The antihelminth drug rafoxanide reverses chromosomal-mediated colistin-resistance in Klebsiella pneumoniae.

The emergence and rapid spread of multi-drug-resistant (MDR) bacteria pose a serious threat to global healthcare. Although the synergistic effect of rafoxanide and colistin was reported, little is known regarding the potential mechanism of this synergy, particularly against chromosomal-mediated colistin-resistant Klebsiella pneumoniae. In the present study, we elucidated the synergistic effect of rafoxanide and colistin against chromosomal-mediated colistin-resistant Klebsiella pneumoniae isolates from human (KP-9) and swine (KP-1) infections. Treatment with 1 mg/L rafoxanide overtly reversed the MIC max to 512-fold. Time-kill assays indicated that rafoxanide acted synergistically with colistin against the growth of KP-1 and KP-9. Mechanistically, we unexpectedly found that the combination destroys the inner-membrane integrity, and ATP synthesis was also quenched, albeit, not via F1F0-ATPase; thereby also inhibiting the activity of efflux pumps. Excessive production of reactive oxygen species (ROS) was also an underlying factor contributing to the bacterial-killing effect of the combination. Transcriptomic analysis unraveled overt heterogeneous expression as treated with both administrations compared with monotherapy. Functional analysis of these differentially expressed genes (DEGs) targeted to the plasma membrane and ATP-binding corroborated phenotypic screening results. These novel findings highlight the synergistic mechanism of rafoxanide in combination with colistin which effectively eradicates chromosomal-mediated colistin-resistant Klebsiella pneumoniae. IMPORTANCE The antimicrobial resistance of Klebsiella pneumoniae caused by the abuse of colistin has increased the difficulty of clinical treatment. A promising combination (i.e., rafoxanide+ colistin) has successfully rescued the antibacterial effect of colistin. However, we still failed to know the potential effect of this combination on chromosome-mediated Klebsiella pneumoniae. Through a series of in vitro experiments, as well as transcriptomic profiling, we confirmed that the MIC of colistin was reduced by rafoxanide by destroying the inner-membrane integrity, quenching ATP synthesis, inhibiting the activity of the efflux pump, and increasing the production of reactive oxygen species. In turn, the expression of relevant colistin resistance genes was down-regulated. Collectively, our study revealed rafoxanide as a promising colistin adjuvant against chromosome-mediated Klebsiella pneumoniae.

Therapeutic Switching of Rafoxanide: a New Approach To Fighting Drug-Resistant Bacteria and Fungi.

Control and management of life-threatening bacterial and fungal infections are a global health challenge. Despite advances in antimicrobial therapies, treatment failures for resistant bacterial and fungal infections continue to increase. We aimed to repurpose the anthelmintic drug rafoxanide for use with existing therapeutic drugs to increase the possibility of better managing infection and decrease treatment failures. For this purpose, we evaluated the antibacterial and antifungal potential of rafoxanide. Notably, 70% (70/100) of bacterial isolates showed multidrug resistance (MDR) patterns, with higher prevalence among human isolates (73.5% [50/68]) than animal ones (62.5% [20/32]). Moreover, 22 fungal isolates (88%) were MDR and were more prevalent among animal (88.9%) than human (87.5%) sources. We observed alarming MDR patterns among bacterial isolates, i.e., Klebsiella pneumoniae (75% [30/40; 8 animal and 22 human]) and Escherichia coli (66% [40/60; 12 animal and 28 human]), and fungal isolates, i.e., Candida albicans (86.7% [13/15; 4 animal and 9 human]) and Aspergillus fumigatus (90% [9/10; 4 animal and 5 human]), that were resistant to at least one agent in three or more different antimicrobial classes. Rafoxanide had antibacterial and antifungal activities, with minimal inhibitory concentration (MICs) ranging from 2 to 128 µg/mL. Rafoxanide at sub-MICs downregulated the mRNA expression of resistance genes, including E. coli and K. pneumoniae blaCTX-M-1, blaTEM-1, blaSHV, MOX, and DHA, C. albicans ERG11, and A. fumigatus cyp51A. We noted the improvement in the activity of ß-lactam and antifungal drugs upon combination with rafoxanide. This was apparent in the reduction in the MICs of cefotaxime and fluconazole when these drugs were combined with sub-MIC levels of rafoxanide. There was obvious synergism between rafoxanide and cefotaxime against all E. coli and K. pneumoniae isolates (fractional inhibitory concentration index [FICI] values ≤ 0.5). Accordingly, there was a shift in the patterns of resistance of 16.7% of E. coli and 22.5% of K. pneumoniae isolates to cefotaxime and those of 63.2% of C. albicans and A. fumigatus isolates to fluconazole when the isolates were treated with sub-MICs of rafoxanide. These results were confirmed by in silico and mouse protection assays. Based on the in silico study, one possible explanation for how rafoxanide reduced bacterial resistance is through its inhibitory effects on bacterial and fungal histidine kinase enzymes. In short, rafoxanide exhibited promising results in overcoming bacterial and fungal drug resistance. IMPORTANCE The drug repurposing strategy is an alternative approach to reducing drug development timelines with low cost, especially during outbreaks of disease caused by drug-resistant pathogens. Rafoxanide can disrupt the abilities of bacterial and fungal cells to adapt to stress conditions. The coadministration of antibiotics with rafoxanide can prevent the failure of treatment of both resistant bacteria and fungi, as the resistant pathogens could be made sensitive upon treatment with rafoxanide. From our findings, we anticipate that pharmaceutical companies will be able to utilize new combinations against resistant pathogens.

Rafoxanide, a salicylanilide anthelmintic, interacts with human plasma protein transthyretin.

Transthyretin (TTR) is a carrier protein for thyroid hormone thyroxine (T4 ) in plasma, placental cytosol, and cerebrospinal fluid. While the potential toxicity of small molecules that compete with T4 for binding to TTR should be carefully studied, these small molecules can also serve as anti-ATTR amyloidosis drugs by stabilizing the TTR structure. Here, we demonstrated that rafoxanide, an EU-approved anthelmintic drug for domesticated animals, binds to the T4 -binding site of TTR. An intrinsic fluorescence quenching assay showed that rafoxanide also binds to the thyroid hormone-related proteins, including serum albumin and thyroid hormone receptor ß. Rafoxanide strongly inhibited TTR amyloidogenesis in fibrillization assay, but the binding of rafoxanide to TTR was interfered with in human plasma, probably due to interactions with thyroid hormone-related proteins. Protein crystallography provided clues for the optimization of binding affinity and selectivity. Our findings emphasize the importance of considering rafoxanide as both a possible thyroid-disrupting chemical and a lead compound for the development of new ATTR amyloidosis inhibitors.

Insight into the Antibacterial Action of Iodinated Imine, an Analogue of Rafoxanide: a Comprehensive Study of Its Antistaphylococcal Activity.

In this study, we have focused on a multiparametric microbiological analysis of the antistaphylococcal action of the iodinated imine BH77, designed as an analogue of rafoxanide. Its antibacterial activity against five reference strains and eight clinical isolates of Gram-positive cocci of the genera Staphylococcus and Enterococcus was evaluated. The most clinically significant multidrug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and vancomycin-resistant Enterococcus faecium, were also included. The bactericidal and bacteriostatic actions, the dynamics leading to a loss of bacterial viability, antibiofilm activity, BH77 activity in combination with selected conventional antibiotics, the mechanism of action, in vitro cytotoxicity, and in vivo toxicity in an alternative animal model, Galleria mellonella, were analyzed. The antistaphylococcal activity (MIC) ranged from 15.625 to 62.5 µM, and the antienterococcal activity ranged from 62.5 to 125 µM. Its bactericidal action; promising antibiofilm activity; interference with nucleic acid, protein, and peptidoglycan synthesis pathways; and nontoxicity/low toxicity in vitro and in vivo in the Galleria mellonella model were found to be activity attributes of this newly synthesized compound. In conclusion, BH77 could be rightfully minimally considered at least as the structural pattern for future adjuvants for selected antibiotic drugs. IMPORTANCE Antibiotic resistance is among the largest threats to global health, with a potentially serious socioeconomic impact. One of the strategies to deal with the predicted catastrophic future scenarios associated with the rapid emergence of resistant infectious agents lies in the discovery and research of new anti-infectives. In our study, we have introduced a rafoxanide analogue, a newly synthesized and described polyhalogenated 3,5-diiodosalicylaldehyde-based imine, that effectively acts against Gram-positive cocci of the genera Staphylococcus and Enterococcus. The inclusion of an extensive and comprehensive analysis for providing a detailed description of candidate compound-microbe interactions allows the valorization of the beneficial attributes linked to anti-infective action conclusively. In addition, this study can help with making rational decisions about the possible involvement of this molecule in advanced studies or may merit the support of studies focused on related or derived chemical structures to discover more effective new anti-infective drug candidates.

Repurposing Anthelmintics: Rafoxanide- and Copper-Functionalized SBA-15 Carriers against Methicillin-Resistant Staphylococcus aureus.

The development of materials that can more efficiently administer antimicrobial agents in a controlled manner is urgently needed due to the rise in microbial resistance to traditional antibiotics. While new classes of antibiotics are developed and put into widespread usage, existing, inexpensive compounds can be repurposed to fight bacterial infections. Here, we present the synthesis of amine-functionalized SBA-15 mesoporous silica nanomaterials with physisorbed rafoxanide (RFX), a commonly used salicylanilide anthelmintic, and anchored Cu(II) ions that exhibit enhanced antimicrobial efficacy against the pathogenic bacterium Staphylococcus aureus. The synthesized nanomaterials are structurally characterized by a combination of physicochemical, thermal, and optical methods. Additionally, release studies are carried out in vitro to determine the effects of pH and the synthetic sequence used to produce the materials on Cu(II) ion release. Our results indicate that SBA-15 mesoporous silica nanocarriers loaded with Cu(II) and RFX exhibit 10 times as much bactericidal action against wild-type S. aureus as the nanocarrier loaded with only RFX. Furthermore, the synthetic sequence used to produce the nanomaterials could significantly affect (enhance) their bactericidal efficacy.

Discovery of rafoxanide as a novel agent for the treatment of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancer cases, is associated with a poor outcome. Rafoxanide is an anthelmintic drug that inhibits tumor growth in certain malignancies. However, its impact on NSCLC remains unknown. In this study, we examined the effect of rafoxanide on NSCLC and dissected the underlying mechanism. The results showed that rafoxanide significantly inhibited the growth, invasion, and migration of NSCLC cells. Besides, rafoxanide can induce NSCLC cell apoptosis and cell cycle arrest in a dose-dependent manner. RNA-seq analysis revealed that genes associated with endoplasmic reticulum stress (ER) stress responses were activated. Mechanistically, we found Rafoxanide can induce ER stress and activate the unfolded protein response (UPR). Apoptosis was activated by excessive ER stress, and autophagy was activated to partially alleviate ER stress. In vivo, we found that rafoxanide inhibited the growth of A549 and H1299 xenograft mouse models without severe side effects. Collectively, the present study indicates that rafoxanide may be a candidate drug for the treatment of NSCLC.

Rafoxanide sensitizes colorectal cancer cells to TRAIL-mediated apoptosis.

Colorectal cancer (CRC) remains a leading causes of cancer-related death in the world, mainly due to the lack of effective treatment of advanced disease. TNF-related apoptosis-inducing ligand (TRAIL)-driven cell death, a crucial event in the control of tumor growth, selectively targets malignant rather than non-transformed cells. However, the fact that cancer cells, including CRC cells, are either intrinsically resistant or acquire resistance to TRAIL, represents a major hurdle to the use of TRAIL-based strategies in the clinic. Agents able to overcome CRC cell resistance to TRAIL have thus great therapeutic potential and many researchers are making efforts to identify TRAIL sensitizers. The anthelmintic drug rafoxanide has recently emerged as a potent anti-tumor molecule for different cancer types and we recently reported that rafoxanide restrained the proliferation of CRC cells, but not of normal colonic epithelial cells, both in vitro and in a preclinical model mimicking sporadic CRC. As these findings were linked with the induction of endoplasmic reticulum stress, a phenomenon involved in the regulation of various components of the TRAIL-driven apoptotic pathway, we sought to determine whether rafoxanide could restore the sensitivity of CRC cells to TRAIL. Our data show that rafoxanide acts as a selective TRAIL sensitizer in vitro and in a syngeneic experimental model of CRC, by decreasing the levels of c-FLIP and survivin, two key molecules conferring TRAIL resistance. Collectively, our data suggest that rafoxanide could potentially be deployed as an anti-cancer drug in the combinatorial approaches aimed at overcoming CRC cell resistance to TRAIL-based therapies.

Repositioning rafoxanide to treat Gram-negative bacilli infections.

OBJECTIVES: Repurposing drugs provides a new approach to the fight against MDR Gram-negative bacilli (MDR-GNB). Rafoxanide, a veterinary antihelminthic drug, has shown antibacterial activity in vitro against Gram-positive bacteria. We aimed to analyse the in vitro and in vivo efficacy of rafoxanide in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) GNB. METHODS: A collection of Col-S and Col-R Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae were used. Chequerboard and time-kill curve analyses were performed to determine the synergy between rafoxanide and colistin. Changes in membrane structure and permeability were analysed using transmission electron microscopy and fluorescence assays. A murine peritoneal sepsis model using Col-R strains of these pathogens was performed to study the efficacy of rafoxanide (10 mg/kg/24 h, IV), colistimethate sodium (CMS) (20 mg/kg/8 h, intraperitoneally) and rafoxanide (10 mg/kg/24 h, IV) plus CMS (20 mg/kg/8 h, intraperitoneally) for 72 h. RESULTS: Rafoxanide showed MICs ≥256 mg/L for all Col-S and Col-R strains. Chequerboard and time-kill curve analyses showed that rafoxanide (1 mg/L) is more synergistic with colistin against Col-R than Col-S strains. Col-R, but not Col-S, strains treated with rafoxanide demonstrated higher membrane permeabilization. Transmission electron microscopy visualization confirmed that Col-R strains suffer morphological changes. In the murine peritoneal sepsis model with Col-R strains, rafoxanide plus CMS, compared with CMS alone, increased mouse survival to 53.8% and 73.3%, and reduced bacterial loads in tissues and blood between 2.34 and 4.99 log10 cfu/g or mL, respectively. CONCLUSIONS: Rafoxanide repurposing, as monotherapy and in combination with CMS, may address the urgent need for new treatments for infections caused by MDR-GNB.

Rafoxanide promotes apoptosis and autophagy of gastric cancer cells by suppressing PI3K /Akt/mTOR pathway.

Rafoxanide is commonly used as anti-helminthic medicine in veterinary medicine, a main compound of salicylanilide. Previous studies have reported that rafoxanide, as an inhibitor of BRAF V600E mutant protein, inhibits the growth of colorectal cancer, multiple myeloma, and skin cancer. However, its therapeutic effect on gastric cancer (GC) and the potential mechanism has not been investigated. Here, we have found that rafoxanide inhibited the proliferation of GC cells in vitro, arrested the cell cycle in the G0/G1 phase, and promoted apoptosis and autophagy in GC cells. Treatment with specific autophagy inhibitor 3-methyladenine drastically inhibited the apoptotic cell death effect by suppressing the switch from autophagy to apoptosis. Mechanistically, we found that rafoxanide inhibited the growth of GC cells in vitro by inhibiting the activity of the PI3K/Akt/mTOR signaling pathway. This process induced autophagy, which essentially resulted in the apoptosis of GC cells. Results from subcutaneous implanted tumor models in nude mice also indicated that rafoxanide inhibited the growth of GC cells in vivo. Taken together, our findings revealed that rafoxanide inhibited the growth of GC cells both in vitro and vivo, indicating a potential drug candidate for the treatment of GC.

Induction of endoplasmic reticulum stress and inhibition of colon carcinogenesis by the anti-helmintic drug rafoxanide.

Colorectal cancer (CRC) remains one of the leading causes of mortality worldwide. Drug repositioning is a promising approach for new cancer therapies, as it provides the opportunity to rapidly advance potentially promising agents into clinical trials. The FDA-approved anti-helminthic drug rafoxanide was recently reported to antagonize the oncogenic function of the BRAF V600E mutant protein, commonly found in CRCs, as well as to inhibit the proliferation of skin cancer cells. These observations prompted us to investigate the potential anti-cancer effects of rafoxanide in CRC models. We found rafoxanide inhibited proliferation in CRC cells, but not in normal colonic epithelial cells. Rafoxanide's anti-proliferative action was associated with marked reduction in cyclin D1 protein levels and accumulation of cells in the G0/G1 phase. These effects relied on selective induction of the endoplasmic reticulum stress (ERS) response in CRC cells and were followed by caspase-dependent cell death. Systemic administration of rafoxanide to Apcmin/+ mice induced to develop CRCs caused ERS activation, proliferation inhibition and apoptosis induction in the neoplastic cells. Collectively, our data suggest rafoxanide might be repurposed as an anti-cancer drug for the treatment of CRC.

Synergistic combinations of anthelmintic salicylanilides oxyclozanide, rafoxanide, and closantel with colistin eradicates multidrug-resistant colistin-resistant Gram-negative bacilli.

Repurposing nonantibiotic drugs for antimicrobial therapy presents a viable approach to drug discovery. Development of therapeutic strategies that overcome existing resistance mechanisms is important especially against those bacterial infections in which treatment options are limited, such as against multidrug-resistant Gram-negative bacilli. Herein, we provide in vitro data that suggest the addition of anthelmintic salicylanilides, including oxyclozanide, rafoxanide, and closantel, in colistin therapy to treat multidrug-resistant colistin-susceptible but more importantly colistin-resistant Gram-negative bacilli. As a stand-alone agent, the three salicylanilides suffered from limited outer membrane permeation in Pseudomonas aeruginosa, with oxyclozanide also susceptible to efflux. Synergy was apparent for the combinations against multidrug-resistant clinical isolates of P. aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae. Susceptibility breakpoints for colistin, but also with polymyxin B, were reached upon addition of 1 µg ml-1 of the corresponding salicylanilide against colistin-resistant Gram-negative bacilli. Furthermore, enhanced bacterial killing was observed in all combinations. Our data corroborate the repositioning of the three salicylanilides as adjuvants to counter resistance to the antibiotic of last resort colistin. Our findings are timely and relevant since the global dissemination of plasmid-mediated colistin resistance had been realized.

Repositioning salicylanilide anthelmintic drugs to treat adenovirus infections.

The repositioning of drugs already approved by regulatory agencies for other indications is an emerging alternative for the development of new antimicrobial therapies. The repositioning process involves lower risks and costs than the de novo development of novel antimicrobial drugs. Currently, infections by adenovirus show a steady increment with a high clinical impact in immunosuppressed and immunocompetent patients. The lack of a safe and efficacious drug to treat these infections supports the search for new antiviral drugs. Here we evaluated the anti-adenovirus activity of niclosanide, oxyclozanide, and rafoxanide, three salicylanilide anthelmintic drugs. Also, we carried out the cytotoxicity evaluation and partial characterization of the mechanism of action of these drugs. The salicylanilide anthelmintic drugs showed significant anti-adenovirus activity at low micromolar concentrations with little cytotoxicity. Moreover, our mechanistic assays suggest differences in the way the drugs exert anti-adenovirus activity. Niclosamide and rafoxanide target transport of the HAdV particle from the endosome to the nuclear envelope, whilst oxyclozanide specifically targets adenovirus immediately early gene E1A transcription. Data suggests that the studied salicylanilide anthelmintic drugs could be suitable for further clinical evaluation for the development of new antiviral drugs to treat infections by adenovirus in immunosuppressed patients and in immunocompetent individuals with community-acquired pneumonia.

Rafoxanide, an organohalogen drug, triggers apoptosis and cell cycle arrest in multiple myeloma by enhancing DNA damage responses and suppressing the p38 MAPK pathway.

Rafoxanide is used in veterinary medicine for the treatment of fascioliasis. We previously repositioned the drug as the inhibitor of B-Raf V600E, but its anti-tumor effect in human cancer has never been reported. In this study, we investigated the effects of rafoxanide in multiple myeloma (MM) in vitro and in vivo. We found that rafoxanide inhibited cell proliferation and overcame the protective effect of the bone marrow (BM) microenvironment on MM cells. Rafoxanide induced cell apoptosis by reducing mitochondrial membrane potential (MMP) and regulating the caspase pathway, while having no apparent toxic effect on normal cells. Rafoxanide also inhibited DNA synthesis and caused cell cycle arrest by regulating the cdc25A-degradation pathway. In addition, rafoxanide enhanced the DNA damage response by up-regulating the expression of γ-H2AX, and suppressed activation of the p38 MAPK pathway by down-regulating p38 MAPK phosphorylation and Stat1 phosphorylation. Rafoxanide treatment inhibited tumor growth, with no significant side effects, in an MM mouse xenograft model. Combination of rafoxanide with bortezomib or lenalidomide significantly induced synergistic cytotoxicity in MM cells. Finally, rafoxanide had anti-proliferation effect on both wild type and B-Raf V600E mutated MM cells. And the weaker anti-MM activity of rafoxanide than vemurafenib may indicate other potential mechanisms besides targeting B-Raf V600E mutation. Collectively, our results provide a rationale for use of this drug in MM treatment.

Discovery of rafoxanide as a dual CDK4/6 inhibitor for the treatment of skin cancer.

Since cyclin­dependent kinases 4/6 (CDK4/6) play pivotal roles in cell cycle regulation and are overexpressed in human skin cancers, CDK4/6 inhibitors are potentially effective drugs for skin cancer. In the present study, we present a mixed computational and experimental study attempting to repurpose approved small­molecule drugs as dual CDK4/6 inhibitors for skin cancer treatment. We performed structure­based virtual screening using the docking software idock, targeting an ensemble of CDK4/6 structures. We identified and selected nine compounds with significant predicted scores, and evaluated their cytotoxic effects in vitro in A375 and A431 human skin cancer cell lines. Rafoxanide was found to exhibit the highest cytotoxic effects (IC50: 1.09 µM for A375 and 1.31 µM for A431 cells). Consistent with the expected properties of CDK4/6 inhibitors, rafoxanide significantly increased the G1 phase population. Notably, we revealed that rafoxanide specifically decreased the expression of CDK4/6, cyclin D, retinoblastoma protein (Rb) and the phosphorylation of CDK4/6 and Rb. Furthermore, the anticancer effect of rafoxanide was demonstrated in vivo in BALB/C nude mice subcutaneously xenografted with human skin cancer A375 cells. Rafoxanide (40 mg/kg, i.p.) exhibited significant antitumor activity, comparable to that of oxaliplatin (5 mg/kg, i.p.). The combined administration of rafoxanide and oxaliplatin produced a synergistic therapeutic effect. To the best of our knowledge, the present study is the first to indicate that rafoxanide inhibits CDK4/6 activity and is a potential candidate drug for the treatment of human skin cancer.

Repurposing the anthelmintic drug niclosamide to combat Helicobacter pylori.

There is an urgent need to discover novel antimicrobial therapies. Drug repurposing can reduce the time and cost risk associated with drug development. We report the inhibitory effects of anthelmintic drugs (niclosamide, oxyclozanide, closantel, rafoxanide) against Helicobacter pylori strain 60190 and pursued further characterization of niclosamide against H. pylori. The MIC of niclosamide against H. pylori was 0.25 µg/mL. Niclosamide was stable in acidic pH and demonstrated partial synergy with metronidazole and proton pump inhibitors, such as omeprazole and pantoprazole. Niclosamide administration at 1 × MIC concentration, eliminated 3-log10 CFU of H. pylori adhesion/invasion to AGS cells. Interestingly, no resistance developed even after exposure of H. pylori bacteria to niclosamide for 30 days. The cytotoxic assay demonstrated that niclosamide is not hemolytic and has an IC50 of 4 µg/mL in hepatic and gastric cell lines. Niclosamide administration decreased transmembrane pH as determined by DiSC3(5) assay indicating that the mechanism of action of the anti-H. pylori activity of niclosamide was the disruption of H. pylori proton motive force. Niclosamide was effective in the Galleria mellonella-H. pylori infection model (p = 0.0001) and it can be develop further to combat H. pylori infection. However, results need to be confirmed with other H. pylori and clinical strains.

Repurposing niclosamide for intestinal decolonization of vancomycin-resistant enterococci.

Enterococci are commensal micro-organisms present in the gastrointestinal tract of humans. Although normally innocuous to the host, strains of enterococcus exhibiting resistance to vancomycin (VRE) have been associated with high rates of infection and mortality in immunocompromised patients. Decolonization of VRE represents a key strategy to curb infection in highly-susceptible patients. However, there is a dearth of decolonizing agents available clinically that are effective against VRE. The present study found that niclosamide, an anthelmintic drug, has potent antibacterial activity against clinical isolates of vancomycin-resistant Enterococcus faecium (minimum inhibitory concentration 1-8 µg/mL). E. faecium mutants exhibiting resistance to niclosamide could not be isolated even after multiple (10) serial passages. Based upon these promising in-vitro results and the limited permeability of niclosamide across the gastrointestinal tract (when administered orally), niclosamide was evaluated in a VRE colonization-reduction murine model. Remarkably, niclosamide outperformed linezolid, an antibiotic used clinically to treat VRE infections. Niclosamide was as effective as ramoplanin in reducing the burden of vancomycin-resistant E. faecium in the faeces, caecal content and ileal content of infected mice after only 8 days of treatment. Linezolid, in contrast, was unable to decrease the burden of VRE in the gastrointestinal tract of mice. The results obtained indicate that niclosamide warrants further evaluation as a novel decolonizing agent to suppress VRE infections.

Rafoxanide and Closantel Inhibit SPAK and OSR1 Kinases by Binding to a Highly Conserved Allosteric Site on Their C-terminal Domains.

SPAK and OSR1 are two protein kinases that have emerged as attractive targets in the discovery of novel antihypertensive agents due to their role in regulating electrolyte balance in vivo. Herein we report the identification of an allosteric pocket on the highly conserved C-terminal domains of these two kinases, which influences their activity. We also show that some known WNK signaling inhibitors bind to this allosteric site. Using in silico screening, we identified the antiparasitic agent rafoxanide as a novel allosteric inhibitor of SPAK and OSR1. Collectively, this work will facilitate the rational design of novel SPAK and OSR1 kinase inhibitors that could be useful antihypertensive agents.

Repositioning organohalogen drugs: a case study for identification of potent B-Raf V600E inhibitors via docking and bioassay.

Drug repositioning has been attracting increasingly attention for its advantages of reducing costs and risks. Statistics showed that around one quarter of the marketed drugs are organohalogens. However, no study has been reported, to the best of our knowledge, to aim at efficiently repositioning organohalogen drugs, which may be attributed to the lack of accurate halogen bonding scoring function. Here, we present a study to show that two organohalogen drugs were successfully repositioned as potent B-Raf V600E inhibitors via molecular docking with halogen bonding scoring function, namely D(3)DOCKxb developed in our lab, and bioassay. After virtual screening by D(3)DOCKxb against the database CMC (Comprehensive Medicinal Chemistry), 3 organohalogen drugs that were predicted to form strong halogen bonding with B-Raf V600E were purchased and tested with ELISA-based assay. In the end, 2 of them, rafoxanide and closantel, were identified as potent inhibitors with IC50 values of 0.07 µM and 1.90 µM, respectively, which are comparable to that of vemurafenib (IC50: 0.17 µM), a marketed drug targeting B-Raf V600E. Single point mutagenesis experiments confirmed the conformations predicted by D(3)DOCKxb. And comparison experiment revealed that halogen bonding scoring function is essential for repositioning those drugs with heavy halogen atoms in their molecular structures.

An improved thyroid hormone reporter assay to determine the thyroid hormone-like activity of amiodarone, bithionol, closantel and rafoxanide.

A number of environmental chemicals have been reported to exhibit thyroid hormone-like activity. Since thyroid hormones play a crucial role in development, it is important to identify chemicals in the environment that are capable of endocrine disruption of thyroid hormone homeostasis. In order to detect thyroid hormone-like activity, the growth of pituitary cell lines has been commonly used as a sensitive marker, albeit with limited specificity to thyroid hormones. Reporter gene assays using the thyroid hormone responsive element (TRE) connected to the luciferase reporter gene have also been developed. Thus far however, this type of assay appears to have limited sensitivity compared to cell growth assays. In the present study, we developed a highly sensitive TRE reporter gene assay by using a pituitary cell line, MtT/E-2, and by culturing cells in a serum-free medium. Our assay was developed in order to detect T3 activity at a concentration of 10(-11)M. This assay identified thyroid hormone-like activity from the antiarrhythmic drug, amiodarone, and from three anti-parasitic drugs, bithionol, closantel and rafoxanide, all commonly used in veterinary medicine. Thyroid hormone-like activity of these compounds was further confirmed by the induction of BCL3 gene expression in MtT/E-2, which is known to be regulated by thyroid hormones. Our improved assay was proved to be a sensitive tool for assessing thyroid hormone-like activity of environmental chemicals.

Effect of three anthelmentics on disposition kinetics of florfenicol in goats.

The pharmacokinetic aspects of florfenicol (FLO) were investigated via intravenous (I.V.) and intramuscular (I.M.) injections in five goats at a dose of 20 mg kg(-1) b.wt. Animals were pre-treated with albendazole orally in a dose of 2.5 mg kg(-1) b.wt, ivermectin or rafoxanide subcutaneously in a dose 0.2 and 7.5 mg kg(-1) b.wt, respectively. Florfenicol was injected intramuscularly two hours following anthelmentic administration and blood samples were taken by jugular venapuncture at standardized intervals. The concentrations of florfenicol (FLO) in serum were determined by high performance liquid chromatography (HPLC) method. The obtained results revealed that ivermectin administration did not induce a significant difference in serum florfenicol concentration between anthelmentic-treated and non-treated goats. On the other hand, goats pre-treated with rafoxanide or albendazole showed a significant decrease in serum florfenicol level as compared to non-anthementic treated goats. The absorption half-life (t(½ab)), C(max), AUMC, AUC and systemic bioavailability (F%) are significantly decreased, whereas elimination half-life (t(½el)) and MRT are increased in goats pre-treated by the three tested anthementics.