Publications by authors named "Fitzgerald S Lao"

10 Publications

  • Page 1 of 1

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphar.2021.668609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085554PMC
April 2021

Identification of Compounds With Glucocorticoid Sparing Effects on Suppression of Chemokine and Cytokine Production by Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Front Pharmacol 2020 17;11:607713. Epub 2020 Dec 17.

Department of Medicine, University of California San Diego, La Jolla, CA, United States.

In recent years target based drug discovery has expanded our therapeutic armamentarium in the treatment of inflammatory and autoimmune diseases. Despite these advances and adverse effects, glucocorticoids remain reliable agents that are used in many of these diseases. The anti-inflammatory mechanisms of glucocorticoids include the suppression of transcription factor activity like nuclear factor kappa B (NF-κB). By reanalyzing data from two prior high throughput screens (HTS) that utilized a NF-κB reporter construct in THP-1 cells, we identified 1824 small molecule synthetic compounds that demonstrated NF-κB suppressive activities similar to the glucocorticoids included in the original >134,000 compound libraries. These 1824 compounds were then rescreened for attenuating NF-κB activity at 5 and 16 h after LPS stimuli in the NF-κB THP-1 reporter cells. After a "Top X" selection approach 122 hit compounds were further tested for toxicity and suppression of LPS induced CXCL8 release in THP-1 cells. Excluding cytotoxic compounds, the remaining active compounds were grouped into chemotype families using Tanimoto based clustering. Promising representatives from clustered chemotype groups were commercially purchased for further testing. Amongst these index compounds a lead chemotype: 1-pyrazolo [3,4 ] pyrimidin-4-amine, effectively suppressed CXCL8, and TNF production by THP-1 cells when stimulated with LPS, TNF or IL-1ß. Extending these studies to primary cells, these lead compounds also reduced IL-6 and CXCL8 production by TNF stimulated fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. Importantly a lead 1-pyrazolo [3,4 ] pyrimidin-4-amine compound demonstrated synergistic effects with dexamethasone when co-administered to TNF stimulated THP-1 cells and RA FLS in suppressing chemokine production. In summary, a cell based HTS approach identified lead compounds that reduced NF-κB activity and chemokine secretion induced by potent immunologic stimuli, and one lead compound that acted synergistically with dexamethasone as an anti-inflammatory agent showing a dose-sparing effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphar.2020.607713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773657PMC
December 2020

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2018/8091283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199861PMC
February 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/2472555218774308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697428PMC
October 2018

Innate immune protection against infectious diseases by pulmonary administration of a phospholipid-conjugated TLR7 ligand.

J Innate Immun 2014 1;6(3):315-24. Epub 2013 Nov 1.

Rebecca and John Moores UCSD Cancer Center, University of California San Diego, La Jolla, Calif., USA.

Pulmonary administration of Toll-like receptor (TLR) ligands protects hosts from inhaled pathogens. However, systemic side effects induced by TLR stimulation limit clinical development. Here, a small-molecule TLR7 ligand conjugated with phospholipid, 1V270 (also designated TMX201), was tested for innate immune activation and its ability to prevent pulmonary infection in mice. We hypothesized that phospholipid conjugation would increase internalization by immune cells and localize the compound in the lungs, thus avoiding side effects due to systemic cytokine release. Pulmonary 1V270 administration increased innate cytokines and chemokines in bronchial alveolar lavage fluids, but neither caused systemic induction of cytokines nor B cell proliferation in distant lymphoid organs. 1V270 activated pulmonary CD11c+ dendritic cells, which migrated to local lymph nodes. However, there was minimal cell infiltration into the pulmonary parenchyma. Prophylactic administration of 1V270 significantly protected mice from lethal infection with Bacillus anthracis, Venezuelan equine encephalitis virus and H1N1 influenza virus. The maximum tolerated dose of 1V270 by pulmonary administration was 75 times the effective therapeutic dose. Therefore, pulmonary 1V270 treatment can protect the host from different infectious agents by stimulating local innate immune responses while exhibiting an excellent safety profile.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000355217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972367PMC
May 2015

Synthesis and characterization of PEGylated toll like receptor 7 ligands.

Bioconjug Chem 2011 Mar 21;22(3):445-54. Epub 2011 Feb 21.

Moores Cancer Center, University of California San Diego, La Jolla California 92093-0820, USA.

Toll-like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid, or poly(ethylene glycol) (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in existing biologics and some low molecular weight compounds. PEGylation could be a feasible method to alter the pharmacokinetics and pharmacodynamics of TLR7 ligands. In this study, we systematically studied the influence of PEG chain length on the in vitro and in vivo properties of potent TLR7 ligands. PEGylation increased solubility of the TLR7 ligands and modulated protein binding. Adding a 6-10 length PEG to the TLR7 ligand reduced its potency toward induction of interleukin (IL)-6 by murine macrophages in vitro and IL-6 and tumor necrosis factor (TNF) in vivo. However, PEGylation with 18 or longer chain restored, and even enhanced, the agonistic activity of the drug. In human peripheral blood mononuclear cells, similar effects of PEGylation were observed for secretion of proinflammatory cytokines, IL-6, IL-12, TNF-α, IL-1β, and type 1 interferon, as well as for B cell proliferation. In summary, these studies demonstrate that conjugation of PEG chains to a synthetic TLR ligand can impact its potency for cytokine induction depending on the size of the PEG moiety. Thus, PEGylation may be a feasible approach to regulate the pharmacological properties of TLR7 ligands.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bc1004813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063487PMC
March 2011
-->