Insights into activity and inhibition from the crystal structure of human O-GlcNAcase.

O-GlcNAc hydrolase (OGA) catalyzes removal of ßα-linked N-acetyl-D-glucosamine from serine and threonine residues. We report crystal structures of Homo sapiens OGA catalytic domain in apo and inhibited states, revealing a flexible dimer that displays three unique conformations and is characterized by subdomain α-helix swapping. These results identify new structural features of the substrate-binding groove adjacent to the catalytic site and open new opportunities for structural, mechanistic and drug discovery activities.

Discovery of 6-(Fluoro-(18)F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine ([(18)F]-MK-6240): A Positron Emission Tomography (PET) Imaging Agent for Quantification of Neurofibrillary Tangles (NFTs).

Neurofibrillary tangles (NFTs) made up of aggregated tau protein have been identified as the pathologic hallmark of several neurodegenerative diseases including Alzheimer's disease. In vivo detection of NFTs using PET imaging represents a unique opportunity to develop a pharmacodynamic tool to accelerate the discovery of new disease modifying therapeutics targeting tau pathology. Herein, we present the discovery of 6-(fluoro-(18)F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine, 6 ([(18)F]-MK-6240), as a novel PET tracer for detecting NFTs. 6 exhibits high specificity and selectivity for binding to NFTs, with suitable physicochemical properties and in vivo pharmacokinetics.

A potent and selective metabotropic glutamate receptor 4 positive allosteric modulator improves movement in rodent models of Parkinson's disease.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGluR4) have been proposed as a novel therapeutic approach for the treatment of Parkinson's disease. However, evaluation of this proposal has been limited by the availability of appropriate pharmacological tools to interrogate the target. In this study, we describe the properties of a novel mGluR4 PAM. 5-Methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine (ADX88178) enhances glutamate-mediated activation of human and rat mGluR4 with EC(50) values of 4 and 9 nM, respectively. The compound is highly selective for mGluR4 with minimal activities at other mGluRs. Oral administration of ADX88178 in rats is associated with high bioavailability and results in cerebrospinal fluid exposure of >50-fold the in vitro EC(50) value. ADX88178 reverses haloperidol-induced catalepsy in rats at 3 and 10 mg/kg. It is noteworthy that this compound alone has no impact on forelimb akinesia resulting from a bilateral 6-hydroxydopamine lesion in rats. However, coadministration of a low dose of L-DOPA (6 mg/kg) enabled a robust, dose-dependent reversal of the forelimb akinesia deficit. ADX88178 also increased the effects of quinpirole in lesioned rats and enhanced the effects of L-DOPA in MitoPark mice. It is noteworthy that the enhancement of the actions of L-DOPA was not associated with an exacerbation of L-DOPA-induced dyskinesias in rats. ADX88178 is a novel, potent, and selective mGluR4 PAM that is a valuable tool for exploring the therapeutic potential of mGluR4 modulation. The use of this novel tool molecule supports the proposal that activation of mGluR4 may be therapeutically useful in Parkinson's disease.

Structure-dependent impairment of intracellular apolipoprotein E4 trafficking and its detrimental effects are rescued by small-molecule structure correctors.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.

PISA, a novel pharmacodynamic assay for assessing poly(ADP-ribose) polymerase (PARP) activity in situ.

INTRODUCTION: Poly ADP-ribose polymerase (PARP) maintains genomic integrity by repairing DNA strand breaks, however over-activation of PARP following neural tissue injury is hypothesized to cause neuronal death. Therefore, PARP inhibitors have potential for limiting neural injury under certain conditions. A reliable method for assessing PARP activity in brain is critical for development of novel inhibitors with CNS activity. We developed the PARP In Situ Activity (PISA) assay to provide a direct, quantitative assessment of CNS PARP activity in vitro or in vivo. METHODS: The assay utilized brain sections from rats with striatal kainic acid (KA) lesions and 3H- or biotinylated NAD+ as the substrate to assess PARP activity. Following optimization of the assay, it was used to assess in vitro and in vivo efficacy of known and novel PARP inhibitors. The assay also was used to assess PARP activity in male and female gonad-intact and ovariectomized rats. RESULTS: Using 3H-NAD+ as the substrate, PARP activity was greater (p<0.01) in tissue from KA-lesioned vs. non-lesioned rats. Using biotinylated NAD+ it was revealed that PARP activity was present ipsilateral to the KA injection site, and labeling was blocked by incubation with excess unlabeled NAD+ or PARP inhibitors. The PARP inhibitor, 3-aminobenzamide and several novel inhibitors reduced (p<0.01) polymerase activity in vitro. Furthermore, the inhibitor MRLSD303 reduced (p<0.001) PARP activity in vivo in both male and female rats. Finally, administration of the novel PARP inhibitor MRLIT115 dose-dependently reduced (p<0.001) polymerase activity in vivo. DISCUSSION: The PISA assay provides a direct, quantitative method for assessing PARP activity in vitro and provides critical information on factors underlying in vivo efficacy of chemical inhibitors including brain penetration and target engagement. These findings support use of the PISA assay as a screening tool for testing efficacy of PARP inhibitors in brain.

Binding modes of dihydroquinoxalinones in a homology model of bradykinin receptor 1.

We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.

Discovery of a potent, non-peptide bradykinin B1 receptor antagonist.

Bradykinin (BK) plays an important role in the pathophysiological processes accompanying pain and inflammation. Selective bradykinin B1 receptor antagonists have been shown to be anti-nociceptive in animal models and could be novel therapeutic agents for the treatment of pain and inflammation. We have explored chemical modifications in a series of dihydroquinoxalinone sulfonamides to evaluate the effects of various structural changes on biological activity. The optimization of a screening lead compound, facilitated by a homology model of the BK B1 receptor, culminated in the discovery of a potent human BK B1 receptor antagonist. Results from site-directed mutagenesis studies and experiments in an animal pain model are presented.