Chloroxine inhibits pancreatic cancer progression through targeted antagonization of the PI3K/AKT/mTOR signaling pathway / La cloroxina inhibe la progresión del cáncer de páncreas mediante la antagonización dirigida de la vía de señalización PI3K/AKT/mTOR

Background: Patients with pancreatic cancer have a dismal prognosis due to tumor cell infiltration and metastasis. Many reports have documented that EMT and PI3K–AKT–mTOR axis control pancreatic cancer cell infiltration and metastasis. Chloroxine is an artificially synthesized antibacterial compound that demonstrated anti-pancreatic cancer effects in our previous drug-screening trial. We have explored the impact of chloroxine on pancreatic cancer growth, infiltration, migration, and apoptosis. Methods: The proliferation of pancreatic cancer cell lines (PCCs) treated with chloroxine was assessed through real-time cell analysis (RTCA), colony formation assay, CCK-8 assay, as well as immunofluorescence. Chloroxine effects on the infiltrative and migratory capacities of PCCs were assessed via Transwell invasion and scratch experiments. To assess the contents of EMT- and apoptosis-associated proteins in tumor cells, we adopted Western immunoblotting as well as immunofluorescence assays, and flow cytometry to determine chloroxine effects on PCCs apoptosis. The in vivo chloroxine antineoplastic effects were explored in nude mice xenografts. Results: Chloroxine repressed pancreatic cancer cell growth, migration, and infiltration in vitro, as well as in vivo, and stimulated apoptosis of the PCCs. Chloroxine appeared to inhibit PCC growth by Ki67 downregulation; this targeted and inhibited aberrant stimulation of the PI3K–AKT–mTOR signaling cascade, triggered apoptosis in PCC via mitochondria-dependent apoptosis, and modulated the EMT to inhibit PCC infiltration and migration. Conclusions: Chloroxine targeted and inhibited the PI3K–AKT–mTOR cascade to repress PCCs growth, migration, as well as invasion, and triggered cellular apoptosis. Therefore, chloroxine may constitute a potential antineoplastic drug for the treatment of pancreatic cancer.(AU)

Chloroxine inhibits pancreatic cancer progression through targeted antagonization of the PI3K/AKT/mTOR signaling pathway.

BACKGROUND: Patients with pancreatic cancer have a dismal prognosis due to tumor cell infiltration and metastasis. Many reports have documented that EMT and PI3K-AKT-mTOR axis control pancreatic cancer cell infiltration and metastasis. Chloroxine is an artificially synthesized antibacterial compound that demonstrated anti-pancreatic cancer effects in our previous drug-screening trial. We have explored the impact of chloroxine on pancreatic cancer growth, infiltration, migration, and apoptosis. METHODS: The proliferation of pancreatic cancer cell lines (PCCs) treated with chloroxine was assessed through real-time cell analysis (RTCA), colony formation assay, CCK-8 assay, as well as immunofluorescence. Chloroxine effects on the infiltrative and migratory capacities of PCCs were assessed via Transwell invasion and scratch experiments. To assess the contents of EMT- and apoptosis-associated proteins in tumor cells, we adopted Western immunoblotting as well as immunofluorescence assays, and flow cytometry to determine chloroxine effects on PCCs apoptosis. The in vivo chloroxine antineoplastic effects were explored in nude mice xenografts. RESULTS: Chloroxine repressed pancreatic cancer cell growth, migration, and infiltration in vitro, as well as in vivo, and stimulated apoptosis of the PCCs. Chloroxine appeared to inhibit PCC growth by Ki67 downregulation; this targeted and inhibited aberrant stimulation of the PI3K-AKT-mTOR signaling cascade, triggered apoptosis in PCC via mitochondria-dependent apoptosis, and modulated the EMT to inhibit PCC infiltration and migration. CONCLUSIONS: Chloroxine targeted and inhibited the PI3K-AKT-mTOR cascade to repress PCCs growth, migration, as well as invasion, and triggered cellular apoptosis. Therefore, chloroxine may constitute a potential antineoplastic drug for the treatment of pancreatic cancer.

Cloxyquin activates hTRESK by allosteric modulation of the selectivity filter.

The TWIK-related spinal cord K+ channel (TRESK, K2P18.1) is a K2P channel contributing to the maintenance of membrane potentials in various cells. Recently, physiological TRESK function was identified as a key player in T-cell differentiation rendering the channel a new pharmacological target for treatment of autoimmune diseases. The channel activator cloxyquin represents a promising lead compound for the development of a new class of immunomodulators. Identification of cloxyquin binding site and characterization of the molecular activation mechanism can foster the future drug development. Here, we identify the cloxyquin binding site at the M2/M4 interface by mutational scan and analyze the molecular mechanism of action by protein modeling as well as in silico and in vitro electrophysiology using different permeating ion species (K+ / Rb+). In combination with kinetic analyses of channel inactivation, our results suggest that cloxyquin allosterically stabilizes the inner selectivity filter facilitating the conduction process subsequently activating hTRESK.

Activation of TRESK background potassium channels by cloxyquin exerts protective effects against excitotoxic-induced brain injury and neuroinflammation in neonatal rats.

We investigated effects of activation of TRESK channels by selective activator cloxyquin on excitotoxic-induced brain injury and neuroinflammation involving brain mast cells and inflammatory cytokines in neonatal rats. Three different doses of cloxyquin (0.2, 1 and 5 mg/kg) were studied in ibotenate-induced perinatal brain injury (PBI) in P5 rat-pups. Cerebral lesions and mast cells in coronal brain sections were evaluated. Concentrations of activin A, IL-1ß, IL-6 and IL-10 in brain homogenates were measured using ELISA. Cloxyquin dose-dependently exerted protective effects against excitotoxic-induced neonatal brain injury and neuroinflammation. TRESK channels may be a promising new target for the treatment of PBIs.

Nanocrystalline chloroxine possesses broad-spectrum antimicrobial activities and excellent skin tolerability in mice.

Background: Antimicrobial submicrometer particles are being studied as promising interventions against a wide range of skin conditions, such as fungal or bacterial infections. Aims: To submicronize chloroxine, the crystalline compound 5,7-dichloro-8-hydroxyquinoline, by nanoprecipitation and characterize the resulting assemblies. Methods: The chloroxine particles were stabilized by a nonionic surfactant and were studied by a broth microdilution assay against 20 medically important bacteria and fungi. The intervention was studied using a murine model of skin irritation. Results & conclusion: Chloroxine nanoparticles with a diameter of 600-800 nm exhibit good tolerability in terms of skin irritation in vivo and good antimicrobial activity. Thus, the fabricated formulation shows great promise for interventions for both cutaneous infection control and prophylaxis.

Repurposing clinically approved drugs for COVID-19 treatment targeting SARS-CoV-2 papain-like protease.

COVID-19 is a disease caused by SARS-CoV-2, which has led to more than 4 million deaths worldwide. As a result, there is a worldwide effort to develop specific drugs for targeting COVID-19. Papain-like protease (PLpro) is an attractive drug target because it has multiple essential functions involved in processing viral proteins, including viral genome replication and removal of post-translational ubiquitination modifications. Here, we established two assays for screening PLpro inhibitors according to protease and anti-ISGylation activities, respectively. Application of the two screening techniques to the library of clinically approved drugs led to the discovery of tanshinone IIA sulfonate sodium and chloroxine with their IC50 values of lower than 10 µM. These two compounds were found to directly interact with PLpro and their molecular mechanisms of binding were illustrated by docking and molecular dynamics simulations. The results highlight the usefulness of the two developed screening techniques for locating PLpro inhibitors.

Chloroxine overrides DNA damage tolerance to restore platinum sensitivity in high-grade serous ovarian cancer.

High-grade serous cancer (HGSC) accounts for ~67% of all ovarian cancer deaths. Although initially sensitive to platinum chemotherapy, resistance is inevitable and there is an unmet clinical need for novel therapies that can circumvent this event. We performed a drug screen with 1177 FDA-approved drugs and identified the hydroxyquinoline drug, chloroxine. In extensive validation experiments, chloroxine restored sensitivity to both cisplatin and carboplatin, demonstrating broad synergy in our range of experimental models of platinum-resistant HGSC. Synergy was independent of chloroxine's predicted ionophore activity and did not relate to platinum uptake as measured by atomic absorption spectroscopy. Further mechanistic investigation revealed that chloroxine overrides DNA damage tolerance in platinum-resistant HGSC. Co-treatment with carboplatin and chloroxine (but not either drug alone) caused an increase in γH2AX expression, followed by a reduction in platinum-induced RAD51 foci. Moreover, this unrepaired DNA damage was associated with p53 stabilisation, cell cycle re-entry and triggering of caspase 3/7-mediated cell death. Finally, in our platinum-resistant, intraperitoneal in vivo model, treatment with carboplatin alone resulted in a transient tumour response followed by tumour regrowth. In contrast, treatment with chloroxine and carboplatin combined, was able to maintain tumour volume at baseline for over 4 months. In conclusion, our novel results show that chloroxine facilitates platinum-induced DNA damage to restore platinum sensitivity in HGSC. Since chloroxine is already licensed, this exciting combination therapy could now be rapidly translated for patient benefit.

Drug screening to identify compounds to act as co-therapies for the treatment of Burkholderia species.

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics. It is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to many antibiotics, requiring at least two weeks of intravenous treatment with ceftazidime, imipenem or meropenem followed by 6 months of orally delivered co-trimoxazole. This places a large treatment burden on the predominantly middle-income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z' factor of 0.68. We screened a library of 61,250 compounds and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six "best in class" compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours, compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, an initial study demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy to treat infections caused by these bacteria.

Mechanical factors tune the sensitivity of mdx muscle to eccentric strength loss and its protection by antioxidant and calcium modulators.

BACKGROUND: Dystrophin deficiency sensitizes skeletal muscle of mice to eccentric contraction (ECC)-induced strength loss. ECC protocols distinguish dystrophin-deficient from healthy, wild type muscle, and test the efficacy of therapeutics for Duchenne muscular dystrophy (DMD). However, given the large lab-to-lab variability in ECC-induced strength loss of dystrophin-deficient mouse skeletal muscle (10-95%), mechanical factors of the contraction likely impact the degree of loss. Therefore, the purpose of this study was to evaluate the extent to which mechanical variables impact sensitivity of dystrophin-deficient mouse skeletal muscle to ECC. METHODS: We completed ex vivo and in vivo muscle preparations of the dystrophin-deficient mdx mouse and designed ECC protocols within physiological ranges of contractile parameters (length change, velocity, contraction duration, and stimulation frequencies). To determine whether these contractile parameters affected known factors associated with ECC-induced strength loss, we measured sarcolemmal damage after ECC as well as strength loss in the presence of the antioxidant N-acetylcysteine (NAC) and small molecule calcium modulators that increase SERCA activity (DS-11966966 and CDN1163) or lower calcium leak from the ryanodine receptor (Chloroxine and Myricetin). RESULTS: The magnitude of length change, work, and stimulation duration ex vivo and in vivo of an ECC were the most important determinants of strength loss in mdx muscle. Passive lengthening and submaximal stimulations did not induce strength loss. We further showed that sarcolemmal permeability was associated with muscle length change, but it only accounted for a minimal fraction (21%) of the total strength loss (70%). The magnitude of length change also significantly influenced the degree to which NAC and small molecule calcium modulators protected against ECC-induced strength loss. CONCLUSIONS: These results indicate that ECC-induced strength loss of mdx skeletal muscle is dependent on the mechanical properties of the contraction and that mdx muscle is insensitive to ECC at submaximal stimulation frequencies. Rigorous design of ECC protocols is critical for effective use of strength loss as a readout in evaluating potential therapeutics for muscular dystrophy.

RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays.

Elevated cytoplasmic [Ca2+] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca2+ leak from sarcoplasmic reticulum stores via dysregulated ryanodine receptor (RyR) channels. Consequently, RyR is recognized as a high-value target for drug discovery to treat such pathologies. Using a FRET-based high-throughput screening assay that we previously reported, we identified small-molecule compounds that modulate the skeletal muscle channel isoform (RyR1) interaction with calmodulin and FK506 binding protein 12.6. Two such compounds, chloroxine and myricetin, increase FRET and inhibit [3H]ryanodine binding to RyR1 at nanomolar Ca2+. Both compounds also decrease RyR1 Ca2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by > 50% but only slightly affected Ca2+ release in excitation-contraction coupling, which is essential for normal muscle contraction. This report demonstrates a pipeline that effectively filters small-molecule RyR1 modulators towards clinical relevance.

Evaluation of ct-DNA and HSA binding propensity of antibacterial drug chloroxine: Multi-spectroscopic analysis, atomic force microscopy and docking simulation.

In the present study, the binding interactions of chloroxine, an antibacterial drug and antibiotic agent with calf thymus-deoxyribonucleic acid (ct-DNA) and human serum albumin (HSA) have been deliberated under simulative physiological conditions (pH = 7.40) employing multiple biophysical, atomic force microscopy and molecular modeling approaches. The ct-DNA binding properties of chloroxine exhibit that it binds to ct-DNA through a groove binding mode, and the binding constant values were computed employing the absorption and emission spectral data. The fluorescence study shows the presence of the static quenching mechanism in the ct-DNA- chloroxine interaction. These results are further supported by UV-vis spectra. Large complexes contain the ct-DNA chains with an average size of 225.45 nm were observed by employing AFM for chloroxine -ct-DNA. The results revealed that the fluorescence quenching of albumin by chloroxine was a static quenching process as a result of albumin-chloroxine (1:1) complex. The distance between chloroxine and albumin was obtained based on the Förster's theory of non-radiative energy transfer. The results of AFM, synchronous and three-dimensional fluorescence spectra all revealed that chloroxine induced the conformational changes of albumin. Molecular docking technology represents the binding of chloroxine to the major groove of ct-DNA and site I (subdomain II A) of albumin.

Discovery of novel halogenated 8-hydroxyquinoline-based anti-MRSA agents: In vitro and QSAR studies.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has been considered to be one of global health problems due to limited classes of effective antimicrobial drugs. Herein, 8-hydroxyquinoline (8HQ) and its derivatives (1-7) were investigated for their anti-MRSA and antioxidant activities. Cloxyquin (2), a halogenated 8HQ, exerted the highest antimicrobial activity (MIC50 ≤ 5.57 µM) with high safety index, whereas an amino-derivative 7 showed the strongest antioxidant activity. Additionally, quantitative structure-activity relationship (QSAR) study demonstrated that mass, polarizability, topological charge, and van der Waals volume are essential properties governing the anti-MRSA activity. Taken together, cloxyquin was highlighted as a promising compound for further development as a novel anti-MRSA agent. QSAR findings would also benefit for further rational design of novel 8HQ-based compounds to combat the MRSA resistance.

Development of p53-targeting drugs that increase radioresistance in normal tissues.

Radiation damage to normal tissues is a serious concern in radiation therapy. Advances in radiotherapeutic technology have improved the dose distribution of the target volumes and risk organs, but damage to risk organs that are located within the irradiation field still limits the allowable prescription dose. To overcome this dose-limiting toxicity, and to further improve the efficacy of radiotherapy, the development of drugs that protect normal tissues but not cancer tissues from the effects of radiation are expected to be developed based on molecular target-based drugs. p53 is a well-known transcription factor that is closely associated with radiation-induced cell death. In radiation-injured tissues, p53 induces apoptosis in hematopoietic lineages, whereas it plays a radioprotective role in the gastrointestinal epithelium. These facts suggest that p53 inhibitor would be effective for radioprotection of the hematopoietic system, and that a drug that upregulates the radioprotective functions of p53 would enhance the radioresistance of gastrointestinal tissues. In this review, we summarize recent progress regarding the prevention of radiation injury by regulating p53 and provide new strategic insights into the development of radioprotectors in radiotherapy. J. Med. Invest. 66 : 219-223, August, 2019.

Cloxiquine, a traditional antituberculosis agent, suppresses the growth and metastasis of melanoma cells through activation of PPARγ.

Melanoma is one of the most aggressive skin cancers and 5-year survival rate is only 4.6% for metastatic melanoma patients. Current therapies, especially those involving clinical chemotherapy drugs, have achieved remarkable advances. However, their side effects, such as bone marrow suppression, limit the effectiveness of available pharmacological therapies. Therefore, exploring new antimelanoma drugs with less toxicity is critical for the treatment of melanoma. In the present study, we aimed to identify the antimelanoma drugs with ability to repress the proliferation of melanoma cells by using a high-content screening of FDA-approved drug libraries. We found that cloxiquine (CLQ), a traditional antituberculosic drug, exhibited strong inhibitory effects on the growth and metastasis of melanoma cells both in vivo and in vitro. In contrast, CLQ at the tested doses did not show any apparent toxicity in normal melanocytes and in the liver. At the metabolic level, treatment with CLQ decreased glycolysis, thus potentially inhibiting the "Warburg effect" in B16F10 cells. More importantly, combination of CLQ and 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, did not show a synergistic effect on the tumor growth and metastasis, indicating that inhibition of glycolysis is potentially involved in mediating CLQ's antimelanoma function. Bioinformatics analyses revealed that peroxisome proliferator-activated receptor-gamma (PPARγ) served as a potential CLQ target. Mechanistically, CLQ stimulated the transcription and nuclear contents of PPARγ. Furthermore, the specific PPARγ inhibitor GW9662 or PPARγ shRNA partially abolished the effects of CLQ. Collectively, our findings demonstrate that CLQ has a great potential in the treatment of melanoma through activation of PPARγ.

Chemically Modified Derivatives of the Activator Compound Cloxyquin Exert Inhibitory Effect on TRESK (K2P18.1) Background Potassium Channel.

Cloxyquin has been reported as a specific activator of TRESK [TWIK-related spinal cord K+ channel (also known as K2P18.1)] background potassium channel. In this study, we have synthetized chemically modified analogs of cloxyquin and tested their effects on TRESK and other K2P channels. The currents of murine K2P channels, expressed heterologously in Xenopus oocytes, were measured by two-electrode voltage clamp, whereas the native background K+ conductance of mouse dorsal root ganglion (DRG) neurons was examined by the whole-cell patch-clamp method. Some of the analogs retained the activator character of the parent compound, but, more interestingly, other derivatives inhibited mouse TRESK current. The inhibitor analogs (A2764 and A2793) exerted state-dependent effects. The degree of inhibition by 100 µM A2764 (77.8% ± 3.5%, n = 6) was larger in the activated state of TRESK (i.e., after calcineurin-dependent stimulation) than in the resting state of the channel (42.8% ± 11.5% inhibition, n = 7). The selectivity of the inhibitor compounds was tested on several K2P channels. A2793 inhibited TWIK-related acid-sensitive K+ channel (TASK)-1 (100 µM, 53.4% ± 13, 5%, n = 5), while A2764 was more selective for TRESK, it only moderately influenced TREK-1 and TWIK-related alkaline pH-activated K+ channel. The effect of A2764 was also examined on the background K+ currents of DRG neurons. A subpopulation of DRG neurons, prepared from wild-type animals, expressed background K+ currents sensitive to A2764, whereas the inhibitor did not affect the currents in the DRG neurons of TRESK-deficient mice. Accordingly, A2764 may prove to be useful for the identification of TRESK current in native cells, and for the investigation of the role of the channel in nociception and migraine. SIGNIFICANCE STATEMENT: TRESK background potassium channel is a potential pharmacological target in migraine and neuropathic pain. In this study, we have identified a selective inhibitor of TRESK, A2764. This compound can inhibit TRESK in native cells, leading to cell depolarization and increased excitability. This new inhibitor may be of use to probe the role of TRESK channel in migraine and nociception.

Evaluation of the in vitro and in vivo antileishmanial activity of a chloroquinolin derivative against Leishmania species capable of causing tegumentary and visceral leishmaniasis.

The treatment against leishmaniasis presents problems, since the currently used drugs are toxic and/or have high costs. In addition, parasite resistance has increased. As a consequence, in this study, a chloroquinolin derivative, namely 7-chloro-N,N-dimethylquinolin-4-amine or GF1059, was in vitro and in vivo tested against Leishmania parasites. Experiments were performed to evaluate in vitro antileishmanial activity and cytotoxicity, as well as the treatment of infected macrophages and the inhibition of infection using pre-treated parasites. This study also investigated the GF1059 mechanism of action in L. amazonensis. Results showed that the compound was highly effective against L. infantum and L. amazonensis, presenting a selectivity index of 154.6 and 86.4, respectively, against promastigotes and of 137.6 and 74.3, respectively, against amastigotes. GF1059 was also effective in the treatment of infected macrophages and inhibited the infection of these cells when parasites were pre-incubated with it. The molecule also induced changes in the parasites' mitochondrial membrane potential and cell integrity, and caused an increase in the reactive oxygen species production in L. amazonensis. Experiments performed in BALB/c mice, which had been previously infected with L. amazonensis promastigotes, and thus treated with GF1059, showed that these animals presented significant reductions in the parasite load when the infected tissue, spleen, liver, and draining lymph node were evaluated. GF1059-treated mice presented both lower parasitism and low levels of enzymatic markers, as compared to those receiving amphotericin B, which was used as control. In conclusion, data suggested that GF1059 can be considered a possible therapeutic target to be tested against leishmaniasis.

The Synthesis of ³H-Labelled 8-Azido-N6-Benzyladenine and Related Compounds for Photoaffinity Labelling of Cytokinin-Binding Proteins.

The biology of the group of plant hormones termed cytokinins is reviewed to reveal areas where further studies of cytokinin-binding proteins could be significant. Such areas include: inhibition of human tumour cell growth by cytokinin ribosides, the role of cytokinins in the development of diverse micro-organisms including the cyanobacteria and Mycobacterium tuberculosis, the very rapid responses of plant cells to exogenous cytokinins, and other aspects of cytokinin plant biology. Photoaffinity labelling (PAL) coupled to the recent advances in HPLC of proteins and mass spectral analysis and sequencing of proteins, may have relevance to these areas. To facilitate PAL, we present experimental details for two methods for synthesis of 8-azido-N6-benzyladenine, which has the azido affinity group in the preferred position of the purine ring. Synthesis from [2-³H]adenosine yielded the above-mentioned PAL reagent with ³H in the purine ring and also gave labelled 9-riboside and 8-azido-N6,9-dibenzyladenine. 8-Azido-N6-benzyladenine was also prepared from 6,8-dichloropurine by a facile synthesis, which would allow a label to be sited in the benzyl group where substituents can also be introduced to vary cytokinin activity. The use of inactive cytokinin analogues in assessing the significance of PAL is discussed.

Validation of a UPLC-PDA method to study the content and stability of 5-chloro 8-hydroxyquinoline and 5,7-dichloro 8-hydroxyquinoline in medicated feed used in swine farming.

A new, rapid, simple and specific method to determine 5-chloro 8-hydroxyquinoline (5-HQ) and 5,7-dichloro 8-hydroxyquinoline (5,7-HQ) stability in swine feed was optimized and validated. A system consisting of an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm), a mobile phase of acetonitrile-0.1% o-phosphoric acid (55:45 v/v) with a 0.5 mL/min flow rate, and a PDA detector (247 nm) were used. The retention times of 5-HQ and 5,7-HQ, were 0.77 min and 1.6 min, respectively. The pure drug was subjected to acid and alkali hydrolysis, chemical oxidation and UV light degradation to perform forced degradation studies. 5,7-HQ was more susceptible to degradation than 5-HQ. The figures of merit of the method (linearity, accuracy, precision, and robustness) were determined. The method was successfully applied to estimate the stability of both analytes in medicated feed.

Insight into the Molecular Interaction of Cloxyquin (5-chloro-8-hydroxyquinoline) with Bovine Serum Albumin: Biophysical Analysis and Computational Simulation.

Cloxyquin is a potential therapeutic compound possessing various bioactivities, especially antibacterial, antifungal, cardioprotective, and pain relief activities. Herein, the interaction mechanism between cloxyquin and bovine serum albumin (BSA) has been elucidated in order to fulfill its pharmacokinetic and pharmacodynamic gaps essential for further development as a therapeutic drug. Multi-spectroscopic and biophysical model analysis suggested that cloxyquin interacts with BSA via a static process by ground-state complex formation. Its binding behavior emerged as a biphasic fashion with a moderate binding constant at the level of 104 M-1. Thermodynamic analysis and molecular docking simulation concurrently revealed that hydrophobic interaction is a major driving force for BSA-cloxyquin complexation. Binding of cloxyquin tends to slightly enlarge the monomeric size of BSA without a significant increase of aggregate fraction. Cloxyquin preferentially binds into the fatty acid binding site 5 (FA5) of the BSA via hydrophobic interaction amongst its quinoline scaffold and Phe550, Leu531, and Leu574 residues of BSA. The quinoline ring and hydroxyl moiety of cloxyquin also form the π-π interaction and the hydrogen bond with Phe506. Our data indicate a potential function of serum albumin as a carrier of cloxyquin in blood circulation.

Lysosome Membrane Permeabilization and Disruption of the Molecular Target of Rapamycin (mTOR)-Lysosome Interaction Are Associated with the Inhibition of Lung Cancer Cell Proliferation by a Chloroquinoline Analog.

Lysosomes degrade cellular proteins and organelles and regulate cell signaling by providing a surface for the formation of critical protein complexes, notably molecular target of rapamycin (mTOR) complex 1 (mTORC1). Striking differences in the lysosomes of cancer versus normal cells suggest that they could be targets for drug development. Although the lysomotropic drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have been widely investigated, studies have focused on their ability to inhibit autophagy. We synthesized a novel compound, called EAD1, which is structurally related to CQ but is a 14-fold more potent inhibitor of cell proliferation. Here we find that EAD1 causes rapid relocation, membrane permeabilization (LMP), and deacidification of lysosomes, and it induces apoptosis and irreversibly blocks proliferation of human lung cancer H460, H520, H1299, HCC827, and H1703 cells. EAD1 causes dissociation of mTOR from lysosomes and increases mTOR's perinuclear versus cytoplasmic localization, changes previously shown to inactivate mTORC1. The effect on mTOR was not seen with HCQ, even at >10-fold greater concentrations. Phosphorylation of a downstream target of mTORC1, ribosomal protein S6, was inhibited by EAD1. Although EAD1 also inhibited autophagy, it retained full antiproliferative activity in autophagy-deficient H1650 lung cancer cells, which have a biallelic deletion of Atg7, and in H460 Atg7-knockout cells. As Atg7 is critical for the canonical autophagy pathway, it is likely that inhibition of autophagy is not how EAD1 inhibits cell proliferation. Further studies are needed to determine the relationship of LMP to mTORC1 disruption and their relative contributions to drug-induced cell death. These studies support the lysosome as an underexplored target for new drug development.