piperidines and methylamine

Using the available structural information of the chemokine receptor CXCR4, we present hit finding and hit exploration studies that make use of virtual fragment screening, design, synthesis and structure-activity relationship (SAR) studies. Fragment 2 was identified as virtual screening hit and used as a starting point for the exploration of 31 N-substituted piperidin-4-yl-methanamine derivatives to investigate and improve the interactions with the CXCR4 binding site. Additionally, subtle structural ligand changes lead to distinct interactions with CXCR4 resulting in a full to partial displacement of CXCL12 binding and competitive and/or non-competitive antagonism. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and binding model studies were used to identify important hydrophobic interactions that determine binding affinity and indicate key ligand-receptor interactions.

Topics: Binding Sites; Chemokine CXCL12; Ligands; Methylamines; Models, Molecular; Peptide Fragments; Piperidines; Protein Binding; Quantitative Structure-Activity Relationship; Receptors, CXCR4

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor (NR) superfamily and regulate development, growth, and metabolism. Upon binding thyroid hormone, TR undergoes a conformational change that allows the release of corepressors and the recruitment of coactivators, which in turn regulate target gene transcription. Although a number of TR antagonists have been developed, most are analogs of the endogenous hormone that inhibit ligand binding. In a screen for inhibitors that block the association of TRβ with steroid receptor coactivator 2 (SRC2), we identified a novel methylsulfonylnitrobenzoate (MSNB)-containing series that blocks this interaction at micromolar concentrations. Here we have studied a series of MSNB analogs and characterized their structure activity relationships. MSNB members do not displace thyroid hormone T3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPARγ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TRβ antagonism. Side-chain alkylamine substituents showed better inhibitory activity than arylamine substituents. Mass spectrum analysis suggested that MSNB inhibitors bind irreversibly to Cys-298 within the AF-2 cleft of TRβ to disrupt SRC2 association.

Topics: Aniline Compounds; HEK293 Cells; Hep G2 Cells; Humans; Methylamines; Nitrobenzoates; Nuclear Receptor Coactivator 2; Piperidines; Protein Binding; Receptors, Thyroid Hormone; Reverse Transcriptase Polymerase Chain Reaction; Structure-Activity Relationship

Monoethylcholine (MECH) enters motor nerve terminals where it is made into acetylmonoethylcholine (AMECH). AMECH opens endplate channels for about half of the average duration observed where they are opened by acetylcholine (ACH). Therefore when AMECH is present in a quantum the endplate currents decay more rapidly. MECH has been used to measure quantal turnover in motor nerve terminals. We find that the incorporation of AMECH into quanta is blocked by vesamicol, an inhibitor of ACH transport into synaptic vesicles. AMECH is incorporated more rapidly when acetylcholinesterase is inhibited, when the choline uptake inhibitor, hemicholinium-3, is present or when extracellular Na+ (required for active CH uptake) is replaced with methylamine. This suggests that in the absence of these inhibitors CH obtained from released ACH is recycled. Therefore, experiments on the rate of incorporation of MECH are misleading unless CH recycling is prevented. Previous work also suggested that MECH is incorporated at a faster rate into those quanta which are released by stimulation than into those released spontaneously. We conclude that quanta released spontaneously and following nerve stimulation probably come from the same pool. The distribution of t1/2's during the incorporation of MECH can be accounted for in the framework of recent studies of the recycling of synaptic vesicles. We conclude that false transmitter is a valuable tool for studying the loading of quanta, but that there are several complications to be considered when trying to use it to measure the turnover of the population of quanta.

Topics: Animals; Choline; Cholinesterase Inhibitors; Edrophonium; Electric Stimulation; Hemicholinium 3; Methylamines; Mice; Mice, Inbred Strains; Neostigmine; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Neurotransmitter Agents; Neurotransmitter Uptake Inhibitors; Piperidines; Prodrugs; Rana pipiens

Uptake of acetylcholine (ACh) by synaptic vesicles isolated from the electric organ of Torpedo was induced with an artificially imposed proton gradient. The gradient was formed by hyposmotic lysis and resealing of vesicles in a low pH buffer to form vesicular ghosts followed by sudden elevation of the pH of the ghost suspension. [3H]ACh accumulated rapidly, the proton gradient collapsed spontaneously within 5 min as monitored by [14C]methylamine uptake, and the accumulated ACh leaked out of the ghosts after 5 min. Vesamicol blocked both uptake and efflux of the [3H]ACh, demonstrating that both processes are mediated by the ACh transporter. The protonophore nigericin also blocked uptake very potently. Specific uptake was titrated with variable concentrations of [3H]ACh. It exhibited Km and Vmax values of approximately 200-500 microM and 7-30 nmol [3H]ACh/mg at 5 min, respectively, which are values close to those commonly observed for ATP-dependent uptake by intact vesicles. Specific uptake by ghosts was titrated with variable internal pH and constant external pH. It exhibited maximal uptake between internal pH 4.5 and 5.5. The dependence was very steep and could be fit best by assuming that the active form of the transporter requires protonation of two internal sites of apparent pK value of 5.3 +/- 0.2. A similar result was obtained when the uptake was titrated with variable internal pH with a constant thermodynamic driving force maintained by keeping the external pH approximately 2.6 units higher. The origin of the transport inhibition that sets in at very low internal pH values is not clear.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Animals; Biological Transport, Active; Carrier Proteins; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Potentials; Membrane Transport Proteins; Methylamines; Nigericin; Piperidines; Synaptic Vesicles; Torpedo; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Topics: alpha-Macroglobulins; Animals; Calcium; Endocytosis; Kinetics; Low Density Lipoprotein Receptor-Related Protein-1; Macrophages; Methylamines; Mice; Penfluridol; Pimozide; Piperidines; Receptors, Immunologic