Design, synthesis, and biological evaluation of N-arylpiperazine derivatives as interferon inducers.

Type I Interferon (IFN) signaling plays an important role in the immune defense system against virus infection and in the innate immune response, thus IFNs are widely used as anti-viral agents and treatment for immune disorder or cancer. However, there is a growing demand for novel small-molecule IFN inducer due to tolerance, toxicity, or short duration of action following direct administration of IFNs. In this study, we assessed arylpiperazine (ARP) as a new core skeleton of IFN inducer. To investigate structure-activity relationship, we designed and synthesized a series of ARP analogues and evaluated the ability to stimulate IFN response in THP-1 human monocyte cells. Compound 5i was identified as a potent type I IFN inducer as it significantly increased cytokine secretion and increased expression of various IFN-stimulating genes which are representative biomarkers of type I IFN pathway. Our results suggested a beneficial therapeutic potential of 5i as an anti-viral agent.

Nanoparticle-cell-nanoparticle communication by stigmergy to enhance poly(I:C) induced apoptosis in cancer cells.

Nanoparticle-cell-nanoparticle communication by stigmergy was demonstrated using two capped nanodevices. The first community of nanoparticles (i.e.S(RA)IFN) is loaded with 9-cis-retinoic acid and capped with interferon-γ, whereas the second community of nanoparticles (i.e.S(sulf)PIC) is loaded with sulforhodamine B and capped with poly(I:C). The uptake of S(RA)IFN by SK-BR-3 breast cancer cells enhanced the expression of TLR3 receptor facilitating the subsequent uptake of S(sulf)PIC and cell killing.

A cell-based high throughput screening assay for the discovery of cGAS-STING pathway agonists.

Stimulator of interferon genes (STING) is an endoplasmic reticulum transmembrane protein that serves as a molecular hub for activation of interferon and inflammatory cytokine response by multiple cellular DNA sensors. Not surprisingly, STING has been demonstrated to play an important role in host defense against microorganisms and pharmacologic activation of STING is considered as an attractive strategy to treat viral diseases and boost antitumor immunity. In light of this we established a HepAD38-derived reporter cell line that expresses firefly luciferase in response to the activation of cyclic GMP-AMP synthase (cGAS)-STING pathway for high throughput screening (HTS) of small molecular human STING agonists. This cell-based reporter assay required only 4 h treatment with a reference STING agonist to induce a robust luciferase signal and was demonstrated to have an excellent performance in HTS format. By screening 16,000 compounds, a dispiro diketopiperzine (DSDP) compound was identified to induce cytokine response in a manner dependent on the expression of functional human STING, but not mouse STING. Moreover, we showed that DSDP induced an interferon-dominant cytokine response in human skin fibroblasts and peripheral blood mononuclear cells, which in turn potently suppressed the replication of yellow fever virus, dengue virus and Zika virus. We have thus established a robust cell-based assay system suitable for rapid discovery and mechanistic analyses of cGAS-STING pathway agonists. Identification of DSDP as a human STING agonist enriches the pipelines of STING-targeting drug development for treatment of viral infections and cancers.

Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF.

Selective delivery of drugs to tumor cells can increase potency and reduce toxicity. In this study, we describe a novel recombinant chimeric protein, dsRBEC, which can bind polyIC and deliver it selectively into EGFR over-expressing tumor cells. dsRBEC, comprises the dsRNA binding domain (dsRBD) of human PKR (hPKR), which serves as the polyIC binding moiety, fused to human EGF (hEGF), the targeting moiety. dsRBEC shows high affinity towards EGFR and triggers ligand-induced endocytosis of the receptor, thus leading to the selective internalization of polyIC into EGFR over-expressing tumor cells. The targeted delivery of polyIC by dsRBEC induced cellular apoptosis and the secretion of IFN-ß and other pro-inflammatory cytokines. dsRBEC-delivered polyIC is much more potent than naked polyIC and is expected to reduce the toxicity caused by systemic delivery of polyIC.

[Stimulating Type I interferon response with small molecules: revival of an old idea]. / Stimuler la réponse interféron de type I avec des petites molécules : le renouveau d'une vieille idée.

Type I interferons play a central role in the establishment of an innate immune response against viral infections and tumor cells. Shortly after their discovery in 1957, several groups have looked for small molecules capable of inducing the expression of these cytokines with therapeutic applications in mind. A set of active compounds in mice were identified, but because of their relative inefficiency in humans for reasons not understood at the time, these studies fell into oblivion. In recent years, the characterization of pathogen recognition receptors and the signaling pathways they activate, together with the discovery of plasmacytoid dendritic cells, have revolutionized our understanding of innate immunity. These discoveries and the popularization of high-throughput screening technologies have renewed the interest for small molecules that can induce type I interferons. Proofs about their therapeutic potency in humans are expected very soon.

Phenolic Compounds from the Roots of Rhodiola crenulata and Their Antioxidant and Inducing IFN-γ Production Activities.

In the present study, two new phenolic compounds 1 and 11, a pair of lignan isomers 12 and 13 with their absolute configurations established for the first time, were isolated from the ethanol extract of the roots of Rhodiola crenulata, together with 13 known phenolic compounds, and their structures were elucidated via NMR, HRESIMS, UV, IR and CD analyses. All the isolated compounds were evaluated for their in vitro antioxidant activities using the 2,2-diphenyl-1-picryhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays. Ten of them exhibited significant antioxidant activities compared to ascorbic acid. Furthermore, the inducibilities of the isolated compounds to IFN-γ production were also assessed. Compounds 1, 8, 9, 12, 13, 14 and 15 could moderately stimulate IFN-γ expression.

Structural Basis for Polymer Packing and Solvation Properties of the Organogermanium Crystalline Polymer Propagermanium and Its Derivatives.

Of organogermanium compounds known to have an immunostimulatory action, propagermanium [PGe; 3-oxygermylpropionic acid polymer, (C3 H5 GeO3.5 )n] is the only one used as a pharmaceutical agent, to treat the hepatitis B virus in Japan. However, because of lack of information about its structure, PGe has been confused with a polymeric solid, repagermanium (RGe, Ge-132, poly-trans-[(2-carboxyethyl) germasesquioxane], (C18 H30 Ge6 O21 )n), which has the same essential formula as PGe. To clarify this issue, the structure of PGe was analyzed using X-ray diffraction (XRD). PGe has a polymeric ladder-shaped structure of a concatenated eight-membered ring composed of Ge-O bonds, which is clearly distinguished from the infinite sheet structure in RGe. Moreover, we observed temperature or moisture-dependent transformations among these compounds using powder XRD. For instance, PGe was easily dissolved in water, and transformed to RGe by exposure to water vapor, but transformed into another straight-chain structure when exposed to aqueous solution. As a result of these findings, PGe was indicated to have labile polymer packing against RGe. These characteristics of PGe may affect pharmaceutical properties such as respective stability and solubility, which indicate its unique impact on physiological activity.

[Spectral characteristics and monosaccharide composition of interferon-inducing antiviral polysaccharide from Heliantnus tuberosus L].

This article presents the results of spectral analysis, monosaccharide composition and interferon-inducing properties of the polysaccharide from Heliantnus tuberosus L. Based on the spectral characteristics and the monosaccharide composition it was concluded that polysaccharide belongs to glucan class, presumably--ß-glucan. It has been shown that the polysaccharide complex of the Heliantnus tuberosus L. cell wall exhibits interferon-inducing properties both in experiments in vitro, and in vivo. It has been supposed that in polysaccharide stimulated models interferon is produced for all three species--α, ß, γ. Antiviral and therapeutic effects of polysaccharide were shown.

Designing improved poly lactic-co-glycolic acid microspheres for a malarial vaccine: incorporation of alginate and polyinosinic-polycytidilic acid.

Vaccination using proteins and peptides is currently gaining importance. One of the major drawbacks of this approach is the lack of an efficient immune response when the antigens are administered without adjuvants. In this study, we have taken the advantage of a combined adjuvant system in order to improve the immunogenicity of the SPf66 malarial antigen. For that purpose, we have combined poly (lactic-co-glycolic) acid microspheres, alginate, and polyinosinic polycytidilic acid. Our results show that microspheres can enhance the IgG production obtained with Freund's complete adjuvant. We have attributed this improvement to the presence of polyinosinic polycytidilic acid, since formulations comprising this adjuvant overcame the immune response from the others. In addition, our microspheres produced both IgG1 and IgG2a, leading to mixed Th1/Th2 activation, optimal for malaria vaccination. In conclusion, we have designed a preliminary formulation with a high potential for the treatment of malaria.

Discovery of a highly potent series of TLR7 agonists.

The discovery of a series of highly potent and novel TLR7 agonist interferon inducers is described. Structure-activity relationships are presented, along with pharmacokinetic studies of a lead molecule from this series of N9-pyridylmethyl-8-oxo-3-deazapurine analogues. A rationale for the very high potency observed is offered. An investigation of the clearance mechanism of this class of compounds in rat was carried out, resulting in aldehyde oxidase mediated oxidation being identified as a key component of the high clearance observed. A possible solution to this problem is discussed.

Quantitative determination of the composition of multi-shell calcium phosphate-oligonucleotide nanoparticles and their application for the activation of dendritic cells.

Biodegradable calcium phosphate nanoparticles as carriers for the immunoactive toll-like receptor ligands CpG and polyinosinic-polycytidylic acid for the activation of dendritic cells (DC) combined with the viral antigen hemagglutinin (HA) were prepared. A purification method based on ultracentrifugation and ultrasonication was developed to separate the nanoparticles from dissolved biomolecules. The number of biomolecules, i.e., oligonucleotides and peptide, incorporated into the nanoparticles was quantitatively determined by UV-spectroscopy, using fluorescent derivatives of the biomolecules. The immunostimulatory effects of purified calcium phosphate nanoparticles on DC were studied, i.e., cytokine production and activation of the cells in terms of the upregulation of surface molecules. Purified calcium phosphate nanoparticles, i.e., without dissolved biomolecules, are capable of inducing adaptive immunity by activation of DC. Immunostimulatory effects of purified calcium phosphate nanoparticles on DC were demonstrated by increased expression of co-stimulatory molecules and MHC II and by cytokine secretion. In addition, DC treated with purified functionalized calcium phosphate nanoparticles induced an antigen-specific T-cell response in vitro.

Induction of IFN-γ by a highly branched 1,3-ß-d-glucan from Aureobasidium pullulans in mouse-derived splenocytes via dectin-1-independent pathways.

We have previously elucidated the precise structure of a unique type of 1,3-ß-D-glucan, AP-FBG (Aureobasidium pullulans-fermented ß-D-glucan), from the fungus A. pullulans and found that AP-FBG strongly induced the production of various cytokines in DBA/2 mouse-derived splenocytes in vitro. However, the mechanism(s) of action of AP-FBG on in vitro mouse primary cells have not been characterized in detail. Herein, we report that the production of IFN-γ in DBA/2 mouse-derived splenocytes by AP-FBG was not inhibited following treatment with an anti-dectin-1 neutralizing antibody. In addition, AP-FBG not only failed to activate dectin-1-mediated signaling pathways, examined by a reporter gene assay but also failed to bind to dectin-1, a pivotal receptor for 1,3-ß-D-glucan. Taken together, AP-FBG induced cell activation via dectin-1-independent pathways.

[The interferon-induced and antiviral activities of dipeptides].

Five phosphodipeptides were synthesized; two of them (H-Lys-Ala(P) and H-Pro-Ala(P) had interferon-induced activity. These dipeptides at millimolar concentrations (10(-4)) and 10(-5) M) induced the synthesis of late (40-hour) interferon in human peripheral blood lymphocytes. The dipeptides H-Lys-Ala(P) and H-Pro-Ala(P) showed a protective antiviral activity in in vivo studies when singly intraperitoneally administered to mice 2 hours before inoculation with murine encephalomyocarditis virus.

Small-molecule interferon inducers. Toward the comprehension of the molecular determinants through ligand-based approaches.

Hepatitis C is becoming an increasingly common cause of mortality especially in the HIV-coinfected group. Due to the efficacy of interferon (IFN) based therapy in the treatment of hepatitis C, various compounds possessing IFN-inducing activity have been hitherto reported. In the present study, we describe how steric, electrostatic, hydrophobic, and hydrogen-bonding interactions might influence the biological activity of a published set of IFN inducers, using a three-dimensional quantitative structure-activity relationship (3-D QSAR) approach. Analyses were conducted evaluating different series of compounds structurally related to 8-hydroxyadenines and 1H-imidazo[4,5-c]quinolines. A ligand-based alignment protocol in combination with the GRID/GOLPE approach was applied: 62 3-D QSAR models were derived using different GRID probes and several training sets. Performed 3-D QSAR investigations proved to be of good statistical value displaying r2, q2CV-LOO, and cross-validated SDEP values of 0.73, 0.61, 0.61 and 0.89, 0.64, 0.58 using the OH or the DRY probe, respectively. Additionally, the predictive performance was evaluated using an external test set of 20 compounds. Analyses of the resulting models led to the definition of a pharmacophore model that can be of interest to explain the observed affinities of known compounds as well as to design novel low molecular weight IFN inducers (IFNIs). To the best of our knowledge, this is the first 3-D QSAR application on IFN-inducing agents.

[Interferon-stimulating activity of muramoyl dipeptide glycosides].

The uninvestigated interferon (IFN)-inducing capacity of glycoside derivatives of N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP) has been studied. Most MDP glycosides tested in vitro were more active than the reference preparation MDP. Under the in vivo conditions three studied preparations: MDP, a-heptyl-MDP and beta-(2-methyl-3-phenylchromonyl-7)-MDP stimulated production of considerable amount of circulating IFN that evidences for the promising character of their further investigation as preparations immunostimulators.

[New generation of drugs for prevention of viral infections].

Drugs that recently (last 2-3 years) have been widely used for prevention of the most prevalent and still imperfectly controlled viral infections. Special attention was attended to use of interferons and other nonspecific cytokines as main factors of innate immunity as well as to new inducers of endogenous interferon (kagocel, allokyn). Conclusion was made that under the conditions of availabilityof wide spectrum of antiviral drugs, their clinical effectiveness is determined by scientifically founded algorithm of their use.

Synthesis and structure-activity-relationships of 1H-imidazo[4,5-c]quinolines that induce interferon production.

1H-Imidazo-[4,5-c]quinolines were prepared while investigating novel nucleoside analogues as potential antiviral agents. While these compounds showed no direct antiviral activity when tested in a number of cell culture systems, some demonstrated potent inhibition of virus lesion development in an intravaginal guinea pig herpes simplex virus-2 assay. We have determined that the in vivo antiviral activity can be attributed to the ability of these molecules to induce the production of cytokines, especially interferon (IFN), in this model. Subsequently, we found that the compounds also induce in vitro production of IFN in human peripheral blood mononuclear cells (hPBMCs). The in vitro results reported herein and the in vivo results reported previously led to the discovery of imiquimod, 26, which was developed as a topical agent and has been approved for the treatment of genital warts, actinic keratosis, and superficial basal cell carcinoma.

Recombinant single-chain canine interleukin 12 induces interferon gamma mRNA expression in peripheral blood mononuclear cells of dogs with visceral leishmaniasis.

Canine visceral leishmaniasis poses important concerns for public health and veterinary medicine in many areas of the world. Resistance to it seems to be associated with cellular specific immune responses of the so-called Th1 type. Interleukin-12 (IL-12) is one of the most potent inducers of Th1 type of immune responses to co-administered antigens. Herein, the cloning of canine IL-12, as a single-chain fusion protein (sccaIL-12), and its expression in biologically active form in COS-7 cells is reported. Supernatants from these cells stimulated the expression of comparable amounts of interferon gamma mRNA in peripheral blood mononuclear cells from dogs with natural visceral leishmaniasis. In addition, after stimulation with sccaIL-12, there was no difference between interferon gamma mRNA expressions in peripheral blood mononuclear cells of dogs with visceral leishmaniasis and from normal healthy control animals.

Germane facts about germanium sesquioxide: I. Chemistry and anticancer properties.

This paper reviews the history, chemistry, safety, toxicity, and anticancer effects of the organogermanium compound bis (2-carboxyethylgermanium) sesquioxide (CEGS). A companion review follows, discussing the inaccuracies in the scientific record that have prematurely terminated research on clinical uses of CEGS. CEGS is a unique organogermanium compound first made by Mironov and coworkers in Russia and, shortly thereafter, popularized by Asai and his colleagues in Japan. Low concentrations of germanium occur in nearly all soils, plants and animal life; natural occurrence of the CEGS form is postulated but not yet demonstrated. The literature demonstrating its anticancer effect is particularly strong: CEGS induces interferon-gamma (IFN-gamma), enhances natural killer cell activity, and inhibits tumor and metastatic growth--effects often detectable after a single oral dose. In addition, oral consumption of CEGS is readily assimilated and rapidly cleared from the body without evidence of toxicity. Given these findings, the absence of human clinical trials of CEGS is unexpected. Possible explanations of why the convincing findings from animal research have not been used to support clinical trials are discussed. Clinical trials on CEGS are recommended.