Nanoscale potassium sensing based on valinomycin-anchored fluorescent gold nanoclusters.

Gold nanoclusters are a smart platform for sensing potassium ions (K+). They have been synthesized using bovine serum albumin (BSA) and valinomycin (Val) to protect and cap the nanoclusters. The nanoclusters (Val-AuNCs) produced have a red emission at 616 nm under excitation with 470 nm. In the presence of K+, the valinomycin polar groups switch to the molecule's interior by complexing with K+, forming a bracelet structure, and being surrounded by the hydrophobic exterior conformation. This structure allows a proposed fluorometric method for detecting K+ by switching between the Val-AuNCs' hydrophilicity and hydrophobicity, which induces the aggregation of gold nanoclusters. As a result, significant quenching is seen in fluorescence after adding K+. The quenching in fluorescence in the presence of K+ is attributed to the aggregation mechanism. This sensing technique provides a highly precise and selective sensing method for K+ in the range 0.78 to 8 µM with LOD equal to 233 nM. The selectivity of Val-AuNCs toward K+ ions was investigated compared to other ions. Furthermore, the Val-AuNCs have novel possibilities as favorable sensor candidates for various imaging applications. Our detection technique was validated by determining K+ ions in postmortem vitreous humor samples, which yielded promising results.

Dissecting antibiotic effects on the cell envelope using bacterial cytological profiling: a phenotypic analysis starter kit.

Phenotypic analysis assays such as bacterial cytological profiling (BCP) have become increasingly popular for antibiotic mode of action analysis. A plethora of dyes, protein fusions, and reporter strains are available and have been used for this purpose, enabling both rapid mode of action categorization and in-depth analysis of antibiotic mechanisms. However, non-expert researchers may struggle choosing suitable assays and interpreting results. This is a particular problem for antibiotics that have multiple or complex targets, such as the bacterial cell envelope. Here, we set out to curate a minimal set of accessible and affordable phenotypic assays that allow distinction between membrane and cell wall targets, can identify dual-action inhibitors, and can be implemented in most research environments. To this end, we employed BCP, membrane potential, fluidity, and cell wall synthesis assays. To assess specificity and ease of interpretation, we tested three well-characterized and commercially available reference antibiotics: the potassium ionophore valinomycin, the lipid II-binding glycopeptide vancomycin, and the dual-action lantibiotic nisin, which binds lipid II and forms a membrane pore. Based on our experiments, we suggest a minimal set of BCP, a membrane-potentiometric probe, and fluorescent protein fusions to MinD and MreB as basic assay set and recommend complementing these assays with Laurdan-based fluidity measurements and a PliaI reporter fusion, where indicated. We believe that our results can provide guidance for researchers who wish to use phenotypic analysis for mode of action studies but do not possess the specialized equipment or expert knowledge to employ the full breadth of possible techniques.IMPORTANCEPhenotypic analysis assays using specialized fluorescence fusions and dyes have become increasingly popular in antibiotic mode of action analysis. However, it can be difficult to implement these methods due to the need for specialized equipment and/or the complexity of bacterial cell biology and physiology, making the interpretation of results difficult for non-experts. This is especially problematic for compounds that have multiple or pleiotropic effects, such as inhibitors of the bacterial cell envelope. In order to make phenotypic analysis assays accessible to labs, whose primary expertise is not bacterial cell biology, or with limited equipment and resources, a set of simple and broadly accessible assays is needed that is easy to implement, execute, and interpret. Here, we have curated a set of assays and strains that does not need highly specialized equipment, can be performed in most labs, and is straightforward to interpret without knowing the intricacies of bacterial cell biology.

Theoretical investigation of hydroxylated analogues of valinomycin as potassium transporter.

Valinomycin is a potent ionophore known for its ability to transport potassium ions across biological membranes. The study focuses on the hydroxylated analogues of valinomycin (HyVLMs) and compares their energy profiles and capabilities for transporting potassium ions across phospholipid membranes. Using metadynamics, we investigated the energy profiles of wildtype valinomycin (VLM_1) and its three hydroxylated analogues (VLM_2, VLM_3, and VLM_4). We observed that all analogues exhibited energy maxima in the centre of the membrane and preferred positions below the phospholipid heads. Furthermore, the entry barriers for membrane penetration were similar among the analogues, suggesting that the hydroxyl group did not significantly affect their passage through the membrane. Transition state calculations provided insights into the ability of valinomycin analogues to capture potassium ions, with VLM_4 showing the lowest activation energy and VLM_2 displaying the highest. Our findings contribute to understanding the mechanisms of potassium transport by valinomycin analogues and highlight their potential as ionophores. The presence of the hydroxyl group is of particular importance because it paves the way for subsequent chemical modifications and the synthesis of new antiviral agents with reduced intrinsic toxicity.

Nigericin Induces Apoptosis in Primary Effusion Lymphoma Cells by Mitochondrial Membrane Hyperpolarization and ß-Catenin Destabilization.

BACKGROUND/AIM: Primary effusion lymphoma (PEL) is classified as a rare non-Hodgkin's B-cell lymphoma that is caused by Kaposi's sarcoma-associated herpesvirus (KSHV); PEL cells are latently infected with KSHV. PEL is frequently resistant to conventional chemotherapies. Therefore, the development of novel therapeutic agents is urgently required. Nigericin, a H+ and K+ ionophore, possesses unique pharmacological effects. However, the effects of nigericin on PEL cells remain unknown. MATERIALS AND METHODS: We examined the cytotoxic effects of the K+ ionophores, nigericin, nonactin, and valinomycin, on various B-lymphoma cells including PEL. We also evaluated ionophore-induced changes in signaling pathways involved in KSHV-induced oncogenesis. Moreover, the effects of nigericin on mitochondrial membrane potential and viral reactivation in PEL were analyzed. RESULTS: Although the three tested ionophores inhibited the proliferation of several B-lymphoma cell lines, nigericin inhibited the proliferation of PEL cells compared to KSHV-negative cells. In PEL cells, nigericin disrupted the mitochondrial membrane potential and caused the release of cytochrome c, which triggered caspase-9-mediated apoptosis. Nigericin also induced both an increase in phosphorylated p38 MAPK and proteasomal degradation of ß-catenin. Combination treatment of nigericin with the p38 MAPK inhibitor SB203580 potentiated the cytotoxic effects towards PEL cells, compared to either compound alone. Meanwhile, nigericin did not influence viral replication in PEL cells. CONCLUSION: Nigericin induces apoptosis in PEL cells by mitochondrial dysfunction and down-regulation of Wnt/ß-catenin signaling. Thus, nigericin is a novel drug candidate for treating PEL without the risk of de novo KSHV infection.

Single-Molecule Electrical Currents Associated with Valinomycin Transport of K.

A quantitative description of ionophore-mediated ion transport is important in understanding ionophore activity in biological systems and developing ionophore applications. Herein, we describe the direct measurement of the electrical current resulting from K+ transport mediated by individual valinomycin (val) ionophores. Step fluctuations in current measured across a 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayer suspended over a ∼400 nm radius glass nanopore result from dynamic partitioning of val between the bilayer and torus region, effectively increasing or decreasing the total number of val present in the membrane. In our studies, approximately 30 val are present in the membrane on average with a val entering or leaving the bilayer approximately every 50 s, allowing measurement of changes in electrical current associated with individual val. The single-molecule val(K+) transport current at 0.1 V applied potential is (1.3 ± 0.6) × 10-15 A, consistent with estimates of the transport kinetics based on large val ensembles. This methodology for analyzing single ionophore transport is general and can be applied to other carrier-type ionophores.

Optimization of fermentation medium and conditions for enhancing valinomycin production by Streptomyces sp. ZJUT-IFE-354.

Valinomycin is a cyclodepsipeptide antibiotic with a broad spectrum of biological activities, such as antiviral, antitumor, and antifungal activities. However, the low yield of valinomycin often limits its applications in medicine, agriculture, and industry. In our previous report, Streptomyces sp. ZJUT-IFE-354 was identified as a high-yielding strain of valinomycin. In this study, Plackett-Burman design (PBD) and response surface methodology (RSM) were used to optimize components of medium. The optimal medium contained 31 g/L glucose, 22 g/L soybean meal, and 1.6 g/L K2HPO4·3H2O, which could generate 262.47 ± 4.28 mg/L of valinomycin. Then, the culture conditions were optimized by a one-factor-at-a-time (OFAT) approach. The optimal conditions for the strain included a seed age of 24 h, an inoculum size of 8% (v/v), an incubation temperature of 28 °C, an initial pH of 7.2, an elicitor of 0.1% Bacillus cereus feeding at 24 h cultivation, and the feeding of 0.6% L-valine at 36 h cultivation. The final valinomycin production increased to 457.23 ± 9.52 mg/L, which was the highest yield ever reported. It highlights that RSM and OFAT may be efficient methods to enhance valinomycin production by Streptomyces sp. ZJUT-IFE-354.

Stapled NRPS enhances the production of valinomycin in Escherichia coli.

Nonribosomal peptides (NRPs) are a large family of secondary metabolites with notable bioactivities, which distribute widely in natural resources across microbes and plants. To obtain these molecules, heterologous production of NRPs in robust surrogate hosts like Escherichia coli represent a feasible approach. However, reconstitution of the full biosynthetic pathway in a host often leads to low productivity, which is at least in part due to the low efficiency of enzyme interaction in vivo except for the well-known reasons of metabolic burden (e.g., expression of large NRP synthetases-NRPSs with molecular weights of >100 kDa) and cellular toxicity on host cells. To enhance the catalytic efficiency of large NRPSs in vivo, here we propose to staple NRPS enzymes by using short peptide/protein pairs (e.g., SpyTag/SpyCatcher) for enhanced NRP production. We achieve this goal by introducing a stapled NRPS system for the biosynthesis of the antibiotic NRP valinomycin in E. coli. The results indicate that stapled valinomycin synthetase (Vlm1 and Vlm2) enables higher product accumulation than those two free enzymes (e.g., the maximum improvement is nearly fourfold). After further optimization by strain and bioprocess engineering, the final valinomycin titer maximally reaches about 2800 µg/L, which is 73 times higher than the initial titer of 38 µg/L. We expect that stapling NRPS enzymes will be a promising catalytic strategy for high-level biosynthesis of NRP natural products.

Ionophore Antibiotics Inhibit Type II Feline Coronavirus Proliferation In Vitro.

Feline coronaviruses (FCoVs) infect cats worldwide and cause severe systemic diseases, such as feline infectious peritonitis (FIP). FIP has a high mortality rate, and drugs approved by the Food and Drug Administration have been ineffective for the treatment of FIP. Investigating host factors and the functions required for FCoV replication is necessary to develop effective drugs for the treatment of FIP. FCoV utilizes an endosomal trafficking system for cellular entry after binding between the viral spike (S) protein and its receptor. The cellular enzymes that cleave the S protein of FCoV to release the viral genome into the cytosol require an acidic pH optimized in the endosomes by regulating cellular ion concentrations. Ionophore antibiotics are compounds that form complexes with alkali ions to alter the endosomal pH conditions. This study shows that ionophore antibiotics, including valinomycin, salinomycin, and nigericin, inhibit FCoV proliferation in vitro in a dose-dependent manner. These results suggest that ionophore antibiotics should be investigated further as potential broad-spectrum anti-FCoV agents.

An evaluation of a new high-sensitivity PrestoBlue assay for measuring cell viability and drug cytotoxicity using EA.hy926 endothelial cells.

INTRODUCTION: Commercially-available resazurin-based reagents used for cell viability assessment contain varying amounts of resorufin; these may contribute to differences in autofluorescence, signal-to-background (S/B) ratio and the dynamic range of the assay. OBJECTIVES: This in vitro study compares the sensitivity of a new, high-sensitivity PrestoBlue (hs-PB) assay with standard PrestoBlue (PB) in assessing the efficacy of valinomycin and antimycin A in human vascular endothelial EA.hy926 cells, as well as cell viability. METHODS: The metabolic activity of EA.hy926 was evaluated based on resorufin fluorescence (PB assays) or formazan absorbance (MTT assay). RESULTS: The hs-PB assay demonstrated lower resorufin autofluorescence than the PB, resulting in a ≥ 1.4-fold increase in S/B ratio in hs-PB compared to PB. Valinomycin was more potent cytotoxic agent than antimycin A. The hs-PB, PB and MTT produced similar IC50 values for valinomycin. Antimycin A showed significantly higher potency in the MTT than in the resazurin-based assays. The EA.hy926 cells demonstrated higher metabolic activity in the presence of the antimycin A solvent - DMSO. CONCLUSION: All the examined methods may be used interchangeably to analyze drug cytotoxicity. Any differences in drug cytotoxicity observed between the assays may be due to relatively low drug potency and/or the influence of solvent on metabolism of assay reagent. The hs-PB assay appears to more effectively detect cell viability and produce a stronger signal than its conventional counterpart.

Recent advances in, and challenges of, designing OMA1 drug screens.

The proteases of the mitochondrial inner membrane are challenging yet highly desirable drug targets for complex, multifactorial diseases prevalent mainly in the elderly. Among them, OMA1 with its substrates OPA1 and DELE1 safeguards mitochondrial homeostasis at the intersection of energy metabolism and apoptosis, which may have relevance for neurodegeneration, malignancy and heart failure, among other diseases. Little is known about OMA1. Its structure has not been solved and we are just beginning to understand the enzyme's context-dependent regulation. OMA1 appears dormant under physiological conditions as judged by OPA1's processing pattern. The protease is rapidly activated, however, when cells experience stress or undergo apoptosis. Intriguingly, genetic OMA1 ablation can delay or even prevent apoptosis in animal models for diseases that can be broadly categorized as ischemia-reperfusion related disorders. Three groups have reported their efforts implementing OMA1 drug screens. This article reviews some of the technical challenges encountered in these assays and highlights what can be learned for future screening campaigns, and about the OMA1 protease more broadly. OMA1 does not exists in a vacuum and potent OMA1 inhibitors are needed to tease apart OMA1's intricate interactions with the other mitochondrial proteases and enzymes. Furthermore, OMA1 inhibitors hold the promise of becoming a new class of cytoprotective medicines for disorders influenced by dysfunctional mitochondria, such as heart failure or Alzheimer's Disease.

Efficient production of valinomycin by the soil bacterium, Streptomyces sp. ZJUT-IFE-354.

A novel strain with antifungal activity against Sclerotinia sclerotiorum was isolated from soil, and identified as Streptomyces sp. ZJUT-IFE-354 using morphological and 16S rDNA sequence analysis. The bioactive metabolite produced by strain ZJUT-IFE-354 was identified and characterized as valinomycin by spectroscopic and chemical methods. The yield of valinomycin was 191.26 mg/L from the culture of Streptomyces sp. ZJUT-IFE-354, which was the highest yield to our knowledge. The in vitro antifungal activity of valinomycin against S. sclerotiorum was investigated as 0.056 ± 0.012 (EC50) and 0.121 ± 0.023 µg/mL (EC95), respectively, which was approximately 10.696- and 30.960-fold more active than that of carbendazim. The results from scanning electron microscopy, cell membrane permeability, and D-sorbitol and ergosterol assay indicated that valinomycin exerted the antifungal activity probably by increasing permeability of fungal cell membrane, leading to mycelial electrolyte leakage, and eventually resulting in the death of S. sclerotiorum. Thus, valinomycin may be a promising antifungal agent to control S. sclerotiorum. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03055-5.

Discovery of Novel Cyclic Ethers with Synergistic Antiplasmodial Activity in Combination with Valinomycin.

With drug resistance threatening our first line antimalarial treatments, novel chemotherapeutics need to be developed. Ionophores have garnered interest as novel antimalarials due to their theorized ability to target unique systems found in the Plasmodium-infected erythrocyte. In this study, during the bioassay-guided fractionation of the crude extract of Streptomyces strain PR3, a group of cyclodepsipeptides, including valinomycin, and a novel class of cyclic ethers were identified and elucidated. Further study revealed that the ethers were cyclic polypropylene glycol (cPPG) oligomers that had leached into the bacterial culture from an extraction resin. Molecular dynamics analysis suggests that these ethers are able to bind cations such as K+, NH4+ and Na+. Combination studies using the fixed ratio isobologram method revealed that the cPPGs synergistically improved the antiplasmodial activity of valinomycin and reduced its cytotoxicity in vitro. The IC50 of valinomycin against P. falciparum NF54 improved by 4-5-fold when valinomycin was combined with the cPPGs. Precisely, it was improved from 3.75 ± 0.77 ng/mL to 0.90 ± 0.2 ng/mL and 0.75 ± 0.08 ng/mL when dosed in the fixed ratios of 3:2 and 2:3 of valinomycin to cPPGs, respectively. Each fixed ratio combination displayed cytotoxicity (IC50) against the Chinese Hamster Ovary cell line of 57-65 µg/mL, which was lower than that of valinomycin (12.4 µg/mL). These results indicate that combinations with these novel ethers may be useful in repurposing valinomycin into a suitable and effective antimalarial.

RETRACTED: Liposomal valinomycin mediated cellular K+ leak promoting apoptosis of liver cancer cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the corresponding author. It has been found that Fig 2B contains manipulated components, and Fig 5A partially overlaps with Fig 6 of a published paper authored by Mirza Muhammad Faran Ashraf Baig, et, al., The effective transfection of a low dose of negatively charged drug-loaded DNA-nanocarriers into cancer cells via scavenger receptors, J. Pharm. Anal. 11 (2021) 174-182, https://doi.org/10.1016/j.jpha.2020.10.003. The corresponding author indicated that they cannot guarantee the integrity of the images in the manuscript, as well as the conclusions of the paper. As a result, the Editor-in-Chief has decided to retract the paper. The corresponding author deeply regrets the circumstances and apologizes to the scientific community for not having detected this prior to publication.

Membrane hyperpolarization abolishes calcium oscillations that prevent induced acrosomal exocytosis in human sperm.

Sperm capacitation is essential to gain fertilizing capacity. During this process, a series of biochemical and physiological modifications occur that allow sperm to undergo acrosomal exocytosis (AE). At the molecular level, hyperpolarization of the sperm membrane potential (Em) takes place during capacitation. This study shows that human sperm incubated under conditions that do not support capacitation (NC) can become ready for an agonist stimulated AE by pharmacologically inducing Em hyperpolarization with Valinomycin or Amiloride. To investigate how Em hyperpolarization promotes human sperm's ability to undergo AE, live single-cell imaging experiments were performed to simultaneously monitor changes in [Ca2+ ]i and the occurrence of AE. Em hyperpolarization turned [Ca2+ ]i dynamics in NC sperm from spontaneously oscillating into a sustained slow [Ca2+ ]i increase. The addition of progesterone (P4) or K+ to Valinomycin-treated sperm promoted that a significant number of cells displayed a transitory rise in [Ca2+ ]i which then underwent AE. Altogether, our results demonstrate that Em hyperpolarization is necessary and sufficient to prepare human sperm for the AE. Furthermore, this Em change decreased Ca2+ oscillations that block the occurrence of AE, providing strong experimental evidence of the molecular mechanism that drives the acquisition of acrosomal responsiveness.

The Nonribosomal Peptide Valinomycin: From Discovery to Bioactivity and Biosynthesis.

Valinomycin is a nonribosomal peptide that was discovered from Streptomyces in 1955. Over the past more than six decades, it has received continuous attention due to its special chemical structure and broad biological activities. Although many research papers have been published on valinomycin, there has not yet been a comprehensive review that summarizes the diverse studies ranging from structural characterization, biogenesis, and bioactivity to the identification of biosynthetic gene clusters and heterologous biosynthesis. In this review, we aim to provide an overview of valinomycin to address this gap, covering from 1955 to 2020. First, we introduce the chemical structure of valinomycin together with its chemical properties. Then, we summarize the broad spectrum of bioactivities of valinomycin. Finally, we describe the valinomycin biosynthetic gene cluster and reconstituted biosynthesis of valinomycin. With that, we discuss possible opportunities for the future research and development of valinomycin.

Effects of valinomycin doping on the electrical and structural properties of planar lipid bilayers supported on polyelectrolyte multilayers.

Supported Lipid Bilayers (SLBs) on Polyelectrolyte Multilayers (PEMs) have large potential as models for developing sensor devices. SLBs can be designed with receptors and channels, which benefit from the biological environment of the lipid layers, to create a sensing interface for ions and biomarkers. PEMs assembled by the Layer-by-Layer (LBL) technique and used as supports for a lipid bilayer enable an easy integration of the bilayer on almost any surface and device. For electrochemical sensors, LBL assembly enables nanoscale tunable separation of the lipid bilayer from the electrode surface, avoiding undesired effects of the electrode surface on the lipid bilayers. We study the fabrication of valinomycin-doped SLBs on PEMs as a model system for biophysical studies and for selective ion sensing. SLBs are fabricated from dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS) 50:50 vesicles doped with valinomycin, as a K+-selective carrier. SLBs were deposited on electrodes coated with poly(allyl amine hydrochloride) (PAH) and poly(styrene sodium sulfonate) (PSS) multilayers. Lipid bilayer formation was monitored by using Quartz Crystal Microbalance with Dissipation (QCMD) technique and Atomic Force Microscopy (AFM). Electrochemical impedance spectroscopy (EIS) and potentiometric measurements were performed to assess K+ selectivity over other ions and the potential of valinomycin-doped SLBs for K+-sensing.

Examination of microcystin neurotoxicity using central and peripheral human neurons.

Microcystins (MC) are a group of cyanobacterial toxins that comprises MC-LF and other cyclic heptapeptides, best known as potent hepatotoxicants. Cell culture and epidemiological studies suggest that MC might also affect the nervous system when there is systemic exposure, e.g., via drinking water or food. We asked whether in vitro studies with human neurons could provide estimates on the neurotoxicity hazard of MC-LF. First, we used LUHMES neurons, a well-established test system for neurotoxicants and neuropathological processes. These central nervous system cells express OATP1A2, a presumed carrier of MC-LF, and we observed selective neurite toxicity in the µM range (EC20 = 3.3 µM ≈ 3.3 µg/mL). Transcriptome changes pointed towards attenuated cell maintenance and biosynthetic processes. Prolonged exposure for up to four days did not increase toxicity. As a second model, we used human dorsal root ganglia-like neurons. These peripheral nervous system cells represent parts of the nervous system not protected by the blood-brain barrier in humans. Toxicity was observed in a similar concentration range (EC20 = 7.4 µM). We conclude that MC-LF poses a potential neurotoxic hazard in humans. The adverse effect concentrations observed here were orders of magnitude higher than those presumed to be encountered after normal nutritional or environmental exposure. However, the low µM concentrations found to be toxic are close to levels that may be reached after very excessive algae supplement intake.

Valinomycin as a potential antiviral agent against coronaviruses: A review.

Human coronaviruses (HCoVs), including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been resulting in global epidemics with heavy morbidity and mortality. Unfortunately, there are currently no specific medicines that can better treat these coronaviruses. Drug repurposing is an effective and economical strategy for drug discovery from existing drugs, natural products, and synthetic compounds. In this review, the broad-spectrum antiviral activity of valinomycin (VAL), especially its activity against coronaviruses such as SARS-CoV, MERS-CoV, human coronavirus OC43 (HCoV-OC43), were summarized, it highlights that VAL has tremendous potential for use as a novel antiviral agent against SARS-CoV-2.

Don't sugar coat the COVID (only the vasculature).

This issue of the Biomedical Journal acquaints us with the compelling hypothesis that the vascular glycocalyx lies at the intersection of severe COVID-19 risk factors and damages, and the ways used by artificial intelligence to predict interactions between SARS-CoV-2 and human proteins. Furthermore, we explore the antiviral potential of valinomycin and the long list of COVID-19-related clinical trials, and learn how (not) to fix a broken femoral head. Last but not least, we get to enjoy the tale of the cellular oxygen-sensing system as well as the role of the host complement system during Leptospira infection, and learn that SARS-CoV-2 can sometimes come with a pathogenic plus one.

Study of mitophagy and ATP-related metabolomics based on ß-amyloid levels in Alzheimer's disease.

The aggregation of ß-amyloid (Aß) peptide in Alzheimer's disease (AD) is characterized by mitochondrial dysfunction and mitophagy impairment. Mitophagy is a homeostatic mechanism by which autophagy selectively eliminates damaged mitochondria. Valinomycin is a respiratory chain inhibitor that activates mitophagy via the PINK1/Parkin signaling pathway. However, the mechanism underlying the association between mitophagy and valinomycin in Aß formation has not been explored. Here, we demonstrate that genetically modified (N2a/APP695swe) cells overexpressing a mutant amyloid precursor protein (APP) serve as an in vitro model of AD for studying mitophagy and ATP-related metabolomics. Our results prove that valinomycin induced a time-dependent increase in the mitophagy activation of N2a/APP695swe cells as indicated by increased levels of PINK1, Parkin, and LC3II as well as increased the colocalization of Parkin-Tom20 and fewer mitochondria (indicated by decreased Tom20 levels). Valinomycin significantly decreased Aß1-42 and Aß1-40 levels after 3 h of treatment. ATP levels and ATP-related metabolites were significantly increased at this time. Our findings suggest that the elimination of impaired mitochondria via valinomycin-induced mitophagy ameliorates AD by decreasing Aß and improving ATP levels.