ABSTRACT
End binding protein 1 (EB1) is a key element in the complex network of protein-protein interactions at microtubule (MT) growing ends, which has a fundamental role in MT polymerisation. EB1 is an important protein target as it is involved in regulating MT dynamic behaviour, and has been associated with several disease states, such as cancer and neuronal diseases. Diverse EB1 binding partners are recognised through a conserved four amino acid motif, (serine-X-isoleucine-proline) which exists within an intrinsically disordered region. Here we report the use of a multidisciplinary computational and experimental approach for the discovery of the first small molecule scaffold which targets the EB1 recruiting domain. This approach includes virtual screening (structure- and ligand-based design) and multiparameter compound selection. Subsequent studies on the selected compounds enabled the elucidation of the NMR structures of the C-terminal domain of EB1 in the free form and complexed with a small molecule. These structures show that the binding site is not preformed in solution, and ligand binding is fundamental for the binding site formation. This work is a successful demonstration of the combination of modelling and experimental methods to enable the discovery of compounds which bind to these challenging systems.
Subject(s)
Drug Discovery/methods , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Protein Interaction Maps/drug effects , Amino Acid Motifs , Binding Sites , Humans , Isoleucine/chemistry , Microtubule-Associated Proteins/chemistry , Proline/chemistry , Protein Binding/drug effects , Protein Interaction Domains and Motifs , Serine/chemistryABSTRACT
Avaliou-se o fracionamento dos carboidratos e das proteínas das gramíneas tropicais tifton 85 (Cynodon dactylon cv Tiffon 85), braquiária (Brachiaria brizantha cv Marandu) e tanzânia (Panicum maximum Jacq. cv Tanzânia), cortadas aos 28, 35 e 54 dias de idade. Determinaram-se os teores de matéria seca (MS), matéria orgânica (MO), proteína bruta (PB), extrato etéreo (EE), fibra em detergente neutro corrigida para cinzas e proteína (FDN CP), fibra em detergente ácido (FDA), lignina (LIG), nitrogênio insolúvel em detergente neutro (NIDN) e nitrogênio insolúvel em detergente ácido (NIDA). Para PB determinaram-se as frações A, B1+B2, B3 e C, e para os carboidratos as frações A+B1, B2 e C. Foi utilizado o delineamento inteiramente ao acaso, em arranjo em parcelas (gramíneas) subdivididas (idades de corte). Houve efeito significativo (P<0,05) da idade de corte sobre os teores de MS, FDN CP, NIDN e NIDA. Os teores de PB apresentaram declínio com o avanço da idade. O capim-marandu apresentou teores elevados para as frações A+B1 e B2 dos carboidratos (25,5 e 34,8 por cento dos carboidratos totais, respectivamente) e para as frações A e B1+B2 das proteínas (28,1 por cento aos 35 dias e 53,8 por cento da PB aos 28 dias, respectivamente).
The carbohydrates and proteins fractions of tropical grasses Tifton 85 (Cynodon dactylon cv Tifton 85), brachiaria (Brachiaria brizantha cv Marandu), and Tanzania (Panicum maximum Jacq. cv Tanzania) were evaluated at cut ages of 28, 35, and 54 days. Contents of dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), fiber in neutral detergent corrected for ashes and protein (NDF CP), fiber in acid detergent (ADF), lignin (LIG), neutral detergent insoluble nitrogen (NDIN), and acid detergent insoluble nitrogen (ADIN) were determined. For CP, the A, B1+B2, B3, and C fractions were determined. For carbohydrates, the fractions A+B1, B2, and C were measured. A completely randomized split-plot design was used. Effect of cut age on DM, NDF CP, NDIN and ADIN was observed. The CP contents decreased with the progress of the age for the three grasses. The Marandu grass presented high percentage of the fractions A+B1 and B2 for the total carbohydrate (25.5 and 34.8 percent, respectively) and of the fractions A and B1+B2 for the proteins (28.1 percent CP at 35 days and 53.8 percent CP at 28 days, respectively).
