The M5 muscarinic acetylcholine receptor is suggested to be a potential pharmacotherapeutic target for the treatment of drug abuse. We describe herein the discovery of a series of M5-preferring orthosteric antagonists based on the scaffold of 1,2,5,6-tetrahydropyridine-3-carboxylic acid. Compound 56, the most selective compound in this series, possesses an 11-fold selectivity for the M5 over M1 receptor, and shows little activity at M2–M4. This compound, although exhibiting modest affinity (Ki = 2.24 μM) for the [3H]N-methylscopolamine binding site on the M5 receptor, is potent (IC50 = 0.45 nM) in inhibiting oxotremorine-evoked [3H]DA release from rat striatal slices. Further, a homology model of human M5 receptor based on the crystal structure of the rat M3 receptor was constructed, and docking studies of compounds 28 and 56 were performed in an attempt to understand the possible binding mode of these novel analogues to the receptor.

Background: Stable isotope/chemical probe mass spectrometry was used to monitor cancer cell metabolism of exogenous isoprenols. Results: Efficient use of exogenous isoprenols for protein isoprenylation was undiminished by genetic or pharmacological inhibition of HMG-CoA reductase. Conclusion: Exogenous isoprenols are metabolized independently of the mevalonate pathway. Significance: This study identifies and quantitates a pathway of isoprenol metabolism with potential relevance to cancer progression.

Farnesylation is an important post-translational modification essential for proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of analogue transfer to peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple turnover activity depends on the analogue structure. Analogues where the first isoprene is replaced by a benzyl group and an analogue where each isoprene is replaced by an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single turnover rate constant for alkylation does depend on the Ca1a2X peptide sequence. These results suggest that the isoprenoid transition state conformation is preferred over the inactive E•FPP• Ca1a2X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for design of novel inhibitors.

By linking two or three mecamylamine or 2,2,6,6-tetramethylpiperidine (TMP) molecules together via a linear lipophilic bis-methylene linker or a specially designed conformationally restricted tris-linker, a series of bis- and tris-tertiary amine analogs has been synthesized and evaluated as potent antagonists at nAChRs mediating nicotine-evoked [3H]dopamine release from rat striatal slices. Compounds 7e, 14b and 16 demonstrated high potency in decreasing nicotine-evoked [3H]dopamine release (IC50=2.2, 46, and 107 nM, respectively. The preliminary structure-activity data obtained with these new analogs suggest the importance of the length of the methylene linker in the bis-analog series. Such bis-tertiary amino analogs may provide a new strategy for the design of drugable ligands that have high inhibitory potency against nAChRs mediating nicotine-evoked dopamine release in striatum, which have been suggested to be target receptors of interest in the development of potential smoking cessation therapies.

Background Dolichyl phosphate-linked mono- and oligosaccharides (DLO) are essential intermediates in protein N-glycosylation, C- and O-mannosylation and GPI anchor biosynthesis. While many membrane proteins in the endoplasmic reticulum (ER) involved in the assembly of DLOs are known, essential proteins believed to be required for the transbilayer movement (flip-flopping) and proteins potentially involved in the regulation of DLO synthesis remain to be identified. Methods The synthesis of a series of Dol-P derivatives composed of citronellyl-based photoprobes with benzophenone groups equipped with alkyne moieties for Huisgen “click” chemistry is now described to utilize as tools for identifying ER proteins involved in regulating the biosynthesis and transbilayer movement of lipid intermediates. In vitro enzymatic assays were used to establish that the photoprobes contain the critical structural features recognized by pertinent enzymes in the dolichol pathway. ER proteins that photoreacted with the novel probes were identified by MS. Results The potential of the newly designed photoprobes, m-PAL-Cit-P and p-PAL-Cit-P, for identifying previously unidentified Dol-P-interacting proteins is supported by the observation that they are enzymatically mannosylated by Man-P-Dol synthase (MPDS) from Chinese Hamster Ovary (CHO) cells at an enzymatic rate similar to that for Dol-P. MS analyses reveal that DPM1, ALG14 and several other yeast ER proteins involved in DLO biosynthesis and lipid-mediated protein O-mannosylation photoreacted with the novel probes. Conclusion The newly-designed photoprobes described in this paper provide promising new tools for the identification of yet to be identified Dol-P interacting ER proteins in yeast and mammalian cells, including the Dol-P flippase required for the “re-cycling” of the glycosyl carrier lipid from the lumenal monolayer of the ER to the cytoplasmic leaflet for new rounds of DLO synthesis.