Nitroxoline and its derivatives are potent inhibitors of metallo-ß-lactamases.

Carbapenemases such as metallo-ß-lactamases (MBLs) are spreading among Gram-negative bacterial pathogens. Infections due to these multidrug-resistant bacteria constitute a major global health challenge. Therapeutic strategies against carbapenemase producing bacteria include ß-lactamase inhibitor combinations. Nitroxoline is a broad-spectrum antibiotic with restricted indication for urinary tract infections. In this study, we report on nitroxoline as an inhibitor of MBLs. We investigate the structure-activity relationships of nitroxoline derivatives considering in vitro MBL inhibitory potency in a fluorescence based assay using purified recombinant MBLs, NDM-1 and VIM-1. We investigated the most potent nitroxoline derivative in combination with imipenem against clinical isolates as well as transformants producing MBL by broth microdilution and time-kill kinetics. Our findings demonstrate that nitroxoline derivatives are potent MBL inhibitors and in combination with imipenem overcome MBL-mediated carbapenem resistance.

Rationally repurposed nitroxoline inhibits preclinical models of Epstein-Barr virus-associated lymphoproliferation.

Repurposing of currently used drugs for new indications benefits from known experience with those agents. Rational repurposing can be achieved when newly uncovered molecular activities are leveraged against diseases that utilize those mechanisms. Nitroxoline is an antibiotic with metal-chelating activity used to treat urinary tract infections. This small molecule also inhibits the function of bromodomain and extraterminal (BET) proteins that regulate oncogene expression in cancer. Lymphoproliferation driven by the Epstein-Barr virus (EBV) depends on these same proteins. We therefore tested the efficacy of nitroxoline against cell culture and small animal models of EBV-associated lymphoproliferation. Nitroxoline indeed reduces cell and tumor growth. Nitroxoline also acts faster than the prototype BET inhibitor JQ1. We suggest that this rational repurposing may hold translational promise.

Selective inhibition mechanism of nitroxoline to the BET family: Insight from molecular simulations.

Although the proteins in bromodomain and extra-terminal domain (BET) family are promising therapy drug targets for numerous human diseases, the binding effectiveness is interfered by the competition from non-BET protein BRD9. In this study, molecular docking, molecular dynamics simulations, binding free energy calculations and per-residue energy decomposition methods were employed to clarify the selective inhibition mechanism of nitroxoline. The results showed that the different cavity volume of effective embedding inhibitor and the changes in conserved residues were associated with the significant higher selectivity of inhibitor nitroxoline for BET family than non-BET protein (BRD9). In addition, the non-polar interactions occurred in Phe83, Val87 at ZA loop, and the polar interaction appeared in Met132, Asn135 at BC loop. Therefore, when designing a new inhibitor, it could better improve the inhibitor activity by introducing the heteroatom conjugated pyridine-like moiety and the strong electron-withdrawing nitro-like moiety. Overall, this study not only clarified the molecular mechanism of the selective inhibition of nitroxoline, but also provided insight into designing more effective BET inhibitors in next step.

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.

Targeted Nanoparticles for Co-delivery of 5-FU and Nitroxoline, a Cathepsin B Inhibitor, in HepG2 Cells of Hepatocellular Carcinoma.

BACKGROUND: The first choice of treatment in Hepatocellular Carcinoma (HCC) is 5-fluorouracil (5-FU). Nitroxoline (NIT), a potent inhibitor of Cathepsin B, impairs tumor progression by decreased extracellular matrix degradation. The objective of the current project was designed to target nanoparticles for co-delivery of 5-FU and NIT in order to enhance the 5-FU cytotoxic effects and reduce the metastatic properties of HepG2 cells. METHODS: 5-FU and NIT were loaded in chitosan-chondroitin nanoparticles. To target the CD44 receptors of HepG2 cells, Hyaluronic Acid (HA) was conjugated to the chondroitin by adipic acid dihydrazide and the conjugation was confirmed by FTIR and 1HNMR. After physicochemical characterization and optimization of the processing variables, MTT assay was done on HepG2 and NIH3T3 cell lines to determine the cytotoxic properties of HA targeted nanoparticles. Migration of the cells was studied to compare the co-delivery of the drugs with each drug alone. RESULTS: The optimized nanoparticles showed the particle size of 244.7±16.3nm, PDI of 0.30±0.03, drug entrapment efficiency of 46.3±5.0% for 5-FU and 75.1±0.9% for NIT. The drug release efficiency up to 8 hours was about 37.6±0.9% for 5-FU and 62.9±0.7% for NIT. The co-delivery of 5-FU and NIT in targeted nanoparticles showed significantly more cytotoxicity than the mixture of the two free drugs, non-targeted nanoparticles or each drug alone and reduced the IC50 value of 5-FU from 3.31±0.65µg/ml to 0.17±0.03µg/ml and the migration of HepG2 cells was also reduced to five-fold. CONCLUSION: Co-delivery of 5-FU and NIT by HA targeted chitosan-chondroitin nanoparticles may be promising in HCC.

Organoruthenated Nitroxoline Derivatives Impair Tumor Cell Invasion through Inhibition of Cathepsin B Activity.

Lysosomal cysteine peptidase cathepsin B (catB) is an important tumor-promoting factor involved in tumor progression and metastasis representing a relevant target for the development of new antitumor agents. In the present study, we synthesized 11 ruthenium compounds bearing either the clinical agent nitroxoline that was previously identified as potent selective reversible inhibitor of catB activity or its derivatives. We demonstrated that organoruthenation is a viable strategy for obtaining highly effective and specific inhibitors of catB endo- and exopeptidase activity, as shown using enzyme kinetics and microscale thermophoresis. Furthermore, we showed that the novel metallodrugs by catB inhibition significantly impair processes of tumor progression in in vitro cell based functional assays at low noncytotoxic concentrations. Generally, by using metallodrugs we observed an improvement in catB inhibition, a reduction of extracellular matrix degradation and tumor cell invasion in comparison to free ligands, and a correlation with the reactivity of the monodentate halide leaving ligand.

Nitroxoline induces cell apoptosis by inducing MDM2 degradation in small-cell lung cancer.

The proto-oncogene MDM2 is a nuclear-localized E3 ubiquitin ligase, which promotes tumor formation by targeting tumor suppressor proteins, such as p53, for proteasomal degradation. In this study, the anti-infective drug nitroxoline (NXQ) was screened out to effectively inhibit cell survival of small-cell lung cancer (SCLC) cells, and induce SCLC cell apoptosis by suppressing antiapoptotic proteins (such as Bcl-2 and MCL1) and upregulating proapoptotic protein Bim. In the mechanistic study, NXQ was found to downregulate MDM2 expression by inducing its proteasomal degradation, and thus upregulated p53 expression, which was a substrate protein of MDM2. Moreover, overexpression of MDM2 decreased the cytotoxicity of NXQ on SCLC cells. These results demonstrated that NXQ displayed anti-SCLC activity by suppressing MDM2 expression, which suggested that anti-infective NXQ had potential for SCLC treatment by targeting the MDM2/p53 axis.

Rational design of 5-((1H-imidazol-1-yl)methyl)quinolin-8-ol derivatives as novel bromodomain-containing protein 4 inhibitors.

Bromodomain-containing protein 4 (BRD4), an epigenetic reader of acetyl lysine, has emerged as a promising therapeutic target for many diseases including cancer, inflammation and heart failure. Our previous study reported that nitroxoline, an FDA approved antibiotic, showed potential BRD4 inhibitory activity and antiproliferation activity against leukemia cell lines. In this study, we further explored the structure-activity relationship (SAR) around nitroxoline and employed our previously developed machine learning based activity scoring function BRD4LGR for further analysis. To improve the cellular level activity, physico-chemical properties were optimized using computational approaches. Then the candidates were tested for their ADME/T profiles. Finally, based on this rational hit-to-lead optimization strategy, 3 drug-like BRD4 inhibitors were obtained, with different profiles on cell line selectivity for multiple myeloma, leukemia and triple negative breast cancer. Further mechanism study showed these compounds could down-regulate c-Myc to inhibit cancer cell growth. This work illustrates the application of multiple computer-aided drug design techniques in a hit-to-lead optimization scenario, and provides novel potent BRD4 inhibitors with different phenotype propensities for future cancer treatment.

Albumin-Stabilized Metal-Organic Nanoparticles for Effective Delivery of Metal Complex Anticancer Drugs.

Many metal-organic complexes showed potent anticancer efficacy, but their clinical applications were limited by the lack of administration route because of their poor solubility. To make metal-organic nanoparticles (MONPs) comprising metal complex drugs is a new formulation strategy for their administration. Herein, we developed a facile synthesis of an MONP composed of bovine serum albumin (BSA), Cu2+, and an anticancer agent, 5-nitro-8-hydroxyquinoline (NQ) with albumin as a nanoreactor. The resultant BSA/Cu/NQ nanoparticle (BSA/Cu/NQ NP) showed good stability in different physiological buffers and could target tumors through the enhanced permeability and retention effect and receptor-mediated cellular uptake. As the BSA/Cu/NQ NP could be readily and efficiently internalized by cancer cells, it showed much higher cytotoxic cancer cells than the NQ + Cu(II) complex and NQ. Therefore, the treatment with BSA/Cu/NQ NP noticeably enhanced the anticancer efficacy without causing systemic toxicity, indicating that such a facile preparation method has great potential to prepare other metal complex nanoparticles for drug delivery.

Antitrypanosomatid Pharmacomodulation at Position 3 of the 8-Nitroquinolin-2(1H)-one Scaffold Using Palladium-Catalysed Cross-Coupling Reactions.

An antikinetoplastid pharmacomodulation study at position 3 of the recently described hit molecule 3-bromo-8-nitroquinolin-2(1H)-one was conducted. Twenty-four derivatives were synthesised using the Suzuki-Miyaura cross-coupling reaction and evaluated in vitro on both Leishmania infantum axenic amastigotes and Trypanosoma brucei brucei trypomastigotes. Introduction of a para-carboxyphenyl group at position 3 of the scaffold led to the selective antitrypanosomal hit molecule 3-(4-carboxyphenyl)-8-nitroquinolin-2(1H)-one (21) with a lower reduction potential (-0.56 V) than the initial hit (-0.45 V). Compound 21 displays micromolar antitrypanosomal activity (IC50 =1.5 µm) and low cytotoxicity on the human HepG2 cell line (CC50 =120 µm), having a higher selectivity index (SI=80) than the reference drug eflornithine. Contrary to results previously obtained in this series, hit compound 21 is inactive toward L. infantum and is not efficiently bioactivated by T. brucei brucei type I nitroreductase, which suggests the existence of an alternative mechanism of action.

Novel 8-nitroquinolin-2(1H)-ones as NTR-bioactivated antikinetoplastid molecules: Synthesis, electrochemical and SAR study.

To study the antiparasitic 8-nitroquinolin-2(1H)-one pharmacophore, a series of 31 derivatives was synthesized in 1-5 steps and evaluated in vitro against both Leishmania infantum and Trypanosoma brucei brucei. In parallel, the reduction potential of all molecules was measured by cyclic voltammetry. Structure-activity relationships first indicated that antileishmanial activity depends on an intramolecular hydrogen bond (described by X-ray diffraction) between the lactam function and the nitro group, which is responsible for an important shift of the redox potential (+0.3 V in comparison with 8-nitroquinoline). With the assistance of computational chemistry, a set of derivatives presenting a large range of redox potentials (from -1.1 to -0.45 V) was designed and provided a list of suitable molecules to be synthesized and tested. This approach highlighted that, in this series, only substrates with a redox potential above -0.6 V display activity toward L. infantum. Nevertheless, such relation between redox potentials and in vitro antiparasitic activities was not observed in T. b. brucei. Compound 22 is a new hit compound in the series, displaying both antileishmanial and antitrypanosomal activity along with a low cytotoxicity on the human HepG2 cell line. Compound 22 is selectively bioactivated by the type 1 nitroreductases (NTR1) of L. donovani and T. brucei brucei. Moreover, despite being mutagenic in the Ames test, as most of nitroaromatic derivatives, compound 22 was not genotoxic in the comet assay. Preliminary in vitro pharmacokinetic parameters were finally determined and pointed out a good in vitro microsomal stability (half-life > 40 min) and a 92% binding to human albumin.

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.

Discovery of novel BET inhibitors by drug repurposing of nitroxoline and its analogues.

The BET family of bromodomain-containing proteins (BRDs) is believed to be a promising drug target for therapeutic intervention in a number of diseases including cancer, inflammation and cardiovascular diseases. Hence, there is a great demand for novel chemotypes of BET inhibitors. The drug repurposing strategy offers great benefits to find inhibitors with known safety and pharmacokinetic profiles, thus increasing medicinal chemists' interest in recent years. Using the drug repurposing strategy, a BRD4-specific score based virtual screening campaign on an in-house drug library was conducted followed by the ALPHA screen assay test. Nitroxoline, an FDA-approved antibiotic, was identified to effectively disrupt the interaction between the first bromodomain of BRD4 (bromodomain-containing protein 4) and acetylated H4 peptide with IC50 of 0.98 µM. Nitroxoline inhibited all BET family members with good selectivity against non-BET bromodomain-containing proteins, thus it is defined as a selective BET inhibitor. Based on the crystal structure of the nitroxoline-BRD4_BD1 complex, the mechanism of action as well as BET specificity of nitroxoline were determined. Since the anticancer activity of nitroxoline against MLL leukemia, one of the BET related diseases, has not been studied before, we tested whether nitroxoline might serve as a potential repurposing drug candidate for MLL leukemia. Nitroxoline effectively inhibited the proliferation of MLL leukemia cells by inducing cell cycle arrest and apoptosis. The profound efficacy is, at least in part, due to the inhibition of BET and downregulation of target gene transcription. Our discovery of nitroxoline as a BET inhibitor suggests potential application of nitroxoline and its derivatives for clinical translation in BET family related diseases.

The two faces of aldehyde oxidase: Oxidative and reductive transformations of 5-nitroquinoline.

Aldehyde oxidase (AOX) is a cytosolic enzyme responsible for the metabolism of some drugs and drug candidates. AOX catalyzes the oxidative hydroxylation of substrates including several aliphatic and aromatic aldehydes, and nitrogen-containing heterocyclic compounds. AOX is also reported to catalyze the reductive metabolism of nitro-compounds, N-oxides, sulfoxides, isoxazoles, isothiazoles, nitrite and hydroxamic acids. These reductive transformations are not well understood and are generally believed to only occur at low oxygen concentrations. In this study, we used 5-nitroquinoline (5NQ) as a substrate to further understand both the oxidative and the reductive transformations catalyzed by AOX. In vitro reaction of 5NQ with AOX under aerobic conditions generated the oxidized (2-oxo-5-nitroquinoline, 2-oxo-5NQ), the reduced (5-aminoquinoline, 5AQ) and the oxidized/reduced (2-oxo-5-aminoquinoline, 2-oxo-5AQ) metabolites. Interestingly, in human liver cytosol, co-incubation of 5NQ and known AOX oxidative substrates DACA and phthalazine significantly increased the yield of the reduced metabolite, while oxidized metabolites production decreased. These data indicate that 5NQ can be reduced at atmospheric oxygen concentrations and that the reductive transformation occurs at a second site that is kinetically distinct from the oxidative site.

Biologically active perspective synthesis of heteroannulated 8-nitroquinolines with green chemistry approach.

A new class of pyrazolo[4,3-c]quinoline (5a-i, 7a-b) and pyrano[3,2-c]quinoline (9a-i) derivatives were designed and synthesized in moderate to good yields by microwave conditions. To enhance the yield of pyrano[3,2-c]quinoline derivatives, multicomponent one-pot synthesis has been developed. The synthesized compounds were identified by spectral and elemental analyses. Compounds 9a and 9i showed good antibacterial activity against Gram-positive and Gram-negative bacterial strains. All of the new compounds exhibited weak to moderate antioxidant activity, compound 9d exerted significant antioxidant power. The cytotoxicity of these compounds were also evaluated against MCF-7 (breast) and A549 (Lung) cancer cell lines. Most of the compounds displayed moderate to good cytotoxic activity against these cell lines. Compound 9i was found to be significantly active in this assay and also induced cell death by apoptosis. Molecular docking studies were carried out using EGFR inhibitor in order to determine the molecular interactions.

Fluorescence in complexes based on quinolines-derivatives: a search for better fluorescent probes.

Quinoline-derived fluorescent complexes were designed; synthesized by the reaction of 5-nitro-8-hydroxyquinoline and 5-chloro-8-hydroxyquinoline with Al(3+), Mg(2+), Zn(2+), and Cd(2+) salts (1-8); and characterized. The (1)H NMR spectra of complexes 1 and 5, containing Al(3+), were consistent with an octahedral structure having approximate D3 symmetry, and the results supported the favored facial isomer (fac). Data for complexes 2-4 and 6-8 supported the formation of tetrahedral structures. Intense luminescence was detected for complexes 5-8, even with the naked eye, as indicated by quantum yield values of 0.087, 0.094, 0.051, and 0.021, respectively. Furthermore, in contrast to 5-nitro-8-hydroxyquinoline, the 5-chloro-8-hydroxyquinoline ligand exhibited bands at different energies depending on the coordinated metal, which supported its potential application in ionic and biological probes, as well as in cell imaging.

A Selective Small Molecule DNA2 Inhibitor for Sensitization of Human Cancer Cells to Chemotherapy.

Cancer cells frequently up-regulate DNA replication and repair proteins such as the multifunctional DNA2 nuclease/helicase, counteracting DNA damage due to replication stress and promoting survival. Therefore, we hypothesized that blocking both DNA replication and repair by inhibiting the bifunctional DNA2 could be a potent strategy to sensitize cancer cells to stresses from radiation or chemotherapeutic agents. We show that homozygous deletion of DNA2 sensitizes cells to ionizing radiation and camptothecin (CPT). Using a virtual high throughput screen, we identify 4-hydroxy-8-nitroquinoline-3-carboxylic acid (C5) as an effective and selective inhibitor of DNA2. Mutagenesis and biochemical analysis define the C5 binding pocket at a DNA-binding motif that is shared by the nuclease and helicase activities, consistent with structural studies that suggest that DNA binding to the helicase domain is necessary for nuclease activity. C5 targets the known functions of DNA2 in vivo: C5 inhibits resection at stalled forks as well as reducing recombination. C5 is an even more potent inhibitor of restart of stalled DNA replication forks and over-resection of nascent DNA in cells defective in replication fork protection, including BRCA2 and BOD1L. C5 sensitizes cells to CPT and synergizes with PARP inhibitors.

Synthesis of 8-heteroaryl nitroxoline analogues via one-pot sequential Pd-catalyzed coupling reactions.

A series of 8-heteroaryl substituted quinolines were prepared, either by direct C-H arylation of five-membered heteroarenes, or Pd-catalyzed coupling of organoboron reagents with bromoquinolines. The use of (benzo)thiophenyl or (benzo)furanyl boron coupling partners allowed further C-H functionalization on the five-membered heteroaryl ring with aryl bromides in one flask to access a variety of polyconjugated molecular architectures. The developed methodology represents a simple approach towards 8-arylated analogues of the biologically interesting nitroxoline core.

Inhibition of endopeptidase and exopeptidase activity of cathepsin B impairs extracellular matrix degradation and tumour invasion.

Cathepsin B is a lysosomal cysteine protease that is implicated in a number of physiological processes, including protein turnover in lysosomes. Changes in its expression are associated with a variety of pathological processes, including cancer. Due to the structural feature, termed the occluding loop, cathepsin B differs from other cysteine proteases in possessing both, endopeptidase and exopeptidase activity. Here we investigated the impact of both cathepsin B activities on intracellular and extracellular collagen IV degradation and tumour cell invasion using new selective synthetic inhibitors, 2-{[(8-hydroxy-5-nitroquinoline-7-yl)methyl]amino}-acetonitrile (1), 8-(4-methylpiperidin-1-yl)-5-nitroquinoline (2) and 7-[(4-methylpiperidin-1yl)methyl]-5-nitroquinolin-8-ol (3). All three compounds (5 µM) reduced extracellular degradation of collagen IV by MCF-10A neoT cells by 45-70% as determined by spectrofluorimetry and they (50 µM) attenuated intracellular collagen IV degradation by 40-60% as measured with flow cytometry. Furthermore, all three compounds (5 µM) impaired MCF-10A neoT cell invasion by 40-80% as assessed by measuring electrical impedance in real time. Compounds 1 and 3 (5 µM), but not compound 2, significantly reduced the growth of MMTV-PyMT multicellular tumour spheroids. Collectively, these data suggest that the efficient strategy to impair harmful cathepsin B activity in tumour progression may include simultaneous and potent inhibition of cathepsin B endopeptidase and exopeptidase activities.

[¹¹C]NS9531, [¹¹C]NS9762 and [¹¹C]NS6417, specific SERT tracers: pre-clinical evaluation in pigs and optimization of synthesis conditions using [¹¹C]methyl triflate.

INTRODUCTION: NS9531, NS9762 and NS6417 are nitroquinolinyl-diazabicyclo-alkane derivatives that have been developed as inhibitors of serotonin reuptake transporters (SERT) by NeuroSearch A/S. METHODS: IC50 was measured on the up-take of serotonin, dopamine and noradrenaline in synaptosomes prepared from selected rat brain regions. For the pre-clinical evaluation in pigs, [(11)C]NS9531, [(11)C]NS9762 and [(11)C]NS6417 were prepared by N-methylation using [(11)C]methyl iodide. These syntheses were later on optimized regarding: 1) choice of labelled precursor; 2) HPLC purification conditions; and 3) formulation using SPE columns. The synthesis protocols were then fully automated on a GE FXc Pro. Preclinical evaluation was performed by PET studies in landrace pigs before and after treatment with citalopram. RESULTS: IC50 measurements showed that all three compounds have low nanomolar affinity for SERT, and micromolar affinity for DAT and NET. The radiochemical yield (r.y.) of all three ligands from [(11)C]methyl iodide was higher than 30%. From [(11)C]methyl triflate, the r.y. of [(11)C]NS9531 and [(11)C]NS9762 were higher than 80% whereas the r.y. of [(11)C]NS6417 was 65%. Residual precursor amounts in final products could be significantly reduced by the use of [(11)C]methyl triflate, <0.2 µg compared with <10 µg, calculated for a 300 MBq injection at 20 minutes EOS. The optimized conditions gave 2.5-4.5 GBq of products with a specific radioactivity of 20-70 MBq/nmol, residual acetonitrile 15-30 ppm, and pH 6.5-7.1. All three compounds showed a rapid and comparable high pig brain uptake of about 3%, producing PET images of good contrast, and uptake was reduced after pre-administration with citalopram. CONCLUSION: The three (11)C labelled PET tracers could be prepared in medium to high yield and high purity. IC50 measurements showed that the three NS compounds were highly selective, high affinity SERT inhibitors. PET studies in pig showed high brain uptake that could be blocked by citalopram pre-treatment.