Alexidine Dihydrochloride Research Articles

Mechanistic insights into antifungal potential of Alexidine dihydrochloride and hexachlorophene in Candida albicans: a drug repurposing approach.

Candida albicans has been listed in the critical priority group by the WHO in 2022 depending upon its contribution in invasive candidiasis and increased resistance to conventional drugs. Drug repurposing offers an efficient, rapid, and cost-effective solution to develop alternative therapeutics against pathogenic microbes. Alexidine dihydrochloride (AXD) and hexachlorophene (HCP) are FDA approved anti-cancer and anti-septic drugs, respectively. In this study, we have shown antifungal properties of AXD and HCP against the wild type (reference strain) and clinical isolates of C. albicans. The minimum inhibitory concentrations (MIC50) of AXD and HCP against C. albicans ranged between 0.34 and 0.69 µM and 19.66-24.58 µM, respectively. The biofilm inhibitory and eradication concentration of AXD was reported comparatively lower than that of HCP for the strains used in the study. Further investigations were performed to understand the antifungal mode of action of AXD and HCP by studying virulence features like cell surface hydrophobicity, adhesion, and yeast to hyphae transition, were also reduced upon exposure to both the drugs. Ergosterol content in cell membrane of the wild type strain was upregulated on exposure to AXD and HCP both. Biochemical analyses of the exposed biofilm indicated reduced contents of carbohydrate, protein, and e-DNA in the extracellular matrix of the biofilm when compared to the untreated control biofilm. AXD exposure downregulated activity of tissue invading enzyme, phospholipase in the reference strain. In wild type strain, ROS level, and activities of antioxidant enzymes were found elevated upon exposure to both drugs. FESEM analysis of the drug treated biofilms revealed degraded biofilm. This study has indicated mode of action of antifungal potential of alexidine dihydrochloride and hexachlorophene in C. albicans.

In silico approach to identify the potential targets of Alexidine dihydrochloride and Hexachlorophene in human fungal pathogen C. glabrata

Widespread usage of antifungals has led to the development of antifungal resistance, causing a change in the epidemiology of the responsible agents from albicans to non-Candida albicans species. Pharmaceutical repurposing is an alternate strategy that has provided a cost-effective method to address the increasing resistance to antifungal medications. The objective of this work was to examine the antifungal properties of Alexidine dihydrochloride (AXD) and Hexachlorophene (HCP) against a non-Albicans Candida model, C. glabrata. The lowest inhibitory doses of AXD and HCP against C. glabrata were determined by in vitro methods to be 0.69-1.03 µM and 14.75-19.66 µM, respectively. The minimum doses of AXD and HCP that caused fungicidal effects were defined as 1.375 µM and 61.44 µM, respectively. Three proteins involved in crucial physiological pathways, namely cell wall production (Kre1p, Kre2p, Ecm33p), membrane calcium channel (Mid1p, Ecm7p), and ergosterol biosynthesis (Erg5p), were chosen as potential targets for the medications due to their functions in survival and disease development. SWISS MODEL was used to create the 3D structures of predicted targets of C. glabrata. The quality of these structures was assessed using Ramachandran plot statistics. AXD and HCP were analyzed by docking software AutoDock Vina against these targets. The findings of computational investigations have shown that both medicines exhibit interaction affinities with all the selected protein types. The binding energy profiles of AXD and HCP showed that Mid1p had the lowest binding energies at -10.1 kcal/mol and -9.2 kcal/mol, respectively. Kre2p had binding energies of -7.9 kcal/mol and -7.1 kcal/mol, respectively. Erg5p had binding energies of -6.6 kcal/mol and -6.2 kcal/mol, respectively. Ecm7p had binding energies of -6.6 kcal/mol and -6.1 kcal/mol, respectively. Recm7p had binding energies of -4.8 kcal/mol and -7.7 kcal/mol, respectively. These results suggest that these genes are likely targets of the two drugs in C. glabrata.

Inhibitor of cardiolipin biosynthesis-related enzyme MoGep4 confers broad-spectrum anti-fungal activity.

Plant pathogens cause devastating diseases, leading to serious losses to agriculture. Mechanistic understanding of pathogenesis of plant pathogens lays the foundation for the development of fungicides for disease control. Mitophagy, a specific form of autophagy, is important for fungal virulence. The role of cardiolipin, mitochondrial signature phospholipid, in mitophagy and pathogenesis is largely unknown in plant pathogenic fungi. The functions of enzymes involved in cardiolipin biosynthesis and relevant inhibitors were assessed using a set of assays, including genetic deletion, plant infection, lipidomics, chemical-protein interaction, chemical inhibition, and field trials. Our results showed that the cardiolipin biosynthesis-related gene MoGEP4 of the rice blast fungus Magnaporthe oryzae regulates growth, conidiation, cardiolipin biosynthesis, and virulence. Mechanistically, MoGep4 regulated mitophagy and Mps1-MAPK phosphorylation, which are required for virulence. Chemical alexidine dihydrochloride (AXD) inhibited the enzyme activity of MoGep4, cardiolipin biosynthesis and mitophagy. Importantly, AXD efficiently inhibited the growth of 10 plant pathogens and controlled rice blast and Fusarium head blight in the field. Our study demonstrated that MoGep4 regulates mitophagy, Mps1 phosphorylation and pathogenesis in M. oryzae. In addition, we found that the MoGep4 inhibitor, AXD, displays broad-spectrum antifungal activity and is a promising candidate for fungicide development.

A laboratory investigation on the effect of biguanide- and pyridine-derived antiseptics on the adhesion of resin composites to dentin.

Optimal bonding of adhesive restorations to dentin is crucial to prevent microleakage and enhance the survival of root-filled teeth. The aim of this study was to investigate the effect of chlorhexidine (CHX), alexidine (ALX) and octenidine dihydrochloride (OCT) on the bond strength of resin composites to coronal dentin. Human coronal dentin specimens were treated with 2% CHX, 0.1% ALX, 0.1% OCT or saline then restored with traditional or bulk-fill resin composites. The adhesion strength between the resin and dentin was measured using the microtensile bond strength and failure mode was determined using a stereomicroscope. Treatment with ALX and OCT resulted in significantly greater μTBS compared with CHX and saline, irrespective of the resin composite used. Alexidine treatment predominantly resulted in mixed failure, while adhesive failures were frequently observed in CHX and saline-treated dentin. In conclusion, final irrigation with ALX or OCT improved the bonding of resin composites to dentin.

Antibacterial and Antibiofilm Properties of the Alexidine Dihydrochloride (MMV396785) against Acinetobacter baumannii.

Antibiotic-resistant Acinetobacter baumannii infections among patients in hospital settings are rising at an alarming rate. The World Health Organization has designated carbapenem-resistant A. baumannii as a priority pathogen for drug discovery. Based on the open drug discovery approach, we screened 400 compounds provided as a Pandemic Response Box by MMV and DNDi to identify compounds with antibacterial and antibiofilm activity against two A. baumannii reference strains using a highly robust resazurin assay. In vitro screening identified thirty compounds with MIC ≤ 50μM having growth inhibitory properties against the planktonic state. Five compounds, with MMV IDs MMV396785, MMV1578568, MMV1578574, MMV1578564, and MMV1579850, were able to reduce metabolically active cells in the biofilm state. Of these five compounds, MMV396785 showed potential antibacterial and antibiofilm activity with MIC, MBIC, and MBEC of 3.125 μM, 12.5, and 25-100 µM against tested A. baumannii strains, respectively, showing biofilm formation inhibition by 93% and eradication of pre-formed biofilms by 60-77.4%. In addition, MMV396785 showed a drastic reduction in the surface area and thickness of biofilms. Further investigations at the molecular level by qRT-PCR revealed the downregulation of biofilm-associated genes when exposed to 50 µM MMV396785 in all tested strains. This study identified the novel compound MMV396785 as showing potential in vitro antibacterial and antibiofilm efficacy against A. baumannii.

1683. Antifungal Activity of Alexidine Dihydrochloride in a Novel Diabetic Mouse Model of Dermatophytosis

Abstract Background Ringworm caused by the dermatophyte species Trichophyton is the foremost superficial fungal infection worldwide. The US alone records over 5 million outpatient visits due to dermatophytosis, with an annual burden of one billion dollars in associated direct medical costs. Patients with diabetes mellitus exhibit a higher incidence rate and a more severe manifestation of the disease. Infections are often recurrent and recalcitrant to most antifungal drugs and new molecules to combat these discomforting infections are sorely needed. Here, we establish a novel model of dermatophytosis, which is clinically relevant, simple to use, and is robust enough for evaluation of antifungal drugs. Using this model, we demonstrate that an FDA-approved small molecule alexidine dihydrochloride has enhanced efficacy against dermatophytes. Methods For the animal model, ICR mice (equal gender) were rendered diabetic by treatment with a single intraperitoneal dose of streptozotocin, followed by confirmation of glycosuria and ketonuria. The back of the anesthetized diabetic mice was shaved, skin abraded with sand paper, and inoculated with 50 µl of sterile water containing 5x10^7 conidia. Infected lesions were monitored daily, and graded for erythema, edema, and extent of infection over a period of 2 weeks. For drug treatment, infected mice were separated equally into four groups: topical treatment of 20 µg/ml alexidine dihydrochloride, topical 1% terbinafine, 10 mg/kg terbinafine by oral gavage, and a vehicle control. Treatment was started on day 4 post infection and given once daily for 7 days. Lesions were monitored as above and clearance of infection was confirmed by fungal culture and histopathology. Results In the absence of drugs, dry, scaly erythematous legions appeared on day 3 after infection and became progressively worse, persisting for at least 14 days. Terbinafine and Alexidine dihydrochloride were highly efficacious in curbing infections within two days after treatment and no recurrence was observed for up to 10 days post treatment. Conclusion The diabetic mouse model was found to be reproducible and applicable for primary evaluation of the anti-dermatophytic efficacy of topical and oral formulations of antifungal agents. Disclosures All Authors: No reported disclosures.

Antifungal activity of alexidine dihydrochloride in a novel diabetic mouse model of dermatophytosis.

Dermatophytosis is one of the most prevalent fungal infections and a major public health problem worldwide. Recent years have seen a change in the epidemiological patterns of infecting fungi, corresponding to an alarming rise in the prevalence of drug-recalcitrant dermatophyte infections. In patients with diabetes mellitus, dermatophytosis is more severe and recurrent. The potency of promising new antifungal drugs in the pipeline must be expanded to include dermatophytosis. To facilitate this effort, we established a clinically pertinent mouse model of dermatophyte infections, in which diabetic mice were infected with Trichophyton mentagrophytes on abraded skin. The diabetic mouse model was optimized as a simple and robust system for simulating dermatophytoses in diabetic patients. The outcome of infection was measured using clinical and mycological parameters. Infected mice with fungal lesions were treated with oral and topical formulations of terbinafine or topical administration of the FDA-approved and repurposed pan-antifungal drug alexidine dihydrochloride (AXD). In this model, AXD was found to be highly effective, with outcomes comparable to those of the standard of care drug terbinafine.

Phosphatase inhibitors BVT-948 and alexidine dihydrochloride inhibit sexual development of the malaria parasite Plasmodium berghei

BackgroundWith the emergence of resistance to front-line antimalarials, there is an urgent need to develop new medicines, including those targeting sexual development. This study aimed to assess the activity of a panel of phosphatase inhibitors against the sexual development of Plasmodium berghei and evaluate their potential as transmission-blocking agents. MethodsTwenty-five compounds were screened for transmission-blocking activity in vitro using the P. berghei ookinete culture assay. The inhibitory effects on male gametogenesis, gamete-ookinete, and zygote-ookinete formation were evaluated. The transmission-blocking activity of two compounds was evaluated using an in vivo mosquito feeding assay. Their cytotoxic effects were assessed on the human cell line HepG2. ResultsTwelve compounds inhibited P. berghei ookinete formation with an IC50 < 10 μM. Two compounds, BVT-948 and alexidine dihydrochloride, significantly inhibited different developmental stages from gametogenesis through ookinete maturation. They also showed a substantial in vivo transmission-blocking activity by the mosquito feeding assay. ConclusionsSome phosphatase inhibitors effectively inhibited Plasmodium sexual development and exhibited evident transmission-blocking activity, suggesting that phosphatases are valid targets for antimalarial development.

Drug repurposing against Candida auris: A systematic review.

Candida auris is a drug-resistant pathogen with several reported outbreaks. The treatment of C. auris infections is difficult due to a limited number of available antifungal drugs. Thus, finding alternative drugs through repurposing approaches would be clinically beneficial. A systematic search in PubMed, Scopus and Web of Science databases, as well as Google Scholar up to 1 November 2021, was conducted to find all articles with data regarding the antifungal activity of non-antifungal drugs against the planktonic and biofilm forms of C. auris. During database and hand searching, 290 articles were found, of which 13 were eligible for inclusion in the present study. Planktonic and biofilm forms have been studied in 11 and 8 articles (with both forms examined in 6 articles), respectively. In total, 22 and 12 drugs/compounds have been reported as repositionable against planktonic and biofilm forms of C. auris, respectively. Antiparasitic drugs, with the dominance of miltefosine, were the most common repurposed drugs against both forms of C. auris, followed by anticancer drugs (e.g. alexidine dihydrochloride) against the planktonic form and anti-inflammatory drugs (e.g. ebselen) against the biofilm form of the fungus. A collection of other drugs from various classes have also shown promising activity against C. auris. Following drug repurposing approaches, a number of drugs/compounds from various classes have been found to inhibit the planktonic and biofilm forms of C. auris. Accordingly, drug repurposing is an encouraging approach for discovering potential alternatives to conventional antifungal agents to combat drug resistance in fungi, especially C. auris.

Modelling and targeting mitochondrial protein tyrosine phosphatase 1: a computational approach.

The present research scintillates on the homology modelling of rat mitochondrial protein tyrosine phosphatase 1 (PTPMT1) and targeting its activity using flavonoids through a computational docking approach. PTPMT1 is a dual-specificity phosphatase responsible for protein phosphorylation and plays a vital role in the metabolism of cardiolipin biosynthesis, insulin regulation, etc. The inhibition of PTPMT1 has also shown enhanced insulin levels. The three-dimensional structure of the protein is not yet known. The homology modelling was performed using SWISS-MODEL and Geno3D webservers to compare the efficiencies. The PROCHECK for protein modelled using SWISS-MODEL showed 91.6% of amino acids in the most favoured region, 0.7% residues in the disallowed region that was found to be significant compared to the model built using Geno3D. 210 common flavonoids were docked in the modelled protein using the AutoDock 4.2.6 along with a control drug alexidine dihydrochloride. Our results show promising candidates that bind protein tyrosine phosphatase 1, including, prunin (- 8.66kcal/mol); oroxindin (- 8.56kcal/mol); luteolin 7-rutinoside (- 8.47kcal/mol); 3(2H)-isoflavenes (- 8.36kcal/mol); nicotiflorin (- 8.29kcal/mol), ranked top in the docking experiments. We predicted the pharmacokinetic and Lipinski properties of the top ten compounds with the lowest binding energies. To further validate the stability of the modelled protein and docked complexes molecular dynamics simulations were performed using Desmond, Schrodinger for 150ns in conjunction with MM-GBSA. Thus, flavonoids could act as potential inhibitors of PTPMT1, and further, in-vitro and in-vivo studies are essential to complete the drug development process.

CYCLIC FATIGUE RESISTANCE OF ROTATE, EDGEFILE PLATINUM, AND K3XF FILES EXPOSED TO INTRACANAL TEMPERATURE IN AN S-SHAPED CANAL

Aim: To evaluate the bond strength of resin-based sealer to dentin after using different final irrigation solutions, one of which is alexidine dihydrochloride (ALX). Material and method: A total of 90 human maxillary central teeth were used. The groups were designed according to the final irrigation solution applied, as follows: Group 1: 17% ethylenediamine tetra-acetic acid (EDTA), Group 2: QMix, Group 3: 2% chlorhexidine gluconate (CHX) , Group 4: 1% ALX, and Group 5: Distilled water (Control group). All canals were obturated with gutta-percha and AH Plus. For bonding strength measurement, three horizontal sections of 2 mm thickness were obtained from each sample towards the coronal to apical. Push-out force was applied until bond failure occurred. Modes of failure were classified by examining each sample using a stereomicroscope as follows: adhesive (sealer-dentin or sealer-gutta-percha interface), cohesive (failure within sealer or dentin), mixed (failure in both the sealer and dentin). The Kruskal-Wallis H test was used for comparison among the groups (p < 0.05). Results: The mean highest values were obtained from the coronal, middle and apical regions, respectively (p < 0.05). The highest significant values for bonding strength was obtained in the EDTA group and the lowest in the control group (p < 0.001). There are no statistical differences among QMix, CHX and ALX groups, except for the coronal third, where the bond strength for the QMix was higher compared to the other two groups (p=0.001). All samples showed the cohesive failure type in most. Conclusion: Bonding strength is high when EDTA is used in any of the three parts of the root canal. ALX showed similar bond strengths to CHX. Key Words : AH Plus, alexidine, bond strength, chlorhexidine, EDTA, Qmix

Insight into the Reaction of Alexidine with Sodium Hypochlorite: A Potential Error in Endodontic Treatment.

Therapeutic success in endodontic treatment depends on successful infection control. Alexidine dihydrochloride (ALX) was recently proposed as a potential alternative to 2% chlorhexidine (CHX) as it possesses similar antimicrobial properties, expresses substantivity and does not produce p-chloroaniline (PCA) when mixed with sodium hypochlorite (NaOCl). However, the products released in this reaction have not been described to date. The aim of this study was to identify detected chemical compounds formed in the reaction of ALX and NaOCl with the ultra-high-performance liquid chromatography–mass spectrophotometry (UHPLC-MS) method and assess whether precipitates and PCA are formed in this reaction. Solutions of ALX were mixed with the equivalent volume of 2% and 5.25% (w/v) NaOCl solutions. As control, 2% (w/v) CHX was mixed with 2% and 5.25% (w/v) NaOCl. Samples were subjected to the UHPLC-MS analysis. The mixture of ALX and NaOCl resulted in a yellowish precipitate formation, the amount of which depended on NaOCl concentration. Interaction of ALX and NaOCl resulted in the production of aliphatic amines. No PCA was formed when NaOCl was mixed with ALX. However, for the first time, we identified the possible products of the interaction. The interaction between NaOCl and ALX results in the formation of aliphatic amines; therefore, these compounds should not be mixed during endodontic treatment.

Genome-wide CRISPR-Cas9 knockout library screening identified PTPMT1 in cardiolipin synthesis is crucial to survival in hypoxia in liver cancer

Hypoxia, low oxygen (O2), is a key feature of all solid cancers, including hepatocellular carcinoma (HCC). Genome-wide CRISPR-Cas9 knockout library screening is used to identify reliable therapeutic targets responsible for hypoxic survival in HCC. We find that protein-tyrosine phosphatase mitochondrial 1 (PTPMT1), an important enzyme for cardiolipin (CL) synthesis, is the most significant gene and ranks just after hypoxia-inducible factor (HIF)-1α and HIF-1β as crucial to hypoxic survival. CL constitutes the mitochondrial membrane and ensures the proper assembly of electron transport chain (ETC) complexes for efficient electron transfer in respiration. ETC becomes highly unstable during hypoxia. Knockout of PTPMT1 stops the maturation of CL and impairs the assembly of ETC complexes, leading to further electron leakage and ROS accumulation at ETC in hypoxia. Excitingly, HCC cells, especially under hypoxic conditions, show great sensitivity toward PTPMT1 inhibitor alexidine dihydrochloride (AD). This study unravels the protective roles of PTPMT1 in hypoxic survival and cancer development.

Abstract PO-031: Repurposing of alexidine dihydrochloride as an apoptosis-initiator and cell cycle inhibitor in human pancreatic cancer

Abstract Purpose: Highly aggressive and resistant to chemotherapy, pancreatic cancers are the fourth leading cause of cancer-related deaths in the western world. The absence of effective chemotherapeutics is leading researchers to develop novel drugs or repurpose existing chemicals. Alexidine dihydrochloride (AD), an orally bioavailable bis-biguanide compound, is an apoptosis stimulating reagent. It induces mitochondrial damage by inhibiting a mitochondrial-specific protein tyrosine phosphatase, PTPMT1. The aim of this study was to test AD as a novel compound to induce apoptosis in a human pancreatic adenocarcinoma cell lines, Panc-1, MIA PaCa-2, AsPC-1, and Psn-1. Methods: After IC50 value of the AD was determined by cytotoxicity assay, apoptosis was observed by a variety of methods including the detection of early apoptosis marker Annexin V and the proteomic profile screening by apoptosis array. Multicaspase and mitochondrial depolarization were measured and changes in cell cycle were analyzed. Results: AD is found to initiate apoptosis by activating the intrinsic pathway and inhibit cell cycle in pancreatic cancer cell lines. Conclusions: As a conclusion, considering its anti-cancer properties and bioavailability, Alexidine dihydrochloride can be considered as a potential candidate against pancreatic adenocarcinomas. Citation Format: Ezgi Kasikci, Esra Aydemir, Bekir Mustafa Yogurtcu, Fikrettin Sahin, Omer Faruk Bayrak. Repurposing of alexidine dihydrochloride as an apoptosis-initiator and cell cycle inhibitor in human pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-031.

Repurposing of Alexidine Dihydrochloride as an Apoptosis Initiator and Cell Cycle Inhibitor in Human Pancreatic Cancer.

Highly aggressive and resistant to chemotherapy, pancreatic cancers are the fourth leading cause of cancer-related deaths in the western world. The absence of effective chemotherapeutics is leading researchers to develop novel drugs or repurpose existing chemicals. Alexidine Dihydrochloride (AD), an orally bioavailable bis-biguanide compound, is an apoptosis stimulating reagent. It induces mitochondrial damage by inhibiting a mitochondrial-specific protein tyrosine phosphatase, PTPMT1. The aim of this study was to test AD as a novel compound to induce apoptosis in a human pancreatic adenocarcinoma cell lines, Panc-1, MIA PaCa-2, AsPC-1, and Psn-1. After the IC50 value of the AD was determined by cytotoxicity assay, apoptosis was observed by a variety of methods, including the detection of early apoptosis marker Annexin V and the proteomic profile screening by apoptosis array. Multicaspase and mitochondrial depolarization were measured, and changes in the cell cycle were analyzed. AD is found to initiate apoptosis by activating the intrinsic pathway and inhibit the cell cycle in pancreatic cancer cell lines. In conclusion, considering its anti-cancer properties and bioavailability, Alexidine dihydrochloride can be considered as a potential candidate against pancreatic adenocarcinomas.

Susceptibility of Contact Lens-Related Pseudomonas aeruginosa Keratitis Isolates to Multipurpose Disinfecting Solutions, Disinfectants, and Antibiotics.

PurposeThis study analyzed the susceptibilities of 17 contact lens (CL)-related keratitis isolates of Pseudomonas aeruginosa from Australia to antibiotics, multipurpose contact lens disinfecting solutions (MPDS), and disinfectants through minimum inhibitory (MIC) and minimum bactericidal concentrations.MethodsAntibiotics included ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime, and polymyxin B. The MPDS OPTI-FREE PureMoist, Complete RevitaLens OcuTec, Biotrue, and Renu Advanced Formula and the constituent disinfectants; alexidine dihydrochloride, polyquaternium-1, polyaminopropyl biguanide, and myristamidopropyl dimethylamine (Aldox) were analyzed. The combined susceptibility of disinfectants based on the MPDS formulation was assessed through fractional inhibitory concentration.ResultsAll isolates were susceptible to levofloxacin and gentamicin, 2/17 were resistant to ciprofloxacin; 1/17 was resistant to tobramycin, piperacillin, and polymyxin; and 3/17 were resistant to ceftazidime whereas 12/17 were resistant to imipenem. Of the four MPDSs, for Renu Advanced Formula 8/17 strains have an MIC ≤ 11.36 for OPTI-FREE PureMoist 14/17 strains have an MIC ≤ 11.36% for Complete RevitaLens 9/17 strains have an MIC ≤ 11.36, and for Biotrue 5/17 strains have MIC = 11.36. All strains were killed by 100% MPDS. At the concentrations used in the MPDSs, individual disinfectants were not active. From three tested isolates, no synergy was found in dual combinations of disinfectants. However, synergy was found for triple combination of disinfectants for three tested strains.ConclusionsAustralian CL-related isolates of P aeruginosa were susceptible to most antibiotics. There was variability in susceptibility to different MPDS. Individual disinfectant excipients had limited activity. The combination of the disinfectants showed synergy, antagonism, and no interaction.Translational RelevanceThis study will help to choose better preventive and treatment measures for microbial keratitis.

Subpopulation targeting of pyruvate dehydrogenase and GLUT1 decouples metabolic heterogeneity during collective cancer cell invasion

Phenotypic heterogeneity exists within collectively invading packs of tumor cells, suggesting that cellular subtypes cooperate to drive invasion and metastasis. Here, we take a chemical biology approach to probe cell:cell cooperation within the collective invasion pack. These data reveal metabolic heterogeneity within invasive chains, in which leader cells preferentially utilize mitochondrial respiration and trailing follower cells rely on elevated glucose uptake. We define a pyruvate dehydrogenase (PDH) dependency in leader cells that can be therapeutically exploited with the mitochondria-targeting compound alexidine dihydrochloride. In contrast, follower cells highly express glucose transporter 1 (GLUT1), which sustains an elevated level of glucose uptake required to maintain proliferation. Co-targeting of both leader and follower cells with PDH and GLUT1 inhibitors, respectively, inhibits cell growth and collective invasion. Taken together, our work reveals metabolic heterogeneity within the lung cancer collective invasion pack and provides rationale for co-targeting PDH and GLUT1 to inhibit collective invasion.

Identification of repositionable drugs with novel antimycotic activity by screening the Prestwick Chemical Library against emerging invasive moulds

ObjectivesThe incidence of severe filamentous fungal infections has increased over the past decade. Some of these filamentous fungi are resistant to available antifungals; it is thus urgent to find new compounds that are active against such life-threatening pathogens. MethodsIn this study, 1280 drugs (Prestwick Chemical Library) were tested against six multidrug-resistant (MDR) filamentous fungi, includingAspergillus, Fusarium, Scedosporium/Lomentospora, Rhizopus and Lichtheimia species. ResultsSeveral hits were identified that induced fungal growth inhibition ≥70%. Among the non-antifungal compounds that were effective against the clinical moulds tested in this study, clioquinol, alexidine dihydrochloride, hexachlorophene and thonzonium bromide displayed a broad activity against all strains tested. ConclusionThis study enriches the potential antifungal options that can be used against MDR invasive fungal diseases.

Abstract 1239: Structure activity relationship of alexidine analogs for the inhibition of collective invasion

Abstract Lung cancer is the leading cause of cancer-related deaths in the US with an overall 5-year survival of only 15-20%. Metastatic disease is a major contributor to poor survival and novel therapeutics that can selectively target invasive tumor cell populations are lacking. Metabolic reprogramming contributes to tumor progression with effective therapies targeting cancer metabolism available. However, it remains unclear whether targeting cancer metabolism will prove valuable in inhibiting invasive cancer populations. Recent studies have demonstrated anticancer activity by the mitochondria-targeting bioactive small molecule alexidine dihydrochloride. Our group has previously found that alexidine specifically inhibits the a highly invasive subpopulation of H1299 previous identified in our lab by decoupling and decreasing collective invasion. Here we report the structure activity relationship (SAR) between analogs of alexidine on the inhibition of collective invasion in NSCLC cell lines. For these studies, we utilized an in vitro 3D invasion model where NSCLC cell lines were allowed to form spheroids, and then were embedded into Matrigel and allowed to invade over 48h in the presence or absence of the analogs. Using this technique, we identified two novel alexidine analogs with improved efficacy at inhibiting the invasion of H1299 NSCLC cells. Additionally, we examined the phosphorylation of pyruvate dehydrogenase (PDH), the gatekeeper for the entrance of pyruvate into the TCA cycle, following treatment with the alexidine analogs. We observed a significant correlation between PDH phosphorylation and inhibition of invasion following treatment with the analogs. In conclusion, we have identified two novel alexidine analogs with improved inhibition of invasion in NSCLC cell lines in an in vitro model. Citation Format: Jamie Arnst, Rachel Commander, Adam Marcus. Structure activity relationship of alexidine analogs for the inhibition of collective invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1239.

Analysis of polyhexamethylene biguanide and alexidine in contact lens solutions using capillary electrophoresis, ultra-performance liquid chromatography and quadrupole time of flight mass spectrometry

Polymeric biguanides, as well as quaternary ammonium compounds, are ubiquitous antimicrobial agents in healthcare. Due to the highly cationic and polymeric nature of these compounds and the complex matrices in which they are found, the analytical characterization of products containing them remains challenging. In this work an efficient, sensitive, and high-resolution separation protocol was developed to perform quantitative measurements (sub-mg L−1) of alexidine dihydrochloride (ADH) and polyhexamethylene biguanide (PHMB) in commercial multipurpose contact lens solutions (MPS). Initially, contactless conductivity (C4D) detection was explored, but lacked adequate selectivity and sensitivity to quantify PHMB or ADH in commercial MPS. To overcome these limitations, an alternative approach using solid phase extraction (SPE) followed by separation with reversed phase ultra-performance liquid chromatography (RP-UPLC) was developed for both ADH and PHMB separation and detection. The most sensitive and reliable method investigated utilized standard additions to compensate for matrix effects. For ADH, concentration values measured with the presented method were consistent with data provided by the MPS manufacturer (1.6 mg L−1) within 0.10 mg L−1. PHMB quantification in MPS products was successful at concentrations <1 mg L−1 with quantitative reproducibility better than 2% RSD. Comparison of blind sample testing using the RP-UPLC method showed strong correlation (R2 = 0.939) of PHMB concentrations with results obtained by the United States Food and Drug Administration using a published HPLC-Evaporative light scattering detection (ELSD) assay. A significant advantage of this method is the ability to partially resolve PHMB polydispersity, which to date has been minimally studied and explained. By coupling with electrospray mass spectrometry (MS), a general trend was observed for increased retention as a function of PHMB chain length. The improved robustness and reproducibility of UV detection versus ELSD coupled with the superior resolving power of UPLC is an asset to the detection and characterization of PHMB and ADH. In addition to quality control of MPS, this method has potential application to the analyses skin wipes, wound dressings and other medical products where understanding how manufacturing processes lead to differences in polydispersity is important to maximize the antimicrobial properties while minimizing toxicologic effects.