RESUMEN
Fatty acid binding protein 5 (FABP5) is a promising target for development of inhibitors to help control pain and inflammation. In this work, computer-based docking (DOCK6 program) was employed to screen â¼2 M commercially available compounds to FABP5 based on an X-ray structure complexed with the small molecule inhibitor SBFI-26 previously identified by our group (also through virtual screening). The goal was discovery of additional chemotypes. The screen resulted in the purchase of 78 candidates, which led to the identification of a new inhibitor scaffold (STK-0) with micromolar affinity and apparent selectivity for FABP5 over FABP3. A second similarity-based screen resulted in three additional hits (STK-15, STK-21, STK-22) from which preliminary SAR could be derived. Notably, STK-15 showed comparable activity to the SBFI-26 reference under the same assay conditions (1.40 vs 0.86 µM). Additional molecular dynamics simulations, free energy calculations, and structural analysis (starting from DOCK-generated poses) revealed that R enantiomers (dihydropyrrole scaffold) of STK-15 and STK-22 have a more optimal composition of functional groups to facilitate additional H-bonds with Arg109 of FABP5. This observation suggests enantiomerically pure compounds could show enhanced activity. Overall, our study highlights the utility of using similarity-based screening methods to discover new inhibitor chemotypes, and the identified FABP5 hits provide a strong starting point for future efforts geared to improve activity.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Proteínas de Unión a Ácidos Grasos/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Supervivencia Celular/efectos de los fármacos , Cristalización , Cristalografía por Rayos X , Ciclobutanos/química , Ciclobutanos/farmacología , Ácidos Dicarboxílicos/química , Ácidos Dicarboxílicos/farmacología , Proteína 3 de Unión a Ácidos Grasos/antagonistas & inhibidores , Proteína 3 de Unión a Ácidos Grasos/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Enlace de Hidrógeno , Ligandos , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Interfaz Usuario-ComputadorRESUMEN
The increasing use of medical marijuana highlights the importance of developing a better understanding of cannabinoid metabolism. Phytocannabinoids, including ∆9-tetrahydrocannabinol (THC), are metabolized and inactivated by cytochrome P450 enzymes primarily within the liver. The lipophilic nature of cannabinoids necessitates mechanism(s) to facilitate their intracellular transport to metabolic enzymes. Here, we test the central hypothesis that liver-type fatty acid binding protein (FABP1) mediates phytocannabinoid transport and subsequent inactivation. Using X-ray crystallography, molecular modeling, and in vitro binding approaches we demonstrate that FABP1 accommodates one molecule of THC within its ligand binding pocket. Consistent with its role as a THC carrier, biotransformation of THC was reduced in primary hepatocytes obtained from FABP1-knockout (FABP1-KO) mice. Compared to their wild-type littermates, administration of THC to male and female FABP1-KO mice potentiated the physiological and behavioral effects of THC. The stark pharmacodynamic differences were confirmed upon pharmacokinetic analyses which revealed that FABP1-KO mice exhibit reduced rates of THC biotransformation. Collectively, these data position FABP1 as a hepatic THC transport protein and a critical mediator of cannabinoid inactivation. Since commonly used medications bind to FABP1 with comparable affinities to THC, our results further suggest that FABP1 could serve a previously unrecognized site of drug-drug interactions.
Asunto(s)
Dronabinol/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Animales , Sitios de Unión , Biotransformación , Células Cultivadas , Cristalografía por Rayos X , Dronabinol/administración & dosificación , Proteínas de Unión a Ácidos Grasos/química , Femenino , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos MolecularesRESUMEN
Fatty acid binding proteins (FABPs) serve as critical modulators of endocannabinoid signaling by facilitating the intracellular transport of anandamide and whose inhibition potentiates anandamide signaling. Our previous work has identified a novel small-molecule FABP inhibitor, α-truxillic acid 1-naphthyl monoester (SB-FI-26, 3) that has shown efficacy as an antinociceptive and anti-inflammatory agent in rodent models. In the present work, we have performed an extensive SAR study on a series of 3-analogs as novel FABP inhibitors based on computer-aided inhibitor drug design and docking analysis, chemical synthesis and biological evaluations. The prediction of binding affinity of these analogs to target FABP3, 5 and 7 isoforms was performed using the AutoDock 4.2 program, using the recently determined co-crystal structures of 3 with FABP5 and FABP7. The compounds with high docking scores were synthesized and evaluated for their activities using a fluorescence displacement assay against FABP3, 5 and 7. During lead optimization, compound 3l emerged as a promising compound with the Ki value of 0.21⯵M for FABP 5, 4-fold more potent than 3 (Ki, 0.81⯵M). Nine compounds exhibit similar or better binding affinity than 3, including compounds 4b (Ki, 0.55⯵M) and 4e (Ki, 0.68⯵M). Twelve compounds are selective for FABP5 and 7 with >10⯵M Ki values for FABP3, indicating a safe profile to avoid potential cardiotoxicity concerns. Compounds 4f, 4j and 4k showed excellent selectivity for FABP5 and would serve as other new lead compounds. Compound 3a possessed high affinity and high selectivity for FABP7. Compounds with moderate to high affinity for FABP5 displayed antinociceptive effects in mice while compounds with low FABP5 affinity lacked in vivo efficacy. In vivo pain model studies in mice revealed that exceeding hydrophobicity significantly affects the efficacy. Thus, among the compounds with high affinity to FABP5 in vitro, the compounds with moderate hydrophobicity were identified as promising new lead compounds for the next round of optimization, including compounds 4b and 4j. For select cases, computational analysis of the observed SAR, especially the selectivity of new inhibitors to particular FABP isoforms, by comparing docking poses, interaction map, and docking energy scores has provided useful insights.