The 8-hydroxyquinoline derivative, clioquinol, is an alpha-1 adrenoceptor antagonist.

Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) is an antimicrobial agent whose actions as a zinc or copper ionophore and an iron chelator revived the interest in similar compounds for the treatment of fungal and bacterial infections, neurodegeneration and cancer. Recently, we reported zinc ionophores, including clioquinol, cause vasorelaxation in isolated arteries through mechanisms that involve sensory nerves, endothelium and vascular smooth muscle. Here, we report that clioquinol also uniquely acts as a competitive alpha-1 (α1) adrenoceptor antagonist. We employed ex vivo functional vascular contraction and pharmacological techniques in rat isolated mesenteric arteries, receptor binding assays using stabilized solubilized α1 receptor variants, or wild-type human α1-adrenoceptors transfected in COS-7 cells (African green monkey kidney fibroblast-like cells), and molecular dynamics homology modelling based on the recently published α1A adrenoceptor cryo-EM and α1B crystal structures. At higher concentrations, all ionophores including clioquinol cause a non-competitive antagonism of agonist-mediated contraction due to intracellular zinc delivery, as reported previously. However, at lower concentration ranges, clioquinol has an additional mechanism of competitively inhibiting α1-adrenoceptors that contributes to decreasing vascular contractility. Molecular dynamic simulation showed that clioquinol binds stably to the orthosteric binding site (Asp106) of the receptor, confirming the structural basis for competitive α1-adrenoceptor antagonism by clioquinol.

Synergistic activity of clioquinol with voriconazole and amphotericin B against fungi of interest in eye infections.

Keratoplasty represents a risk factor for fungal eye infections, despites the antibacterial actives in the corneal tissue preservation means, it does not contain active substances with antifungal action. Among the most commonly associated fungal agents are the species belonging to the genera Fusarium and Candida. These agents can trigger an infectious process characterized by swift progression associated with high rates of morbidity, causing irreversible damage. Polyene and azole antifungals are the main agents of ocular therapy, however, they demonstrate some limitations, such as their toxicity and fungal resistance. In this context, drug repositioning and the combination of antifungals may be an alternative. Hence, the goal of this study was to investigate the potential activity of clioquinol (CLQ), a derivative of 8-hydroxyquinoline with previously described antifungal activity, along with its triple and quadruple combinations with antifungal agents commonly used in ophthalmic fungal therapy, natamycin (NAT), voriconazole (VRC), and amphotericin B (AMB), against main fungal pathogens in eye infections. The MICs for CLQ ranged from 0.25 to 2.0 µg/mL, for NAT from 4.0 to 32.0 µg/mL, for AMB it ranged from 0.25 to 16.0 µg/mL and for VRC from 0.03125 to 512.0 µg/mL. Among the tested combinations, the VRC-AMB-CLQ combination stands out, which showed a synergistic effect for more than 50 % of the tested strains and did not present antagonistic results against any of them. Toxicity data were similar to those antifungals already used, even with lower potential toxicity. Therefore, both clioquinol and the triple combination VCR-AMB-CLQ exhibited promising profiles for use as active components in corneal tissue preservation medium.

In vitro evaluation of the efficacy of 8-hydroxyquinoline derivatives for the control of Phaeomoniella chlamydospora, the causative agent of Petri disease in grapevines.

AIM: This study evaluates the in vitro efficacy of 8-hydroxyquinoline (8HQ) derivatives in controlling the phytopathogenic fungus Phaeomoniella chlamydospora. METHODS AND RESULTS: The in vitro tests assessed the susceptibility to the minimum inhibitory concentration (MIC), checkerboard assay, mycelial growth (MG) inhibition, and EC50 determination. Among the seven agricultural fungicides tested, tebuconazole (TEB) displayed the lowest MIC, 1.01 µg mL-1, followed by captan (CAP), thiophanate methyl (TM), and mancozeb with MICs of 4.06, 5.46, and 10.62 µg mL-1, respectively. The 8HQ derivatives used in this study were clioquinol and PH 151 (PH) with MICs of 1.09 and 2.02 µg mL-1, respectively. PH associated with TEB and CAP showed synergism and inhibited 95.8% of MG at the highest dose. TEB inhibited 100% of MG at the three highest doses, while associated with PH exhibited the lowest EC50 (0.863 + 0.0381 µg mL-1). CONCLUSIONS: We concluded that the 8HQ derivatives tested controlled effectively the P. chlamydospora in vitro. PH associated with CAP and TEB exhibited a synergistic effect. The association between PH and TM was considered indifferent. IMPACT STATEMENT: This study expands the list of active ingredients tested against P. chlamydospora, with the PH 151 and clioquinol derivatives being tested for the first time. The in vitro efficacy and synergistic action with other fungicides suggest a potential use as a grapevine wound protectant. This association makes it possible to reduce doses and increase the potency of both drugs, reducing the risk of resistance development and harm to humans and the environment.

Understanding Alzheimer's Disease and its Metal Chelation Therapeutics: A Narrative Review.

The neurodegenerative disorders are age-related illnesses that cause the morphology or activity of neurons to deteriorate over time. Alzheimer's disease is the most frequent neurodegenerative illness in the long run. The rate of advancement might vary, even though it is a progressive neurological illness. Various explanations have been proposed, however the true etiology of Alzheimer's disease remains unclear. Most pharmacological interventions are based on the cholinergic theory, that is earliest idea. In accordance with the amyloid hypothesis, the buildup of beta-amyloid in brain regions is the primitive cause of illness. There is no proof that any one strategy is useful in avoiding Alzheimer's disease, though some epidemiological studies have suggested links within various modifiable variables, such as cardiovascular risk, diet and so on. Different metals like zinc, iron, and copper are naturally present in our bodies. In metal chelation therapy drugs are used to jam the metal ions from combining with other molecules in the body. Clioquinol is one of the metal chelation drugs used by researchers. Research on metal chelation is still ongoing. In the present review, we go over the latest developments in prevalence, incidence, etiology, or pathophysiology of our understanding of Alzheimer's disease. Additionally, a brief discussion on the development of therapeutic chelating agents and their viability as Alzheimer's disease medication candidates is presented. We also assess the effect of clioquinol as a potential metal chelator.

Broad susceptibility of Candida auris strains to 8-hydroxyquinolines and mechanisms of resistance.

The fungal pathogen Candida auris represents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in healthcare settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targeting C. auris and narrowed our focus to five Food and Drug Administration (FDA)-approved compounds with inhibitory concentrations under 10 µM for C. auris and significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors of C. auris. To pursue these compounds further, we exposed C. auris to clioquinol in an extended experimental evolution study and found that C. auris developed only twofold to fivefold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulator CAP1 (causing upregulation of the drug transporter MDR1) and in the drug transporter CDR1. These mutations had only modest effects on resistance to traditional antifungal agents, and the CDR1 mutation rendered C. auris more susceptible to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might prevent C. auris from developing resistance to this established antifungal agent. IMPORTANCE The rapidly emerging fungal pathogen Candida auris represents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated 8-hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains of C. auris. Although this compound has been identified in previous screens, we extended the analysis by showing that C. auris developed only modest twofold to fivefold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500-fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated 8-hydroxyquinolines are working inside the fungal cell and should be developed further to combat C. auris and other fungal pathogens. Lohse and colleagues characterize a class of compounds that inhibit the fungal pathogen C. auris. Unlike many other antifungal drugs, C. auris does not readily develop resistance to this class of compounds.

Development of Clioquinol Platinum(IV) Conjugates as Autophagy-Targeted Antimetastatic Agents.

A series of autophagy-targeted antimetastatic clioquinol (CLQ) platinum(IV) conjugates were designed and prepared by incorporating an autophagy activator CLQ into the platinum(IV) system. Complex 5 with the cisplatin core bearing dual CLQ ligands with potent antitumor properties was screened out as a candidate. More importantly, it displayed potent antimetastatic properties both in vitro and in vivo as expected. Mechanism investigation manifested that complex 5 induced serious DNA damage to increase γ-H2AX and P53 expression and caused mitochondria-mediated apoptosis through the Bcl-2/Bax/caspase3 pathway. Then, it promoted prodeath autophagy by suppressing PI3K/AKT/mTOR signaling and activating the HIF-1α/Beclin1 pathway. The T-cell immunity was elevated by restraining the PD-L1 expression and subsequently increasing CD3+ and CD8+ T cells. Ultimately, metastasis of tumor cells was suppressed by the synergistic effects of DNA damage, autophagy promotion, and immune activation aroused by CLQ platinum(IV) complexes. Key proteins VEGFA, MMP-9, and CD34 tightly associated with angiogenesis and metastasis were downregulated.

Clioquinol induces autophagy by down-regulation of calreticulin in human neurotypic SH-SY5Y cells.

Clioquinol (CQ) is considered as a promising drug of neurodegenerative diseases. However, the underlying mechanism is unclear. Our previous study has proved that CQ induces S-phase cell cycle arrest through the elevation of intracellular calcium concentration ([Ca2+]i) with high levels of SERCA2. Furthermore, it could induce autophagy in an intracellular calcium independent manner in human neurotypic SH-SY5Y cells. In this study, the involvement of calreticulin (CRT) in autophagy induced by CQ was investigated. Our results illustrated the endoplasmic reticulum (ER) stress induced by CQ and DTT led to the cell death in different manners. DTT, an ER stress positive control, induced UPR accompanied with up-regulation of CRT and apoptosis, while CQ inhibited UPR accompanied with down-regulation of CRT，resulting in autophagy. Then, overexpression of CRT was shown to cause UPR and decrease [Ca2+]i, leading to cell apoptosis and inhibition of S-phase arrest induced by CQ. While the UPR was alleviated and autophagy was further enhanced in CRT deficient cells by using targeted siRNA. Meanwhile, down-regulation of CRT resulted in [Ca2+]i overload and induction of S-phase arrest. Finally, we found that the effect of CQ on the HT22 cells was similar to that on the SH-SY5Y cells. Our data showed for the first time that CQ decreased expression of CRT, leading to autophagy, an increase of [Ca2+]i, and cell S-phase arrest in the neurotypic cells. The present study describes the cellular signal pathways regulating autophagy by CQ and highlights the potential therapeutic application of CQ in neurodegenerative disorders.

Discovery of novel hybrids containing clioquinol-1-benzyl-1,2,3,6-tetrahydropyridine as multi-target-directed ligands (MTDLs) against Alzheimer's disease.

Based on the multitarget strategy, a series of novel clioquinol-1-benzyl-1,2,3,6-tetrahydropyridine hybrids were identified for the potential treatment of Alzheimer's disease (AD). Biological evaluation in vitro revealed that these hybrids exhibited significant inhibitory activities toward acetylcholinesterase (AChE). The optimal compound, 19n, exhibited excellent AChE inhibitory potency (IC50 = 0.11 µM), appropriate metal chelating functions, modulation of AChE- and metal-induced Aß aggregation, neuroprotection against okadaic acid-induced mitochondrial dysfunction and ROS damage, and interesting properties that reduced p-Tau levels in addition to no toxicity on SH-SY5Y cells observed at a concentration up to 50 µM. Most importantly, compound 19n was more well tolerated (>1200 mg/kg) than donepezil (LD50 = 28.124 mg/kg) in vivo. Moreover, compound 19n demonstrated marked improvements in cognitive and spatial memory in two AD mice models (scopolamine-induced and Aß1-42-induced) and suppressed inflammation induced by Aß1-42 in the cortex. The multifunctional profiles of compound 19n demonstrate that it deserves further investigation as a promising lead in the development of innovatively multifunctional drugs for Alzheimer's disease.

The Zinc Ionophore Clioquinol Reduces Parkinson's Disease Patient-Derived Brain Extracts-Induced Neurodegeneration.

Parkinson's disease (PD) is pathologically characterized by intracellular α-synuclein-rich protein aggregates, named Lewy bodies (LB), and by the progressive loss of dopaminergic neurons in the substantia nigra. Several heavy metals, including zinc (Zn), have been suggested to play a role in PD progression, although the exact role of Zn in neurodegeneration remains to be fully elucidated. To address this gap, we investigated the effects of Zn modulation on the progression of degeneration in mice injected with PD patient-derived LB-extracts carrying toxic α-synuclein aggregates. Zn modulation was achieved using either a clioquinol-enriched diet, a Zn ionophore that redistributes cellular Zn, or a Zn-enriched diet that increases Zn levels. Clioquinol treatment significantly prevented dopaminergic neurodegeneration and reduced α-synuclein-associated pathology in LB-injected mice, while no differences were observed with Zn supplementation. Biochemical analyses further demonstrate that the expression levels of vesicle-specific Zn transporter ZnT3 in the striatum of LB-injected mice treated with clioquinol were decreased, suggesting an intracellular redistribution of Zn. Additionally, we found that clioquinol modulates the autophagy-lysosomal pathway by enhancing lysosomal redistribution within the neuronal compartments. Collectively, we found that in vivo pharmacological chelation of Zn, by dampening Zn-mediated cytotoxicity, can result in an overall attenuation of PD-linked lysosomal alterations and dopaminergic neurodegeneration. The results support zinc chelation as a disease-modifying strategy for treating PD.

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.

Dysregulation of Streptococcus pneumoniae zinc homeostasis breaks ampicillin resistance in a pneumonia infection model.

Streptococcus pneumoniae is the primary cause of community-acquired bacterial pneumonia with rates of penicillin and multidrug-resistance exceeding 80% and 40%, respectively. The innate immune response generates a variety of antimicrobial agents to control infection, including zinc stress. Here, we characterize the impact of zinc intoxication on S. pneumoniae, observing disruptions in central carbon metabolism, lipid biogenesis, and peptidoglycan biosynthesis. Characterization of the pivotal peptidoglycan biosynthetic enzyme GlmU indicates a sensitivity to zinc inhibition. Disruption of the sole zinc efflux pathway, czcD, renders S. pneumoniae highly susceptible to ß-lactam antibiotics. To dysregulate zinc homeostasis in the wild-type strain, we investigated the safe-for-human-use ionophore 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2). PBT2 rendered wild-type S. pneumoniae strains sensitive to a range of antibiotics. Using an invasive ampicillin-resistant strain, we demonstrate in a murine pneumonia infection model the efficacy of PBT2 + ampicillin treatment. These findings present a therapeutic modality to break antibiotic resistance in multidrug-resistant S. pneumoniae.

Synthesis of new clioquinol derivatives as potent α-glucosidase inhibitors; molecular docking, kinetic and structure-activity relationship studies.

Diabetes mellitus is a chronic metabolic disorder with increasing prevalence and long-term complications. The aim of this study was to identify α-glucosidase inhibitory compounds with potential anti-hyperglycemic activity. For this purpose, a series of new clioquinol derivatives 2a-11a was synthesized, and characterized by various spectroscopic techniques. The enzyme inhibitory activities of the resulting derivatives were assessed using an in-vitro mechanism-based assay. All the tested compounds 2a-11a of the series showed a significant α-glucosidase inhibition with IC50 values 43.86-325.81 µM, as compared to the standard drug acarbose 1C50: 875.75 ± 2.08 µM. Among them, compounds 4a, 5a, 10a, and 11a showed IC50 values of 105.51 ± 2.41, 119.24 ± 2.37, 99.15 ± 2.06, and 43.86 ± 2.71 µM, respectively. Kinetic study of the active analogues showed competitive, non-competitive, and mixed-type inhibitions. Furthermore, the molecular docking study was performed to elucidate the binding interactions of most active analogues with the various sites of α-glucosidase enzyme. The results indicate that these compounds have the potential to be further studied as new anti-diabetic agents.

Clioquinol Decreases Levels of Phosphorylated, Truncated, and Oligomerized Tau Protein.

The neuropathological hallmarks of Alzheimer's disease (AD) are senile plaques (SPs), which are composed of amyloid ß protein (Aß), and neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau protein. As bio-metal imbalance may be involved in the formation of NFT and SPs, metal regulation may be a direction for AD treatment. Clioquinol (CQ) is a metal-protein attenuating compound with mild chelating effects for Zn2+ and Cu2+, and CQ can not only detach metals from SPs, but also decrease amyloid aggregation in the brain. Previous studies suggested that Cu2+ induces the hyperphosphorylation of tau. However, the effects of CQ on tau were not fully explored. To examine the effects of CQ on tau metabolism, we used a human neuroblastoma cell line, M1C cells, which express wild-type tau protein (4R0N) via tetracycline-off (TetOff) induction. In a morphological study and ATP assay, up to 10 µM CQ had no effect on cell viability; however, 100 µM CQ had cytotoxic effects. CQ decreased accumulation of Cu+ in the M1C cells (39.4% of the control), and both total and phosphorylated tau protein. It also decreased the activity of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) (37.3% and 60.7% levels of the control, respectively), which are tau kinases. Of note, activation of protein phosphatase 2A (PP2A), which is a tau phosphatase, was also observed after CQ treatment. Fractionation experiments demonstrated a reduction of oligomeric tau in the tris insoluble, sarkosyl soluble fraction by CQ treatment. CQ also decreased caspase-cleaved tau, which accelerated the aggregation of tau protein. CQ activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Although further studies are needed to elucidate the mechanisms responsible for the effects of CQ on tau, CQ may shed light on possible AD therapeutics.

Clioquinol Attenuates Pulmonary Fibrosis through Inactivation of Fibroblasts via Iron Chelation.

Strict control of iron homeostasis is critical for the maintenance of normal lung function. Iron accumulates in the lungs of patients with idiopathic pulmonary fibrosis (PF), but the characteristics of iron metabolism in the pathogenesis of PF and related targeting therapeutics are not well studied. In this study, we investigated the cellular and molecular characteristics of iron metabolism in fibrotic lungs and further explored the efficacy of clioquinol (CQ) for the treatment of PF as well as its functional mechanism. Iron aggregates accumulated in the lungs of patients with idiopathic PF, and FTL (ferritin light chain) transcripts were increased in their pulmonary fibroblasts. In the bleomycin (BLM)-induced PF (BLM-PF) mouse model, pulmonary iron accumulation is a very early and concomitant event of PF. Labile iron pool levels in both fibroblasts and macrophages from the BLM-PF model were elevated, and iron metabolism was dysregulated. CQ attenuated PF induced by BLM and FITC, and iron-saturated CQ did not alleviate BLM-PF. Furthermore, CQ inhibited the activation of fibroblasts, including proliferation, fibrotic differentiation, proinflammatory cytokine secretion, and migration. In conclusion, our study demonstrated that CQ, acting as an iron chelator, attenuates experimental PF through inactivation of fibroblasts, providing support for targeting iron metabolism as a basis for PF treatment.

Clioquinol inhibits cell growth in a SERCA2-dependent manner.

Clioquinol has been reported to act as a potential therapy for neurodegenerative diseases and cancer. However, the underlying mechanism is unclear. We have previously reported that clioquinol induces S-phase cell cycle arrest through the elevation of calcium levels in human neurotypic SH-SY5Y cells. In this study, different types of cells were observed to detect if the effect of clioquinol on intracellular calcium levels is cell type-specific. The Cell Counting Kit-8 assay showed that clioquinol exhibited varying degrees of concentration-dependent cytotoxicity in different cell lines, and that the growth inhibition caused by it was not related to cell source or carcinogenesis. In addition, the inhibition of cell growth by clioquinol was positively associated with its effect on intracellular calcium content ([Ca2+ ]i ). Furthermore, the elevation of [Ca2+ ]i induced by clioquinol led to S-phase cell cycle arrest. Similar to our previous studies, the increase in [Ca2+ ]i was attributed to changes in the expression levels of the calcium pump SERCA2. Comparison of expression levels of SERCA2 between cell lines showed that cells with high levels of SERCA2 were more sensitive to clioquinol. In addition, analysis using UALCAN and the Human Protein Atlas also showed that the expression of SERCA2 in the corresponding human tissues was similar to that of the cells tested in this study, suggesting potential in the application of clioquinol in the future. In summary, our results expand the understanding of the molecular mechanism of clioquinol and provide an important strategy for the rational use of clioquinol.

A Combination of Clioquinol, Zinc and Copper Increases the Abundance and Function of Breast Cancer Resistance Protein in Human Brain Microvascular Endothelial Cells.

Modulating the abundance of the blood-brain barrier (BBB) efflux transporter breast cancer resistance protein (BCRP) has the potential to impact brain levels of drugs and endogenous substrates. Studies have demonstrated that the metal ionophore clioquinol (CQ) increases BBB abundance of P-glycoprotein (P-gp), an effect associated with increased endothelial cell levels of Cu2+. This study therefore assessed whether human brain endothelial (hCMEC/D3) cell abundance and function of BCRP is modulated by CQ. hCMEC/D3 cells were treated with CQ, Zn2+ and Cu2+ (CZC) (0.5 µM, 0.5 µM, 0.1 µM, respectively) for 24 h and BCRP mRNA and protein abundance was determined by Western blot and qPCR, respectively. After a series of optimisation studies assessing specificity of bodipy prazosin (BP) and Ko143 as a substrate and inhibitor of BCRP, respectively, the impact of CZC on BP uptake was assessed. While CZC did not increase mRNA expression of BCRP, BCRP abundance was increased 1.8 ± 0.1-fold; this was associated with a 68.1 ± 3.3% reduction in accumulation of BP in hCMEC/D3 cells. This is the first study to demonstrate that augmenting metal ion availability enhances protein abundance and function of BCRP at the BBB, which may be exploited to modulate CNS access of therapeutics and endogenous substrates.

In silico and in vitro analysis of FAK/MMP signaling axis inhibition by VO-clioquinol in 2D and 3D human osteosarcoma cancer cells.

The study of novel mechanisms of action of vanadium compounds is critical to elucidating the role and importance of these kinds of compounds as antitumor and antimetastatic agents. This work deals with in silico and in vitro studies of one clioquinol oxidovanadium(iv) complex [VO(clioquinol)2], VO(CQ)2, and its regulation of FAK. In particular, we focus on elucidating the relationship of the FAK inhibition, MMP activity and antimetastatic effects of the complex in human bone cancer cells.

Prolongation of metallothionein induction combats Aß and α-synuclein toxicity in aged transgenic Caenorhabditis elegans.

Neurodegenerative disorders (ND) like Alzheimer's (AD), Parkinson's (PD), Huntington's or Prion diseases share similar pathological features. They are all age dependent and are often associated with disruptions in analogous metabolic processes such as protein aggregation and oxidative stress, both of which involve metal ions like copper, manganese and iron. Bush and Tanzi proposed 2008 in the 'metal hypothesis of Alzheimer's disease' that a breakdown in metal homeostasis is the main cause of NDs, and drugs restoring metal homeostasis are promising novel therapeutic strategies. We report here that metallothionein (MT), an endogenous metal detoxifying protein, is increased in young amyloid ß (Aß) expressing Caenorhabditis elegans, whereas it is not in wild type strains. Further MT induction collapsed in 8 days old transgenic worms, indicating the age dependency of disease outbreak, and sharing intriguing parallels to diminished MT levels in human brains of AD. A medium throughput screening assay method was established to search for compounds increasing the MT level. Compounds known to induce MT release like progesterone, ZnSO4, quercetin, dexamethasone and apomorphine were active in models of AD and PD. Thioflavin T, clioquinol and emodin are promising leads in AD and PD research, whose mode of action has not been fully established yet. In this study, we could show that the reduction of Aß and α-synuclein toxicity in transgenic C. elegans models correlated with the prolongation of MT induction time and that knockdown of MT with RNA interference resulted in a loss of bioactivity.

Synergistic association of clioquinol with antifungal drugs against biofilm forms of clinical Fusarium isolates.

BACKGROUND: The influence of biofilm on the complexity of fungal diseases has been reported in recent years, especially in non-invasive mycoses such as keratitis and onychomycosis. The difficulty in treating cases of fusariosis in the human medical clinic exemplifies this situation, because when Fusarium spp. are present in the form of biofilm, the permeation of antifungal agents is compromised. OBJECTIVES: This study proposes an association of clioquinol, an inhibitor of fungal cells with antifungal drugs prescribed to combat fusariosis in humans. METHODS: Susceptibility was assessed by microdilution in broth. Formation of biofilm by staining with violet crystal. Inhibition and removal of biofilm using the MTT colorimetric reagent. Time-kill combination, hypoallergenicity test, cytotoxicity test and toxicity prediction by computer analysis were also performed. RESULTS: Clioquinol associated with voriconazole and ciclopirox inhibited biofilm formation. Possibly, clioquinol acts in the germination and elongation of hyphae, while voriconazole prevents cell adhesion and ciclopirox the formation of the extracellular polymeric matrix. The CLIO-VRC association reduced the biofilm formation by more than 90%, while the CLIO-CPX association prevented over 95%. None of the association was irritating, and over 90% of the leucocytes remained viable. Computational analysis does not reveal toxicity relevant to CLIO, whereas VRC and CPX showed some risks for systemic use, but suitable for topical formulations. CONCLUSIONS: The combination of CLIO-VRC or CLIO-CPX proved to be a promising association strategy in the medical clinic, both in combating fungal keratitis and onychomycosis, since they prevent the initial process of establishing an infection, the formation of biofilm.

In vitro antidermatophytic synergism of double and triple combination of clioquinol with ciclopirox and terbinafine.

BACKGROUND: Dermatophytoses are the most frequent fungal infections worldwide and there have been described clinical resistance to the commonly used antifungals. Clioquinol is an antimicrobial that had the oral formulations withdrawn from the market in the 70s due to the report of neurotoxicity and recently has been considered as an effective alternative for the treatment of dermatophytosis. OBJECTIVES: To evaluate the effect of the double and triple association between clioquinol with terbinafine and ciclopirox on clinical isolates of dermatophytes. The cytotoxicity of these associations on human leukocytes was also verified. METHODS: Checkerboard method was used to evaluate the interaction between antifungal agents. Time-kill assay was used to verify fungicidal action and evaluate the combination with greater effect for TRU47 isolate. Cell viability was assessed by loss of integrity of the leukocyte membrane in order to verify the toxicity. RESULTS: Synergistic interaction was observed in 42% of isolates when terbinafine was associated with clioquinol and in 50% of isolates when ciclopirox was associated with clioquinol. The triple association resulted in synergistic interaction for 75% of the isolates. Clioquinol + terbinafine and triple combination were more effective for TRU47 isolate, and the combinations exhibited a time-dependent fungicidal effect. Furthermore, the results of cell viability demonstrated that clioquinol and terbinafine combination is not cytotoxic to human leukocytes. CONCLUSIONS: Clioquinol in combination with antifungals in the treatment of dermatophytosis can be a therapeutic strategy to overcome problems related to resistance, action spectrum and toxicity of the antifungal drugs used in the clinic.