A New "Non-Traditional" Antibacterial Drug Fluorothiazinone-Clinical Research in Patients with Complicated Urinary Tract Infections.

In order to combat resistance, it is necessary to develop antimicrobial agents that act differently from conventional antibiotics. Fluorothiazinone, 300 mg tablet (The Gamaleya National Research Center), is an original antibacterial drug based on a new small molecule T3SS and flagellum inhibitor. A total of 357 patients with complicated urinary tract infections (UTIs) were divided into two groups and given Fluorothiazinone 1200 mg/day or a placebo for 7 days to evaluate the efficacy and safety of the drug. Additionally, all patients were given Cefepime 2000 mg/day. Fluorothiazinone with Cefepime showed superiority over placebo/Cefepime based on the assessment of the proportion of patients with an overall outcome in the form of a cure after 21 days post-therapy (primary outcome), overall outcome in cure rates, clinical cure rates, and microbiological efficacy at the end of therapy and after 21 days post-therapy (secondary outcomes). In patients who received Fluorothiazinone, the rate of infection recurrences 53 and 83 days after the end of the therapy was lower by 18.9%, compared with patients who received placebo. Fluorothiazinone demonstrated a favorable safety profile with no serious unexpected adverse events reported. The results showed superiority of the therapy with Fluorothiazinone in combination with Cefepime compared with placebo/Cefepime in patients with cUTIs.

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.

Study of the Antibacterial Activity of Superhydrophilic and Superhydrophobic Copper Substrates against Multi-Drug-Resistant Hospital-Acquired Pseudomonas aeruginosa Isolates.

The global spread of multidrug-resistant (MDR) hospital-acquired pathogens is a serious problem for healthcare units. The challenge of the spreading of nosocomial infections, also known as hospital-acquired pathogens, including Pseudomonas aeruginosa, must be addressed not only by developing effective drugs, but also by improving preventive measures in hospitals, such as passive bactericidal coatings deposited onto the touch surfaces. In this paper, we studied the antibacterial activity of superhydrophilic and superhydrophobic copper surfaces against the P. aeruginosa strain PA103 and its four different polyresistant clinical isolates with MDR. To fabricate superhydrophilic and superhydrophobic coatings, we subjected the copper surfaces to laser processing with further chemosorption of fluorooxysilane to get a superhydrophobic substrate. The antibacterial activity of superhydrophilic and superhydrophobic copper surfaces was shown, with respect to both the collection strain PA103 and polyresistant clinical isolates of P. aeruginosa, and the evolution of the decontamination of a bacterial suspension is presented and discussed. The presented results indicate the promising potential of the exploitation of superhydrophilic coatings in the manufacture of contact surfaces for healthcare units, where the risk of infection spread and contamination by hospital-acquired pathogens is extremely high.

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.

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.

Small Molecule Inhibitor of Type Three Secretion System Belonging to a Class 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones Improves Survival and Decreases Bacterial Loads in an Airway Pseudomonas aeruginosa Infection in Mice.

Pseudomonas aeruginosa is a cause of high mortality in burn, immunocompromised, and surgery patients. High incidence of antibiotic resistance in this pathogen makes the existent therapy inefficient. Type three secretion system (T3SS) is a leading virulence system of P. aeruginosa that actively suppresses host resistance and enhances the severity of infection. Innovative therapeutic strategies aiming at inhibition of type three secretion system of P. aeruginosa are highly attractive, as they may reduce the severity of clinical manifestations and improve antibacterial immune responses. They may also represent an attractive therapy for antibiotic-resistant bacteria. Recently our laboratory developed a new small molecule inhibitor belonging to a class 2,4-disubstituted-4H-[1,3, 4]-thiadiazine-5-ones, Fluorothiazinon (FT), that effectively suppressed T3SS in chlamydia and salmonella in vitro and in vivo. In this study, we evaluate the activity of FT towards antibiotic-resistant clinical isolates of P. aeruginosa expressing T3SS effectors ExoU and ExoS in an airway infection model. We found that FT reduced mortality and bacterial loads and decrease lung pathology and systemic inflammation. In addition, we show that FT inhibits the secretion of ExoT and ExoY, reduced bacteria cytotoxicity, and increased bacteria internalization in vitro. Overall, FT shows a strong potential as an antibacterial therapy of antibiotic-resistant P. aeruginosa infection.