Molecular Periphery Design Allows Control of the New Nitrofurans Antimicrobial Selectivity.

A series of 13 new 3-substituted 5-(5-nitro-2-furyl)-1,2,4-oxadiazoles was synthesized from different aminonitriles. All compounds were screened in the disc diffusion test at a 100 µg/mL concentration to determine the bacterial growth inhibition zone presence and diameter, and then the minimum inhibitory concentrations (MICs) were determined for the most active compounds by serial dilution. The compounds showed antibacterial activity against ESKAPE bacteria, predominantly suppressing the growth of 5 species out of the panel. Some compounds had similar or lower MICs against ESKAPE pathogens compared to ciprofloxacin, nitrofurantoin, and furazidin. In particular, 3-azetidin-3-yl-5-(5-nitro-2-furyl)-1,2,4-oxadiazole (2h) inhibited S. aureus at a concentration lower than all comparators. Compound 2e (5-(5-nitro-2-furyl)-3-[4-(pyrrolidin-3-yloxy)phenyl]-1,2,4-oxadiazole) was active against Gram-positive ESKAPE pathogens as well as M. tuberculosis. Differences in the molecular periphery led to high selectivity for the compounds. The induced-fit docking (IFD) modeling technique was applied to in silico research. Molecular docking results indicated the targeting of compounds against various nitrofuran-associated biological targets.

The Nitrofuran-Warhead-Equipped Spirocyclic Azetidines Show Excellent Activity against Mycobacterium tuberculosis.

A series of 21 new 7'H-spiro[azetidine-3,5'-furo [3,4-d]pyrimidine]s substituted at the pyrimidine ring second position were synthesized. The compounds showed high antibacterial in vitro activity against M. tuberculosis. Two compounds had lower minimum inhibitory concentrations against Mtb (H37Rv strain) compared with isoniazid. The novel spirocyclic scaffold shows excellent properties for anti-tuberculosis drug development.

Detection of nitrofurazone with metal-organic frameworks and reduced graphene oxide composites: insights from molecular dynamics simulations.

Two-dimensional metal-organic framework (MOF) composites were produced by incorporating Fe-MOFs into reduced graphene oxide (rGO) nanosheets to form Fe-MOF/rGO composites by hydrothermal synthesis. SEM, TEM, XRD, XPS, and measurements of contact angles were used to characterize the composites. TEM studies revealed that the rod-like-shaped Fe-MOFs were extensively dispersed on the rGO sheets. Incorporating Fe-MOF into rGO significantly improves performance due to the large surface area, chemical stability, and high electrical conductivity. The response signals for the electrochemical sensing performance of Fe-MOF/rGO-modified electrodes to nitrofurazone (NFZ) were significantly enhanced. Differential pulse voltammetry was used to detect the NFZ, and the MOF/rGO sensor possesses a lower detection limit (0.77µM) with two dynamic ranges from 0.6-60 to 128-499.3 µM and high sensitivity (1.909 µA·mM-1·cm-2). Moreover, the anti-interference properties of the sensor were quite reproducible and stable. To understand the mechanism responsible for the enhanced sensing performance of the composite, grand canonical Monte Carlo calculations were performed for Fe-MOF/rGO composites with five unit cells of Fe-MOF and four layers of rGO. We attributed the improvement to the fact that the interface between the Fe-MOF and rGO absorbed increased NFZ molecules. The findings reported herein confirm that such Fe-MOF/rGO composites have significantly improved electrochemical performance and practical applicability of sensing nitrofurazone.

Development and validation of a rapid LC-MS/MS method for the confirmatory analysis of the bound residues of eight nitrofuran drugs in meat using microwave reaction.

A rapid analytical method was developed and validated for the analysis of eight bound nitrofurans in animal tissue, shortening laboratory turnaround times from 4 to 2 days. The majority of methodologies for nitrofuran analysis focus on the detection of only four drugs (nitrofurantoin, furazolidone, furaltadone and nitrofurazone), and is time-consuming given the 16-h overnight derivatisation step and a double liquid-liquid extraction. In this study, the narrow scope of analysis was addressed by including further four important nitrofuran drugs (nifursol, nitrofuroxazide, nifuraldezone and nitrovin). Full chromatographic separation was achieved for the metabolites of all eight nitrofurans, using phenyl-hexyl column chemistry and a rigorous optimisation of the mobile phase additives and gradient profile. The conventional, lengthy sample preparation was substantially shortened by replacing the traditional overnight water bath derivatisation with a rapid 2-h microwave-assisted reaction, followed by a modified-QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction. This confirmatory method was fully validated in accordance with the new 2021/808/EC legislation, and was shown to perform satisfactorily when applied to incurred tissues. The decision limit (CCα) for the eight analytes ranged between 0.013 and 0.200 µg kg-1, showing abundant sensitivity given that the current RPA for nitrofurans is 0.5 µg kg-1. This innovative method can play a major role in the surveillance of the illegal use of nitrofuran drugs.

[Rationale for choosing an antibiotic for the treatment of cystitis: recommendations of clinical pharmacologists: A review].

In recent years, the harmonization of domestic and foreign clinical recommendations for the treatment of cystitis has been achieved. Nitrofurans and fosfomycin trometamol are recommended as first line therapy antibiotics, and oral 3rd generation of cephalosporins are recommended as alternative antibiotics; fluoroquinolones are excluded from the recommended medications due to an unfavorable safety profile. The main rationale for inclusion of antibiotics in the recommendations as a first line therapy of cystitis is the level of resistance of uropathogens to antibiotics, primarily Escherichia coli. Stable low level of resistance of E. coli in Russia was noted to nitrofurans and fosfomycin (5%), higher to cephalosporins. Among nitrofurans, furazidine is characterized by higher activity against E. coli compared to nitrofurantoin. The potassium salt of furazidine in dosage form with magnesium carbonate is preferred, since it is characterized by higher bioavailability and provides a therapeutic level of concentrations in urine above the MIC during the entire dosing period. Due to the global increase in the resistance of uropathogens observed in recent years, experts have begun to pay more and more attention to the ecological safety of antimicrobial therapy in order to minimize the risk of concomitant (collateral) damage, contributing to the selection of multi-drug resistant strains of microorganisms. In the latest WHO document of 2021, experts divided antibiotics into three groups (ACCESS, WATCH, RESERVE) according to the priority of choice. The ACCESS group of drugs for the treatment of cystitis includes nitrofurantoin and furazidine as agents with minimal collateral effect, while fosfomycin trometamol and cephalosporins are listed in the WATCH group. Thus, from the standpoint of ecological safety, WHO experts recommend prescribing nitrofurans in the treatment of cystitis in the first line of therapy.

[Lower urinary tract infections in female problems and mistakes].

Lower urinary tract infections in women represent an urgent medical and social problem due to their high prevalence, frequent recurrences, and the need for an interdisciplinary approach to diagnosis and treatment. Timely and adequate diagnosis of the causes of urinary disorders in women with the determination of risk factors, concomitant diseases and isolation of pathogens allows to choose a rational therapy that may alleviate patients symptoms, improve the quality of life, and prevent the transformation of the disease to a chronic form. It is extermely important that treatment should not contribute to the growth of antibiotic resistance of pathogens, not disrupt the normal microbiome, and not aggravate the disturbances of the normal vaginal and intestinal microflora in women.

In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria.

BACKGROUND: Antimicrobial combinations have been proven as a promising approach in the confrontation with multi-drug resistant bacterial pathogens. In the present study, we identify and characterize a synergistic interaction of broad-spectrum nitroreductase-activated prodrugs 5-nitrofurans, with a secondary bile salt, Sodium Deoxycholate (DOC) in growth inhibition and killing of enterobacteria. RESULTS: Using checkerboard assay, we show that combination of nitrofuran furazolidone (FZ) and DOC generates a profound synergistic effect on growth inhibition in several enterobacterial species including Escherichia coli, Salmonella enterica, Citrobacter gillenii and Klebsiella pneumoniae. The Fractional Inhibitory Concentration Index (FICI) for DOC-FZ synergy ranges from 0.125 to 0.35 that remains unchanged in an ampicillin-resistant E. coli strain containing a ß-lactamase-producing plasmid. Findings from the time-kill assay further highlight the synergy with respect to bacterial killing in E. coli and Salmonella. We further characterize the mechanism of synergy in E. coli K12, showing that disruption of the tolC or acrA genes that encode components of multidrug efflux pumps causes, respectively, a complete or partial loss, of the DOC-FZ synergy. This finding indicates the key role of TolC-associated efflux pumps in the DOC-FZ synergy. Overexpression of Nitric Oxide-detoxifying enzyme Hmp results in a three-fold increase in FICI for DOC-FZ interaction, suggesting a role of nitric oxide in the synergy. We further demonstrate that DOC-FZ synergy is largely independent of NfsA and NfsB, the two major activation enzymes of the nitrofuran prodrugs. CONCLUSIONS: This study is to our knowledge the first report of nitrofuran-deoxycholate synergy against Gram-negative bacteria, offering potential applications in antimicrobial therapeutics. The mechanism of DOC-FZ synergy involves FZ-mediated inhibition of TolC-associated efflux pumps that normally remove DOC from bacterial cells. One possible route contributing to that effect is via FZ-mediated nitric oxide production.

Nitrofuran drugs beyond redox cycling: Evidence of Nitroreduction-independent cytotoxicity mechanism.

Nitrofurans (5-nitro-2-hydrazonylfuran as pharmacophore) are a group of widely used antimicrobial drugs but also associated to a variety of side effects. The molecular mechanisms that underlie the cytotoxic effects of nitrofuran drugs are not yet clearly understood. One-electron reduction of 5-nitro group by host enzymes and ROS production via redox cycling have been attributed as mechanisms of cell toxicity. However, the current evidence suggests that nitrofuran ROS generation by itself is uncapable to explain the whole toxic effects associated to nitrofuran consumption, proposing a nitro-reduction independent mechanism of toxicity. In the present work, a series of nitrated and non-nitrated derivatives of nitrofuran drugs were synthesized and evaluated in vitro for their cytotoxicity, ROS-producing capacity, effect on GSH-S-transferase and antibacterial activity. Our studies showed that in human cells non-nitrated derivatives were less toxic than parental drugs but, unexpectedly preserved the ability to generate intracellular ROS in similar amounts to nitrofurans despite not entering into a redox cycle mechanism. In addition, some non-nitrated derivatives although being uncapable to generate ROS exhibited the highest cell toxicity among all derivatives. Inhibition of cytosolic glutathione-S-transferase activity by some derivatives was also observed. Finally, only nitrofuran derivatives displayed antibacterial effect. Results suggest that the combined 2-hydrazonylfuran moiety, redox cycling of 5-nitrofuran, and inhibitory effects on antioxidant enzymes, would be finally responsible for the toxic effects of the studied nitrofurans on mammalian cells.

Single-step synthesis and in vitro anti-mycobacterial activity of novel nitrofurantoin analogues.

The emergence of drug-resistant tuberculosis (DR-TB) as well as the requirement for long, expensive and toxic drug regimens impede efforts to control and eliminate TB. Therefore, there's a need for effective and affordable anti-mycobacterial agents which can shorten the duration of therapy and are active against Mycobacterium tuberculosis (Mtb) in both active and latent phases. Nitrofurantoin (NFT) is a hypoxic agent with activity against a myriad of anaerobic pathogens and, like the first-line TB drug, rifampicin (RIF), kills non-replicating bacilli. However, the poor ability of NFT to cross host cell membranes and penetrate tissue means that it does not reach therapeutic concentrations. To improve TB efficacy of NFT, a series of NFT analogues was synthesized and evaluated in vitro for anti-mycobacterial activity against the laboratory strain, Mtb H37Rv, and for potential cytotoxicity using human embryonic kidney (HEK-293) and Chinese hamster ovarian (CHO) cells. The NFT analogues showed good safety profiles, enhanced anti-mycobacterial potency, improved lipophilicity, as well as reduced protein binding affinity. Analogue 9 which contains an eight carbon aliphatic chain was the most active, equipotent to isoniazid (INH), a major front-line agent, with MIC90 = 0.5 µM, 30-fold more potency than the parent drug, nitrofurantoin (MIC90 = 15 µM), and 100-fold more selective towards mycobacteria. Therefore, 9 was identified as a validated hit for further investigation in the urgent search for new, safe and affordable TB drugs.

Cobalt molybdate nanorods decorated on boron-doped graphitic carbon nitride sheets for electrochemical sensing of furazolidone.

A nanorod-like structured CoMoO4 embedded on boron doped-graphitic carbon nitride composite (CoMoO4/BCN) has been developed by a simple sonochemical method for electrochemical detection of furazolidone (FUZ). Interestingly, the impedance of CoMoO4/BCN fabricated screen-printed carbon electrode (SPCE) possesses a lower resistance charge transfer (Rct), which favors superior electrochemical detection of FUZ. Such CoMoO4/BCN/SPCE exhibits an ultralow detection limit of 1.6 nM with a concentration range of 0.04-408.9 µM, and high sensitivity of 11.6 µA µM-1 cm-2 by DPV method. In addition, biological and water samples were used for demonstration of practical application of CoMoO4/BCN/SPCE towards electrochemical detection of FUZ, and the result exhibits a satisfactory recovery.Graphical abstract.

Novel 5-Nitrofuran-Activating Reductase in Escherichia coli.

The global spread of multidrug-resistant enterobacteria warrants new strategies to combat these pathogens. One possible approach is the reconsideration of "old" antimicrobials, which remain effective after decades of use. Synthetic 5-nitrofurans such as furazolidone, nitrofurantoin, and nitrofurazone are such a class of antimicrobial drugs. Recent epidemiological data showed a very low prevalence of resistance to this antimicrobial class among clinical Escherichia coli isolates in various parts of the world, forecasting the increasing importance of its uses to battle antibiotic-resistant enterobacteria. However, although they have had a long history of clinical use, a detailed understanding of the 5-nitrofurans' mechanisms of action remains limited. Nitrofurans are known as prodrugs that are activated in E. coli by reduction catalyzed by two redundant nitroreductases, NfsA and NfsB. Furazolidone, nevertheless, retains relatively significant antibacterial activity in the nitroreductase-deficient ΔnfsA ΔnfsBE. coli strain, indicating the presence of additional activating enzymes and/or antibacterial activity of the unreduced form. Using genome sequencing, genetic, biochemical, and bioinformatic approaches, we discovered a novel 5-nitrofuran-activating enzyme, AhpF, in E. coli The discovery of a new nitrofuran-reducing enzyme opens new avenues for overcoming 5-nitrofuran resistance, such as designing nitrofuran analogues with higher affinity for AhpF or screening for adjuvants that enhance AhpF expression.

Nanocarbon material-supported conducting poly(melamine) nanoparticle-modified screen-printed carbon electrodes for highly sensitive determination of nitrofuran drugs by adsorptive stripping voltammetry.

The toxicity of nitrofuran drugs has attracted great attention, and the reported electroanalytical methods suffered limited sensitivity. In this work, a sensitive electrochemical assay in the cathodic region is developed to determine four nitrofuran derivatives, including nitrofurantoin (NFT), nitrofurazone (NFZ), furaltadone (FTD), and furazolidone (FZD). The screen-printed carbon electrode (SPCE) was used as the electrode substrate, and the sensing surface was composed of multi-walled carbon nanotube (MWCNT) and conducting poly(melamine) (PME). The overoxidation-pretreated MWCNTs affect the surface morphology of the electrodeposited PME and, thus, the interaction with nitrofuran drugs. The characteristics of the nanocomposite-modified electrode surfaces were well characterized by field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and surface water contact angle experiments. The nanocomposite-modified electrodes exhibited excellent adsorption and electrochemical reduction of nitrofurans by cyclic voltammetry. The proposed assay exhibited a linear range of sub-micro to micro molar concentrations for the four drugs under the optimized differential pulse voltammetric (DPV) technique. The detection limits were found to be in the nanomolar ranges. The developed assay was applied to detect NFT in two real samples, and the results showed good recoveries that ranged from 99.0 to 104.8% and 98.0 to 103.2% for milk and lake water samples, respectively. Graphical abstract ᅟ.

New nitrofurans amenable by isocyanide multicomponent chemistry are active against multidrug-resistant and poly-resistant Mycobacterium tuberculosis.

A set of structurally diverse N-amino δ-lactams decorated with a 5-nitro-2-furyl moiety was synthesized using isocyanide-based multicomponent chemistry and evaluated for antibacterial activity. Three compounds displayed a selective and potent (MIC 22-33µM) inhibition of M. tuberculosis H37Rv strain growth, while other Gram-positive (MRSA and E. faecium) or Gram-negative (E. coli, P. aeruginosa, A. baumannii, K. pneumoniae) pathogens were not affected. The compounds also displayed moderate-low cytotoxicity, as demonstrated in cell line viability assays. Several multidrug- and poly-resistant patient-derived M. tuberculosis strains were found to be susceptible to treatment with these compounds. The three most potent compounds share a significant structural similarity which provides a basis for further scaffold-hopping analog design.

Depletion kinetics of semicarbazide in giant river prawn (Macrobrachium rosenbergii) following nitrofurazone oral administration and its occurrence in an aquaculture farm.

Semicarbazide (SEM), a marker residue used to monitor the use of prohibited drug nitrofurazone (NFZ), is commonly found in wild crustaceans, implying the natural origin. However, the difference between endogenous and exogenous SEM has rarely been investigated. So, tissue-bound SEM was determined in samples collected from giant river prawns cultured in an aquaculture farm and in samples from an experiment where giant river prawns were fed twice a day with NFZ at 30 mg/kg for 5 days. At day 10 of drug withdrawal, muscle SEM of the NFZ-fed prawn was 17.78 ng/g and depleted to 1.18 ng/g at day 90 (half-life 20.31 days) which was significantly higher than the control prawn (usually ≤ 0.1 ng/g). In contrast, the average SEM in the shell was independent of NFZ treatment. SEM was not found in the aquaculture farm samples, implying that the SEM in cultured prawn did not originate from SEM contamination.

Aqueous fate of furaltadone: Kinetics, high-resolution mass spectrometry - based elucidation and toxicity assessment of photoproducts.

Furaltadone (FTD) is an antibiotic belonging to the nitrofurans group. It has been broadly used in livestock and aquaculture for therapeutic purposes, as well as for stimulating promotion. Although the European Union has imposed restrictions on the use of FTD since 1995 due to concerns regarding its toxicity, in many cases FTD has been excessively and/or illegally applied in productive animals in developing countries, because of its high efficacy and low-cost. Unlike other nitrofuran compounds, the hydrolytic and photolytic behavior of FTD in natural aquatic systems has not been thoroughly investigated. To this end, hydrolysis in different pH values and photolysis in aquatic environment, including lake, river and sea water have been both examined. Hydrolysis was found to have an insignificant impact on degradation of FTD in the aquatic environment relevant pH values, whereas indirect photolysis proved to be the main route of its elimination. The identification of tentative photoproducts (PPs) was performed using ultra high performance liquid chromatography coupled to hybrid LTQ/Orbitrap high resolution mass spectrometry. A possible pathway for photolytic transformation of FTD was proposed. Additionally, in silico simulations were used to evaluate the toxicity such as the mutagenicity of FTD and PPs. Complementary to the low-cost and time-limited simulations, an in vitro method (Vibrio Fischeri bioluminescence) was also used to assess ecotoxicity.

Exposure Assessment of Nitrofuran Metabolites in Fish and Honey Produced in Armenia: A Pilot Investigation.

In Armenia, the presence of nitrofuran residues in food products is unacceptable for both domestic sales and export. However, food may contain nitrofuran metabolites (NMs) due to the illegal use of these drugs in the agrofarming practice. This study aimed to identify NMs as the marker residues for nitrofurans in fish and honey produced in Armenia and assess the potential health risks associated with consuming these foods. The commodities studied were natural honey and three species of farmed fish produced by various regions nationwide. Concentrations of the marker metabolites (3-amino-2-oxazolidinone (AOZ), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD), and semicarbazide (SEM)) were determined through an enzyme-linked immunosorbent assay (ELISA) and verified using liquid chromatography-mass spectrometry (LC-MS/MS). Consumer groups were identified based on their average daily intake of foods. Health risk was assessed by calculating the margin of exposure (MOE). Reference values for health risk assessment were obtained from the European Food Safety Authority (EFSA). Results showed that 33.3% of fish samples and 44.4% of honey samples contained NMs, the mean concentrations ranging from 0.05 µg/kg to 0.52 µg/kg. All MOE values obtained were over 10,000, indicating that the detected concentrations of NMs in fish and honey produced in Armenia pose no health risk to consumers. However, these results highlight the illicit use of highly toxic substances and the need for improved control of farming practices.

Fast screening method to determine metabolites of nitrofurans in chicken meat using partitioned dispersive liquid-liquid microextraction combined with HPLC/DAD.

Methods have been developed for the hydrolysis and derivatisation of protein-bound metabolites of nitrofurans and for the extraction of 2-nitrobenzaldehyde derivatives of the metabolites from chicken meat. In this work, the time needed for hydrolysis and derivatisation was reduced from the conventional 16 h to 90 min. Based on partitioned dispersed liquid-liquid microextraction, a method for extracting 2-nitrobenzaldehyde derivatives of metabolites from crude chicken meat has been developed. Under the optimised experimental conditions, enrichment factors (EFs) ranging from 92.8 to 208.9 were obtained. The method was linear over the range of 10-600 µg kg-1 with determination coefficients (r2) between 0.9979 and 0.9996. Intraday and interday repeatability expressed as a percentage RSD, ranged from 2.2% to 11.2%, and 2.7% to 12.4%, respectively. LOD of 1.07-2.25 µg kg-1 and LOQ of 3.09-6.2 µg kg-1 were obtained. The proposed method was applied in the analysis of metabolites of nitrofurans in chicken meat obtained from farmers using them for their domestic consumption and proved free of the analytes. A recovery of 85.2-109.4% with a %RSD ranging between 3.4% and 13.7% was obtained at three spiking levels. The proposed method was successfully further applied for the analysis of target analytes in chicken meat samples purchased from different supermarkets around Roodeport, Gauteng (South Africa). There was no target analyte detected in the analysed samples. Therefore, the developed methods can be used for monitoring the corresponding metabolites of nitrofurans in chicken meat.

Survey of Meat Collected from Commercial Broiler Processing Plants Suggests Low Levels of Semicarbazide Can Be Created during Immersion Chilling.

ABSTRACT: Semicarbazide (SEM) is routinely employed as an indicator for the use of nitrofurazone, a banned antimicrobial. The validity of SEM as a nitrofurazone marker has been scrutinized because of other possible sources of the compound. Nonetheless, a U.S. trade partner rejected skin-on chicken thighs because of SEM detection and suspected nitrofurazone use. Because nitrofurazone has been banned in U.S. broiler production since 2003, we hypothesized that incidental de novo SEM formation occurs during broiler processing. To assess this possibility, raw leg quarters were collected from 23 commercial broiler processing plants across the United States and shipped frozen to our laboratory, where liquid chromatography-mass spectrometry was used to quantitatively assess for SEM. Leg quarter samples were collected at four points along the processing line: hot rehang (transfer from the kill line to the evisceration line), prechill (before the chilling process), postchill (immediately following chilling), and at the point of pack. Thigh meat with skin attached was removed from 535 leg quarters and analyzed in triplicate for SEM concentrations. The concentrations ranged from 0 to 2.67 ppb, with 462 (86.4%) of 535 samples below the regulatory decision level of 0.5 ppb of SEM. The 73 samples over the 0.5-ppb limit came from 21 plants; 53 (72.6%) of positive samples were in meat collected after chilling (postchill or point of pack). The difference in both prevalence and concentration of SEM detected before and after chilling was highly significant (P < 0.0001). These data support our hypothesis that SEM detection in raw broiler meat is related to de novo creation of the chemical during processing.

From Anti-infective Agents to Cancer Therapy: A Drug Repositioning Study Revealed a New Use for Nitrofuran Derivatives.

BACKGROUND: The progression of ovarian cancer seems to be related to HDAC1, HDAC3, and HDAC6 activity. A possible strategy for improving therapies for treating ovarian carcinoma, minimizing the preclinical screenings, is the repurposing of already approved pharmaceutical products as inhibitors of these enzymes. OBJECTIVE: This work was aimed to implement a computational strategy for identifying new HDAC inhibitors for ovarian carcinoma treatment among approved drugs. METHOD: The CHEMBL database was used to construct training, test, and decoys sets for performing and validating HDAC1, HDAC3 and HDAC6 3D-QSAR models obtained by using the FLAP program. Docking and MD simulations were used in combination with the generated models to identify novel potential HDAC inhibitors. Cell viability assays and Western blot analyses were performed on normal and cancer cells for a direct evaluation of the anti-proliferative activity and an in vitro estimation of HDAC inhibition of the compounds selected through in silico screening. RESULT: The best quantitative prediction was obtained for the HDAC6 3D-QSAR model. The screening of approved drugs highlighted a new potential use as HDAC inhibitors for some compounds, in particular nitrofuran derivatives, usually known for their antibacterial activity and frequently used as antimicrobial adjuvant therapy in cancer treatment. Experimental evaluation of these derivatives highlighted a significant antiproliferative activity against cancer cell lines overexpressing HDAC6, and an increase in acetylated alpha-tubulin levels. CONCLUSION: Experimental results support the hypothesis of potential direct interaction of nitrofuran derivatives with HDACs. In addition to the possible repurposing of already approved drugs, this work suggests the nitro group as a new zinc-binding group, able to interact with the catalytic zinc ion of HDACs.

Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures.

Histoplasma capsulatum is a fungus that causes histoplasmosis. The increased evolution of microbial resistance and the adverse effects of current antifungals help new drugs to emerge. In this work, fifty-four nitrofurans and indoles were tested against the H. capsulatum EH-315 strain. Compounds with a minimum inhibitory concentration (MIC90) equal to or lower than 7.81 µg/mL were selected to evaluate their MIC90 on ATCC G217-B strain and their minimum fungicide concentration (MFC) on both strains. The quantification of membrane ergosterol, cell wall integrity, the production of reactive oxygen species, and the induction of death by necrosis-apoptosis was performed to investigate the mechanism of action of compounds 7, 11, and 32. These compounds could reduce the extracted sterol and induce necrotic cell death, similarly to itraconazole. Moreover, 7 and 11 damaged the cell wall, causing flaws in the contour (11), or changing the size and shape of the fungal cell wall (7). Furthermore, 7 and 32 induced reactive oxygen species (ROS) formation higher than 11 and control. Finally, the cytotoxicity was measured in two models of cell culture, i.e., monolayers (cells are flat) and a three-dimensional (3D) model, where they present a spheroidal conformation. Cytotoxicity assays in the 3D model showed a lower toxicity in the compounds than those performed on cell monolayers. Overall, these results suggest that derivatives of nitrofurans and indoles are promising compounds for the treatment of histoplasmosis.