Chloride Conductance Research Articles

Drug Repurposing for Cystic Fibrosis: Identification of Drugs That Induce CFTR-Independent Fluid Secretion in Nasal Organoids.

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including β2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

Bicarbonate defective CFTR variants increase risk for chronic pancreatitis: A meta-analysis.

IntroductionCystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in pancreatic ductal fluid secretion by mediating Cl- and HCO3- ion transport across the apical membrane. Severe CFTR mutations that diminish chloride conductance cause cystic fibrosis (CF) if both alleles are affected, whereas heterozygous carrier status increases risk for chronic pancreatitis (CP). It has been proposed that a subset of CFTR variants characterized by a selective bicarbonate conductance defect (CFTRBD) may be associated with CP but not CF. However, a rigorous genetic analysis of the presumed association has been lacking.AimsTo investigate the role of heterozygous CFTRBD variants in CP by meta-analysis of published case-control studies.Materials and methodsA systematic search was conducted in the MEDLINE, Embase, Scopus, and CENTRAL databases for published studies that reported the CFTRBD variants p.R74Q, p.R75Q, p.R117H, p.R170H, p.L967S, p.L997F, p.D1152H, p.S1235R, and p.D1270N in CP patients and controls.ResultsTwenty-two studies were eligible for quantitative synthesis. Combined analysis of the 9 CFTRBD variants indicated enrichment in CP patients versus controls (OR = 2.31, 95% CI = 1.17–4.56). Individual analysis of CFTRBD variants revealed no association of p.R75Q with CP (OR = 1.12, 95% CI = 0.89–1.40), whereas variants p.R117H and p.L967S were significantly overrepresented in cases relative to controls (OR = 3.16, 95% CI = 1.94–5.14, and OR = 3.88, 95% CI = 1.32–11.47, respectively). The remaining 6 low-frequency variants gave inconclusive results when analyzed individually, however, their pooled analysis indicated association with CP (OR = 2.08, 95% CI = 1.38–3.13).ConclusionHeterozygous CFTRBD variants, with the exception of p.R75Q, increase CP risk about 2-4-fold.

682 Cystic fibrosis transmembrane conductance regulator variants in Fischer rat thyroid cells exhibit distinct bicarbonate to chloride conductance ratios that are affected by forskolin and ivacaftor

682 Cystic fibrosis transmembrane conductance regulator variants in Fischer rat thyroid cells exhibit distinct bicarbonate to chloride conductance ratios that are affected by forskolin and ivacaftor

400 Novel solute carrier family 26 member 9 inhibitor reveals that solute carrier family 26 member 9 is not the constitutive airway chloride conductance

400 Novel solute carrier family 26 member 9 inhibitor reveals that solute carrier family 26 member 9 is not the constitutive airway chloride conductance

Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect

A significant challenge to making targeted cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q, with a potential role contributing to intrapore chloride ion permeation, in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using an ion transport assay and forskolin-induced swelling assay, respectively. CFTR potentiators (VX-770, GLPG1837, and VX-445) and correctors (VX-809, VX-445, with or without VX-661) were tested. Data from R352Q-CFTR were compared with data of 20 participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function that was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics simulations of R352Q-CFTR were carried out, which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico molecular dynamics simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

In a Degraded Stream, Increased Salt Concentration is Correlated with Decreased EPT Diversity

Many streams in the Northern Panhandle of West Virginia are biologically impaired due to historical and concurrent anthropogenic activities, including mining, agriculture, and urbanization. These activities alter water chemistry which affects the benthic macroinvertebrate communities. While taxa like Ephemeroptera, Plecoptera, and Trichoptera (EPT taxa) are sensitive to water pollution, other taxa like tipulids, beetles, and chironomid midges are more pollution tolerant. However, responses to specific chemical pollutants are taxa specific and even some EPT are relatively pollution tolerant. We investigated how increases in chloride (Cl) and specific conductance (SPC) affected the macroinvertebrate community in a stream already heavily impacted by mining, urbanization and agriculture. Five upstream sampling sites were placed in a degraded stream above the outfall of an active coal mine, while five additional sites were placed downstream of the discharge. Water chemistry was measured in each site, and macroinvertebrates were collected with kick nets, leaf packs and a visual collection of unique habitats following the Stream Monitoring Information Exchange (SMIE) protocol. We found that temperature, SPC, and Cl were significantly higher downstream of the mine outfall with SPC and Cl more than doubling. SMIE water quality scores and whole community Shannon-Wiener Diversity Index scores were not significantly different between upstream and downstream sites but EPT diversity was significantly higher upstream of the mine. This data helps determine taxa specific responses to changes in water chemistry, specifically SPC and Cl, and highlights how streams already afflicted with poor water quality can lose additional taxa when the concentrations of salts increases.

Systemic bis-phosphinic acid derivative restores chloride transport in Cystic Fibrosis mice

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) are responsible for Cystic Fibrosis (CF). The most common CF-causing mutation is the deletion of the 508th amino-acid of CFTR (F508del), leading to dysregulation of the epithelial fluid transport in the airway’s epithelium and the production of a thickened mucus favoring chronic bacterial colonization, sustained inflammation and ultimately respiratory failure. c407 is a bis-phosphinic acid derivative which corrects CFTR dysfunction in epithelial cells carrying the F508del mutation. This study aimed to investigate c407 in vivo activity in the F508del Cftrtm1Eur murine model of CF. Using nasal potential difference measurement, we showed that in vivo administration of c407 by topical, short-term intraperitoneal and long-term subcutaneous route significantly increased the CFTR dependent chloride (Cl−) conductance in F508del Cftrtm1Eur mice. This functional improvement was correlated with a relocalization of F508del-cftr to the apical membrane in nasal epithelial cells. Importantly, c407 long-term administration was well tolerated and in vitro ADME toxicologic studies did not evidence any obvious issue. Our data provide the first in vivo preclinical evidence of c407 efficacy and absence of toxicity after systemic administration for the treatment of Cystic Fibrosis.

Impact of Gender and Age at Lung Transplantation on Long-Term Survival of Patients with Cystic Fibrosis

<h3>Purpose</h3> Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator; these mutations impair chloride conduction, which reduces water on the cell surface and leads to accumulation of viscous mucous in the lungs. Accumulated mucous drives chronic infections, bronchiectasis, and eventually, respiratory failure requiring lung transplant (LT). We aimed to study the impact of gender and age at the time of LT on long-term survival of patients with CF. <h3>Methods</h3> Lung transplant recipients with CF who received post-LT care at our center between July 1, 2007 and April 30, 2021 were included. Primary outcome was death or redo LT. Cox proportional hazard analysis, Kaplan-Meier method, and log-rank tests were used for data analysis. <h3>Results</h3> A total of 59 LT recipients met inclusion criteria. The median age at primary LT was 28 (22, 36) years, 36 (61%) patients were ≥25 years old, and 30 (51%) were male. At the end of the study period, 21 (35.6%) LT recipients died and 17 (28.8%) required redo LT with a median lung allograft survival of 33.4 (16.1, 66.4) months. The remaining 21 patients (36%) were alive at a median of 68 (28, 112) months after primary LT. The risk of death trended lower and the probability of survival trended higher in male patients than in female patients (HR: 0.487 [95% CI: 0.221-1.075], p=0.075 and 66.7% vs 44.8%, p=0.069, respectively, Figure 1A). Compared to age <25 years at LT, age ≥25 years was associated with a lower risk of death and an increased probability of survival (HR: 0.466 [95% CI: 0.215-1.007], p=0.052 and 63.9% vs 43.5%, p=0.047, respectively, Figure 1B). <h3>Conclusion</h3> In our study, LT recipients with CF ≥25 years at LT had significantly higher survival than younger patients, and survival trended higher for male recipients than female recipients. These results identify an "at-risk" population of LT recipients who may warrant closer attention after LT. Large multicenter trials with adequate power are needed.

Interleukin-1 beta is a potential mediator of airway nitric oxide deficiency in cystic fibrosis

Airway nitric oxide (NO) deficiency is a hallmark of cystic fibrosis (CF), but the reasons for the reduced NO production in CF airways are unclear. Interleukin (IL)-1 pathway activation plays a role in early CF lung disease and is also involved in the regulation of NO synthase activity. Treatment of CF patients with the CFTR-targeting drug ivacaftor, among other beneficial effects, results in an increase in airway NO levels. In this longitudinal observational trial, we show that ivacaftor therapy leads to a significant reduction in sputum IL-1β concentration but not in other IL-1- or Th17-associated cytokines. IL-1β concentrations were closely linked to improvement in pulmonary function, measures of NO metabolism in sputum and exhaled NO. These data therefore suggest a potential interaction between transepithelial chloride conductance, IL-1β and airway NO production.

Excitability properties of mouse and human skeletal muscle fibres compared by muscle velocity recovery cycles

Mouse models of skeletal muscle channelopathies are not phenocopies of human disease. In some cases (e.g. Myotonia Congenita) the phenotype is much more severe, whilst in others (e.g. Hypokalaemic periodic paralysis) rodent physiology is protective. This suggests a species’ difference in muscle excitability properties. In humans these can be measured indirectly by the post-impulse changes in conduction velocity, using Muscle Velocity Recovery Cycles (MVRCs). We performed MVRCs in mice and compared their muscle excitability properties with humans. Mouse Tibialis Anterior MVRCs (n = 70) have only one phase of supernormality (increased conduction velocity), which is smaller in magnitude (p = 9 × 10−21), and shorter in duration (p = 3 × 10−24) than human (n = 26). This abbreviated supernormality is followed by a period of late subnormality (reduced velocity) in mice, which overlaps in time with the late supernormality seen in human MVRCs. The period of late subnormality suggests increased t-tubule Na+/K+-pump activity. The subnormal phase in mice was converted to supernormality by blocking ClC-1 chloride channels, suggesting relatively higher chloride conductance in skeletal muscle. Our findings help explain discrepancies in phenotype between mice and humans with skeletal muscle channelopathies and potentially other neuromuscular disorders. MVRCs are a valuable new tool to compare in vivo muscle membrane properties between species and will allow further dissection of the molecular mechanisms regulating muscle excitability.

Microscopic Characterization of the Chloride Permeation Pathway in the Human Excitatory Amino Acid Transporter 1 (EAAT1).

Excitatory amino acid transporters (EAATs) are glutamate transporters that belong to the solute carrier 1A (SLC1A) family. They couple glutamate transport to the cotransport of three sodium (Na+) ions and one proton (H+) and the counter-transport of one potassium (K+) ion. In addition to this coupled transport, binding of cotransported species to EAATs activates a thermodynamically uncoupled chloride (Cl-) conductance. Structures of SLC1A family members have revealed that these transporters use a twisting elevator mechanism of transport, where a mobile transport domain carries substrate and coupled ions across the membrane, while a static scaffold domain anchors the transporter in the membrane. We recently demonstrated that the uncoupled Cl- conductance is activated by the formation of an aqueous pore at the domain interface during the transport cycle in archaeal GltPh. However, a pathway for the uncoupled Cl- conductance has not been reported for the EAATs, and it is unclear if such a pathway is conserved. Here, we employ all-atom molecular dynamics (MD) simulations combined with enhanced sampling, free-energy calculations, and experimental mutagenesis to approximate large-scale conformational changes during the transport process and identified a Cl--conducting conformation in human EAAT1 (hEAAT1). Sampling the large-scale structural transitions in hEAAT1 allowed us to capture an intermediate conformation formed during the transport cycle with a continuous aqueous pore at the domain interface. The free-energy calculations performed for the conduction of Cl- and Na+ ions through the captured conformation highlight the presence of two hydrophobic gates that control low-barrier movement of Cl- through the aqueous pathway. Overall, our findings provide insights into the mechanism by which a human neurotransmitter transporter supports functional duality of active transport and passive Cl- permeation and confirm the commonality of this mechanism in different members of the SLC1A family.

Mechanisms of inhibition and activation of extrasynaptic \u03b1\u03b2 GABAA receptors

Type A GABA (γ-aminobutyric acid) receptors represent a diverse population in the mammalian brain, forming pentamers from combinations of α-, β-, γ-, δ-, ε-, ρ-, θ- and π-subunits1. αβ, α4βδ, α6βδ and α5βγ receptors favour extrasynaptic localization, and mediate an essential persistent (tonic) inhibitory conductance in many regions of the mammalian brain1,2. Mutations of these receptors in humans are linked to epilepsy and insomnia3,4. Altered extrasynaptic receptor function is implicated in insomnia, stroke and Angelman and Fragile X syndromes1,5, and drugs targeting these receptors are used to treat postpartum depression6. Tonic GABAergic responses are moderated to avoid excessive suppression of neuronal communication, and can exhibit high sensitivity to Zn2+ blockade, in contrast to synapse-preferring α1βγ, α2βγ and α3βγ receptor responses5,7–12. Here, to resolve these distinctive features, we determined structures of the predominantly extrasynaptic αβ GABAA receptor class. An inhibited state bound by both the lethal paralysing agent α-cobratoxin13 and Zn2+ was used in comparisons with GABA–Zn2+ and GABA-bound structures. Zn2+ nullifies the GABA response by non-competitively plugging the extracellular end of the pore to block chloride conductance. In the absence of Zn2+, the GABA signalling response initially follows the canonical route until it reaches the pore. In contrast to synaptic GABAA receptors, expansion of the midway pore activation gate is limited and it remains closed, reflecting the intrinsic low efficacy that characterizes the extrasynaptic receptor. Overall, this study explains distinct traits adopted by αβ receptors that adapt them to a role in tonic signalling.

Translating in vitro CFTR rescue into small molecule correctors for cystic fibrosis using the Library of Integrated Network-based Cellular Signatures drug discovery platform.

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The common ΔF508‐CFTR mutation results in protein misfolding and proteasomal degradation. If ΔF508‐CFTR trafficks to the cell surface, its anion channel function may be partially restored. Several in vitro strategies can partially correct ΔF508‐CFTR trafficking and function, including low‐temperature, small molecules, overexpression of miR‐138, or knockdown of SIN3A. The challenge remains to translate such interventions into therapies and to understand their mechanisms. One approach for connecting such interventions to small molecule therapies that has previously succeeded for CF and other diseases is via mRNA expression profiling and iterative searches of small molecules with similar expression signatures. Here, we query the Library of Integrated Network‐based Cellular Signatures using transcriptomic signatures from previously generated CF expression data, including RNAi‐ and low temperature‐based rescue signatures. This LINCS in silico screen prioritized 135 small molecules that mimicked our rescue interventions based on their genomewide transcriptional perturbations. Functional screens of these small molecules identified eight compounds that partially restored ΔF508‐CFTR function, as assessed by cAMP‐activated chloride conductance. Of these, XL147 rescued ΔF508‐CFTR function in primary CF airway epithelia, while also showing cooperativity when administered with C18. Improved CF corrector therapies are needed and this integrative drug prioritization approach offers a novel method to both identify small molecules that may rescue ΔF508‐CFTR function and identify gene networks underlying such rescue.

CLN7 is an organellar chloride channel regulating lysosomal function.

Neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive lysosomal storage diseases. One variant form of late-infantile NCL (vLINCL) is caused by mutations of a lysosomal membrane protein CLN7, the function of which has remained unknown. Here, we identified CLN7 as a novel endolysosomal chloride channel. Overexpression of CLN7 increases endolysosomal chloride currents and enlarges endolysosomes through a Ca2+/calmodulin-dependent way. Human CLN7 and its yeast homolog exhibit characteristics of chloride channels and are sensitive to chloride channel blockers. Moreover, CLN7 regulates lysosomal chloride conductance, luminal pH, and lysosomal membrane potential and promotes the release of lysosomal Ca2+ through transient receptor potential mucolipin 1 (TRPML1). Knocking out CLN7 causes pathological features that are similar to those of patients with vLINCL, including retinal degeneration and autofluorescent lipofuscin. The pathogenic mutations in CLN7 lead to a decrease in chloride permeability, suggesting that reconstitution of lysosomal Cl− homeostasis may be an effective strategy for the treatment of vLINCL.

The Chloride Conductance Inhibitor NS3623 Enhances the Activity of a Non-selective Cation Channel in Hyperpolarizing Conditions

Handbooks of physiology state that the strategy adopted by red blood cells (RBCs) to preserve cell volume is to maintain membrane permeability for cations at its minimum. However, enhanced cation permeability can be measured and observed in specific physiological and pathophysiological situations such as in vivo senescence, storage at low temperature, sickle cell anemia and many other genetic defects affecting transporters, membrane or cytoskeletal proteins. Among cation pathways, cation channels are able to dissipate rapidly the gradients that are built and maintained by the sodium and calcium pumps. These situations are very well-documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. In addition, non-selective cation (NSC) channels present in the RBC membrane have proven difficult to molecular identification and functional characterization. For instance, NSC channel activity can be elicited by Low Ionic Strength conditions (LIS): the associated change in membrane potential triggers its opening in a voltage dependent manner. But, whereas this depolarizing media produces a spectacular activation of NSC channel, Gárdos channel-evoked hyperpolarization's have been shown to induce sodium entry through a pathway thought to be conductive and termed Pcat. Using the CCCP method, which allows to follow fast changes in membrane potential, we show here (i) that hyperpolarization elicited by Gárdos channel activation triggers sodium entry through a conductive pathway, (ii) that chloride conductance inhibition unveils such conductive cationic conductance, (iii) that the use of the specific chloride conductance inhibitor NS3623 (a derivative of Neurosearch compound NS1652), at concentrations above what is needed for full anion channel block, potentiates the non-selective cation conductance. These results indicate that a non-selective cation channel is likely activated by the changes in the driving force for cations rather than a voltage dependence mechanism per se.

Potassium-induced potentiation of subtetanic force in rat skeletal muscles: influences of β2-activation, lactic acid, and temperature.

Moderate elevations of extracellular K+ concentration ([K+]o) occur during exercise and have been shown to potentiate force during contractions elicited with subtetanic frequencies. Here, we investigated whether lactic acid (reduced chloride conductance), β2-adrenoceptor activation, and increased temperature would influence the potentiating effect of potassium in slow- and fast-twitch muscles. Isometric contractions were elicited by electrical stimulation at various frequencies in isolated rat soleus and extensor digitorum longus (EDL) muscles incubated at normal (4 mM) or elevated K+, in combination with salbutamol (5 μM), lactic acid (18.1 mM), 9-anthracene-carboxylic acid (9-AC; 25 μM), or increased temperature (30-35°C). Elevating [K+]o from 4 mM to 7 mM (soleus) and 10 mM (EDL) potentiated isometric twitch and subtetanic force while slightly reducing tetanic force. In EDL, salbutamol further augmented twitch force (+27 ± 3%, P < 0.001) and subtetanic force (+22 ± 4%, P < 0.001). In contrast, salbutamol reduced subtetanic force (-28 ± 6%, P < 0.001) in soleus muscles. Lactic acid and 9-AC had no significant effects on isometric force of muscles already exposed to moderate elevations of [K+]o. The potentiating effect of elevated [K+]o was still well maintained at 35°C. Addition of salbutamol exerts a further force-potentiating effect in fast-twitch but not in slow-twitch muscles already potentiated by moderately elevated [K+]o, whereas lactic acid, 9-AC, or increased temperature does not exert any further augmentation. However, the potentiating effect of elevated [K+]o was still maintained in the presence of these, thus emphasizing the positive influence of moderately elevated [K+]o for contractile performance during exercise.

Elevated newborn serum immunoreactive trypsinogen associated with a congenital pancreatic cyst

Congenital abdominal cysts have a broad differential diagnosis. Pancreatic cysts are rare lesions that offer a diagnostic and surgical challenge to surgeons. We report on a case of a large left upper quadrant cyst detected in utero , which remained of unclear etiology despite cross-sectional imaging at birth. Intraoperative findings confirmed its pancreatic origin. In hindsight, a pre-operative elevated immunoreactive trypsinogen, performed as part of universal cystic fibrosis (CF) newborn screening, may have suggested the diagnosis. False-positive CF screening tests may be unnoticed by a surgical team assessing infants with an abdominal mass. As universal testing for cystic fibrosis increases in Canada and worldwide, physicians should review a patient's CF screening history. Elevated immunoreactive trypsinogen levels with normal sweat chloride and cystic fibrosis transmembrane conductance regulator (CFTR) detection tests may orient towards a pancreatic etiology when assessing a patient with a congenital abdominal cyst of unclear etiology. • Investigate false-positive cystic fibrosis screening tests when assessing patients with a congenital abdominal cyst. • High immunoreactive trypsinogen levels on cystic fibrosis screening tests may be a marker of congenital pancreatic cysts. • Negative sweat chloride test and genetic testing were not enough to rule out a pancreatic etiology in our case.

Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody.

The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system, and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face, and one to the cytosolic. Two are specific to VGLUT2, whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function, presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.

Abstract 2452: Metformin administration in hypoglycemia compromises cancer cell viability in glioblastoma initiating cells

Abstract Cancer cells show higher energy and biomolecules requirements than healthy cells, making the targeting of cancer metabolic dysfunctions an appealing therapeutic approach. Unfortunately, tackling the tumor metabolism is a daunting task, due to the ability of cancer cells to re-organize their metabolism switching between glycolysis and oxidative phosphorylation. Cancer metabolic plasticity and re-programming is partially supported by altered ionic channels expression and activity, our laboratory has described Chloride Intracellular Channel 1 (CLIC1) as a marker of malignancy in tumor cells and a possible therapeutic target. CLIC1 is a metamorphic protein able to switch between cytoplasmic and transmembrane forms (tmCLIC1), the latter characterized by chloride conductance. TmCLIC1 was found to be overexpressed in several solid tumors where supports cancer proliferation and growth, while it is mainly absent in healthy cells. Recently, it has been demonstrated that tmCLIC1 activity is affected by biguanide compounds, in particular metformin. Metformin was recently repurposed as an anticancer drug with unclear mechanisms. We demonstrated that in hypoglycemia, metformin displays increased toxic effect on cancer cells, inducing apoptosis. Metformin inhibits Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A), and hypoglycemia upregulates PP2A subunit B56δ targeting the Glycogen Synthase Kinase 3 Beta (GSK3b). Metformin treatment in combination with hypoglycemia leads to de-phosphorylation of GSK3b protein resulting in degradation of induced myeloid leukemia cell differentiation protein (MCL-1), a pro-survival protein, and cell death (Elgendy et al., Cancer Cell 2019). In primary glioblastoma cell lines enriched in tumor-initiating-cells (GSCs), metformin has proven to be effective in reducing cell growth without affecting viability (Gritti et al., Oncotarget 2014). Here, we are going to show studies trying to establish whether CLIC1 is a component of the established PP2A-mediated signaling network. Our work aims to clarify metformin molecular mechanism of action and to assess a new potential therapeutic approach for solid tumors in which tmCLIC1 is found to be overexpressed. Citation Format: Francesca Cianci, Riccardo Cazzoli, Ivan Verduci, Saverio Minucci, Michele Mazzanti. Metformin administration in hypoglycemia compromises cancer cell viability in glioblastoma initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2452.

Mono-and Binuclear Cobalt(II) Mixed Ligand Complexes of 2,2’-Bipyridine and Ethylenediamine: Synthesis, Characterization and Biological Application

Antibiotic resistance of pathogens to the current commercially available drugs is a serious problem. To curve this problem, the discovery of active compounds with a new mode of action is insistent. In line with this, we report two new complexes; mononuclear [Co(L1)2(L2)(H2O)]Cl2 and binuclear [Co2(L1)4(L2)(H2O)2]Cl4 using precursor complex, [Co(L1)2(H2O)2]Cl2, synthesized before; where L1: 2,2’-bipyridine and L2: ethylenediamine. The precursor complex was prepared from CoCl2.6H2O and L1 in ethanol, treated with a different mole of L2 under optimized reaction conditions to give the corresponding mono-and binuclear cobalt(II) mixed ligand complexes. These complexes were characterized using the spectroscopic technique (ICP-OES, UV-Vis, FT-IR) and physicochemical methods (chloride determination, thermal analysis, and conductance measurement). Their antibacterial activities were also tested against two Gram-negative (Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae)) and two Gram-positive (Staphylococcus aureus (S. aureus), Streptococcus pyogenes (S. pyogenes)) bacteria using the disc diffusion method. The new complexes showed better activities against K. pneumoniae than the reference Gentamycin. Furthermore, [Co(L1)2(L2)(H2O)]Cl2 demonstrated better activity than Gentamycin against S. aureus and E. coli.