Publications by authors named "Fumi Sato-Kaneko"

10 Publications

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Structure-activity relationship studies in substituted sulfamoyl benzamidothiazoles that prolong NF-κB activation.

Bioorg Med Chem 2021 Aug 30;43:116242. Epub 2021 May 30.

Moores Cancer Center, University of California San Diego, La Jolla, CA 92093-0809, USA.

In the face of emerging infectious diseases, there remains an unmet need for vaccine development where adjuvants that enhance immune responses to pathogenic antigens are highly desired. Using high-throughput screens with a cell-based nuclear factor κB (NF-κB) reporter assay, we identified a sulfamoyl benzamidothiazole bearing compound 1 that demonstrated a sustained activation of NF-κB after a primary stimulus with a Toll-like receptor (TLR)-4 agonist, lipopolysaccharide (LPS). Here, we explore systematic structure-activity relationship (SAR) studies on compound 1 that indicated the sites on the scaffold that tolerated modification and yielded more potent compounds compared to 1. The selected analogs enhanced release of immunostimulatory cytokines in the human monocytic cell line THP-1 cells and murine primary dendritic cells. In murine vaccination studies, select compounds were used as co-adjuvants in combination with the Food and Drug Administration approved TLR-4 agonistic adjuvant, monophosphoryl lipid A (MPLA) that showed significant enhancement in antigen-specific antibody titers compared to MPLA alone. Additionally, our SAR studies led to identification of a photoaffinity probe which will aid the target identification and mechanism of action studies in the future.
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http://dx.doi.org/10.1016/j.bmc.2021.116242DOI Listing
August 2021

Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection.

Proc Natl Acad Sci U S A 2021 Jun;118(23)

Moores Cancer Center, University of California San Diego, La Jolla, CA 92093-0809;

Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors-such as aging, pregnancy, and genetic polymorphisms-influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure-activity relationship studies, 2F52 [-benzyl--(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.
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http://dx.doi.org/10.1073/pnas.2025718118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201894PMC
June 2021

Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release.

Front Pharmacol 2021 16;12:668609. Epub 2021 Apr 16.

Moores Cancer Center, University of California, San Diego, La Jolla, CA, United States.

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z' factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.
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http://dx.doi.org/10.3389/fphar.2021.668609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085554PMC
April 2021

A Novel Synthetic Dual Agonistic Liposomal TLR4/7 Adjuvant Promotes Broad Immune Responses in an Influenza Vaccine With Minimal Reactogenicity.

Front Immunol 2020 19;11:1207. Epub 2020 Jun 19.

Moores Cancer Center, University of California, San Diego, La Jolla, CA, United States.

The limited efficacy of seasonal influenza vaccines is usually attributed to ongoing variation in the major antigenic targets for protective antibody responses including hemagglutinin (HA) and neuraminidase (NA). Hence, vaccine development has largely focused on broadening antigenic epitopes to generate cross-reactive protection. However, the vaccine adjuvant components which can accelerate, enhance and prolong antigenic immune responses, can also increase the breadth of these responses. We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus HA, inducing rapid, and sustained antibody responses that are protective against influenza viruses in homologous and heterologous murine challenge models. To further enhance adjuvant efficacy, we performed a structure-activity relationship study for the TLR4 ligand, -cyclohexyl-2-((5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-]indol-2-yl)thio)acetamide (CHNOS; ), and identified the 8-(furan-2-yl) substituted pyrimido[5,4-]indole analog (CHNOS; ) as a derivative with higher potency in activating both human and mouse TLR4-NF-κB reporter cells and primary cells. In a prime-boost immunization model using inactivated influenza A virus [IIAV; A/California/04/2009 (H1N1)pdm09], used as adjuvant induced higher serum anti-HA and anti-NA IgG1 levels compared to , and also increased the anti-NA IgG2a responses. In combination with a TLR7 ligand, , induced equivalent levels of anti-NA and anti-HA IgG1 to . However, the combination of induced 10-fold higher anti-HA and anti-NA IgG2a levels compared to . A stable liposomal formulation of was developed, which synergistically enhanced anti-HA and anti-NA IgG1 and IgG2a responses without demonstrable reactogenicity after intramuscular injection. Notably, vaccination with IIAV plus the liposomal formulation of protected mice against lethal homologous influenza virus (H1N1)pdm09 challenge and reduced lung viral titers and cytokine levels. The combination adjuvant induced a greater diversity in B cell clonotypes of immunoglobulin heavy chain (IGH) genes in the draining lymph nodes and antibodies against a broad spectrum of HA epitopes encompassing HA head and stalk domains and with cross-reactivity against different subtypes of HA and NA. This novel combination liposomal adjuvant contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.
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http://dx.doi.org/10.3389/fimmu.2020.01207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318308PMC
April 2021

Structure-Activity Relationship Studies To Identify Affinity Probes in Bis-aryl Sulfonamides That Prolong Immune Stimuli.

J Med Chem 2019 11 25;62(21):9521-9540. Epub 2019 Oct 25.

Moores Cancer Center , University of California, San Diego , La Jolla , California 92093-0695 , United States.

Agents that safely induce, enhance, or sustain multiple innate immune signaling pathways could be developed as potent vaccine adjuvants or coadjuvants. Using high-throughput screens with cell-based nuclear factor κB (NF-κB) and interferon stimulating response element (ISRE) reporter assays, we identified a bis-aryl sulfonamide bearing compound that demonstrated sustained NF-κB and ISRE activation after a primary stimulus with lipopolysaccharide or interferon-α, respectively. Here, we present systematic structure-activity relationship (SAR) studies on the two phenyl rings and amide nitrogen of the sulfonamide group of compound focused toward identification of affinity probes. The murine vaccination studies showed that compounds and when used as coadjuvants with monophosphoryl lipid A (MPLA) showed significant enhancement in antigen ovalbumin-specific immunoglobulin responses compared to MPLA alone. SAR studies pointed to the sites on the scaffold that can tolerate the introduction of aryl azide, biotin, and fluorescent rhodamine substituents to obtain several affinity and photoaffinity probes which will be utilized in concert for future target identification and mechanism of action studies.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00870DOI Listing
November 2019

Discovery of a Novel Microtubule Targeting Agent as an Adjuvant for Cancer Immunotherapy.

Biomed Res Int 2018 10;2018:8091283. Epub 2018 Oct 10.

Moores Cancer Center, University of California San Diego, La Jolla 92093, USA.

For an activating immunotherapy such as adjuvants, a compound that can prolong immune stimulation may enhance efficacy. We leveraged data from two prior high throughput screens with NF-B and interferon reporter cell lines to identify 4-chromene-3-carbonitriles as a class of compounds that prolonged activation in both screens. We repurchased 23 of the most promising candidates. Out of these compounds we found to be the most effective agent in stimulating the release of cytokines and chemokines from immune cells, including murine primary bone marrow derived dendritic cells. Mechanistically, inhibited tubulin polymerization, and its effect on immune cell activation was abolished in cells mutated in the beta-tubulin gene () encoding the site where colchicine binds. Treatment with resulted in mitochondrial depolarization followed by mitogen-activated protein kinase activation. Because tubulin polymerization modulating agents have been used for chemotherapy to treat malignancy and activated cytokine responses, we hypothesized that could be effective for cancer immunotherapy. Intratumoral injection of delayed tumor growth in a murine syngeneic model of head and neck cancer. When combined with PD-1 blockade, tumor growth slowed in the injected tumor nodule and there was an abscopal effect in an uninjected nodule on the contralateral flank, suggesting central antitumor immune activation. Thus, we identified a new class of tubulin depolymerizing agent that acts as both an innate and an adaptive immune activating agent and that limits solid tumor growth when used concurrently with a checkpoint inhibitor.
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http://dx.doi.org/10.1155/2018/8091283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199861PMC
February 2019

Induction of oligoclonal CD8 T cell responses against pulmonary metastatic cancer by a phospholipid-conjugated TLR7 agonist.

Proc Natl Acad Sci U S A 2018 07 2;115(29):E6836-E6844. Epub 2018 Jul 2.

Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093-0695;

Recent advances in cancer immunotherapy have improved patient survival. However, only a minority of patients with pulmonary metastatic disease respond to treatment with checkpoint inhibitors. As an alternate approach, we have tested the ability of systemically administered 1V270, a toll-like receptor 7 (TLR7) agonist conjugated to a phospholipid, to inhibit lung metastases in two variant murine 4T1 breast cancer models, as well as in B16 melanoma, and Lewis lung carcinoma models. In the 4T1 breast cancer models, 1V270 therapy inhibited lung metastases if given up to a week after primary tumor initiation. The treatment protocol was facilitated by the minimal toxic effects exerted by the phospholipid TLR7 agonist compared with the unconjugated agonist. 1V270 exhibited a wide therapeutic window and minimal off-target receptor binding. The 1V270 therapy inhibited colonization by tumor cells in the lungs in an NK cell dependent manner. Additional experiments revealed that single administration of 1V270 led to tumor-specific CD8 cell-dependent adaptive immune responses that suppressed late-stage metastatic tumor growth in the lungs. T cell receptor (TCR) repertoire analyses showed that 1V270 therapy induced oligoclonal T cells in the lungs and mediastinal lymph nodes. Different animals displayed commonly shared TCR clones following 1V270 therapy. Intranasal administration of 1V270 also suppressed lung metastasis and induced tumor-specific adaptive immune responses. These results indicate that systemic 1V270 therapy can induce tumor-specific cytotoxic T cell responses to pulmonary metastatic cancers and that TCR repertoire analyses can be used to monitor, and to predict, the response to therapy.
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http://dx.doi.org/10.1073/pnas.1803281115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055176PMC
July 2018

Identification of Compounds That Prolong Type I Interferon Signaling as Potential Vaccine Adjuvants.

SLAS Discov 2018 10 11;23(9):960-973. Epub 2018 May 11.

4 Department of Medicine, University of California, San Diego, La Jolla, CA, USA.

Vaccines are reliant on adjuvants to enhance the immune stimulus, and type I interferons (IFNs) have been shown to be beneficial in augmenting this response. We were interested in identifying compounds that would sustain activation of an endogenous type I IFN response as a co-adjuvant. We began with generation of a human monocytic THP-1 cell line with an IFN-stimulated response element (ISRE)-β-lactamase reporter construct for high-throughput screening. Pilot studies were performed to optimize the parameters and conditions for this cell-based Förster resonance energy transfer (FRET) reporter assay for sustaining an IFN-α-induced ISRE activation signal. These conditions were confirmed in an initial pilot screen, followed by the main screen for evaluating prolongation of an IFN-α-induced ISRE activation signal at 16 h. Hit compounds were identified using a structure enrichment strategy based on chemoinformatic clustering and a naïve "Top X" approach. A select list of confirmed hits was then evaluated for toxicity and the ability to sustain IFN activity by gene and protein expression. Finally, for proof of concept, a panel of compounds was used to immunize mice as co-adjuvant with a model antigen and an IFN-inducing Toll-like receptor 4 agonist, lipopolysaccharide, as an adjuvant. Selected compounds significantly augmented antigen-specific immunoglobulin responses.
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http://dx.doi.org/10.1177/2472555218774308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697428PMC
October 2018

Combination immunotherapy with TLR agonists and checkpoint inhibitors suppresses head and neck cancer.

JCI Insight 2017 09 21;2(18). Epub 2017 Sep 21.

Moores Cancer Center, UCSD, La Jolla, California, USA.

Checkpoint inhibitors have demonstrated efficacy in patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). However, the majority of patients do not benefit from these agents. To improve the efficacy of checkpoint inhibitors, intratumoral (i.t.) injection with innate immune activators, TLR7 and TLR9 agonists, were tested along with programmed death-1 receptor (PD-1) blockade. The combination therapy suppressed tumor growth at the primary injected and distant sites in human papillomavirus-negative (HPV-negative) SCC7 and MOC1, and HPV-positive MEER syngeneic mouse models. Abscopal effects and suppression of secondary challenged tumor suggest that local treatment with TLR agonists in combination with anti-PD-1 provided systemic adaptive immunity. I.t. treatment with a TLR7 agonist increased the ratio of M1 to M2 tumor-associated macrophages (TAMs) and promoted the infiltration of tumor-specific IFNγ-producing CD8+ T cells. Anti-PD-1 treatment increased T cell receptor (TCR) clonality of CD8+ T cells in tumors and spleens of treated mice. Collectively, these experiments demonstrate that combination therapy with i.t. delivery of TLR agonists and PD-1 blockade activates TAMs and induces tumor-specific adaptive immune responses, leading to suppression of primary tumor growth and prevention of metastasis in HNSCC models.
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http://dx.doi.org/10.1172/jci.insight.93397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621908PMC
September 2017

Identification of Biologically Active Pyrimido[5,4-b]indoles That Prolong NF-κB Activation without Intrinsic Activity.

ACS Comb Sci 2017 08 13;19(8):533-543. Epub 2017 Jul 13.

Moores UCSD Cancer Center, University of California San Diego , La Jolla, California 92093, United States.

Most vaccine adjuvants directly stimulate and activate antigen presenting cells but do not sustain immunostimulation of these cells. A high throughput screening (HTS) strategy was designed to identify compounds that would sustain NF-κB activation by a stimulus from the Toll-like receptor (TLR)4 ligand, lipopolysaccharide (LPS). Several pilot studies optimized the parameters and conditions for a cell based NF-κB reporter assay in human monocytic THP-1 cells. The final assay evaluated prolongation of LPS induced NF-κB activation at 12 h. The dynamic range of the assay was confirmed in a pilot screen of 14 631 compounds and subsequently in a main extensive screen with 166 304 compounds. Hit compounds were identified using an enrichment strategy based on unsupervised chemoinformatic clustering, and also by a naı̈ve "Top X" approach. A total of 2011 compounds were then rescreened for levels of coactivation with LPS at 5 h and 12 h, which provided kinetic profiles. Of the 407 confirmed hits, compounds that showed correlation of the kinetic profiles with the structural similarities led to identification of four chemotypes: pyrimido[5,4-b]indoles, 4H-chromene-3-carbonitriles, benzo[d][1,3]dioxol-2-ylureas, and tetrahydrothieno[2,3-c]pyridines, which were segregated by 5 h and 12 h kinetic characteristics. Unlike the TLR4 agonistic pyrimidoindoles identified in previous studies, the revealed pyrimidoindoles in the present work did not intrinsically stimulate TLR4 nor induce NF-κB but rather prolonged NF-κB signaling induced by LPS. A 42-member combinatorial library was synthesized which led to identification of potent N3-alkyl substituted pyrimidoindoles that were not only active in vitro but also enhanced antibody responses in vivo when used as a coadjuvant. The novel HTS strategy led to identification of compounds that are intrinsically quiescent but functionally prolong stimulation by a TLR4 ligand and thereby potentiate vaccine efficacy.
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http://dx.doi.org/10.1021/acscombsci.7b00080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841913PMC
August 2017
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