gene expression than control cells from healthy donors. In the presence of IL-2, whose concentration in the serum of EBV-T/NK-LPDs was higher than that of healthy donors, CD137 protein expression was upregulated in the patients' cells whereas not in control cells from healthy donors. <i>In vitro</i> EBV infection of MOLT4 cells resulted in induction of endogenous CD137 expression. Transient expression of <i>LMP1</i>, which was enhanced by IL-2 in EBV-T/NK-LPDs cells, induced endogenous <i>CD137</i> gene expression in T and NK-cell lines. In order to examine <i>in vivo</i> CD137 expression, we used EBV-T/NK-LPDs xenograft models generated by intravenous injection of patients' cells. We identified EBV-positive and CD8-positive T cells, as well as CD137 ligand-positive cells, in their tissue lesions. In addition, we detected CD137 expression on the EBV infected cells from the lesions of the models by immune-fluorescent staining. Finally, CD137 stimulation suppressed etoposide-induced cell death not only in the EBV-positive T- or NK-cell lines, but also in the patients' cells. These results indicate that upregulation of CD137 expression through LMP1 by EBV promotes cell survival in T or NK cells leading to development of EBV-positive T/NK-cell neoplasms.</p></div

EBV induces CD137 expression in T cells.

<p><i>In vitro</i> EBV infection assay performed in MOLT4 cells. (A) EBNA1 protein expression was examined by immune fluorescence staining, 48 hours after the infection, the time when CD137 expression was examined. (B) Expression of the <i>CD137</i> gene was examined by RT-PCR. The infection was confirmed by detecting mRNAs of the viral proteins, EBNA1 and LMP1. (C) Transcripts of <i>CD137</i> and <i>GAPDH</i> were quantified by real time RT-PCR. Relative copy number was obtained by normalizing the <i>CD137</i> transcripts to those of those of <i>GAPDH</i>. (D) Surface expression of CD137 of MOLT4 cells and EBV-infected MOLT4 cells was examined by flow cytometry.</p

CD137 expression was upregulated by LMP1 whose expression was enhanced by IL-2 in EBV-T/NK-LPDs cells.

<p>(A) <i>CD137</i> transcription was examined using the assay described. Briefly, MOLT4 cells were transfected with 10 µg of the expression plasmids of the viral proteins, EBNA1, LMP1, LMP2A, LMP2B, or an empty vector as indicated, along with 10 µg of PGL3-4-1BB and 1 µg of pRLSV40. Twelve hours after transfection, the cells were harvested for a dual luciferase assay. Luciferase activity was normalized by <i>Renilla</i> luciferase activity and expressed in arbitrary units. The data are expressed as mean ± S.D. of 3 independent experiments. (B) MOLT4 cells were transfected with 10 µg of the expression plasmids of the viral proteins, EBNA1, LMP1, LMP2A, LMP2B, or an empty vector. Transcripts of CD137 (the upper panel) and GAPDH (the lower panel) in these cells were examined by RT-PCR. Jurkat-CD137 cells were used as a positive control. (C) RNAs were obtained from PBMCs from a EBV-T/NK-LPDs patient (CD4-1) which had been cultured with or without IL-2 for 3 days. Semi-quantitative RT-PCR assay for <i>LMP</i> was performed. Transcripts of <i>LMP1</i> (the upper panel) and <i>GAPDH</i> (the lower panel) were presented. (D) Transcripts of <i>CD137</i> and <i>GAPDH</i> were quantified by real time RT-PCR for the sample of 4C. Relative copy number was obtained by normalizing the <i>CD137</i> transcripts to those of <i>GAPDH</i>.</p

Histopathological specimen from the liver of the xenograft models.

<p>We generated the models by transplanting the cells from CD8-3 patient. Nine mice were examined and the representative data were shown. (A) Hematoxylin and eosin staining showed periportal infiltration of lymphocytes. (B) Immunochemical staining with anti-CD8 antibody (brown) showed that the infiltrating lymphocytes were positive for CD8. (C) <i>In situ</i> hybridization of Epstein–Barr virus-encoded mRNA (EBER) (brown). Infiltration of EBV-positive cells was detected in the periportal space. (D) Immunochemical staining with anti-CD137L antibody (brown) showed that CD137L-positive cells existed in the periportal space although the number of the cells was smaller than that of EBER positive cells. (original magnification, ×400). (E) Immune-fluorescent staining with anti-EBNA1 and anti-CD137 antibodies of cells isolated from the lesions. Mononuclear cells were obtained from the tissue lesions of a model mouse, stained with the antibodies. The cells were analyzed by confocal microscopy.</p

IL-2 concentration of the serum from EBV-T/NK-LPD patients.

<p>The concentration of IL-2 of the serum from EBV-T/NK-LPDs patients and from healthy donors. The lowest detection limit was 0.8 U/ml.</p><p>IL-2 concentration of the serum from EBV-T/NK-LPD patients.</p

Clinical information of the patients' samples subjected to the assay.

<p>M: Male, F: Female.</p><p>EBV: Epstein-Barr virus, PB: peripheral blood.</p><p>sCAEBV: systemic chronic active Epstein-Barr virus infection, HMB: hypersensitivity to mosquito bites (HMB), HV: hydroa vacciniforme-like eruption.</p><p>*The clonality was detected by Southern blotting for EBV terminal repeat.</p><p>Clinical information of the patients' samples subjected to the assay.</p

CD137 expression in Epstein-Barr virus (EBV)-positive T- or NK-cell lines.

<p>(A) Transcripts of <i>CD137</i> (the upper panel) and <i>GAPDH</i> (the lower panel) in EBV- positive T- or NK-cell lines were examined by RT-PCR. EBV negative T-, NK, B-cell lines, and EBV-positive B-cell lines were also obtained for the examination. (B) Surface expression of CD137 was examined by flow cytometry using an antibody to CD137 (open histogram) or isotype-matched control immunoglobulin (gray, shaded histogram). The mean fluorescent intensity of CD137 was normalized by that of isotype-matched control and expressed as mean fluorescence intensity rate (MFIR). Each experiment was independently performed more than 3 times and their average data are presented.</p

CD137 expression in EBV-positive T or NK cells of patients with EBV-T/NK-lymphoproliferative disorders (EBV-T/NK-LPDs).

<p>(A) Transcripts of <i>CD137</i> and <i>GAPDH</i> of freshly isolated EBV-positive cell fractions from 9 EBV-T/NK-LPDs patients, or cells of the same fractions from healthy donors were quantified by real-time RT-PCR. Relative copy number was obtained by normalizing the <i>CD137</i> transcripts to those of <i>GAPDH</i>. The relative copy number of the EBV-T/NK-LPDs patients' cells and healthy donor cells were compared. (B) CD137 protein expression in peripheral blood mononuclear cells (PBMCs) from 15 EBV-T/NK-LPDs patients or 5 healthy donors. PBMCs were cultured with IL-2 for 3 days and examined by flow cytometry. The mean fluorescent intensity of CD137 was normalized by that of isotype-matched control and expressed as MFIR (mean fluorescence intensity rate). (C) A bar graph for the relative MFIRs. Each point represents the MFIR of each sample.</p