Hookworm disease, characterized by severe anemia and cognitive and growth delays, currently affects an estimated 740 million people worldwide. Despite the prevalence of this parasitic disease, few effective drug therapies are in use today, and the heavy reliance upon benzimidazoles highlights the need for the development of novel chemotherapies. Recent work with the trematode parasite Schistosoma mansoni has identified oxadiazole 2-oxides as effective antischistosomal compounds that function by targeting and inhibiting the antioxidant enzyme, thioredoxin glutathione reductase. In this study, a related enzyme, glutathione reductase, from the human hookworm Ancylostoma ceylanicum was identified and characterized, and its in vitro activity in the presence of the oxadiazole 2-oxides was analyzed. Ex vivo worm killing assays were also conducted to establish the relationship between a given compound's effect upon worm survival and inhibition of recombinant glutathione reductase (rAceGR). Finally, the in vivo anthelminthic efficacy of furoxan (Fx) was assessed in the hamster model of hookworm infection. The predicted amino acid sequence of AceGR contained a prototypical glutathione reductase active site sequence, but no thioredoxin reductase consensus sequences, suggesting that the glutathione and thioredoxin pathways of A. ceylanicum are distinct. Although ten of the forty-two oxadiazole 2-oxides tested inhibited rAceGR activity by at least fifty percent, and fifteen compounds were toxic to parasites ex vivo, little overlap existed between these two results. We therefore suggest that AceGR is not the primary target of the oxadiazole 2-oxides in effecting parasite death. Lastly, oral treatment of A. ceylanicuminfected hamsters with furoxan resulted in significantly improved weight gains and reduced intestinal worm burdens compared to vehicle treated controls, supporting continued development of this molecule as a novel anthelminthic.

Elevated cellular reactive species, which can be produced by diabetic serum conditions such as elevated inflammatory cytokines, lipotoxicity or glucotoxicity contribute to islet beta cell dysfunction and cell death. Cellular pathways that result in beta cell oxidative stress are poorly resolved. In this study, stimulation of human donor islets, primary mouse islets or homogeneous beta cell lines with a cocktail of inflammatory cytokines (TNFα, IL-1β, and INFγ) significantly induced NADPH oxidase-1 (NOX-1) gene expression (p<0.05). This pro-inflammatory cytokine cocktail concomitantly induced loss of islet glucose stimulated insulin response (p<0.05), elevated expression of MCP-1 (p<0.01), increased cellular reactive oxygen species (ROS) and induced cell death. Inhibitors of NADPH oxidase, apocynin and diphenyleneiodonium, and a dual selective NOX1/4 inhibitor, blocked ROS generation (p<0.01) and induction of MCP-1 (p<0.05) by pro-inflammatory cytokines in beta cells. It has previously been reported that pro-inflammatory cytokine stimulation induces 12-lipoxygenase (12-LO) expression in human islets. 12-Hydroxyeicosatetraenoic acid (12-HETE), a product of 12-LO activity, stimulated NOX-1 expression in human islets (p<0.05). A novel selective inhibitor of 12-LO blocked induction of NOX-1, production of ROS and pro-caspase 3 cleavage by pro-inflammatory cytokines in INS-1 beta cells (p<0.01). Inhibition was not seen with a structurally related but inactive analog. Importantly, islets from human type 2 diabetic donors have an elevated expression of NOX-1 (p<0.05). This study describes an integrated pathway in beta cells that links beta cell dysfunction induced by pro-inflammatory cytokines with 12-lipoxygenase and NADPH oxidase (NOX-1) activation. Inhibitors of this pathway may provide a new therapeutic strategy to preserve beta cell mass in diabetes.

APE1 is an essential protein that operates in the base excision repair (BER) pathway and is responsible for ≥95% of the total apurinic/apyrimidinic (AP) endonuclease activity in human cells. BER is a major pathway that copes with DNA damage induced by several anticancer agents, including ionizing radiation and temozolomide. Overexpression of APE1 and enhanced AP endonuclease activity have been linked to increased resistance of tumor cells to treatment with monofunctional alkylators, implicating inhibition of APE1 as a valid strategy for cancer therapy. We report herein the results of a focused medicinal chemistry effort around a novel APE1 inhibitor, N-(3-(benzo[d]thiazol-2-yl)-6-isopropyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)acetamide (3). Compound 3 and related analogues exhibit single-digit micromolar activity against the purified APE1 enzyme and comparable activity in HeLa whole cell extract assays and potentiate the cytotoxicity of the alkylating agents methylmethane sulfonate and temozolomide. Moreover, this class of compounds possesses a generally favorable in vitro ADME profile, along with good exposure levels in plasma and brain following intraperitoneal dosing (30 mg/kg body weight) in mice.

Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis, which is a major cause of myocardial infarction and stroke. After activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation because inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured, including platelet aggregation, calcium mobilization, and integrin activation. Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ(3) activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Furthermore, inhibition of 12-LOX had no effect on PKC-mediated aggregation, indicating that 12-LOX is upstream of PKC. These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for antiplatelet therapy.

We report the discovery of novel small molecule inhibitors of platelet-type 12-human lipoxygenase, which display nanomolar activity against the purified enzyme, using a quantitative high-throughput screen (qHTS) on a library of 153607 compounds. These compounds also exhibit excellent specificity, >50-fold selectivity vs the paralogues, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity vs ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic experiments indicate this chemotype is a noncompetitive inhibitor that does not reduce the active site iron. Moreover, chiral HPLC separation of two of the racemic lead molecules revealed a strong preference for the (-)-enantiomers (IC(50) of 0.43 ± 0.04 and 0.38 ± 0.05 μM) compared to the (+)-enantiomers (IC(50) of >25 μM for both), indicating a fine degree of selectivity in the active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their relevance to disease modification.

BACKGROUND: Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(ε)-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(ε)-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. PRINCIPAL FINDINGS: High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. CONCLUSIONS: These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.

There are a variety of lipoxygenases in the human body (hLO), each having a distinct role in cellular biology. Human reticulocyte 15-lipoxygenase-1 (15-hLO-1), which catalyzes the dioxygenation of 1,4-cis,cis-pentadiene-containing polyunsaturated fatty acids, is implicated in a number of diseases including cancer, atherosclerosis, and neurodegenerative conditions. Despite the potential therapeutic relevance of this target, few inhibitors have been reported that are both potent and selective. To this end, we have employed a quantitative high-throughput (qHTS) screen against ∼74000 small molecules in search of reticulocyte 15-hLO-1 selective inhibitors. This screen led to the discovery of a novel chemotype for 15-hLO-1 inhibition, which displays nM potency and is >7500-fold selective against the related isozymes, 5-hLO, platelet 12-hLO, epithelial 15-hLO-2, ovine cyclooxygenase-1, and human cyclooxygenase-2. In addition, kinetic experiments were performed which indicate that this class of inhibitor is tight binding, reversible, and appears not to reduce the active-site ferric ion.

Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 A cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; K(D) = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the "off-target" effect of a small molecule is mediated by an MAI mechanism.

The synthesis of several 1,2,5-oxadiazole-2-oxide (Furoxan) analogues is described herein. These compounds were prepared in an effort to probe the SAR around the phenyl substituent and oxadiazole core for our studies toward thioredoxin-glutathione reductase (TGR) inhibition and anti-schistosomal activity.

Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide. Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic evaluation of the core molecular structure of this chemotype, we define the essential pharmacophore, establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this chemotype versus related reductase enzymes, and present evidence that these agents can be modified to possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between exogenous NO donation and parasite injury is expanded and better defined. The results of these studies verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious antischistosomal agents.