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Angew Chem Int Ed Engl ; 53(42): 11376-80, 2014 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-25196717


Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long-lived states (LLS) in NMR spectroscopy. In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N-terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment. The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants K(D). This property makes the LLS method particularly attractive for the initial steps of fragment-based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods.

Proteínas HSP90 de Choque Térmico/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Adenosina Trifosfato/metabolismo , Sitios de Unión , Evaluación Preclínica de Medicamentos/métodos , Proteínas HSP90 de Choque Térmico/química , Humanos , Ligandos , Unión Proteica
ChemMedChem ; 9(11): 2509-15, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25196781


Transverse and longitudinal relaxation times (T1ρ and T1) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long-lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T1((1)H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D-DNP). Hyperpolarized weak "spy ligands" can be displaced by high-affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput.

Espectroscopía de Resonancia Magnética , Bromuros/química , Dominio Catalítico , Evaluación Preclínica de Medicamentos , Ligandos , Proteínas/química , Proteínas/metabolismo , Tiofenos/química
J Phys Chem B ; 116(50): 14581-91, 2012 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-23190348


Insight into structural and motional features of the C-terminal part of the Human Centrin 2 in complex with the peptide P17-XPC was obtained by using complementary solid-state NMR methods. We demonstrate that the experimental conditions and procedures of sample crystallization determine the quality of solid-state NMR spectra and the internal mobility of the protein. Two-dimensional (2D) (13)C-(13)C and (15)N-(15)N correlation spectra reveal intra- and inter-residue dipolar connectivities and provide partial, site-specific assignments of (13)C and (15)N resonance signals. The secondary structure of the C-ter HsCen2/P17-XPC complex in a microcrystalline state appears similar to that found in solution. Conformational flexibility is probed through relaxation-compensated measurements of dipolar order parameters that exploit the dynamics of cross-polarization in multidimensional experiments. The extracted dipolar coupling constants and relevant order parameters reveal increased backbone flexibility of the loops except for residues involved in coordination with the Ca(2+) cation that stabilizes the hydrophobic pocket containing the peptide P17-XPC.

Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Movimiento , Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Péptidos/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica