RESUMEN
Catechol-O-methyltransferase (COMT) plays an important role in the deactivation of catecholamine neurotransmitters and hormones. Inhibitors of COMT, such as tolcapone and entacapone, are used clinically in the treatment of Parkinson's disease. Discovery of novel inhibitors has been hampered by a lack of suitable assays for high-throughput screening (HTS). Although assays using esculetin have been developed, these are affected by fluorescence, a common property of catechol-type compounds. We have therefore evaluated a new homogenous time-resolved fluorescence (HTRF)-based assay from CisBio (Codolet, France), which measures the production of S-adenosyl-L-homocysteine (SAH). The assay has been run in both HTS and medium-throughput screening (MTS) modes. The assay was established using membranes expressing human membrane-bound COMT and was optimized for protein and time to give an acceptable signal window, good potency for tolcapone, and a high degree of translation between data in fluorescence ratio and data in terms of [SAH] produced. pIC50 values for the hits from the HTS mode were determined in the MTS mode. The assay also proved suitable for kinetic studies such as Km,app determination.
Asunto(s)
Inhibidores de Catecol O-Metiltransferasa/aislamiento & purificación , Catecol O-Metiltransferasa/química , Ensayos Analíticos de Alto Rendimiento/métodos , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Benzofenonas/uso terapéutico , Catecol O-Metiltransferasa/efectos de los fármacos , Inhibidores de Catecol O-Metiltransferasa/química , Inhibidores de Catecol O-Metiltransferasa/uso terapéutico , Catecoles/uso terapéutico , Humanos , Cinética , Nitrofenoles/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , S-Adenosilhomocisteína/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , TolcaponaRESUMEN
AIMS: Lacosamide (LCM; SPM 927, Vimpat®) is an antiepileptic drug (AED) used as adjunctive treatment for adults with partial-onset seizures. LCM has a different mode of action from traditional sodium channel blocking AEDs in that it selectively enhances slow inactivation of sodium channels without affecting fast inactivation. Initial investigations suggested that LCM might have an additional mode of action by binding to the collapsin response mediator protein 2 (CRMP-2), which is further investigated here. METHODS: LCM binding to native and cloned human CRMP-2 was determined using radioligand binding experiments and surface plasmon resonance measurements. RESULTS: No specific binding of [(3) H]LCM (free concentration 100-1450 nM) to isolated or membrane bound human CRMP-2 expressed in mammalian cell systems and bacteria was observed. Surface plasmon resonance analysis also showed that LCM, over a concentration range of 0.39-100 µM, does not specifically bind to human CRMP-2. CONCLUSION: The diverse drug binding methods employed here are well suited to detect specific binding of LCM to CRMP-2 in the micromolar range, yet the results obtained were all negative. Results of this study suggest that LCM does not specifically bind to CRMP-2.