RESUMEN
Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations >10 µM, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition.
Asunto(s)
Antineoplásicos/farmacología , Diterpenos/farmacología , Farnesol/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Ácido Mevalónico/metabolismo , Neoplasias/tratamiento farmacológico , Fosfatos de Poliisoprenilo/metabolismo , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Diterpenos/farmacocinética , Farnesol/análogos & derivados , Farnesol/farmacocinética , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Prenilación de Proteína/efectos de los fármacos , Prenilación de Proteína/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
An efficient, diversity oriented synthesis of homoisoprenoid α-monofluorophosphonates utilizing electrophilic fluorination is presented along with their activity as inhibitors of PPAPDC2 family integral membrane lipid phosphatases. These novel phosphatase-resistant analogues of isoprenoid monophosphates are a platform for further structure-activity relationship studies and provide access to other isoprenoid family members where the phosphate ester oxygen is replaced by a α-monofluoromethylene moiety.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Fosfatos de Poliisoprenilo/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Estructura Molecular , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfatos de Poliisoprenilo/síntesis química , Fosfatos de Poliisoprenilo/química , Relación Estructura-ActividadRESUMEN
A Wittig reaction employing Li(CD3)2CP(C6H5)3 was used to prepare d6-farnesol and d6-geranylgeraniol. Reductive amination of aniline-2,3,4,5,6-d5 was used to prepare the unnatural isoprenoid analogues d5-anilinogeraniol and d5-anilinofarnesol. All of these deuterated isoprenols were elaborated into their diphosphate and cysteine thioether derivatives suitable for use as stable-isotope labeled standards for quantitative mass spectrometric analysis.
Asunto(s)
Cisteína/análogos & derivados , Deuterio/síntesis química , Fosfatos de Poliisoprenilo/síntesis química , Cisteína/síntesis química , Marcaje Isotópico , Espectrometría de Masas/métodosRESUMEN
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.
Asunto(s)
Dopamina/metabolismo , Mecamilamina/farmacología , Nicotina , Antagonistas Nicotínicos/síntesis química , Piperidinas/química , Receptores Nicotínicos/efectos de los fármacos , Animales , Concentración 50 Inhibidora , Estructura Molecular , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacología , Piperidinas/síntesis química , Piperidinas/farmacología , Ratas , Receptores Nicotínicos/metabolismoRESUMEN
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.