Deciphering neuroprotective mechanism of nitroxoline in cerebral ischemia: network pharmacology and molecular modeling-based investigations.

Cerebral ischemia is one of the major causes of death and disability worldwide. Currently, existing approved therapies are based on reperfusion and there is an unmet need to search for drugs with neuroprotective effects. The present study aims to investigate the neuroprotective mechanisms of nitroxoline, a nitro derivative of 8-Hydroxyquinoline, against cerebral ischemia using integrated network pharmacology and molecular docking approaches. Critical analytical tools used were SwissTarget, PharmMapper, BindingDB, DisGeNet, Cytoscape, GeneMANIA, ShinyGo, Metascape, GeneCodis, and Schrodinger GLIDE. Thirty-six overlapping drug and disease targets were identified and used for further analysis. Gene Ontology results showed that nitroxoline enriched the genes involved in biological processes of oxidative stress and apoptotic cell death that are highly implicated in hypoxic injury. KEGG enrichment analysis showed nitroxoline influenced a total of 159 biological pathways, out of which, top pathways involved in cerebral ischemia included longevity regulating pathway, VEGF signaling, EGFR tyrosine kinase inhibitor resistance, IL-17 and HIF-1 pathways, FoxO signaling, and AGE-RAGE pathway. Protein-protein interaction analysis using string database showed PARP1, EGFR, PTEN, BRD4, RAC1, NOS2, MTOR, MAPK3, BCL2, MAPK1, APP, METAP2, MAPK14, SIRT1, PRKAA1, and MCL1 as highly interactive proteins involved in pathogenesis of ischemic stroke regulated by nitroxoline. The highly interactive protein targets were validated by molecular docking studies and molecular dynamic simulations. Amongst all these targets, nitroxoline showed the highest binding affinity towards BRD4 followed by PARP1 and PTEN. Nitroxoline, through network pharmacology analysis, showed a role in regulating proteins, biological processes, and pathways crucial in cerebral ischemia. The current study thus provides a preliminary insight that nitroxoline might be used as a neuroprotectant against cerebral ischemia via modulating the epigenetic reader BRD4 and transcription factors such as RELA, NF-κß1, and SP1. However, further in-vitro and preclinical studies need to be performed for concrete evidence.

A quality-by-design evaluated liquid chromatography method development and validation for the separation and quantification of nitroxoline and its impurities.

A novel, isocratic, sensitive, stability-indicating high-performance liquid chromatography method was developed for the separation and quantification of related substances in nitroxoline (NTL). The chromatographic separation has been achieved on Inertsil ODS-3 V, (250 × 4.6 mm, 5 µm) at 240 nm using ethylenediamine tetraacetic acid buffer and methanol in the ratio of 60:40 v/v as mobile phase. The performance of the method has been checked as per the International Conference on Harmonization guidelines for specificity, linearity, accuracy, precision, and robustness. Regression analysis showed a correlation coefficient value greater than 0.99 for NTL and its three impurities. The detection limit of impurities was in the range of 0.01% (0.05 µg/mL)-0.22% (1.1 µg/mL) indicating the sensitivity of the newly developed method. The accuracy of the method was established based on the recovery obtained between 94.7% and 104.1% for all the impurities. The percentage relative standard deviation obtained for the repeatability was less than 4.0% at the specification level for all impurities. Forced degradation was performed to establish the stability-indicating nature of the method and to know about the degradation products, the quality of a drug substance changes with time under the influence of stress conditions. Thus, the proposed method was validated and found to be specific, sensitive, linear, accurate, precise, reproducible, and beneficial for routine usage.

Application of nitroxoline in urologic oncology - a review of evidence.

The persistence of high incidence and mortality rates associated with urologic cancers underscores the urgent need for effective and safe treatments. Conventional chemotherapy regimens are often limited by their high toxicity, the cancer's drug resistance, and the challenge of managing independently evolving multifocal spread. In this context, a repurposing strategy is particularly enticing. It allows for the introduction of a drug with a known safety profile, thus significantly reducing the costs and time necessary to introduce a new treatment. Nitroxoline (NIT), a drug with a well-established pharmacokinetic profile known for over 50 years and utilised in treating uncomplicated urinary tract infections, has recently garnered attention for its potential oncologic applications. Given the pharmacokinetic properties of NIT, our focus was specifically on urologic cancers in which its excretion profile is most advantageous. We examined all available studies, demonstrating significant effectiveness of NIT in inhibiting angiogenesis, tissue invasion, metastasis formation, and counteracting multidrug resistance. The efficacy and mechanism of action of NIT were found to vary across different cell lines. The findings to date are promising, suggesting that NIT or its derivatives could play a role in oncology, although further research is necessary to fully understand its potential and applicability in cancer treatment.

Successful Treatment of Balamuthia mandrillaris Granulomatous Amebic Encephalitis with Nitroxoline.

A patient in California, USA, with rare and usually fatal Balamuthia mandrillaris granulomatous amebic encephalitis survived after receiving treatment with a regimen that included the repurposed drug nitroxoline. Nitroxoline, which is a quinolone typically used to treat urinary tract infections, was identified in a screen for drugs with amebicidal activity against Balamuthia.

Antimicrobial activity of clioquinol and nitroxoline: a scoping review.

Clioquinol and nitroxoline, two drugs with numerous pharmacological properties fallen into disuse for many decades. The first was considered dangerous due to contraindications and the second mainly because was taken as ineffective, despite its known antibacterial activity. In the last decades, the advances in pharmaceutical chemistry, molecular biology, toxicology and genetics allowed to better understand the cellular action of these compounds, some toxicological issues and/or activity scopes. Thus, a new opportunity for these drugs to be considered as potential antimicrobial agents has arisen. This review contemplates the trajectory of clioquinol and nitroxoline from their emergence to the present day, emphasizing the new studies that indicate the possibility of reintroduction for specific cases.

Efficacy data of halogenated phenazine and quinoline agents and an NH125 analogue to veterinary mycoplasmas.

BACKGROUND: Mycoplasmas primarily cause respiratory or urogenital tract infections impacting avian, bovine, canine, caprine, murine, and reptilian hosts. In animal husbandry, mycoplasmas cause reduced feed-conversion, decreased egg production, arthritis, hypogalactia or agalactia, increased condemnations, culling, and mortality in some cases. Antibiotics reduce transmission and mitigate clinical signs; however, concerning levels of antibiotic resistance in Mycoplasma gallisepticum and M. capricolum isolates exist. To address these issues, we evaluated the minimum inhibitory concentrations (MICs) of halogenated phenazine and quinoline compounds, an N-arylated NH125 analogue, and triclosan against six representative veterinary mycoplasmas via microbroth or agar dilution methods. Thereafter, we evaluated the minimum bactericidal concentration (MBC) of efficacious drugs. RESULTS: We identified several compounds with MICs ≤25 µM against M. pulmonis (n = 5), M. capricolum (n = 4), M. gallisepticum (n = 3), M. alligatoris (n = 3), M. agassizii (n = 2), and M. canis (n = 1). An N-arylated NH125 analogue, compound 21, served as the most efficacious, having a MIC ≤25 µM against all mycoplasmas tested, followed by two quinolines, nitroxoline (compound 12) and compound 20, which were effective against four and three mycoplasma type strains, respectively. Nitroxoline exhibited bactericidal activity among all susceptible mycoplasmas, and compound 21 exhibited bactericidal activity when the MBC was able to be determined. CONCLUSIONS: These findings highlight a number of promising agents from novel drug classes with potential applications to treat veterinary mycoplasma infections and present the opportunity to evaluate preliminary pharmacokinetic indices using M. pulmonis in rodents as an animal model of human infection.

Synthesis and evaluation of a large library of nitroxoline derivatives as pancreatic cancer antiproliferative agents.

Pancreatic cancer (PC) is one of the deadliest carcinomas and in most cases, which are diagnosed with locally advanced or metastatic disease, current therapeutic options are highly unsatisfactory. Based on the anti-proliferative effects shown by nitroxoline, an old urinary antibacterial agent, we explored a large library of newly synthesised derivatives to unravel the importance of the OH moiety and pyridine ring of the parent compound. The new derivatives showed a valuable anti-proliferative effect and some displayed a greater effect as compared to nitroxoline against three pancreatic cancer cell lines with different genetic profiles. In particular, in silico pharmacokinetic data, clonogenicity assays and selectivity indexes of the most promising compounds showed several advantages for such derivatives, as compared to nitroxoline. Moreover, some of these novel compounds had stronger effects on cell viability and/or clonogenic capacity in PC cells as compared to erlotinib, a targeted agent approved for PC treatment.

Turning the Tide against Antibiotic Resistance by Evaluating Novel, Halogenated Phenazine, Quinoline, and NH125 Compounds against Ureaplasma Species Clinical Isolates and Mycoplasma Type Strains.

Escalating levels of antibiotic resistance in mycoplasmas, particularly macrolide resistance in Mycoplasma pneumoniae and M. genitalium, have narrowed our antibiotic arsenal. Further, mycoplasmas lack a cell wall and do not synthesize folic acid, rendering common antibiotics, such as beta-lactams, vancomycin, sulfonamides, and trimethoprim, of no value. To address this shortage, we screened nitroxoline, triclosan, and a library of 20 novel, halogenated phenazine, quinoline, and NH125 analogues against Ureaplasma species and M. hominis clinical isolates from urine. We tested a subset of these compounds (n = 9) against four mycoplasma type strains (M. pneumoniae, M. genitalium, M. hominis, and Ureaplasma urealyticum) using a validated broth microdilution or agar dilution method. Among 72 Ureaplasma species clinical isolates, nitroxoline proved most effective (MIC90, 6.25 µM), followed by an N-arylated NH125 analogue (MIC90, 12.5 µM). NH125 and its analogue had significantly higher MICs against U. urealyticum isolates than against U. parvum isolates, whereas nitroxoline did not. Nitroxoline exhibited bactericidal activity against U. parvum isolates but bacteriostatic activity against the majority of U. urealyticum isolates. Among the type strains, the compounds had the greatest activity against M. pneumoniae and M. genitalium, with 8 (80%) and 5 (71.4%) isolates demonstrating MICs of ≤12.5 µM, respectively. Triclosan also exhibited lower MICs against M. pneumoniae and M. genitalium Overall, we identified a promising range of quinoline, halogenated phenazine, and NH125 compounds that showed effectiveness against M. pneumoniae and M. genitalium and found that nitroxoline, approved for use outside the United States for the treatment of urinary tract infections, and an N-arylated NH125 analogue demonstrated low MICs against Ureaplasma species isolates.

Nitroxoline: an option for the treatment of urinary tract infection with multi-resistant uropathogenic bacteria.

The number of multi-resistant uropathogens is increasing. A multi-morbid patient developed a symptomatic urinary tract infection with two multi-resistant bacteria, namely Klebsiella pneumoniae and Proteus mirabilis. Nitroxoline was the only drug active against both uropathogens. Obviously, nitroxoline can be an option for the therapy of a urinary tract infection with multi-resistant uropathogens.

Cathepsin B inhibitors: Further exploration of the nitroxoline core.

Human cathepsin B is a cysteine protease with many house-keeping functions, such as intracellular proteolysis within lysosomes. Its increased activity and expression have been strongly associated with many pathological processes, including cancers. We present here the design and synthesis of novel derivatives of nitroxoline as inhibitors of cathepsin B. These were prepared either by omitting the pyridine part, or by modifying positions 2, 7, and 8 of nitroxoline. All compounds were evaluated for their ability to inhibit endopeptidase and exopeptidase activities of cathepsin B. For the most promising inhibitors, the ability to reduce extracellular and intracellular collagen IV degradation was determined, followed by their evaluation in cell-based in vitro models of tumor invasion. The presented data show that we have further defined the structural requirements for cathepsin B inhibition by nitroxoline derivatives and provided additional knowledge that could lead to non-peptidic compounds with usefulness against tumor progression.

Structure-based development of nitroxoline derivatives as potential multifunctional anti-Alzheimer agents.

Tremendous efforts have been dedicated to the development of effective therapeutics against Alzheimer's disease, which represents the most common debilitating neurodegenerative disease. Multifunctional agents are molecules designed to have simultaneous effects on different pathological processes. Such compounds represent an emerging strategy for the development of effective treatments against Alzheimer's disease. Here, we report on the synthesis and biological evaluation of a series of nitroxoline-based analogs that were designed by merging the scaffold of 8-hydroxyquinoline with that of a known selective butyrylcholinesterase inhibitor that has promising anti-Alzheimer properties. Most strikingly, compound 8g inhibits self-induced aggregation of the amyloid beta peptide (Aß1-42), inhibits with sub-micromolar potency butyrylcholinesterase (IC50=215 nM), and also selectively complexes Cu(2+). Our study thus designates this compound as a promising multifunctional agent for therapeutic treatment of Alzheimer's disease. The crystal structure of human butyrylcholinesterase in complex with compound 8g is also solved, which suggests ways to further optimize compounds featuring the 8-hydroxyquinoline scaffold.

Novel nitroxoline derivative combating resistant bacterial infections through outer membrane disruption and competitive NDM-1 inhibition.

ABSTRACTNew Delhi metallo-ß-lactamase-1 (NDM-1) has rapidly disseminated worldwide, leading to multidrug resistance and worse clinical prognosis. Designing and developing effective NDM-1 inhibitors is a critical and urgent challenge. In this study, we constructed a library of long-lasting nitroxoline derivatives and identified ASN-1733 as a promising dual-functional antibiotic. ASN-1733 can effectively compete for Ca2+ on the bacterial surface, causing the detachment of lipopolysaccharides (LPS), thereby compromising the outer membrane integrity and permeability and exhibiting broad-spectrum bactericidal activity. Moreover, ASN-1733 demonstrated wider therapeutic applications than nitroxoline in mouse sepsis, thigh and mild abdominal infections. Furthermore, ASN-1733 can effectively inhibit the hydrolytic capability of NDM-1 and exhibits synergistic killing effects in combination with meropenem against NDM-1 positive bacteria. Mechanistic studies using enzymatic experiments and computer simulations revealed that ASN-1733 can bind to key residues on Loop10 of NDM-1, hindering substrate entry into the enzyme's active site and achieving potent inhibitory activity (Ki = 0.22 µM), even in the presence of excessive Zn2+. These findings elucidate the antibacterial mechanism of nitroxoline and its derivatives, expand their potential application in the field of antibacterial agents and provide new insights into the development of novel NDM-1 inhibitors.

Nitroxoline: treatment and prevention of urinary tract infections from the urologist's perspective.

Introduction: Nitroxoline is an old antimicrobial agent with a broad spectrum of pharmacological applications and a unique mechanism of action. However, its use in the treatment and prevention of urinary tract infections (UTIs) has not been popular in the recent past. Recently, nitroxoline is gaining interest, due to frequent drug-resistance in uropathogens. Unfortunately, there are few modern clinical trials assessing this antibiotic. Also, older researchers often do not meet current scientific standards. This review seeks to provide a comprehensive overview of nitroxoline as a viable option in treating uncomplicated lower UTIs. Material and methods: A comprehensive literature search regarding the use of nitroxoline in UTIs was conducted using Pubmed, Cochrane Library and Embase databases. A cross-reference search was also performed. Case reports, editorials and non-peer-reviewed literature were excluded from further analysis. As a result, 21 publications were included in this review. Results: The available literature on nitroxoline's mechanism of action, pharmacokinetics, minimum inhibitory concentrations, in vitro activity and resistance rates strongly suggests that nitroxoline is a potent broad-spectrum antimicrobial agent. Moreover, clinical efficacy of the drug was analyzed - 2 articles proved high eradication rates in women with uncomplicated lower UTIs and 1 reported unsuccessful treatment in geriatric patients with lower complicated and uncomplicated UTIs. Finally, the present data on adverse effects indicate that nitroxoline is well-tolerated. Conclusions: Nitroxoline is an obscure, yet potentially effective and safe antimicrobial agent in uncomplicated lower UTIs. Unfortunately, it is available only in a few countries. Nonetheless, nitroxoline can be useful in urological practice.

Nitroxoline resistance is associated with significant fitness loss and diminishes in vivo virulence of Escherichia coli.

IMPORTANCE: Antimicrobial resistance (AMR) poses a global threat and requires the exploration of underestimated treatment options. Nitroxoline, an effective broad-spectrum antibiotic, does not suffer from high resistance rates in the clinics but surprisingly, it is not heavily used yet. Our findings provide compelling evidence that Nitroxoline resistance renders bacteria unable to cause an infection in vivo, thereby reinvigorating the potential of Nitroxoline in combating AMR.

Induction of Programmed Cell Death in Acanthamoeba culbertsoni by the Repurposed Compound Nitroxoline.

Acanthamoeba is a ubiquitous genus of amoebae that can act as opportunistic parasites in both humans and animals, causing a variety of ocular, nervous and dermal pathologies. Despite advances in Acanthamoeba therapy, the management of patients with Acanthamoeba infections remains a challenge for health services. Therefore, there is a need to search for new active substances against Acanthamoebae. In the present study, we evaluated the amoebicidal activity of nitroxoline against the trophozoite and cyst stages of six different strains of Acanthamoeba. The strain A. griffini showed the lowest IC50 value in the trophozoite stage (0.69 ± 0.01 µM), while the strain A. castellanii L-10 showed the lowest IC50 value in the cyst stage (0.11 ± 0.03 µM). In addition, nitroxoline induced in treated trophozoites of A. culbertsoni features compatibles with apoptosis and autophagy pathways, including chromatin condensation, mitochondrial malfunction, oxidative stress, changes in cell permeability and the formation of autophagic vacuoles. Furthermore, proteomic analysis of the effect of nitroxoline on trophozoites revealed that this antibiotic induced the overexpression and the downregulation of proteins involved in the apoptotic process and in metabolic and biosynthesis pathways.

[Therapy of cystitis with nitroxoline-NitroxWin : Prospective, multicenter, non-interventional study and microbiological resistance surveillance]. / Therapie der Zystitis mit Nitroxolin ­ NitroxWin : Prospektive, multizentrische, nicht-interventionelle Studie und mikrobiologische Untersuchungen zur Resistenzsituation.

BACKGROUND: According to German AWMF S3 guideline nitroxoline is recommended as one of the first-choice antibiotics for treatment of acute uncomplicated cystitis (UC) in women. Under real-world conditions the clinical efficacy of nitroxoline should be checked in a noninterventional, prospective and multicenter study (NIS) and the prevalence of nitroxoline resistance in E. coli be monitored. MATERIALS AND METHODS: Female patients with UC treated with nitroxoline (recommended dosage 250 mg tid for 5 days) were included by urologists, general practitioners (GPs), and internists in family medicine throughout Germany from April-December 2022 and followed for 21-28 days. The diagnosis and course of therapy were judged by the Acute Cystitis Symptom Score (ACSS) questionnaire and laboratory investigations (leukocyturia etc). Separately, a nationwide resistance surveillance was performed during 2019-2020 in collaboration with 23 laboratories to collect urinary E. coli isolates and test their susceptibility to nitroxoline. RESULTS: Of the 316 patients with mean (SD) age of 57.2 (±20.4 [median 62.5]) years who were included in the NIS, 193/248 (86.3%) in the per-protocol group and in 193/263 (81.44%) in the intention-to-treat group were clinically successful. Furthermore, 96% of the patients rated the tolerability of nitroxoline as "very good" or "good". All 272 E. coli isolates tested were susceptible to nitroxoline. CONCLUSIONS: Nitroxoline showed very good clinical results in the NIS, and 100% of the tested E. coli urine isolates were susceptible to nitroxoline. Nitroxoline can still be recommended as one of the first-choice antibiotics for treatment of UC in women.

In silico and in vitro analysis of the mechanisms of action of nitroxoline against some medically important opportunistic fungi.

The increasing resistance to antifungal agents associated with toxicity and interactions turns therapeutic management of fungal infections difficult. This scenario emphasizes the importance of drug repositioning, such as nitroxoline - a urinary antibacterial agent that has shown potential antifungal activity. The aims of this study were to discover the possible therapeutic targets of nitroxoline using an in silico approach, and to determine the in vitro antifungal activity of the drug against the fungal cell wall and cytoplasmic membrane. We explored the biological activity of nitroxoline using PASS, SwissTargetPrediction and Cortellis Drug Discovery Intelligence web tools. After confirmation, the molecule was designed and optimized in HyperChem software. GOLD 2020.1 software was used to predict the interactions between the drug and the target proteins. In vitro investigation evaluated the effect of nitroxoline on the fungal cell wall through sorbitol protection assay. Ergosterol binding assay was carried out to assess the effect of the drug on the cytoplasmic membrane. In silico investigation revealed biological activity with alkane 1-monooxygenase and methionine aminopeptidase enzymes, showing nine and five interactions in the molecular docking, respectively. In vitro results exhibited no effect on the fungal cell wall or cytoplasmic membrane. Finally, nitroxoline has potential as an antifungal agent due to the interaction with alkane 1-monooxygenase and methionine aminopeptidase enzymes, which are not the main human therapeutic targets. These results have potentially revealed a new biological target for the treatment of fungal infections. We also consider that further studies are required to confirm the biological activity of nitroxoline on fungal cells, mainly the confirmation of the alkB gene.

Cathepsin inhibitors nitroxoline and its derivatives inhibit SARS-CoV-2 infection.

The severity of the SARS-CoV-2 pandemic and the recurring (re)emergence of viruses prompted the development of new therapeutic approaches that target viral and host factors crucial for viral infection. Among them, host peptidases cathepsins B and L have been described as essential enzymes during SARS-CoV-2 entry. In this study, we evaluated the effect of potent selective cathepsin inhibitors as antiviral agents. We demonstrated that selective cathepsin B inhibitors, such as the antimicrobial agent nitroxoline and its derivatives, impair SARS-CoV-2 infection in vitro. Antiviral activity observed at early stage of virus entry was cell-type dependent and correlated well with the intracellular content and enzymatic function of cathepsins B or L. Furthermore, tested inhibitors were effective against the ancestral SARS-CoV-2 D614 as well as against the more recent BA.1_4 (Omicron). Taken together, our results highlight the important role of host cysteine cathepsin B in SARS-CoV-2 virus entry and show that cathepsin-specific inhibitors, such as nitroxoline and its derivatives, could be used to treat COVID-19. Finally, these results also suggest that nitroxoline has potential to be further explored as repurposed drug in antiviral therapy.

Repurposing of Nitroxoline as an Alternative Primary Amoebic Meningoencephalitis Treatment.

Among the pathogenic free-living amoebae (FLA), Naegleria fowleri is the etiological agent of a fatal disease known as primary amoebic meningoencephalitis (PAM). Once infection begins, the lesions generated in the central nervous system (CNS) result in the onset of symptoms leading to death in a short period of time. Currently, there is no standardized treatment against the infection, which, due to the high virulence of the parasite, results in a high case fatality rate (>97%). Therefore, it is essential to search for new therapeutic sources that can generate a rapid elimination of the parasite. In recent years, there have already been several successful examples of drug repurposing, such as Nitroxoline, for which, in addition to its known bioactive properties, anti-Balamuthia activity has recently been described. Following this approach, the anti-Naegleria activity of Nitroxoline was tested. Nitroxoline displayed low micromolar activity against two different strains of N. fowleri trophozoites (IC50 values of 1.63 ± 0.37 µM and 1.17 ± 0.21 µM) and against cyst stages (IC50 of 1.26 ± 0.42 µM). The potent anti-parasitic activity compared to the toxicity produced (selectivity index of 3.78 and 5.25, respectively) in murine macrophages and human cell lines (reported in previous studies), together with the induction of programmed cell death (PCD)-related events in N. fowleri make Nitroxoline a great candidate for an alternative PAM treatment.

Chelation in Antibacterial Drugs: From Nitroxoline to Cefiderocol and Beyond.

In the era of escalating antimicrobial resistance, the need for antibacterial drugs with novel or improved modes of action (MOAs) is a health concern of utmost importance. Adding or improving the chelating abilities of existing drugs or finding new, nature-inspired chelating agents seems to be one of the major ways to ensure progress. This review article provides insight into the modes of action of antibacterial agents, class by class, through the perspective of chelation. We covered a wide scope of antibacterials, from a century-old quintessential chelating agent nitroxoline, currently unearthed due to its newly discovered anticancer and antibiofilm activities, over the commonly used antibacterial classes, to new cephalosporin cefiderocol and a potential future class of tetramates. We show the impressive spectrum of roles that chelation plays in antibacterial MOAs. This, by itself, demonstrates the importance of understanding the fundamental chemistry behind such complex processes.