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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