Identification of 3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase.

Plasmodium falciparum's resistance to available antimalarial drugs highlights the need for the development of novel drugs. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. PfDHODH is targeted by the inhibitor DSM265, which binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. In this study, we screened 40,400 compounds from the Kyoto University chemical library against recombinant PfDHODH. These studies led to the identification of 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine and its derivatives as a new class of PfDHODH inhibitor. Moreover, the hit compounds identified in this study are selective for PfDHODH without inhibition of the human enzymes. Finally, this new scaffold of PfDHODH inhibitors showed growth inhibition activity against P. falciparum 3D7 with low toxicity to three human cell lines, providing a new starting point for antimalarial drug development.

Three distinct mechanisms underlying human γδ T cell-mediated cytotoxicity against malignant pleural mesothelioma.

Introduction: Malignant pleural mesothelioma (MPM) is a rare and highly aggressive thoracic tumor with poor prognosis and limited therapeutic options. Although immune checkpoint inhibitors exhibit a promising effect in some patients with unresectable MPM in clinical trials, the majority of MPM patients show only modest response rates to the currently available treatments. It is thus imperative to develop novel and innovative therapeutic modalities for MPM, including immune effector cell-based therapies. Methods: γδ T cells were expanded using tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate (PTA) and interleukin-2, and the therapeutic potential of γδ T cells was examined through analyzing cell surface markers and cellular cytotoxicity against MPM in vitro using a europium chelate-based time-resolved fluorescence assay system and a luciferase-based luminescence assay system. Results and discussion: We successfully expanded γδ T cells from peripheral blood mononuclear cells of healthy donors and MPM patients. γδ T cells expressed natural killer receptors such as NKG2D and DNAM-1 and exhibited a moderate level of cytotoxicity to MPM cells in the absence of antigens. The inclusion of PTA, (E)-4-hydroxy-3- methylbut-2-enyl diphosphate (HMBPP) or zoledronic acid (ZOL) induced a TCR-dependent cytotoxicity in γδ T cells and secreted interferon-γ (IFN-γ). In addition, γδ T cells expressing CD16 exhibited a significant level of cytotoxicity against MPM cells in the presence of an anti-epidermal growth factor receptor (EGFR) mAb, at lower concentrations than in clinical settings, whereas a detectable level of IFN-γ was not produced. Taken together, γδ T cells showed cytotoxic activity against MPM in three distinct mechanisms through NK receptors, TCRs and CD16. Since major histocompatibility complex (MHC) molecules are not involved in the recognition, both autologous and allogeneic γδ T cells could be used for the development of γδ T cell-based adoptive immunotherapy for MPM.

Two Distinct Mechanisms Underlying γδ T Cell-Mediated Regulation of Collagen Type I in Lung Fibroblasts.

Idiopathic pulmonary fibrosis is a chronic intractable lung disease, leading to respiratory failure and death. Although anti-fibrotic agents delay disease progression, they are not considered curative treatments, and alternative modalities have attracted attention. We examined the effect of human γδ T cells on collagen type I in lung fibroblasts. Collagen type I was markedly reduced in a γδ T cell number-dependent manner following treatment with γδ T cells expanded with tetrakis-pivaloxymethyl 2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate (PTA) and interleukin-2. Collagen type I levels remained unchanged on addition of γδ T cells to the culture system through a trans-well culture membrane, suggesting that cell-cell contact is essential for reducing its levels in lung fibroblasts. Re-stimulating γδ T cells with (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) reduced collagen type I levels without cell-cell contact, indicating the existence of HMBPP-induced soluble anti-fibrotic factors in γδ T cells. Adding anti-interferon-γ (IFN-γ)-neutralizing mAb restored collagen type I levels, demonstrating that human γδ T cell-derived IFN-γ reduces collagen type I levels. Conversely, interleukin-18 augmented γδ T cell-induced suppression of collagen type I. Therefore, human γδ T cells reduce collagen levels in lung fibroblasts via two distinct mechanisms; adoptive γδ T cell transfer is potentially a new therapeutic candidate.

Screening of Inhibitors Targeting Heat Shock Protein 47 Involved in the Development of Idiopathic Pulmonary Fibrosis.

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is causally related to fibrotic diseases, including idiopathic pulmonary fibrosis. The identification of Compounds that interfere with the HSP47-collagen interaction is essential for the development of relevant therapeutics. Herein, we prepared human HSP47 as a soluble fusion protein expressed in E. coli and established an assay system for HSP47 inhibitor screening. We screened a natural and synthetic Compound library established at Nagasaki University. Among 1023 Compounds, 13 exhibited inhibitory activity against human HSP47, of which three inhibited its function in a dose-dependent manner. Epigallocatechin-3-O-gallate, one of these three Compounds, is a typical polyphenol Compound derived from tea leaves. Structurally related Compounds were synthesized and examined for their activity, revealing a hydroxyl group at A-ring position 5 as important for its activity. The present findings provide valuable insight for the development of natural product-derived therapeutics for fibrotic diseases, including idiopathic pulmonary fibrosis.

Comparison of a Novel Bisphosphonate Prodrug and Zoledronic Acid in the Induction of Cytotoxicity in Human Vγ2Vδ2 T Cells.

Increasing attention has been paid to human γδ T cells expressing Vγ2Vδ2 T cell receptor (also termed Vγ9Vδ2) in the field of cancer immunotherapy. We have previously demonstrated that a novel bisphosphonate prodrug, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA), efficiently expands peripheral blood Vγ2Vδ2 T cells to purities up to 95-99% in 10-11 days. In the present study, we first examined the effect of PTA on farnesyl diphosphate synthase (FDPS) using liquid chromatography mass spectrometry (LC-MS) to analyze the mechanism underlying the PTA-mediated expansion of Vγ2Vδ2 T cells. We find that the prodrug induced the accumulation of both isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), direct upstream metabolites of FDPS. This indicates that not only IPP but also DMAPP plays an important role in PTA-mediated stimulation of Vγ2Vδ2 T cells. We next analyzed TCR-independent cytotoxicity of Vγ2Vδ2 T cells. When human lung cancer cell lines were challenged by Vγ2Vδ2 T cells, no detectable cytotoxicity was observed in 40 min. The lung cancer cell lines were, however, significantly killed by Vγ2Vδ2 T cells after 4-16 h in an effector-to-target ratio-dependent manner, demonstrating that Vγ2Vδ2 T cell-based cell therapy required a large number of cells and longer time when tumor cells were not sensitized. By contrast, pulsing tumor cell lines with 10-30 nM of PTA induced significant lysis of tumor cells by Vγ2Vδ2 T cells even in 40 min. Similar levels of cytotoxicity were elicited by ZOL at concentrations of 100-300 µM, which were much higher than blood levels of ZOL after infusion (1-2 µM), suggesting that standard 4 mg infusion of ZOL was not enough to sensitize lung cancer cells in clinical settings. In addition, Vγ2Vδ2 T cells secreted interferon-γ (IFN-γ) when challenged by lung cancer cell lines pulsed with PTA in a dose-dependent manner. Taken together, PTA could be utilized for both expansion of Vγ2Vδ2 T cells ex vivo and sensitization of tumor cells in vivo in Vγ2Vδ2 T cell-based cancer immunotherapy. For use in patients, further studies on drug delivery are essential because of the hydrophobic nature of the prodrug.

Synthesis and Immunomodulatory Activity of Fluorine-Containing Bisphosphonates.

Immune checkpoint blockade using anti-PD-1/PD-L1 or anti-CTLA-4 monoclonal antibodies (mAbs) has revolutionized cancer treatment. However, many types of cancer do not respond and for those that do, only a minority of patients achieve durable remissions. Therefore, oncoimmunologists are working to develop adoptive cell therapies for non-hematopoietic tumors by harnessing immune effector cells such as αß T cells and γδ T cells. In contrast to conventional αß T cells that recognize peptides in the context of MHC class I or II molecules, γδ T cells expressing Vγ2Vδ2 T cell receptors (also termed Vγ9Vδ2) are stimulated by isoprenoid metabolites (phosphoantigens) such as isopentenyl diphosphate in a butyrophilin-3A1-dependent manner. Vγ2Vδ2 T cells kill almost all types of tumor cells that have been treated with bisphosphonates. In this study, we synthesized a series of fluorine-containing bisphosphonates based on current drugs and found that they stimulated Vγ2Vδ2 T cell killing of tumor cells. A fluorine-containing prodrug analogue of zoledronate where phosphonate moieties were masked with pivaloyloxymethyl groups markedly enhanced Vγ2Vδ2 T-cell-mediated cytotoxicity, and also promoted the expansion of peripheral blood Vγ2Vδ2 T cells. These results demonstrate that a prodrug of a fluorine-containing zoledronate analogue can sensitize tumor cells for killing as well as expand Vγ2Vδ2 T cells for adoptive cell therapy.

Determination of human γδ T cell-mediated cytotoxicity using a non-radioactive assay system.

The adoptive transfer of immune effector cells, such as CD8+ killer αß T cells, γδ T cells, NK (natural killer) cells, and genetically-modified T cells, has been receiving increasing attention. It is essential to determine cellular cytotoxicity so as to monitor the function and quality of ex vivo-expanded immune effector cells before infusion. The most common method is the [51Cr]-sodium chromate release assay. It is, however, preferable to avoid the use of radioactive materials in clinical laboratories. In order to establish a non-radioactive alternative to the standard radioactive assay, we previously synthesized a chelate-forming prodrug (BM-HT) and demonstrated that a combination of BM-HT and europium (Eu3+) was useful to determine NK cell-mediated cytotoxicity. In the present study, we examined whether or not this improved assay system could be used to determine the cellular cytotoxicity exhibited by Vγ2Vδ2+ γδ T cells. In addition, we compared Eu3+ and terbium (Tb3+) in the measurement of cellular cytotoxicity. Our assay system using BM-HT could be used successfully for the analysis of both γδ T cell receptor (TCR)- and CD16-mediated cytotoxicity. When the intensity of fluorescence was compared between Eu3+ and Tb3+, Tb3+ chelate was more sensitive than Eu3+ chelate, suggesting that the detection system using Tb3+ is superior to Eu3+ when tumor cells are not efficiently labeled with BM-HT. The method established herein is expected to promote the development of novel adoptive cell therapies for cancer.

Live Cell Labeling with Terpyridine Derivative Proligands to Measure Cytotoxicity Mediated by Immune Cells.

Immunotherapy using immune checkpoint inhibitors and CAR-T cells has revolutionized treatment for patients with malignant tumors. However, measuring tumor cell cytotoxicity mediated by immune effector cells in clinical laboratories has been difficult due to the requirement for radioactive substances. In this study, a series of novel terpyridine derivative proligands were synthesized, and a non-radioactive cellular cytotoxicity assay using the newly synthesized compounds was developed for use in preclinical and clinical studies for cancer immunotherapy. Once internalized into target cells, the compounds are hydrolyzed by esterases, resulting in the intracellular accumulation of the negatively charged terpyridine derivatives. When the labeled target cells are recognized and killed by immune effector cells, the integrity of the cell membrane is disrupted, and the terpyridine derivatives are released. Upon combining the culture supernatant with europium (Eu3+ ), the cytotoxicity of immune effector cells for the target cells can be quantitatively determined by measuring the intensity of the Eu3+ /ligand-derived time-resolved fluorescence. Thus, the assay developed in this study would facilitate the development of novel cancer immunotherapies.