Tilorone increases glucose uptake in vivo and in skeletal muscle cells by enhancing Akt2/AS160 signaling and glucose transporter levels.

Skeletal muscle plays a major role in whole-body glucose metabolism. Insulin resistance in skeletal muscle is characterized by decreased insulin-stimulated glucose uptake resulting from impaired intracellular trafficking and decreased glucose transporter 4 (GLUT4) expression. In this study, we illustrated that tilorone, a low-molecular-weight antiviral agent, improves glucose uptake in vitro and in vivo. Tilorone increased bone morphogenetic protein (BMP) signaling in C2C12 myoblasts, the transcription of multiple BMPs (BMP2, BMP4, BMP7, and BMP14), Smad4 expression, and the phosphorylation of BMP-mediated Smad1/5/8. The activation of Akt2/AS160 (TBC1D4) signaling, the critical regulator of GLUT4 translocation, was also increased, as well as the levels of GLUT4 and GLUT1, leading to enhanced uptake of the radioactively labeled glucose analog 18 F-fluoro-2-deoxyglucose (18 FDG). However, this excess glucose content did not result in increased ATP formation by mitochondrial respiration; both basal and ATP-linked respiration were diminished, thereby contributing to the induction of AMPK. In differentiated myotubes, AS160 phosphorylation and 18 FDG uptake also increased. Moreover, tilorone administration further increased insulin-stimulated phosphorylation of Akt2 and glucose uptake of myotubes indicating an insulin-sensitizing effect. Importantly, during in vivo experiments, the systemic administration of tilorone resulted in increased 18 FDG uptake of skeletal muscle, liver, and adipose tissue in C57BL/6 mice. Our results provide new perspectives for the treatment of type 2 diabetes, which has a limited number of treatments that regulate protein expression or translocation.

Effect of Tilorone on the Dynamics of Viral Load and the Levels of Interferons and Interleukin-1ß in the Lung Tissue and Blood Serum of Mice with Experimental Influenza.

We studied the effect of tilorone on the dynamics of IFNα, IFNγ, and IL-1ß levels in the lung tissue and blood serum in relation to viral load in the lungs of BALB/c mice with pneumonia caused by influenza virus A/Aichi/2/68 (H3N2). Tilorone was administered per os in doses of 40, 150, and 540 µg per mouse 6, 30, and 78 h postinfection, which simulated the drug regimen used in the clinic for the treatment of influenza and acute respiratory viral infections in Russia and post-Soviet countries. Tilorone reduced viral load with the maximum amplitude (2-3 lg) after 1-2 administrations. The results of studying the dynamics of the cytokine levels in the infected animals in general support the previous hypothesis that, in repeated dosing, tilorone enhances the IFN response (compensates for its deficiency) at the early stages of acute respiratory viral infections and suppresses (damps) excessive production of IFN and proinflammatory cytokines at the later stages.

Cationic Compounds with SARS-CoV-2 Antiviral Activity and Their Interaction with Organic Cation Transporter/Multidrug and Toxin Extruder Secretory Transporters.

In the wake of the COVID-19 pandemic, drug repurposing has been highlighted for rapid introduction of therapeutics. Proposed drugs with activity against SARS-CoV-2 include compounds with positive charges at physiologic pH, making them potential targets for the organic cation secretory transporters of kidney and liver, i.e., the basolateral organic cation transporters, OCT1 and OCT2; and the apical multidrug and toxin extruders, MATE1 and MATE2-K. We selected several compounds proposed to have in vitro activity against SARS-CoV-2 (chloroquine, hydroxychloroquine, quinacrine, tilorone, pyronaridine, cetylpyridinium, and miramistin) to test their interaction with OCT and MATE transporters. We used Bayesian machine learning models to generate predictions for each molecule with each transporter and also experimentally determined IC50 values for each compound against labeled substrate transport into CHO cells that stably expressed OCT2, MATE1, or MATE2-K using three structurally distinct substrates (atenolol, metformin and 1-methyl-4-phenylpyridinium) to assess the impact of substrate structure on inhibitory efficacy. For the OCTs substrate identity influenced IC50 values, although the effect was larger and more systematic for OCT2. In contrast, inhibition of MATE1-mediated transport was largely insensitive to substrate identity. Unlike MATE1, inhibition of MATE2-K was influenced, albeit modestly, by substrate identity. Maximum unbound plasma concentration/IC50 ratios were used to identify potential clinical DDI recommendations; all the compounds interacted with the OCT/MATE secretory pathway, most with sufficient avidity to represent potential DDI issues for secretion of cationic drugs. This should be considered when proposing cationic agents as repurposed antivirals. SIGNIFICANCE STATEMENT: Drugs proposed as potential COVID-19 therapeutics based on in vitro activity data against SARS-CoV-2 include compounds with positive charges at physiological pH, making them potential interactors with the OCT/MATE renal secretory pathway. We tested seven such molecules as inhibitors of OCT1/2 and MATE1/2-K. All the compounds blocked transport activity regardless of substrate used to monitor activity. Suggesting that plasma concentrations achieved by normal clinical application of the test agents could be expected to influence the pharmacokinetics of selected cationic drugs.

The Antiviral Drug Tilorone Is a Potent and Selective Inhibitor of Acetylcholinesterase.

Acetylcholinesterase (AChE) is an important drug target in neurological disorders like Alzheimer's disease, Lewy body dementia, and Parkinson's disease dementia as well as for other conditions like myasthenia gravis and anticholinergic poisoning. In this study, we have used a combination of high-throughput screening, machine learning, and docking to identify new inhibitors of this enzyme. Bayesian machine learning models were generated with literature data from ChEMBL for eel and human AChE inhibitors as well as butyrylcholinesterase inhibitors (BuChE) and compared with other machine learning methods. High-throughput screens for the eel AChE inhibitor model identified several molecules including tilorone, an antiviral drug that is well-established outside of the United States, as a newly identified nanomolar AChE inhibitor. We have described how tilorone inhibits both eel and human AChE with IC50's of 14.4 nM and 64.4 nM, respectively, but does not inhibit the closely related BuChE IC50 > 50 µM. We have docked tilorone into the human AChE crystal structure and shown that this selectivity is likely due to the reliance on a specific interaction with a hydrophobic residue in the peripheral anionic site of AChE that is absent in BuChE. We also conducted a pharmacological safety profile (SafetyScreen44) and kinase selectivity screen (SelectScreen) that showed tilorone (1 µM) only inhibited AChE out of 44 toxicology target proteins evaluated and did not appreciably inhibit any of the 485 kinases tested. This study suggests there may be a potential role for repurposing tilorone or its derivatives in conditions that benefit from AChE inhibition.

Tilorone: a Broad-Spectrum Antiviral Invented in the USA and Commercialized in Russia and beyond.

For the last 50 years we have known of a broad-spectrum agent tilorone dihydrochloride (Tilorone). This is a small-molecule orally bioavailable drug that was originally discovered in the USA and is currently used clinically as an antiviral in Russia and the Ukraine. Over the years there have been numerous clinical and non-clinical reports of its broad spectrum of antiviral activity. More recently we have identified additional promising antiviral activities against Middle East Respiratory Syndrome, Chikungunya, Ebola and Marburg which highlights that this old drug may have other uses against new viruses. This may in turn inform the types of drugs that we need for virus outbreaks such as for the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Tilorone has been long neglected by the west in many respects but it deserves further reassessment in light of current and future needs for broad-spectrum antivirals.

Tilorone, a Broad-Spectrum Antiviral for Emerging Viruses.

Tilorone is a 50-year-old synthetic small-molecule compound with antiviral activity that is proposed to induce interferon after oral administration. This drug is used as a broad-spectrum antiviral in several countries of the Russian Federation. We have recently described activity in vitro and in vivo against the Ebola virus. After a broad screening of additional viruses, we now describe in vitro activity against Chikungunya virus (CHIK) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV).

Repurposing Quinacrine against Ebola Virus Infection In Vivo.

Quinacrine hydrochloride is a small-molecule, orally bioavailable drug that has been used clinically as an antimalarial and for many other applications. A machine learning model trained on Ebola virus (EBOV) screening data identified quinacrine as a potent (nanomolar) in vitro inhibitor. In the current study, quinacrine 25 mg/kg was shown to protect 70% of mice (statistically significant) from a lethal challenge with mouse-adapted EBOV with once-daily intraperitoneal dosing for 8 days.

The effects of coadministration of tilorone dihydrochloride and culture supernatants from Lactobacillus reuteri on the mouse hepatoma cell line.

CONTEXT: Tilorone dihydrochloride is a therapeutic agent with a different mechanism in cancer. The species of Lactobacillus have an important role in cytotoxic effect. AIMS: Because of unknown effects of tilorone and culture supernatants from Lactobacillus reuteri on hepatoma, the aim of this study is to evaluate apoptotic, cytotoxic, and therapeutic effects of tilorone on mouse hepatoma cell line with and without culture supernatants from L. reuteri. MATERIALS AND METHODS: To do so, after cell line culture, cells were divided into different groups such as negative control, treatment with four doses of tilorone, positive control of supernatant (single dose), and combination therapy groups of different doses of tilorone with supernatant (constant doses), for 48 h. All groups were studied with pathologic tests, biochemical study, tetrazolium dye (3-(4, 5- dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide [MTT]) assay, and absolute real-time-polymerase chain reaction (RT-PCR) were done to assess Bax and Bcl-2 genes expression, as molecular studies. RESULTS: MTT assay results revealed that the tilorone tissue culture IC50 (TCIC50) on the Hepa1-6 cell line was 50 µg/ml. RT-PCR analysis showed that tilorone dihydrochloride induced upregulation and downregulation in expression of Bax and Bcl-2, respectively. Simultaneous, antioxidant effect has also seen in a way that prevented necrosis, in biochemical analysis. These results were dose dependent and statistically significant compared to the control group. CONCLUSIONS: Based on these results, it appeared that this agent could be a good candidate for further evaluation as effective chemotherapy acting through the induction of apoptosis in hepatoma. The cell death caused through bacterial supernatant was rather necrosis than apoptosis.

Tilorone activates NK cells and cytotoxic lymphocytes that kill HLA-negative tumor cells.

Tilorone hydrochloride, a low-molecular-weight synthetic compound, induces interferon production and has been reported to have both antiviral and antitumor activities. Here, we have demonstrated the ability of tilorone to activate NK cells and specific subpopulations of cytotoxic CD4+ and CD8+ T lymphocytes that recognize immune-evasive tumor cells and kill them via the FasL-Fas interaction. We have also performed a comparative analysis of characteristics between lymphocytes activated in the fraction of human peripheral blood mononuclear cells (PBMCs) upon treatment with different stimulants of the immune response: tilorone, innate immunity protein Tag7, and cytokine IL-2, a regulator of adaptive immunity. The results show that all the three stimulants, regardless of their nature, activate lymphocytes that are identical with respect to the spectrum of target cells, phenotype, and mechanism of cytotoxic action However, these stimulants induce different mechanisms of lymphocyte activation at early stages of the immune response. © 2018 IUBMB Life, 71(3):376-384, 2019.

Efficacy of Tilorone Dihydrochloride against Ebola Virus Infection.

Tilorone dihydrochloride (tilorone) is a small-molecule, orally bioavailable drug that is used clinically as an antiviral outside the United States. A machine-learning model trained on anti-Ebola virus (EBOV) screening data previously identified tilorone as a potent in vitro EBOV inhibitor, making it a candidate for the treatment of Ebola virus disease (EVD). In the present study, a series of in vitro ADMET (absorption, distribution, metabolism, excretion, toxicity) assays demonstrated the drug has excellent solubility, high Caco-2 permeability, was not a P-glycoprotein substrate, and had no inhibitory activity against five human CYP450 enzymes (3A4, 2D6, 2C19, 2C9, and 1A2). Tilorone was shown to have 52% human plasma protein binding with excellent plasma stability and a mouse liver microsome half-life of 48 min. Dose range-finding studies in mice demonstrated a maximum tolerated single dose of 100 mg/kg of body weight. A pharmacokinetics study in mice at 2- and 10-mg/kg dose levels showed that the drug is rapidly absorbed, has dose-dependent increases in maximum concentration of unbound drug in plasma and areas under the concentration-time curve, and has a half-life of approximately 18 h in both males and females, although the exposure was ∼2.5-fold higher in male mice. Tilorone doses of 25 and 50 mg/kg proved efficacious in protecting 90% of mice from a lethal challenge with mouse-adapted with once-daily intraperitoneal (i.p.) dosing for 8 days. A subsequent study showed that 30 mg/kg/day of tilorone given i.p. starting 2 or 24 h postchallenge and continuing through day 7 postinfection was fully protective, indicating promising activity for the treatment of EVD.

The effect of amixin and agmatine on cytochrome C release from isolated mitochondria

Mitochondrial nicotinic acetylcholine receptors (nAChRs) control permeability transition pore formation and cytochrome c release in the presence of apoptogenic factors. This study demonstrates that pharmacological agents amixin and agmatine affect mitochondrial nAChR functioning: they slightly suppress cytochrome c release from mouse brain and liver mitochondria stimulated with apoptogenic dose of Са2+ and prevent the effect of α7 nAChR agonist PNU282987. We conclude that mitochondria may be one of therapeutic targets of amixin and agmatine.

An Effective Synthesis Method for Tilorone Dihydrochloride with Obvious IFN-α Inducing Activity.

Tilorone dihydrochloride (1) has great potential for inducing interferon against pathogenic infection. In this paper, we describe a convenient preparation method for 2,7-dihydroxyfluoren-9-one (2), which is a usual pharmaceutical intermediate for preparing tilorone dihydrochloride (1). In the novel method, methyl esterification of 4,4'-dihydroxy-[1,1'-biphenyl]-2-carboxylic acid (4) was carried out under milder conditions with higher yield and played an important role in the preparation of compound 2. The structures of the relative intermediates and target compound were characterized by melting point, IR, MS, and ¹H-NMR. Furthermore, the synthesized tilorone dihydrochloride exhibited an obvious effect on induction of interferon-α (IFN-α) in mice within 12 h, and the peak level was observed until 24 h. This fruitful work has resulted in tilorone dihydrochloride becoming available in large-scale and wide application in clinics, which has a good pharmaceutical development prospects.

Small-molecule screening of PC3 prostate cancer cells identifies tilorone dihydrochloride to selectively inhibit cell growth based on cyclin-dependent kinase 5 expression.

Cyclin-dependent kinase 5 (CDK5) is a potential target for prostate cancer treatment, the enzyme being essential for prostate tumor growth and formation of metastases. In the present study, we identified agents that target prostate cancer cells based on CDK5 expression. CDK5 activity was suppressed by transfection of PC3 prostate cancer cells with a dominant-negative construct (PC3 CDK5dn). PC3 CDK5dn and PC3 control cells were screened for compounds that selectively target cells based on CDK5 expression, utilizing the Johns Hopkins Drug Library. MTS proliferation, clonogenic and 3D growth assays were performed to validate the selected hits. Screening of 3,360 compounds identified rutilantin, ethacridine lactate and cetalkonium chloride as compounds that selectively target PC3 control cells and a tilorone analog as a selective inhibitor of PC3 CDK5dn cells. A PubMed literature study indicated that tilorone may have clinical use in patients. Validation experiments confirmed that tilorone treatment resulted in decreased PC3 cell growth and invasion; PC3 cells with inactive CDK5 were inhibited more effectively. Future studies are needed to unravel the mechanism of action of tilorone in CDK5 deficient prostate cancer cells and to test combination therapies with tilorone and a CDK5 inhibitor for its potential use in clinical practice.

Design, synthesis and antiproliferative evaluation of fluorenone analogs with DNA topoisomerase I inhibitory properties.

A series of 2,7-diamidofluorenones were designed, synthesized, and screened by SRB assay. Some synthesized compounds exhibited antitumor activities in submicromolar range. Ten compounds (3a, 3b, 3c, 3g, 3j, 3l, 4a, 4h, 4i, and 4j) were also selected by NCI screening system and 3c (GI50=1.66 µM) appeared to be the most active agent of this series. Furthermore, 3c attenuated topoisomerase I-mediated DNA relaxation at low micromolar concentrations. These results indicated that fluorenones have potential to be further developed into anticancer drugs.

Discovery of inhibitors of Bacillus anthracis primase DnaG.

Primase DnaG is an essential bacterial enzyme that synthesizes short ribonucleotide primers required for chromosomal DNA replication. Inhibitors of DnaG can serve as leads for development of new antibacterials and biochemical probes. We recently developed a nonradioactive in vitro primase-pyrophosphatase assay to identify and analyze DnaG inhibitors. Application of this assay to DnaG from Bacillus anthracis (Ba DnaG), a dangerous pathogen, yielded several inhibitors, which include agents with DNA intercalating properties (doxorubicin and tilorone) as well as those that do not intercalate into DNA (suramin). A polyanionic agent and inhibitor of eukaryotic primases, suramin, identified by this assay as a low-micromolar Ba DnaG inhibitor, was recently shown to be also a low-micromolar inhibitor of Mycobacterium tuberculosis DnaG (Mtb DnaG). In contrast, another low-micromolar Ba DnaG inhibitor, tilorone, is much more potent against Ba DnaG than against Mtb DnaG, despite homology between these enzymes, suggesting that DnaG can be targeted selectively.

[Effects of diphenyl derivatives on electrophoretic mobility of murine T lymphocytes].

The early changes of electrophoretic mobility (EPM) of murine T lymphocytes induced by structural analogues of amixine-dihydrochloryde 4,4'-bis-[2(diethylamino)ethoxy]diphenyl (compound 1) and dihydrochloryde 2-methoxycarbonil-4,4'-bis-[2(diethylamino)ethoxy]diphenyl (compound 2) were studied by electrophoresis technique. During the interval 0-2 hours all compounds increased the absolute values of EPM in comparison with control. These changes were of the same kind--distinctions were quantitative. Amixine and compound 1 during the interval 2-4 hours additionally increased the EPM. The compound 2, on the contrary, decreased the EPM. It was shown that the opposite effects of the aforementioned compounds were caused by the fact that amixine and compound 1 induce, and compound 2 does not induce IFN production in T lymphocytes in vitro. The results of our experiments are important for understanding of the mechanisms of immunomodulating effect of amixine and its structural analogues.

Synthesis and activity evaluation of tilorone analogs as potential anticancer agents.

Tilorone is an interferon inducer with anticancer activity. Twenty-two novel tilorone analogs were synthesized by improvements of fluorenone skeleton, side chains and amino groups to screen new anticancer agents. In vitro evaluation showed that ten new compounds had better anticancer activities than tilorone. Among them, 2c (IC50 < 7 µM against cancer cell lines and IC50 > 35 µM against non-cancer cell lines) and 5d (IC50 < 10 µM against cancer cell lines and IC50 > 53 µM against non-cancer cell lines) exhibited the best anticancer activities and selectivities. Pharmacophore modeling of highly active compounds was carried out by Molecular Operating Environment (MOE) to generate a visualized model for compound design in future study.

Bone morphogenetic protein-inducer tilorone identified by high-throughput screening is antifibrotic in vivo.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and very few therapeutic options. On the molecular level, patients with IPF have increased amounts of the bone morphogenetic protein (BMP) inhibitor gremlin in their lungs, which results in decreased BMP signaling, and an increase in transforming growth factor-ß signaling. Based on these findings, we hypothesized that restoration of the impaired BMP signaling would offer a novel strategy for the prevention of fibrosis progression or for the treatment of pulmonary fibrosis. We used reporter cell lines and high-throughput screening of a chemical compound library as an approach to finding molecules that increase BMP signaling in lung epithelial cells, without increasing transforming growth factor-ß signaling. The most promising candidate drug was analyzed further by studying its effects on BMP target gene expression, Smad protein phosphorylation, and a mouse model of silica-induced pulmonary fibrosis. The most promising drug candidate, tilorone, induced BMP signaling in the reporter cells and increased the expression of BMP-7 and a BMP target gene, Id3, in lung epithelial A549 cells. In a mouse model of pulmonary fibrosis, tilorone decreased lung hydroxyproline content and the expression of collagen genes Col1A1 and Col3A1. Mice treated with tilorone showed markedly decreased histological changes, compared with untreated mice. These findings indicate that tilorone has biologically significant antifibrotic properties.

Dose-dependent IFN-stimulating and immunomodulating properties of 6H-indolo[2,3-B] quinoxaline derivatives.

Two 6H-indoloquinoxaline derivatives were studied in different doses and schemes of application for their INFgamma-inducing potential and ability to effect functional activity of phagocytic cells. Tested compounds were shown to possess comparable or higher activity than reference drug Amixin in analogous doses. One indoloquinoxaline significantly elevated metabolic activity of macrophages and increased their potential for phagocytosis. Application of multiple treatments and higher doses allowed us to reveal differences between studied derivatives that were not obvious in previous in vivo experiment. Capacity of 6H-indoloquinoxalines to induce vast IFN amounts on in vivo level was demonstrated for the first time.

Effect of tilorone and its analogues on the change of mitochondrial potential of rat hepatocytes.

The influence of tilorone dihydrochloride and its analogues--diphenyl derivatives on the changes of transmembrane potential of mitochondrial membranes of the isolated rat hepatocytes has been estimated. Authors have shown a significant increase in mitochondrial potential thirty minutes after the introduction of the test compounds to the cells using the fluorescent probe JC-1. These results indicate the rapid activation with tilorone and its analog--dihydrochloryde 4,4'-bis-[2-(diethylamino)ethoxy]diphenyl--of the RLR signaling pathway. The final stage of this pathway is the cell production of IFN type I. The authors concluded that there is an increasing of the organelles resistance to the extra/intracellular damaging agents under the influence of the test compounds.