New conjugates based on N4-hydroxycytidine with more potent antiviral efficacy in vitro than EIDD-2801 against SARS-CoV-2 and other human coronaviruses.

The spread of COVID-19 continues due to genetic variation in SARS-CoV-2. Highly mutated variants of SARS-CoV-2 have an increased transmissibility and immune evasion. Due to the emergence of various new variants of the virus, there is an urgent need to develop broadly effective specific drugs for therapeutic strategies for the prevention and treatment of COVID-19. Molnupiravir (EIDD-2801, MK-4482), is an orally bioavailable ribonucleoside analogue of ß-D-N4-hydroxycytidine (NHC), has demonstrated efficacy against SARS-CoV-2 and was recently approved for COVID-19 treatment. To improve antiviral potency of NHC, we developed a panel of NHC conjugates with lipophilic vectors and ester derivatives with amino- and carboxylic-acids. Most of the synthesized compounds had comparable or higher (2-20 times) antiviral activity than EIDD-2801, against different lineages of SARS-CoV-2, MERS-CoV, seasonal coronaviruses OC43 and 229E, as well as bovine coronavirus. For further studies, we assessed the most promising compound in terms of activity, simplicity and cost of synthesis - NHC conjugate with phenylpropionic acid (SN_9). SN_9 has shown high efficacy in prophylactic, therapeutic and transmission models of COVID-19 infection in hamsters. Importantly, SN_9 profoundly inhibited virus replication in the lower respiratory tract of hamsters and transgenic mice infected with the Omicron sublineages XBB.1.9.1, XBB.1.16 and EG.5.1.1. These data indicate that SN_9 represents a promising antiviral drug candidate for COVID-19 treatment, and NHC modification strategies deserve further investigation as an approach to develop prodrugs against various coronaviruses.

Pharmacokinetics, tissue distribution, bioavailability and excretion of the anti-virulence drug Fluorothiazinon in rats and rabbits.

Growing antimicrobial resistance has accelerated the development of anti-virulence drugs to suppress bacterial toxicity without affecting cell viability. Fluorothiazinon (FT), an anti-virulence, type three secretion system and flagella motility inhibitor which has shown promise to suppress drug-resistant pathogens having the potential to enhance the efficacy of commonly prescribed antibiotics when used in combination. In this study we characterized the pharmacokinetics, tissue distribution, bioavailability and excretion of FT in rats and rabbits. FT presented a dose-proportional linear increase in the blood of rats. Tissue distribution profiling confirmed that FT distributes to all organs being substantially higher than in the blood of rats. The bioavailability of FT was higher when administered with starch than with water implying FT should be ideally dosed with food. FT was primarily excreted in the feces in rats and rabbits while negligible amounts are recovered from the urine.

Naphthyl-Substituted Indole and Pyrrole Carboxylic Acids as Effective Antibiotic Potentiators-Inhibitors of Bacterial Cystathionine γ-Lyase.

Over the past decades, the problem of bacterial resistance to most antibiotics has become a serious threat to patients' survival. Nevertheless, antibiotics of a novel class have not been approved since the 1980s. The development of antibiotic potentiators is an appealing alternative to the challenging process of searching for new antimicrobials. Production of H2S-one of the leading defense mechanisms crucial for bacterial survival-can be influenced by the inhibition of relevant enzymes: bacterial cystathionine γ-lyase (bCSE), bacterial cystathionine ß-synthase (bCBS), or 3-mercaptopyruvate sulfurtransferase (MST). The first one makes the main contribution to H2S generation. Herein, we present data on the synthesis, in silico analyses, and enzymatic and microbiological assays of novel bCSE inhibitors. Combined molecular docking and molecular dynamics analyses revealed a novel binding mode of these ligands to bCSE. Lead compound 2a manifested strong potentiating activity when applied in combination with some commonly used antibiotics against multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. The compound was found to have favorable in vitro absorption, distribution, metabolism, excretion, and toxicity parameters. The high effectiveness and safety of compound 2a makes it a promising candidate for enhancing the activity of antibiotics against high-priority pathogens.

Pharmacokinetics, quorum-sensing signal molecules and tryptophan-related metabolomics of the novel anti-virulence drug Fluorothiazinon in a Pseudomonas aeruginosa-induced pneumonia murine model.

Pseudomonas aeruginosa (PA) infection is commonly associated with hospital-acquired infections in patients with immune deficiency and/or severe lung diseases. Managing this bacterium is complex due to drug resistance and high adaptability. Fluorothiazinon (FT) is an anti-virulence drug developed to suppress the virulence of bacteria as opposed to bacterial death increasing host's immune response to infection and improving treatment to inhibit drug resistant bacteria. We aimed to evaluate FT pharmacokinetics, quorum sensing signal molecules profiling and tryptophan-related metabolomics in blood, liver, kidneys, and lungs of mice. Study comprised three groups: a group infected with PA that was treated with 400 mg/kg FT ("infected treated group"); a non-infected group, but also treated with the same single drug dose ("non-infected treated group"); and an infected group that received a vehicle ("infected non-treated group"). PA-mediated infection blood pharmacokinetics profiling was indicative of increased drug concentrations as shown by increased Cmax and AUCs. Tissue distribution in liver, kidneys, and lungs, showed that liver presented the most consistently higher concentrations of FT in the infected versus non-infected mice. FT showed that HHQ levels were decreased at 1 h after dosing in lungs while PQS levels were lower across time in lungs of infected treated mice in comparison to infected non-treated mice. Metabolomics profiling performed in lungs and blood of infected treated versus infected non-treated mice revealed drug-associated metabolite alterations, especially in the kynurenic and indole pathways.

Synthesis and Antiviral Activity of Novel ß-D-N4-Hydroxycytidine Ester Prodrugs as Potential Compounds for the Treatment of SARS-CoV-2 and Other Human Coronaviruses.

The spread of COVID-19 infection continues due to the emergence of multiple transmissible and immune-evasive variants of the SARS-CoV-2 virus. Although various vaccines have been developed and several drugs have been approved for the treatment of COVID-19, the development of new drugs to combat COVID-19 is still necessary. In this work, new 5'-O-ester derivatives of N4-hydroxycytidine based on carboxylic acids were developed and synthesized by Steglich esterification. The antiviral activity of the compounds was assessed in vitro-inhibiting the cytopathic effect of HCoV-229E, and three variants of SARS-CoV-2, on huh-7 and Vero E6 cells. Data have shown that most synthesized derivatives exhibit high activity against coronaviruses. In addition, the relationship between the chemical structure of the compounds and their antiviral effect has been established. The obtained results show that the most active compound was conjugate SN_22 based on 3-methyl phenoxyacetic acid. The results of this study indicate the potential advantage of the chemical strategies used to modify NHC as a promising avenue to be explored in vivo, which could lead to the development of drugs with improved pharmacological properties that potently inhibit SARS-CoV-2.

Antiviral Activity of N1,N3-Disubstituted Uracil Derivatives against SARS-CoV-2 Variants of Concern.

Despite the widespread use of the COVID-19 vaccines, the search for effective antiviral drugs for the treatment of patients infected with SARS-CoV-2 is still relevant. Genetic variability leads to the continued circulation of new variants of concern (VOC). There is a significant decrease in the effectiveness of antibody-based therapy, which raises concerns about the development of new antiviral drugs with a high spectrum of activity against VOCs. We synthesized new analogs of uracil derivatives where uracil was substituted at the N1 and N3 positions. Antiviral activity was studied in Vero E6 cells against VOC, including currently widely circulating SARS-CoV-2 Omicron. All synthesized compounds of the panel showed a wide antiviral effect. In addition, we determined that these compounds inhibit the activity of recombinant SARS-CoV-2 RdRp. Our study suggests that these non-nucleoside uracil-based analogs may be of future use as a treatment for patients infected with circulating SARS-CoV-2 variants.

Preventative treatment with Fluorothiazinon suppressed Acinetobacter baumannii-associated septicemia in mice.

The high prevalence of multidrug-resistant Acinetobacter baumannii has emerged as a serious problem in the treatment of nosocomial infections in the past three decades. Recently, we developed a new small-molecule inhibitor belonging to a class of 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones, Fluorothiazinon (FT, previously called CL-55). FT effectively suppressed the T3SS of Chlamydia spp., Pseudomonas aeruginosa, and Salmonella sp. without affecting bacterial growth in vitro. In this study, we describe that prophylactic use of FT for 4 days prior to challenge with resistant clinical isolates of A. baumannii (ABT-897-17 and 52TS19) suppressed septic infection in mice, resulting in improved survival, limited bacteraemia and decreased bacterial load in the organs of the mice. We show that FT had an inhibitory effect on A. baumannii biofilm formation in vitro and, to a greater extent, on biofilm maturation. In addition, FT inhibited Acinetobacter isolate-induced death of HeLa cells, which morphologically manifested as apoptosis. The mechanism of FT action on A. baumannii is currently being studied. FT may be a promising candidate for the development of a broad-spectrum anti-virulence drug to use in the prevention of nosocomial infections.

Fluorothiazinon, a small-molecular inhibitor of T3SS, suppresses salmonella oral infection in mice.

Therapeutic strategies that target bacterial virulence have received considerable attention. The type III secretion system (T3SS) is important for bacterial virulence and represents an attractive therapeutic target. Recently, we developed a new small-molecule inhibitor belonging to a class 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones, Fluorothiazinon (FT-previously called CL-55). FT effectively suppressed T3SS of Chlamydia spp., Pseudomonas aeruginosa, and Salmonella without affecting bacterial growth in vitro. FT was previously characterized by low toxicity, stability, and therapeutic efficacy in animal models. Salmonella T3SS inhibition by FT was studied using in vitro assays for effector proteins detection and estimation of salmonella replication in peritoneal macrophages. The antibacterial effect of FT in vivo was investigated in murine models of salmonella chronic systemic and acute infection. Oral administration of the virulent strain of Salmonella enterica serovar Typhimurium to mice-induced chronic systemic infection with the pathogen persistence in different lymphoid organs such as spleens, Peyer's plaques, and mesenteric lymph nodes. We found that FT suppressed orally induced salmonella infection both with therapeutic and prophylactic administration. Treatment by FT at a dose of 50 mg/kg for 4 days starting from day 7 post-infection (therapy) as well as for 4 days before infection (prevention) led to practically complete eradication of salmonella in mice. FT shows a strong potential for antibacterial therapy and could be used as a substance in the design of antibacterial drugs for pharmaceutical intervention including therapy of antibiotic-resistant infections.

Lycopene presence in facial skin corneocytes and sebum and its association with circulating lycopene isomer profile: Effects of age and dietary supplementation.

Lycopene is a dietary antioxidant known to prevent skin photodamage. This study aimed to examine age-dependent presence of this carotenoid on the surface of the facial skin and in the serum as well as to measure the same parameters during supplementation with lycopene. Serum samples and samples from facial skin surface were obtained from 60 young (under 25 years old) and 60 middle-aged (over 50 years old) volunteers. Similar samples were taken from 15 middle-aged subjects during 4-week supplementation with lycopene (7 mg/day). Serum lycopene levels and isomer profiles were analyzed by HPLC. Lycopene in desquamated corneocytes and the sebum from facial skin surface was determined using lycopene-specific fluorescent monoclonal antibodies. The results demonstrated that there was no age-related difference in serum lycopene levels, but a higher proportion of (all-E)-lycopene was detected in the "young" group (37.5% vs 26.2% in the "middle-aged" group; p < 0.0001). "Young" volunteers also had a higher lycopene level in both corneocytes (p = 0.0071) and the sebum (p = 0.0139) from the skin surface. Supplementation with lycopene resulted in a sharp increase of lycopene concentrations in both serum and skin surface samples. There was also a clear change in the pattern of lycopene isomers in the serum manifested by a significant increase in the proportion of (all-E)-lycopene (from 22.1% to 44.0% after supplementation, p < 0.0001). It can be concluded that dietary supplementation with lycopene results in its accumulation in the serum and skin. This process is accompanied by significant changes in the circulating lycopene isomer profile which becomes similar to that typical for young individuals.

A small-molecule compound belonging to a class of 2,4-disubstituted 1,3,4-thiadiazine-5-ones suppresses Salmonella infection in vivo.

Therapeutic strategies that target bacterial virulence have received considerable attention. The type III secretion system (T3SS) is important for bacterial virulence and represents an attractive therapeutic target. A novel compound with a predicted T3SS inhibitory activity named CL-55 (N-(2,4-difluorophenyl)-4-(3-ethoxy-4-hydroxybenzyl)-5-oxo-5,6-dihydro-4H-[1,3,4]-thiadiazine-2-carboxamide) was previously characterized by low toxicity, high levels of solubility, stability and specific efficiency toward Chlamydia trachomatis in vitro and in vivo. In this study, we describe the action of CL-55 on Salmonella enterica serovar Typhimurium. We found that CL-55 does not affect Salmonella growth in vitro but suppresses Salmonella infection in vivo. The i.p. injection of CL-55 at a dose of 10 mg kg(-1) for 4 days significantly (500-fold) decreased the numbers of Salmonella in the spleen and peritoneal lavages and increased the survival rates in susceptible (BALB/c, I/St) and resistant (A/Sn) mice. Twelve days of therapy led to complete eradication of Salmonella in mice. Moreover, no pathogen was found 4-6 weeks post treatment. CL-55 was not carcinogenic or mutagenic, did not increase the level of chromosomal aberrations in bone marrow cells and had low toxicity in mice, rats and rabbits. Pharmacokinetic studies have shown that CL-55 rapidly disappears from systemic blood circulation and is distributed in the organs. Our data demonstrates that CL-55 affects S. enterica serovar Typhimurium in vivo and could be used as a substance in the design of antibacterial inhibitors for pharmaceutical intervention of bacterial virulence for infection.

Chlamydia trachomatis promotes 3T3 cell differentiation into adipocytes.

BACKGROUND: There is experimental and clinical evidence showing that some viral and bacterial pathogens are linked to the accumulation of excessive body fat and obesity. OBJECTIVES: The aim of the study was to investigate the ability of C. trachomatis to propagate in the pre-adipocyte cell line and induce its differentiation into fat cells. MATERIAL AND METHODS: 3T3 L1 pre-adipocytes or McCoy cells were plated and infected with C. trachomatis. The cell monolayers were further studied by immunofluorescent and quantitative RT-PCR methods. RESULTS: C. trachomatis can efficiently propagate in 3T3 L1 cells, a mouse pre-adipocyte cell line. The morphological characteristics of chlamydial growth revealed in 3T3 L1 cells with the monoclonal chlamydial MOMP-specific antibody resembled those seen in McCoy cells, a classic cell line used for chlamydial research. The number of chlamydial 16S rRNA copies detectable in the lysates of McCoy and 3T3 cells infected with C. trachomatis was almost identical, suggesting similar efficiency of pathogen propagation in both cell lines. Moreover, there was a significant increase in aP2 mRNA transcript levels as well as moderate induction of SCD-1 mRNA in the total RNA extracted from the infected 3T3 L1 cells 48 h following the pathogen inoculation. The increased expression of the adipogenic markers was also accompanied by lipid droplet accumulation in the C. trachomatis infected 3T3 L1 cells, suggesting their transformation into differentiated adipocytes. CONCLUSIONS: The direct effect of the pathogen on fat cell progenitors observed in this work may explain abnormal fat deposition at the sites of chronic inflammation caused by C. trachomatis.

Chlamydia trachomatis growth inhibition and restoration of LDL-receptor level in HepG2 cells treated with mevastatin.

BACKGROUND: Perihepatitis is rare but consistently occurring extragenital manifestation of untreated Chlamydia trachomatis infection. Despite of possible liver involvement in generalized C. trachomatis infection, the ability of the pathogen to propagate in the hepatic cells and its impact on liver functions is not thoroughly investigated. The effect of mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, on C. trachomatis growth in human hepatoma cell line HepG2 has been studied. Bacterial growth was assessed by immunostaining with FITC-labeled monoclonal antibody against chlamydial lipopolysaccharide and by RT-PCR for two chlamydial genetic markers (16S rRNA and euo). RESULTS: Chlamydial inclusion bodies were seen in approximately 50% of hepatocytes at 48 hours in the post infection period. Lysates obtained from infected hepatocytes were positive in the infective progeny test at 48 and especially in 72 hours after infection initiation. It has been shown that chlamydial infection in hepatocytes also leads to the decline of LDL-receptor mRNA which reflects infection multiplicity rate. Additions of mevastatin (1, 20 and 40 microM) 1 hour before inoculation restored and upregulated LDL-receptor mRNA level in a dose-dependent manner. Mevastatin treatment had no effect on internalization of chlamydial particles. However it reduced drastically the number of chlamydial 16S rRNA and euo transcripts as well as overall infection rate in HepG-2 cells. Complete eradication of infection has been seen by immunofluorescent staining at 40 microM mevastatin concentration, when expression level of chlamydial 16S rRNA and euo was undetectable. Lower concentration of mevastatin (20 microM) promoted euo expression level and the appearance of atypically small chlamydial inclusions, while there was a noticeable reduction in the number of infected cells and 16S rRNA transcripts. CONCLUSIONS: C. trachomatis can efficiently propagate in hepatocytes affecting transcription rate of some liver-specific genes. Ongoing cholesterol synthesis is essential for chlamydial growth in hepatocytes. Inhibitors of cholesterol biosynthesis can supplement conventional strategy in the management of C. trachomatis infection.