Identification of 3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase.

Plasmodium falciparum's resistance to available antimalarial drugs highlights the need for the development of novel drugs. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. PfDHODH is targeted by the inhibitor DSM265, which binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. In this study, we screened 40,400 compounds from the Kyoto University chemical library against recombinant PfDHODH. These studies led to the identification of 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine and its derivatives as a new class of PfDHODH inhibitor. Moreover, the hit compounds identified in this study are selective for PfDHODH without inhibition of the human enzymes. Finally, this new scaffold of PfDHODH inhibitors showed growth inhibition activity against P. falciparum 3D7 with low toxicity to three human cell lines, providing a new starting point for antimalarial drug development.

Evaluation of spice and herb as phyto-derived selective modulators of human retinaldehyde dehydrogenases using a simple in vitro method.

Selective modulation of retinaldehyde dehydrogenases (RALDHs)-the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.

Discovery of selective BPTF bromodomain inhibitors by screening and structure-based optimization.

Bromodomain and PHD finger containing transcription factor (BPTF) is a multidomain protein that regulates the transcription of chromatin and is related to many cancers. Herein, we report the screening-based discovery of Cpd1, a compound with micromolar affinity to the BPTF bromodomain. Through structure-guided optimization, we synthesized a variety of new inhibitors. Among these compounds, Cpd8 and Cpd10 were highly potent and selective inhibitors, with KD values of 428 nM and 655 nM in ITC assays, respectively. The high activity was explained by the cocrystal structure of Cpd8 in complex with the BPTF bromodomain protein. Cpd8 and Cpd10 were able to stabilize the BPTF bromodomain protein in cells in a cellular thermal shift assay (CETSA). Cpd8 downregulated c-MYC expression in A549 cells. All experiments prove that these two compounds are potential BPTF inhibitors.

A pipeline to evaluate inhibitors of the Pseudomonas aeruginosa exotoxin U.

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. In severe P. aeruginosa infections, the phospholipase activity of the type 3 secretion system toxin, ExoU, induces lysis of target host cells and results in the poorest clinical outcomes. We have developed an integrated pipeline to evaluate small molecule inhibitors of ExoU in vitro and in cultured cell models, including a disease-relevant corneal epithelial (HCE-T) scratch and infection model using florescence microscopy and cell viability assays. Compounds Pseudolipasin A, compound A and compound B were effective in vitro inhibitors of ExoU and mitigated P. aeruginosa ExoU-dependent cytotoxicity after infection of HCE-T cells at concentrations as low as 0.5 µM. Addition of the antimicrobial moxifloxacin controlled bacterial load, allowing these assays to be extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration for 24 h, but, when used in combination with either Pseudolipasin A, compound A or compound B, a greater amount of viable cells and scratch healing were observed. Thus, our pipeline provides evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat infections due to ExoU producing P. aeruginosa, as well as the means to identify more potent ExoU inhibitors for future therapeutics.

Inhibition of hyperpolarization-activated cyclic nucleotide-gated channels with natural flavonoid quercetin.

Quercetin is a natural flavonoid which has been reported to be analgesic in different animal models of pain. However, the mechanism underlying the pain-relieving effects is still unclear. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in controlling pacemaker activity in cardiac and nervous systems, making the channel a new target for therapeutic exploration. In this study, we explored a series of flavonoids for their modulation on HCN channels. Among all tested flavonoids, quercetin was the most potent inhibitor for HCN channels with an IC50 value of 27.32 ± 1.19 µM for HCN2. Furthermore, quercetin prominently left shifted the voltage-dependent activation curves of HCN channels and decelerated deactivation process. The results presented herein firstly characterize quercetin as a novel and potent inhibitor for HCN channels, which represents a novel structure for future drug design of HCN channel inhibitors.

The ellagitannin trimer rugosin G inhibits recombinant human histidine decarboxylase.

Rugosin G, an ellagitannin trimer, was isolated from the water-soluble fraction of red rose petals, and its inhibitory activity against recombinant human histidine decarboxylase was investigated. Rugosin G showed potent inhibition compared to ellagitannin monomers and a dimer with macrocyclic structure (oenothein B), suggesting the potent inhibition of rugosin G was attributed to its linear oligomeric conformation. Abbreviations: HDC, histidine decarboxylase; Me2CO, acetone; EtOAc, ethyl acetate.

Selective Inhibition of PAR4 (Protease-Activated Receptor 4)-Mediated Platelet Activation by a Synthetic Nonanticoagulant Heparin Analog.

Objective- PAR4 (protease-activated receptor 4), one of the thrombin receptors in human platelets, has emerged as a promising target for the treatment of arterial thrombotic disease. Previous studies implied that thrombin exosite II, known as a binding site for heparin, may be involved in thrombin-induced PAR4 activation. In the present study, a heparin octasaccharide analog containing the thrombin exosite II-binding domain of heparin was chemically synthesized and investigated for anti-PAR4 effect. Approach and Results- PAR4-mediated platelet aggregation was examined using either thrombin in the presence of a PAR1 antagonist or γ-thrombin, which selectively activates PAR4. SCH-28 specifically inhibits PAR4-mediated platelet aggregation, as well as the signaling events downstream of PAR4 in response to thrombin. Moreover, SCH-28 prevents thrombin-induced ß-arrestin recruitment to PAR4 but not PAR1 in Chinese Hamster Ovary-K1 cells using a commercial enzymatic complementation assay. Compared with heparin, SCH-28 is more potent in inhibiting PAR4-mediated platelet aggregation but has no significant anticoagulant activity. In an in vitro thrombosis model, SCH-28 reduces thrombus formation under whole blood arterial flow conditions. Conclusions- SCH-28, a synthetic small-molecular and nonanticoagulant heparin analog, inhibits thrombin-induced PAR4 activation by interfering with thrombin exosite II, a mechanism of action distinct from other PAR4 inhibitors that target the receptor. The characteristics of SCH-28 provide a new strategy for targeting PAR4 with the potential for the treatment of arterial thrombosis.

Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V.

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.

Alkaloids of Amaryllidaceae as Inhibitors of Cholinesterases (AChEs and BChEs): An Integrated Bioguided Study.

INTRODUCTION: Enzymatic inhibition of acetylcholinesterase (AChE) is an essential therapeutic target for the treatment of Alzheimer's disease (AD) and AChE inhibitors are the first-line drugs for it treatment. However, butyrylcholinesterase (BChE), contributes critically to cholinergic dysfunction associated with AD. Thus, the development of novel therapeutics may involve the inhibition of both cholinesterase enzymes. OBJECTIVE: To evaluate, in an integrated bioguided study, cholinesterases alkaloidal inhibitors of Amaryllidaceae species. METHODOLOGY: The proposed method combines high-performance thin-layer chromatography (HPTLC) with data analysis by densitometry, enzymatic bioautography with different AChEs and BChEs, the detection of bioactive molecules through gas chromatography mass spectrometry (GC-MS) analysis of spots of interest, and theoretical in silico studies. RESULTS: To evaluate the bioguided method, the AChE and BChE inhibitory activities of seven Amaryllidaceae plant extracts were evaluated. The alkaloid extracts of Eucharis bonplandii exhibited a high level of inhibitory activity (IC50  = 0.72 ± 0.05 µg/mL) against human recombinant AChE (hAChE). Regarding human serum BChE (hBChE), the bulb and leaf extracts of Crinum jagus had the highest activity (IC50  = 8.51 ± 0.56 µg/mL and 11.04 ± 1.21 µg/mL, respectively). In the HPTLC spots with high inhibitory activity, several alkaloids were detected using GC-MS, and some of these alkaloids were identified. Galanthamine, galanthamine N-oxide and powelline should be the most prominent inhibitors of substrate accommodation in the active site of the Torpedo californica AChE (TcAChE), hAChE and hBChE enzymes. CONCLUSIONS: These results are evidence of the chemical relevance of the Colombian's Amaryllidaceae species for the inhibition of cholinesterases and as potent sources for the palliative treatment of AD. Copyright © 2017 John Wiley & Sons, Ltd.

Mammalian odorant receptor tuning breadth persists across distinct odorant panels.

The molecular receptive range (MRR) of a mammalian odorant receptor (OR) is the set of odorant structures that activate the OR, while the distribution of these odorant structures across odor space is the tuning breadth of the OR. Variation in tuning breadth is thought to be an important property of ORs, with the MRRs of these receptors varying from narrowly to broadly tuned. However, defining the tuning breadth of an OR is a technical challenge. For practical reasons, a screening panel that broadly covers odor space must be limited to sparse coverage of the many potential structures in that space. When screened with such a panel, ORs with different odorant specificities, but equal tuning breadths, might appear to have different tuning breadths due to chance. We hypothesized that ORs would maintain their tuning breadths across distinct odorant panels. We constructed a new screening panel that was broadly distributed across an estimated odor space and contained compounds distinct from previous panels. We used this new screening panel to test several murine ORs that were previously characterized as having different tuning breadths. ORs were expressed in Xenopus laevis oocytes and assayed by two-electrode voltage clamp electrophysiology. MOR256-17, an OR previously characterized as broadly tuned, responded to nine novel compounds from our new screening panel that were structurally diverse and broadly dispersed across an estimated odor space. MOR256-22, an OR previously characterized as narrowly tuned, responded to a single novel compound that was structurally similar to a previously known ligand for this receptor. MOR174-9, a well-characterized receptor with a narrowly tuned MRR, did not respond to any novel compounds in our new panel. These results support the idea that variation in tuning breadth among these three ORs is not an artifact of the screening protocol, but is an intrinsic property of the receptors.

Structural characterization of nonactive site, TrkA-selective kinase inhibitors.

Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residues from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of-but adjacent to-the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.

Evaluation of the antioxidant properties of tryptophan and its metabolites in in vitro assay.

BACKGROUND: Human milk contains a number of nutrients and bioactive ingredients which play an important role in the growth and development of infants. One important nutrient and bioactive ingredient of human milk is L-tryptophan. L-Tryptophan is an essential aromatic α-amino acid and is required in the diet of children and adult humans. As an essential amino acid, it is needed for protein synthesis and as a precursor of key biomolecules such as serotonin, melatonin, tryptamine, niacin, quinolinic acid and kynurenic acid, nicotinamide adenine dinucleotide. The aim of the study was to evaluate the antioxidant, anti-inflammatory and antiproliferative properties of tryptophan isolated from enzymatic hydrolysates from human milk and its metabolites on human glioma U251 cells and to evaluate the effects of human recombinant (hrIFNγ) on molecular ions of tryptophan and its metabolites in human glial U251 cells. METHODS: The cytotoxicity was determined by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. The antioxidant property was assessed by the oxygen radical scavenging capacity (ORAC) method. The anti-inflammatory effect was determined by the enzyme-linked immunosorbent assay (ELISA) against cytokines IL-6 and TNF-α. The effects of recombinant human (rhIFNγ) on molecular ions of tryptophan and its catabolites were evaluated by mass spectrometry. The tryptophan was isolated from milk peptides following enzymatic digestion, followed by separation by chromatographic and mass spectrometric methods. RESULTS: Tryptophan from human milk exhibited profoundly higher oxygen radical absorption capacity (7,986±468 µm Trolox equivalent (TE)/g) than that of whole human milk (80.4±13.3 µm TE/g). Tryptophan showed a moderate degree of anti-inflammatory activity against TNF-α and IL-6. rhIFNγ inhibited tryptophan metabolism. A low concentration of L-tryptophan (10-25 µg/mL) inhibited nearly 25% of cell growth. When U251 cells were treated with 25 µg/mL L-tryptophan and subsequently challenged with 30 ng/mL of human recombinant IFNγ, a significant inhibitory effect on cell growth was observed. Low concentrations of Xanthurenic acid, L-kynurenine, and 3-OH DL kynurenine were found to inhibit cell growth except melatonin and 3-OH anthranilic acid. Melatonin was a strong inducer of TNF-α in RAW cells, whereas 3-OH kynurenine at 25, 50 and 100 µg/mL inhibited IL-6 in RAW cells. No significant change was observed in the IL-8 profile in tryptophan-treated U251 cells except that L-kynurenine at 10 µg/mL produced significantly high level of an inflammatory cytokine IL-8. Melatonin, 3-OH, DL kynurenine at high concentrations (100 µg/mL) induced proliferation of U251 cells. Melatonin seemed to show synergistic effects with recombinant human IFNγ (rhINFγ) in promoting growth of human glioma cells. While treatment of U251 cells with tryptophan alone and subsequent treatment with rhIFNγ inhibited the growth of human cancer glioma cells, and conversely melatonin combined with rhIFNγ promoted growth of the U251 cells. CONCLUSIONS: The findings from this study suggest that human milk-derived tryptophan and its metabolites possess strong antioxidant properties. Such effects might play a significant role in regulating the cell proliferation and growth of human cancer cells in a concentration-dependent manner.

Molecular Characterization of Babesia bovis M17 Leucine Aminopeptidase and Inhibition of Babesia Growth by Bestatin.

The M17 leucine aminopeptidase (M17LAP) enzymes of the other apicomplexan parasites have been characterized and shown to be inhibited by bestatin. Though Babesia bovis also belongs to the apicomplexan group, it is not known whether its M17LAP could display similar biochemical properties as well as inhibition profile. To unravel this uncertainty, a B. bovis M17LAP (BbM17LAP) gene was expressed in Escherichia coli , and activity of the recombinant enzyme as well as its inhibition by bestatin were evaluated. The inhibitory effect of the compound on growths of B. bovis and Babesia gibsoni in vitro was also determined. The expression of the gene fused with glutathione S-transferase (GST) yielded approximately 81-kDa recombinant BbM17LAP (rBbM17LAP). On probing with mouse anti-rBbM17LAP serum, a green fluorescence was observed on the parasite cytosol on confocal laser microscopy, and a specific band greater than the predicted molecular mass was seen on Western blotting. The Km and Vmax values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 µM, respectively, while the Ki was 2210 ± 358 µM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC50 (50% inhibition concentration) = 131.7 ± 51.43 µM] than B. gibsoni [IC50 = 460.8 ± 114.45 µM] in vitro. The modest inhibition of both the rBbM17LAP activity and Babesia parasites' growth in vitro suggests that this inhibition may involve the endogenous enzyme in live parasites. Therefore, BbM17LAP may be a target of bestatin, though more studies with other aminopeptidase inhibitors are required to confirm this.

Ginsenoside Rb1 inhibits fibrillation and toxicity of alpha-synuclein and disaggregates preformed fibrils.

Compelling evidence indicates that α-synuclein (α-syn) aggregation plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. Identification of compounds that inhibit or reverse the aggregation process may thus represent a viable therapeutic strategy against PD and related disorders. Ginseng is a well-known medicinal plant that has been used in East Asia for more than two thousand years to treat several conditions. It is now understood that the pharmacological properties of ginseng can be attributed to its biologically active components, the ginsenosides, which in turn have been shown to have neuroprotective properties. We therefore sought to determine for the first time, the potential of the most frequently used and studied ginsenosides, namely Rg1, Rg3 and Rb1, as anti-amyloidogenic agents. The effect of Rg1, Rg3 and Rb1 on α-syn aggregation and toxicity was determined by an array of biophysical, biochemical and cell-culture-based techniques. Among the screened ginsenosides, only Rb1 was shown to be a potent inhibitor of α-syn fibrillation and toxicity. Additionally, Rb1 exhibited a strong ability to disaggregate preformed fibrils and to inhibit the seeded polymerization of α-syn. Interestingly, Rb1 was found to stabilize soluble non-toxic oligomers with no ß-sheet content, that were susceptible to proteinase K digestion, and the binding of Rb1 to those oligomers may represent a potential mechanism of action. Thus, Rb1 could represent the starting point for designing new molecules that could be utilized as drugs for the treatment of PD and related disorders.

Probing α4ßδ GABAA receptor heterogeneity: differential regional effects of a functionally selective α4ß1δ/α4ß3δ receptor agonist on tonic and phasic inhibition in rat brain.

In the present study, the orthosteric GABAA receptor (GABAAR) ligand 4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol (Thio-THIP) was found to possess a highly interesting functional profile at recombinant human GABAARs and native rat GABAARs. Whereas Thio-THIP displayed weak antagonist activity at α1,2,5ß2,3γ2S and ρ1 GABAARs and partial agonism at α6ß2,3δ GABAARs expressed in Xenopus oocytes, the pronounced agonism exhibited by the compound at α4ß1δ and α4ß3δ GABAARs was contrasted by its negligible activity at the α4ß2δ subtype. To elucidate to which extent this in vitro profile translated into functionality at native GABAARs, we assessed the effects of 100 µm Thio-THIP at synaptic and extrasynaptic receptors in principal cells of four different brain regions by slice electrophysiology. In concordance with its α6ß2,3δ agonism, Thio-THIP evoked robust currents through extrasynaptic GABAARs in cerebellar granule cells. In contrast, the compound did not elicit significant currents in dentate gyrus granule cells or in striatal medium spiny neurons (MSNs), indicating predominant expression of extrasynaptic α4ß2δ receptors in these cells. Interestingly, Thio-THIP evoked differential degrees of currents in ventrobasal thalamus neurons, a diversity that could arise from differential expression of extrasynaptic α4ßδ subtypes in the cells. Finally, whereas 100 µm Thio-THIP did not affect the synaptic currents in ventrobasal thalamus neurons or striatal MSNs, it reduced the current amplitudes recorded from dentate gyrus granule cells, most likely by targeting perisynaptic α4ßδ receptors expressed at distal dendrites of these cells. Being the first published ligand capable of discriminating between ß2- and ß3-containing receptor subtypes, Thio-THIP could be a valuable tool in explorations of native α4ßδ GABAARs.

Dihydroisoxazole inhibitors of Anopheles gambiae seminal transglutaminase AgTG3.

BACKGROUND: Current vector-based malaria control strategies are threatened by the rise of biochemical and behavioural resistance in mosquitoes. Researching mosquito traits of immunity and fertility is required to find potential targets for new vector control strategies. The seminal transglutaminase AgTG3 coagulates male Anopheles gambiae seminal fluids, forming a 'mating plug' that is required for male reproductive success. Inhibitors of AgTG3 can be useful both as chemical probes of A. gambiae reproductive biology and may further the development of new chemosterilants for mosquito population control. METHODS: A targeted library of 3-bromo-4,5-dihydroxoisoxazole inhibitors were synthesized and screened for inhibition of AgTG3 in a fluorescent, plate-based assay. Positive hits were tested for in vitro activity using cross-linking and mass spectrometry, and in vivo efficacy in laboratory mating assays. RESULTS: A targeted chemical library was screened for inhibition of AgTG3 in a fluorescent plate-based assay using its native substrate, plugin. Several inhibitors were identified with IC50 < 10 µM. Preliminary structure-activity relationships within the library support the stereo-specificity and preference for aromatic substituents in the chemical scaffold. Both inhibition of plugin cross-linking and covalent modification of the active site cysteine of AgTG3 were verified. Administration of an AgTG3 inhibitor to A. gambiae males by intrathoracic injection led to a 15% reduction in mating plug transfer in laboratory mating assays. CONCLUSIONS: A targeted screen has identified chemical inhibitors of A. gambiae transglutaminase 3 (AgTG3). The most potent inhibitors are known inhibitors of human transglutaminase 2, suggesting a common binding pose may exist within the active site of both enzymes. Future efforts to develop additional inhibitors will provide chemical tools to address important biological questions regarding the role of the A. gambiae mating plug. A second use for transglutaminase inhibitors exists for the study of haemolymph coagulation and immune responses to wound healing in insects.

ML297 (VU0456810), the first potent and selective activator of the GIRK potassium channel, displays antiepileptic properties in mice.

The G-protein activated, inward-rectifying potassium (K(+)) channels, "GIRKs", are a family of ion channels (Kir3.1-Kir3.4) that has been the focus of intense research interest for nearly two decades. GIRKs are comprised of various homo- and heterotetrameric combinations of four different subunits. These subunits are expressed in different combinations in a variety of regions throughout the central nervous system and in the periphery. The body of GIRK research implicates GIRK in processes as diverse as controlling heart rhythm, to effects on reward/addiction, to modulation of response to analgesics. Despite years of GIRK research, very few tools exist to selectively modulate GIRK channels' activity and until now no tools existed that potently and selectively activated GIRKs. Here we report the development and characterization of the first truly potent, effective, and selective GIRK activator, ML297 (VU0456810). We further demonstrate that ML297 is active in two in vivo models of epilepsy, a disease where up to 40% of patients remain with symptoms refractory to present treatments. The development of ML297 represents a truly significant advancement in our ability to selectively probe GIRK's role in physiology as well as providing the first tool for beginning to understand GIRK's potential as a target for a diversity of therapeutic indications.

Screening for natural chemoprevention agents that modify human Keap1.

Upregulation of cytoprotective enzymes by therapeutic agents to prevent damage by reactive oxygen species and xenobiotic electrophiles is a strategy for cancer chemoprevention. The Kelch-like ECH-associated protein 1 (Keap1) and its binding partner, transcription factor NF-E2-related factor-2 (NRF2), are chemoprevention targets because of their role in regulating the antioxidant response element (ARE) in response to oxidative stress and exposure to electrophiles. Modification of the sensor protein Keap1 by electrophiles such as the isothiocyanate sulforaphane can direct Nrf2 accumulation in the nucleus and subsequent ARE activation. Since our previous matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS)-based screening method to discover natural products that modify Keap1 does not detect covalent modification of Keap1 by some highly reversible agents such as sulforaphane, a more sensitive screening assay was developed. In this new assay, electrophiles that have reversibly modified Keap1 can be released, trapped, and detected as ß-mercaptoethanol adducts by mass spectrometry. Isoliquiritigenin and sulforaphane, known ARE activators that target Keap1, were used to validate the assay. To determine the ability of the assay to identify electrophiles in complex matrixes that modify Keap1, sulforaphane was spiked into a cocoa extract, and LC-MS/MS using high resolution mass spectrometry with accurate mass measurement was used to identify ß-mercaptoethanol adducts of sulforaphane that had been released from Keap1. This screening assay permits identification of potential chemoprevention agents in complex natural product mixtures that reversibly modify Keap1 but cannot be detected using MALDI-TOF MS.

System-dependent outcomes during the evaluation of drug candidates as inhibitors of cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) enzymes: human hepatocytes versus liver microsomes versus recombinant enzymes.

The ability of a drug to cause clinically significant drug-drug interactions due to direct or metabolism-dependent inhibition of cytochrome P450 (CYP) can generally be predicted from in vitro studies with human liver microsomes (HLM) or recombinant CYP enzymes, as recommended by the FDA and other regulatory agencies. This review highlights some examples of system-dependent inhibition of CYP and uridine diphosphate glucuronosyltransferase (UGT) enzymes. In the case of CYP enzymes, examples are presented where in vitro studies with HLM under-predict or over-predict the degree of inhibition observed in the clinic and where the correct prediction comes from studies with human hepatocytes. Studies with HLM under-predict the ability of gemfibrozil and bupropion to cause clinically significant inhibition of CYP2C8 and CYP2D6, respectively, and over-predict the ability of ezetimibe to cause clinically significant inhibition of CYP3A4. Gemfibrozil and bupropion represent examples of glucuronidation-dependent and reduction-dependent activation to metabolites that inhibit CYP2C8 and CYP2D6, respectively, whereas ezetimibe represents an example of glucuronidation-dependent protection against metabolism-dependent inhibition of CYP3A4. This article illustrates why, when drug candidates are extensively metabolized by non-CYP enzymes, it would be prudent to use human hepatocytes in addition to HLM or recombinant enzymes to evaluate their ability to inhibit CYP enzymes.

Discovery of 1,5-disubstituted pyridones: a new class of positive allosteric modulators of the metabotropic glutamate 2 receptor.

A series of 1,5-disubstituted pyridones was identified as positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 2 (mGluR2) via high throughput screening (HTS). Subsequent SAR exploration led to the identification of several compounds with improved in vitro activity. Lead compound 8 was further profiled and found to attenuate the increase in PCP induced locomotor activity in mice.