RESUMO
Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residues from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of-but adjacent to-the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Receptor trkA/antagonistas & inibidores , Receptor trkA/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Cinética , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Conformação Proteica , Inibidores de Proteínas Quinases/síntese química , Receptor trkA/genética , Receptor trkB/antagonistas & inibidores , Receptor trkB/química , Receptor trkB/genética , Receptor trkC/antagonistas & inibidores , Receptor trkC/química , Receptor trkC/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
As many processes in the preclinical drug discovery process become highly parallel, the need to also produce a large number of different proteins in parallel has become acute, such as for protein crystallization and activity screening. In turn, the requisite DNA constructions to produce these proteins must now be done at a rate that requires automated cloning procedures, each with an intrinsic low failure probability per sample. The high-throughput cloning solutions presented here achieve production of 192 different expression plasmids at a success rate of greater than 95% of the targeted open reading frames. Time for completion of the set by one person is reduced to approximately 11 working days, starting with polymerase chain reactions for a number of source clones and ending with purified expression plasmids. Achievement of this throughput utilizes the following: (1) the Beckman Coulter (Fullerton, CA) Biomek FX liquid handler for most manipulations, (2) Gateway cloning technology (Invitrogen Corp., Carlsbad, CA), and (3) computer programs designed for parallel processing of all sample information, including primer design and the resulting DNA and protein sequence assembly. Exemplary data are presented for discovery of a form of the Rho-kinase that crystallizes (ROCK2).