How to Deal with Weak Interactions in Noncovalent Complexes Analyzed by Electrospray Mass Spectrometry: Cyclopeptidic Inhibitors of the Nuclear Receptor Coactivator 1-STAT6

Mass spectrometry, and especially electrospray ionization, is now an efficient tool to study noncovalent interactions between proteins and inhibitors. It is used here to study the interaction of some weak inhibitors with the NCoA-1/STAT6 protein with KD values in the μM range. High signal intensities corresponding to some nonspecific electrostatic interactions between NCoA-1 and the oppositely charged inhibitors were observed by nanoelectrospray mass spectrometry, due to the use of high ligand concentrations. Diverse strategies have already been developed to deal with nonspecific interactions, such as controlled dissociation in the gas phase, mathematical modeling, or the use of a reference protein to monitor the appearance of nonspecific complexes. We demonstrate here that this last methodology, validated only in the case of neutral sugar–protein interactions, i.e., where dipole–dipole interactions are crucial, is not relevant in the case of strong electrostatic interactions. Thus, we developed a novel strategy based on half-maximal inhibitory concentration (IC50) measurements in a competitive assay with readout by nanoelectrospray mass spectrometry. IC50 values determined by MS were finally converted into dissociation constants that showed very good agreement with values determined in the liquid phase using a fluorescence polarization assay

2- and 3-substituted imidazo [1,2-a] pyrazines as inhibitors of bacterial type IV secretion

A novel series of 8-amino imidazo[1,2-a]pyrazine derivatives has been developed as inhibitors of the VirB11 ATPase HP0525, a key component of the bacterial type IV secretion system. A flexible synthetic route to both 2- and 3-aryl substituted regioisomers has been developed. The resulting series of imidazo[1,2-a]pyrazines has been used to probe the structure–activity relationships of these inhibitors, which show potential as antibacterial agents

A recurrent polyalanine expansion in the transcription factor FOXL2 induces extensive nuclear and cytoplasmic protein aggregation

Blepharophimosis syndrome is an autosomal dominant disease characterised by eyelid malformations, associated or not with premature ovarian failure. It is caused by mutations in the FOXL2 gene, which encodes a forkhead transcription factor containing a polyalanine (polyAla) domain of 14 alanines. Expansions of the polyAla tract from 14 to 24 residues account for 30% of the reported mutations and lead mainly to isolated palpebral defects. We have transfected COS-7 cells with DNA constructs driving the expression of the wildtype and mutant FOXL2 proteins fused to the green fluorescent protein. The polyAla expansion was found to induce the formation of intranuclear aggregates and a mislocalisation of the protein due to extensive cytoplasmic aggregation. These findings were confirmed by immunofluorescence. Co-transfection experiments suggest that the wildtype and mutant proteins can co-aggregate. We propose that the mechanism for the molecular pathogenesis of the polyAla expansions of FOXL2 may be its mislocalisation concomitant with its inclusion into nuclear aggregates. This may diminish the pool of active protein. Potential effects of aggregation on cell viability are under study

MITOGEN AND STRESS-ACTIVATED KINASE-1 DEFICIENCY AND TRANSCRIPTIONAL DYSREGULATION IN HUNTINGTON'S DISEASE

8th Plenary Meeting of the European-Huntingtons-Disease-Network, Barcelona, SPAIN, SEP 19-21, 2014International audienceno abstrac

BENEFICIAL EFFECTS OF STRIATAL RESTORATION OF CYP46A1 EXPRESSION ON CHOLESTEROL METABOLISM AND NEURODEGENERATION IN HUNTINGTON'S DISEASE MOUSE MODEL (R6/2)

8th Plenary Meeting of the European-Huntingtons-Disease-Network, Barcelona, SPAIN, SEP 19-21, 2014International audienceno abstrac

A novel polyalanine expansion in FOXL2: the first evidence for a recessive form of the blepharophimosis syndrome (BPES) associated with ovarian dysfunction

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AAV-CYP46A1 brain administration restores cholesterol metabolism and is neuroprotective in Huntington's disease

Conference on Changing the Face of Modern Medicine - Stem Cells and Gene Therapy, Florence, ITALY, OCT 18-21, 2016International audienceno abstrac

Enantioselective synthesis of alpha-fluorinated beta(2)-amino acids

Copyright © 2008 American Chemical SocietyA methodology for the enantioselective synthesis of α-fluorinated β²-amino acids has been developed from readily available carboxylic acids 3. Conversion to the Evan's oxazolidinone followed by enantioselective fluorination and alkylation gave 7 in high diastereomeric excess (>95%). Subsequent removal of the oxazolidinone and amination at the Bn-protected hydroxyl center gave optically active α-fluorinated β²-amino acids.Michael K. Edmonds, Florian H. M. Graichen, James Gardiner, and Andrew D. Abel

Oral Administration of the Pimelic Diphenylamide HDAC Inhibitor HDACi 4b Is Unsuitable for Chronic Inhibition of HDAC Activity in the CNS In Vivo

<div><p>Histone deacetylase (HDAC) inhibitors have received considerable attention as potential therapeutics for a variety of cancers and neurological disorders. Recent publications on a class of pimelic diphenylamide HDAC inhibitors have highlighted their promise in the treatment of the neurodegenerative diseases Friedreich’s ataxia and Huntington’s disease, based on efficacy in cell and mouse models. These studies’ authors have proposed that the unique action of these compounds compared to hydroxamic acid-based HDAC inhibitors results from their unusual slow-on/slow-off kinetics of binding, preferentially to HDAC3, resulting in a distinctive pharmacological profile and reduced toxicity. Here, we evaluate the HDAC subtype selectivity, cellular activity, absorption, distribution, metabolism and excretion (ADME) properties, as well as the central pharmacodynamic profile of one such compound, HDACi <b>4b</b>, previously described to show efficacy <em>in vivo</em> in the R6/2 mouse model of Huntington’s disease. Based on our data reported here, we conclude that while the <em>in vitro</em> selectivity and binding mode are largely in agreement with previous reports, the physicochemical properties, metabolic and p-glycoprotein (Pgp) substrate liability of HDACi <b>4b</b> render this compound suboptimal to investigate central Class I HDAC inhibition <em>in vivo</em> in mouse per oral administration. A drug administration regimen using HDACi <b>4b</b> dissolved in drinking water was used in the previous proof of concept study, casting doubt on the validation of CNS HDAC3 inhibition as a target for the treatment of Huntington’s disease. We highlight physicochemical stability and metabolic issues with <b>4b</b> that are likely intrinsic liabilities of the benzamide chemotype in general.</p> </div