Edited by Ruma BanerjeeCopper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2. Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo.Mitochondrial dysfunction contributes to the pathogenesis of heart failure, neurodegenerative disorders, myopathies, and diabetes (1). Mitochondria are dynamic, double membranebound organelles with a semi-permeable outer membrane that allows exchange of metabolites between the cytosol and the intermembrane space (IMS).5 In contrast, the inner membrane (IM) that separates the IMS and the matrix is tightly sealed. Thus, numerous transporters are required to provide the matrix with a diverse range of substrates that are necessary to support metabolism, the biogenesis of iron-sulfur clusters, and the assembly of the electron transport chain (ETC) required for oxidative phosphorylation (2).Cytochrome c oxidase (COX) is the terminal electron-accepting complex of the ETC. Mammalian COX contains 14 major subunits, two of which bind three redox centers required for electron transfer (3). The catalytic core consists of the mitochondrially-encoded subunits COX1, COX2, and COX3. COX2 binds the binuclear Cu A site required for accepting electrons from cytochrome c. These electrons are then transferred to the cofactors of COX1, first to heme a and then to the heme a 3 -Cu B site where oxygen is bound. COX biogenesis requires Ͼ25 accessory proteins known as COX assembly factors (1). The overwhelming majority of ETC defects that underlie mitochondrial dysfunction and human disease is caused by pathogenic mutations in accessory factors (1). At least nine of these factors facilitate the insertion of the copper cofactors that are essential for the catalytic competence of the COX holoenzyme (4). In yeast, it has been demonstrated that the copper used for COX assembly comes from the matrix, and this matrix copper pool is conserved in mammals (5, 6). Howe...

Pathogenic free-living amoebae, Balamuthia mandrillaris, Naegleria fowleri, and several Acanthamoeba species are the etiological agents of severe brain diseases, with case mortality rates > 90%. A number of constraints including misdiagnosis and partially effective treatments lead to these high fatality rates. The unmet medical need is for rapidly acting, highly potent new drugs to reduce these alarming mortality rates. Herein, we report the discovery of new drugs as potential anti-amoebic agents. We used the CellTiter-Glo 2.0 high-throughput screening methods to screen the Medicines for Malaria Ventures (MMV) Pandemic Response Box in a search for new active chemical scaffolds. Initially, we screened the library as a single-point assay at 10 and 1 µM. From these data, we reconfirmed hits by conducting quantitative dose–response assays and identified 12 hits against B. mandrillaris, 29 against N. fowleri, and 14 against A. castellanii ranging from nanomolar to low micromolar potency. We further describe 11 novel molecules with activity against B. mandrillaris, 22 against N. fowleri, and 9 against A. castellanii. These structures serve as a starting point for medicinal chemistry studies and demonstrate the utility of phenotypic screening for drug discovery to treat diseases caused by free-living amoebae.

Pathogenic free-living amoebae, Balamuthia mandrillaris, Naegleria fowleri and several Acanthamoeba species are the etiological agents of severe brain diseases, with case mortality rates >90%. A number of constraints including misdiagnosis and partially effective treatments lead to these high fatality rates. The unmet medical need is for rapidly acting, highly potent new drugs to reduce these alarming mortality rates. Herein, we report the discovery of new drugs as potential anti-amoebic agents. We used the CellTiter-Glo 2.0 high-throughput screening methods to screen the Medicines for Malaria Ventures (MMV) Pandemic Response Box in a search for new active chemical scaffolds. Initially we screened the library as a single-point assay at 10 and 1 µM. From these data, we reconfirmed hits by conducting quantitative dose response assays and identified 12 hits against B. mandrillaris, 29 against N. fowleri and 14 against A. castellanii ranging from nanomolar to low micromolar potency. We further describe 11 novel molecules with activity against B. mandrillaris, 22 against N. fowleri and 9 against A. castellanii. These structures serve as a starting point for medicinal chemistry studies and demonstrate the utility of phenotypic screening for drug discovery to treat diseases caused by free-living amoebae.

Naegleria fowleri , also known as the brain-eating amoeba, is ubiquitous in warm freshwater and is an opportunistic pathogen that causes primary amoebic meningoencephalitis. Although few cases are described each year, the disease has a case fatality rate of >97%.

The free-living amoeba, Naegleria fowleri, which typically dwells within warm, freshwater environments, can opportunistically cause Primary Amoebic Meningoencephalitis (PAM), a disease with a mortality rate of >98%, even with the administration of the best available drug regimens. The lack of positive outcomes for PAM has prompted a push for the discovery and development of more effective therapeutics, but most studies only utilize one or two clinical isolates in their drug discovery assays. The inability to assess possible heterogenic responses to drugs among isolates from varying geographical regions hinders progress in the field due to a lack of proven universal efficacy for novel therapeutics. Herein we conducted drug efficacy and growth rate determinations for 11 different clinical isolates, including one obtained from a successful treatment outcome, by applying a previously developed CellTiter-Glo 2.0 screening technique and flow cytometry. We found some significant differences in the susceptibility of these isolates to 7 of 8 different drugs tested, all of which comprise the cocktail that is recommended to physicians by the Centers for Disease Control. We also discovered significant variances in growth rates among isolates which draws attention to the dissidence among the amoebae populations collected from different patients. The findings of this study reiterate the need for inclusion of additional clinical isolates of varying genotypes in drug assays and highlight the necessity for more targeted therapeutics with universal efficacy across N. fowleri isolates. Our data establishes a needed baseline for drug susceptibility among clinical isolates and provides a segue for future combination therapy studies as well as research related to phenotypic or genetic differences that could shed light on mechanisms of action or predispositions to specific drugs.