RESUMO
We report a general approach to engineering multivalent d-proteins with antibody-like activities in vivo. Mirror-image phage display and structure-guided design were utilized to create a d-protein that uses receptor mimicry to antagonize vascular endothelial growth factor A (VEGF-A). Selections against the d-protein form of VEGF-A using phage-displayed libraries of two different domain scaffolds yielded two proteins that bound distinct receptor interaction sites on VEGF-A. X-ray crystal structures of the d-protein/VEGF-A complexes were used to guide affinity maturation and to construct a heterodimeric d-protein VEGF-A antagonist with picomolar activity. The d-protein VEGF-A antagonist prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration and slowed tumor growth in the MC38 syngeneic mouse tumor model with efficacies comparable to those of approved antibody drugs, and in contrast with antibodies, the d-protein was non-immunogenic during treatment and following subcutaneous immunizations.
Assuntos
Antineoplásicos/química , Neoplasias/tratamento farmacológico , Peptídeos/química , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Vasos Retinianos/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Bevacizumab/farmacologia , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Olho/efeitos dos fármacos , Feminino , Humanos , Camundongos , Modelos Moleculares , Biblioteca de Peptídeos , Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Coelhos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
An increasing number of assay detection technologies are routinely used in small molecule drug discovery and lead optimization. These assays range from solid-phase heterogeneous assays such as enzyme-linked immunosorbent assay and dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA, PerkinElmer Life and Analytical Sciences, Boston, MA) to solution phase, bead-based assays such as electrochemiluminescence assay (BioVeris [Gaithersburg, MD] technology) and amplified luminescent proximity homogeneous assay (AlphaScreen, PerkinElmer Life and Analytical Sciences) to completely solution-based homogeneous assays such as time-resolved fluorescence resonance energy transfer and fluorescence polarization. The aim of this study is to compare these assay technologies and assess the advantages and disadvantages of each in the context of our efforts to develop small molecule antagonists to the melanoma inhibitor of apoptosis protein. In this study, seven peptides have been evaluated for their potencies in each assay format. Our results indicate that these assay technologies produce similar relative potencies; however, some methods may be more susceptible to interference than others. Consequently, the choice of the method used frequently depends on a number of factors in addition to assay reproducibility and performance, such as throughput of the assay, cost, compound interference, and ease of use.
Assuntos
Bioensaio/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Peptídeos/antagonistas & inibidores , Peptídeos/química , Proteínas/antagonistas & inibidores , Proteínas/química , Biotina/química , Interpretação Estatística de Dados , Escherichia coli/genética , Escherichia coli/metabolismo , Fluoresceínas/química , Polarização de Fluorescência , Peptídeos/síntese químicaRESUMO
Technology has been developed to display small molecules on phage particles. This innovation enables the generation of libraries of phage-tagged compounds with novel properties that are well suited for in vivo assays.