Photoaffinity Labeling of the Sigma‑1 Receptor with <i>N</i>‑[3-(4-Nitrophenyl)propyl]‑<i>N</i>‑dodecylamine: Evidence of Receptor Dimers

The sigma-1 receptor is a ligand-regulated endoplasmic reticulum (ER) resident chaperone involved in the maintenance of cellular homeostasis. Coupling of the sigma-1 receptor with various ER and/or plasma membrane ion channels is associated with its ability to regulate the locomotor activity and cellular proliferation produced in response to sigma-1 receptor ligands. A number of endogenous small molecules bind to the sigma-1 receptor and have been shown to regulate its activity; these include progesterone, <i>N</i>,<i>N</i>-dimethyltryptamine, d-<i>erythro</i>-sphingosine, and/or other endogenous lipids. We previously reported the synthesis of long chain <i>N</i>-alkylamine derivatives and the characterization of the structure–activity relationship between the chain length of <i>N</i>-alkylamine and affinities at the sigma-1 receptor. Here, we present data demonstrating the photoincorporation of one of these <i>N</i>-alkylamine derivatives, <i>N</i>-[3-(4-nitrophenyl)­propyl]-<i>N</i>-dodecylamine (4-NPPC12), to the sigma-1 receptor. Matrix-assisted laser desorption ionization time-of-flight and tandem mass spectrometry showed that 4-NPPC12 photoinserted at histidine 154 of the derivatized population of the sigma-1 receptor. Interestingly, light-dependent photoinsertion of 4-NPPC12 resulted in an enhanced electrophoretic mobility of only 50% of the derivatized receptor molecules as assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proposed binding and reactivity of 4-NPPC12 evoke a ligand binding model for the sigma-1 receptor that likely involves a receptor dimer and/or oligomer

Noncompetitive Inhibition of Indolethylamine‑<i>N</i>‑methyltransferase by <i>N</i>,<i>N</i>‑Dimethyltryptamine and <i>N</i>,<i>N</i>‑Dimethylaminopropyltryptamine

Indolethylamine-<i>N</i>-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of <i>N</i>,<i>N</i>-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and <i>S</i>-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis–Menten and Lineweaver–Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, <i>N</i>-[2-(1<i>H</i>-indol-3-yl)­ethyl]-<i>N</i>′,<i>N</i>′-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a <i>K</i>_i of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-<i>N</i>-methyltransferase (hPNMT) and human nicotinamide-<i>N</i>-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. <i>In silico</i> analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14) identified an N-terminal helix–loop–helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were −6.34 and −7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT