Longitudinal MRI employing diffusion tensor imaging and T2 mapping approaches has been applied to investigate the mechanisms of white matter damage caused by acute hexachlorophene neurotoxicity in rats in vivo. Male Sprague-Dawley rats were administered hexachlorophene orally once a day for five consecutive days at a dose of 30mg/kg and were monitored in 7T MRI scanner at days 0 (baseline), 3, 6, 13, and 20 following the first hexachlorophene dose. Quantitative T2 maps as well as a number of diffusion tensor parameters (fractional anisotropy, radial and axial diffusivity, apparent diffusion coefficient, and trace) were calculated from corresponding MR images. T2, as well as all diffusion tensor derived parameters (except fractional anisotropy) showed significant changes during the course of neurotoxicity development. These changes peaked at 6days after the first dose of hexachlorophene (one day after the last dose) and recovered to practically baseline levels at the end of observation (20days from the first dose). While such changes in diffusivity and T2 relaxation clearly demonstrate myelin perturbations consistent with edema, the lack of changes of fractional anisotropy suggests that the structure of the myelin sheath was not disrupted significantly by hexachlorophene in this study. This is also confirmed by the rapid recovery of all observed MRI parameters after cessation of hexachlorophene exposure.

The voltage-gated KCNQ1 potassium channel is expressed in cardiac tissues, and coassembly of KCNQ1 with an auxiliary KCNE1 subunit mediates a slowly activating current that accelerates the repolarization of action potential in cardiomyocytes. Mutations of KCNQ1 genes that result in reduction or loss of channel activity cause prolongation of repolarization during action potential, thereby causing long QT syndrome (LQTs). Small molecule activators of KCNQ1/KCNE1 are useful both for understanding the mechanism of the complex activity and for developing therapeutics for LQTs. In this study we report that hexachlorophene (HCP), the active component of the topical anti-infective prescription drug pHisoHex, is a KCNQ1/KCNE1 activator. HCP potently increases the current amplitude of KCNQ1/KCNE1 expressed by stabilizing the channel in an open state with an EC(50) of 4.61 ± 1.29 μM. Further studies in cardiomyocytes showed that HCP significantly shortens the action potential duration at 1 μM. In addition, HCP is capable of rescuing the loss of function of the LQTs mutants caused by either impaired activation gating or phosphatidylinositol-4,5-bisphosphate (PIP2) binding affinity. Our results indicate HCP is a novel KCNQ1/KCNE1 activator and may be a useful tool compound for the development of LQTs therapeutics.

The hexachlorophene (HCP) is a highly lipophilic chlorinated bisphenol present in hygienic and dermatological products. The HCP accumulates preferentially in adipose tissue that is a privileged source of mesenchymal stem cells (MSCs). The evaluation of the potential effects of HCP on MSCs is important for their medical application. Here we examined the effects of HCP on murine adipose tissue-derived stem cells (ADSCs) and human umbilical cord-derived stem cells (UCSCs) in cell culture. We found that 10-4 and 10-5 M HCP inhibits proliferation, osteogenesis and increases apoptosis of ADSCs and UCSCs. While the effect of HCP on proliferation and differentiation potential of these two cell lines was similar, the UCSCs appeared much more resistant to HCP-induced apoptosis than ADSCs. These results suggest that the adipose tissue-derived ADSCs have higher sensitive for HCP than umbilical cord-derived UCSCs and indicate that the umbilical cord can be a preferable source of MSCs for prospective medical applications in the future.

Many studies have shown that the activation of beta-catenin signaling can promote oncogenesis, and it is therefore of interest to find agents that modulate this pathway. Recent work has shown using B lymphoma cells that infection by Epstein-Barr virus (EBV) and expression of its latent membrane protein (LMP)-1, cause increases in the expression of beta-catenin and cellular transformation. Conversely, results from cell-based small molecule screening studies have shown that the antibiotic hexachlorophene can down-regulate beta-catenin in colon cancer cells. Here we report that hexachlorophene also counteracts the elevated beta-catenin levels in EBV-infected B lymphomas. This is associated with restoration in levels of Siah-1 (an E3 ubiquitin ligase that is active in beta-catenin regulation) which had been diminished by LMP-1. Our results suggest that Siah-1 is targeted by both LMP-1 and hexachlorophene with opposite effects. The hexachlorophene modulation of Siah-1 and beta-catenin is independent of p53 and results in reduced expression of cyclin-D1 and c-Myc (target genes of beta-catenin), leading to the growth arrest of B lymphoma cells. From these results we propose that hexachlorophene may provide a novel therapeutic strategy for EBV-infected B lymphoma cells by reducing beta-catenin levels via the restoration of Siah-1.

Hexachlorophene (HCP) is known to induce myelin vacuolation corresponding to intramyelinic edema of nerve fibers in the central and peripheral nervous system in animals. This study investigated the effect of maternal exposure to HCP on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 100, or 300 ppm HCP in the diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, the numbers of T box brain 2(+) progenitor cells and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling(+) apoptotic cells in the hippocampal subgranular zone (SGZ) decreased in female offspring at 300 ppm, which was accompanied by myelin vacuolation and punctate tubulin beta-3 chain staining of nerve fibers in the hippocampal fimbria. In addition, transcript levels of the cholinergic receptor, nicotinic beta 2 (Chrnb2) and B-cell CLL/lymphoma 2 (Bcl2) decreased in the dentate gyrus. HCP-exposure did not alter the numbers of SGZ proliferating cells and reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)-ergic interneuron subpopulations in the dentate hilus on PND 21 and PND 77. Although some myelin vacuolation remained, all other changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77. These results suggest that maternal HCP exposure reversibly decreases type-2b intermediate-stage progenitor cells via the mitochondrial apoptotic pathway in offspring hippocampal neurogenesis at 300 ppm HCP. Neurogenesis may be affected by dysfunction of cholinergic inputs into granule cell lineages and/or GABAergic interneurons as indicated by decreased transcript levels of Chrnb2 and numbers of Chrnb2(+) interneurons caused by myelin vacuolation in the septal-hippocampal pathway.

Maternal hexachlorophene (HCP) exposure causes transient disruption of hippocampal neurogenesis in mouse offspring. We examined epigenetically hypermethylated and downregulated genes related to this HCP-induced disrupted neurogenesis. Mated female mice were dietary exposed to 0 or 100 ppm HCP from gestational day 6 to postnatal day (PND) 21 on weaning. The hippocampal dentate gyrus of male offspring was subjected to methyl-capture sequencing and real-time reverse transcription-polymerase chain reaction analyses on PND 21. Validation analyses on methylation identified three genes, Dlx4, Dmrt1, and Plcb4, showing promoter-region hypermethylation. Immunohistochemically, DLX4+, DMRT1+, and PLCB4+ cells in the dentate hilus co-expressed GAD67, a γ-aminobutyric acid (GABA)ergic neuron marker. HCP decreased all of three subpopulations as well as GAD67+ cells on PND 21. PLCB4+ cells also co-expressed the metabotropic glutamate receptor, GRM1. HCP also decreased transcript level of synaptic plasticity-related genes in the dentate gyrus and immunoreactive granule cells for synaptic plasticity-related ARC. On PND 77, all immunohistochemical cellular density changes were reversed, whereas the transcript expression of the synaptic plasticity-related genes fluctuated. Thus, HCP-exposed offspring transiently reduced the number of GABAergic interneurons. Among them, subpopulations expressing DLX4, DMRT1, or PLCB4 were transiently reduced in number through an epigenetic mechanism. Considering the role of the Dlx gene family in GABAergic interneuron migration and differentiation, the decreased number of DLX4+ cells may be responsible for reducing those GABAergic interneurons regulating neurogenesis. The effect on granule cell synaptic plasticity was sustained until the adult stage, and reduced GABAergic interneurons active in GRM1-PLCB4 signaling may be responsible for the suppression on weaning.

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations account for 35% of the genetic alterations in non-small cell lung cancer (NSCLC). The Src-homology region 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by PTPN11, is closely involved in RAS downstream pathways and development of many tumors by affecting cell proliferation, differentiation, and immunity. Targeting SHP2 with small molecules may be a promising avenue for the treatment of KRAS-mutant (mut) NSCLC. Herein, hexachlorophene (HCP) was identified as a SHP2 inhibitor with an IC50 value of 5.63 ± 0.75 μM through screening of the FDA-approved drug library. HCP specifically inhibited SHP2 rather than other phosphatases. Molecular docking showed that HCP displayed an orientation favorable for nucleophilic attack in the catalytic domain of SHP2. HCP suppressed viability of multiple KRAS-mut and KRAS-wild type cells and induced senescence and apoptosis in KRAS-mut cells. Moreover, HCP reversed epithelial-mesenchymal transition to suppress metastasis in KRAS-mut cells, and inhibited the RAS/MEK/ERK and PI3K/AKT signaling pathways by suppression of SHP2 phosphorylation and formation SHP2/Grb2/Gab1/SOS1 complex. In summary, HCP can act as a specific SHP2 inhibitor to inhibit KRAS-mut NSCLC cell proliferation and metastasis and induce senescence through suppression of the RAF/MEK/ERK and PI3K/AKT pathways. HCP warrants further investigation as a new compound skeleton for the development of selective SHP2 inhibitors for the treatment of NSCLC.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe disease in humans with case-fatality rates of up to 30%. There are currently very limited treatment options for SFTSV infection. We conducted a drug repurposing program by establishing a two-tier test system to rapidly screen a Food and Drug Administration- (FDA)-approved drug library for drug compounds with anti-SFTSV activity in vitro. We identified five drug compounds that inhibited SFTSV replication at low micromolar concentrations, including hexachlorophene, triclosan, regorafenib, eltrombopag, and broxyquinoline. Among them, hexachlorophene was the most potent with an IC50 of 1.3 ± 0.3 µM and a selectivity index of 18.7. Mechanistic studies suggested that hexachlorophene was a virus entry inhibitor, which impaired SFTSV entry into host cells by interfering with cell membrane fusion. Molecular docking analysis predicted that the binding of hexachlorophene with the hydrophobic pocket between domain I and domain III of the SFTSV Gc glycoprotein was highly stable. The novel antiviral activity and mechanism of hexachlorophene in this study would facilitate the use of hexachlorophene as a lead compound to develop more entry inhibitors with higher anti-SFTSV potency and lower toxicity.

Biocides are used without restriction for several purposes. As a consequence, large amounts of biocides are released without any control in the environment, a situation that can challenge the microbial population dynamics, including selection of antibiotic resistant bacteria. Previous work has shown that triclosan selects Stenotrophomonas maltophilia antibiotic resistant mutants overexpressing the efflux pump SmeDEF and induces expression of this pump triggering transient low-level resistance. In the present work we analyze if two other common biocides, benzalkonium chloride and hexachlorophene, trigger antibiotic resistance in S. maltophilia. Bioinformatic and biochemical methods showed that benzalkonium chloride and hexachlorophene bind the repressor of smeDEF, SmeT. Only benzalkonium chloride triggers expression of smeD and its effect in transient antibiotic resistance is minor. None of the hexachlorophene-selected mutants was antibiotic resistant. Two benzalkonium chloride resistant mutants presented reduced susceptibility to antibiotics and were impaired in growth. Metabolic profiling showed they were more proficient than their parental strain in the use of some dipeptides. We can then conclude that although bioinformatic predictions and biochemical studies suggest that both hexachlorophene and benzalkonium chloride should induce smeDEF expression leading to transient S. maltophilia resistance to antibiotics, phenotypic assays showed this not to be true. The facts that hexachlorophene resistant mutants are not antibiotic resistant and that the benzalkonium chloride resistant mutants presenting altered susceptibility to antibiotics were impaired in growth suggests that the risk for the selection (and fixation) of S. maltophilia antibiotic resistant mutants by these biocides is likely low, at least in the absence of constant selection pressure.

SARS-CoV 3CL protease is essential for viral protein processing and is regarded as a good drug target to prevent SARS-CoV replication. In the present study, we established a high-throughput FRET technique for screening for anti-SARS-CoV 3CL protease drugs. Of a thousand existing drugs examined, hexachlorophene was identified as the most potent in inhibiting SARS-CoV 3CL protease. Further characterization showed that it was effective at micromolar concentrations (K(i) = 4 microM). The binding mode was competitive, and the inhibitory effect was dependent on preincubation time. Two other drugs, triclosan and nelfinavir, were about 10 times less potent. The structure-based search and biological evaluation of various hexachlorophene analogues were described. These analogues gave optimal inhibitory activity against SARS-CoV 3CL protease with IC(50) values ranging from 7.6 to 84.5 microM. Optimization of hexachlorophene analogues was shown to provide several active 3CL protease inhibitors that function as potential anti-SARS agents.

3C-like (3CL) protease is essential for the life cycle of severe acute respiratory syndrome-coronavirus (SARS-CoV) and therefore represents a key anti-viral target. A compound library consisting of 960 commercially available drugs and biologically active substances was screened for inhibition of SARS-CoV 3CL protease. Potent inhibition was achieved using the mercury-containing compounds thimerosal and phenylmercuric acetate, as well as hexachlorophene. As well, 1-10 microM of each compound inhibited viral replication in Vero E6 cell culture. Detailed mechanism studies using a fluorescence-based protease assay demonstrated that the three compounds acted as competitive inhibitors (Ki=0.7, 2.4, and 13.7 microM for phenylmercuric acetate, thimerosal, and hexachlorophene, respectively). A panel of metal ions including Zn2+ and its conjugates were then evaluated for their anti-3CL protease activities. Inhibition was more pronounced using a zinc-conjugated compound (1-hydroxypyridine-2-thione zinc; Ki=0.17 microM) than using the ion alone (Ki=1.1 microM).

We previously reported that transplantation of hepatic cell sheets from human bone marrow-derived mesenchymal stem cells (BM-MSCs) with hexachlorophene, a Wnt/β-catenin signaling inhibitor, ameliorated acute liver injury. In a further previous report, we identified IC-2, a newly synthesized derivative of the Wnt/β-catenin signaling inhibitor ICG-001, as a potent inducer of hepatic differentiation of BM-MSCs.

Aβ accumulation plays a central role in the pathogenesis of Alzheimer's disease (AD). Recent studies suggest that the process of Aβ nucleated polymerization is essential for Aβ fibril formation, pathology spreading and toxicity. Therefore, targeting this process represents an effective therapeutic strategy to slow or block disease progression. To discover compounds that might interfere with the Aβ seeding capacity, toxicity and pathology spreading, we screened a focused library of FDA-approved drugs in vitro using a seeding polymerization assay and identified small molecule inhibitors that specifically interfered with Aβ seeding-mediated fibril growth and toxicity. Mitoxantrone, bithionol and hexachlorophene were found to be the strongest inhibitors of fibril growth and protected primary cortical neuronal cultures against Aβ-induced toxicity. Next, we assessed the effects of these three inhibitors in vivo in the mThy1-APPtg mouse model of AD (8-month-old mice). We found that mitoxantrone and bithionol, but not hexachlorophene, stabilized diffuse amyloid plaques, reduced the levels of Aβ42 oligomers and ameliorated synapse loss, neuronal damage and astrogliosis. Together, our findings suggest that targeting fibril growth and Aβ seeding capacity constitutes a viable and effective strategy for protecting against neurodegeneration and disease progression in AD.

In the aim to determine neurotoxicity, new methods are being validated, including tests and test batteries comprising in vitro and in vivo approaches. Alternative test models such as the zebrafish (Danio rerio) embryo have received increasing attention, with minor modifications of the fish embryo toxicity test (FET; OECD TG 236) as a tool to assess behavioral endpoints related to neurotoxicity during early developmental stages. The spontaneous tail movement assay, also known as coiling assay, assesses the development of random movement into complex behavioral patterns and has proven sensitive to acetylcholine esterase inhibitors at sublethal concentrations. The present study explored the sensitivity of the assay to neurotoxicants with other modes of action (MoAs). Here, five compounds with diverse MoAs were tested at sublethal concentrations: acrylamide, carbaryl, hexachlorophene, ibuprofen, and rotenone. While carbaryl, hexachlorophene, and rotenone consistently induced severe behavioral alterations by ~ 30 h post fertilization (hpf), acrylamide and ibuprofen expressed time- and/or concentration-dependent effects. At 37-38 hpf, additional observations revealed behavioral changes during dark phases with a strict concentration-dependency. The study documented the applicability of the coiling assay to MoA-dependent behavioral alterations at sublethal concentrations, underlining its potential as a component of a neurotoxicity test battery.

Cystathionine-β-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the 'Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ∼60μM), tannic acid (IC50: ∼40μM) and benserazide (IC50: ∼30μM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ∼3μM) and NSC67078 (IC50: ∼1μM), while aurintricarboxylic acid (IC50: ∼3μM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ∼1μM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ∼6μM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ∼6μM), tannic acid (IC50: ∼20μM), benserazide (IC50: ∼20μM), and NSC67078 (IC50: ∼0.3μM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ∼300μM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300μM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300μM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100μM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50mg/kg/days.q.) prevented tumor growth. In silico docking simulations showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus that produces severe fever with thrombocytopenia syndrome (SFTS). It is widespread in Japan, South Korea, and Central and Eastern China. The epidemic has developed rapidly through China in recent years. SFTS cases have been reported in 25 provinces in China, mainly distributed in rural areas in mountainous and hilly areas. The infection has a high case fatality rate and no specific treatments or vaccinations. Therefore, early diagnosis and treatment of SFTS infection is important to survival and disease control. In this article, we provide an overview on different aspects of SFTS with an emphasis on management, to explore the current treatment and prophylactic measures further.

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.

This study involved an attempt to establish a new photosafety screening system for dermally-applied chemicals consisting of a reactive oxygen species (ROS) assay and an in vitro skin permeation test. The ROS assay was undertaken to evaluate photoreactivity of six test compounds, acridine (ACD), furosemide (FSM), hexachlorophene (HCP), 8-methoxypsoralen (MOP), norfloxacin (NFX), and promethazine (PMZ), and the in vitro skin permeation test was conducted to obtain steady-state concentration (Css) values of test compounds in removed rat skin. All test compounds were photoreactive based on ROS generation under simulated sunlight exposure. In particular, ROS generation from ACD was high compared with other test compounds, and photoreactivity of ACD was deduced to be potent. The Css values of ACD, HCP, MOP, and PMZ were over 50 μg/mL, and skin exposure to FSM and NFX was found to be extremely low. Upon these findings, ACD was judged to be highly phototoxic. The rank for phototoxic risk of test compounds based on photoreactivity and in vitro skin exposure was mostly in agreement with outcomes on their in vivo phototoxicity in rats. The proposed strategy, an alternative to animal testing, would be efficacious for photosafety evaluation of drug candidates in early stages of pharmaceutical development.

Japanese encephalitis virus (JEV), a major cause of Japanese encephalitisis, is an arbovirus that belongs to the genus Flavivirus of the family Flaviviridae. Currently, there is no effective drugs available for the treatment of JEV infection. Therefore, it is important to establish efficient antiviral screening system for the development of antiviral drugs. In this study, we constructed a full-length infectious clone of eGFP-JEV reporter virus by inserting the eGFP gene into the capsid-coding region of the viral genome. The reporter virus RNA transfected-BHK-21 cells generated robust eGFP fluorescence signals that were correlated well with viral replication. The reporter virus displayed growth kinetics similar to wild type (WT) virus although replicated a little slower. Using a known JEV inhibitor, NITD008, we demonstrated that the reporter virus could be used to identify inhibitors against JEV. Furthermore, an eGFP-JEV-based high throughput screening (HTS) assay was established in a 96-well format and used for screening of 1443 FDA-approved drugs. Sixteen hit drugs were identified to be active against JEV. Among them, five compounds which are lonafarnib, cetylpyridinium chlorid, cetrimonium bromide, nitroxoline and hexachlorophene, are newly discovered inhibitors of JEV, providing potential new therapies for treatment of JEV infection.

Glutamate dehydrogenases (GDHs) play key roles in cellular redox, amino acid, and energy metabolism, thus representing potential targets for pharmacological interventions. Here we studied the functional network provided by the three known glutamate dehydrogenases of the malaria parasite Plasmodium falciparum. The recombinant production of the previously described PfGDH1 as hexahistidyl-tagged proteins was optimized. Additionally, PfGDH2 was cloned, recombinantly produced, and characterized. Like PfGDH1, PfGDH2 is an NADP(H)-dependent enzyme with a specific activity comparable to PfGDH1 but with slightly higher K(m) values for its substrates. The three-dimensional structure of hexameric PfGDH2 was solved to 3.1 Å resolution. The overall structure shows high similarity with PfGDH1 but with significant differences occurring at the subunit interface. As in mammalian GDH1, in PfGDH2 the subunit-subunit interactions are mainly assisted by hydrogen bonds and hydrophobic interactions, whereas in PfGDH1 these contacts are mediated by networks of salt bridges and hydrogen bonds. In accordance with this, the known bovine GDH inhibitors hexachlorophene, GW5074, and bithionol were more effective on PfGDH2 than on PfGDH1. Subcellular localization was determined for all three plasmodial GDHs by fusion with the green fluorescent protein. Based on our data, PfGDH1 and PfGDH3 are cytosolic proteins whereas PfGDH2 clearly localizes to the apicoplast, a plastid-like organelle specific for apicomplexan parasites. This study provides new insights into the structure and function of GDH isoenzymes of P. falciparum, which represent potential targets for the development of novel antimalarial drugs.