RESUMEN
Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors, including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, limitations to developing an effective matriptase inhibitor include weak binding affinity, poor selectivity, and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type-1 (HAI-1), a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase-binding and inhibition domain, whereas the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here, we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains four stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a Ki of 70 ± 5 pm, an increase of 120-fold compared with the natural HAI-1 inhibitor, to our knowledge making it one of the most potent matriptase inhibitors identified to date. Furthermore, the engineered inhibitor demonstrates a protease selectivity profile similar to that of wildtype KD1 but distinct from that of HAI-1. It also inhibits activation of the natural pro-HGF substrate and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KD1.
Asunto(s)
Ingeniería de Proteínas/métodos , Proteínas Inhibidoras de Proteinasas Secretoras/química , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Perros , Humanos , Células de Riñón Canino Madin Darby , Dominios Proteicos , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
In the last decade, numerous growth factors and biomaterials have been explored for the treatment of myocardial infarction (MI). While pre-clinical studies have demonstrated promising results, clinical trials have been disappointing and inconsistent, likely due to poor translatability. In the present study, we investigate a potential myocardial regenerative therapy consisting of a protein-engineered dimeric fragment of hepatocyte growth factor (HGFdf) encapsulated in a shear-thinning, self-healing, bioengineered hydrogel (SHIELD). We hypothesized that SHIELD would facilitate targeted, sustained intramyocardial delivery of HGFdf thereby attenuating myocardial injury and post-infarction remodeling. Adult male Wistar rats (n = 45) underwent sham surgery or induction of MI followed by injection of phosphate buffered saline (PBS), 10 µg HGFdf alone, SHIELD alone, or SHIELD encapsulating 10 µg HGFdf. Ventricular function, infarct size, and angiogenic response were assessed 4 weeks post-infarction. Treatment with SHIELD + HGFdf significantly reduced infarct size and increased both ejection fraction and borderzone arteriole density compared to the controls. Thus, sustained delivery of HGFdf via SHIELD limits post-infarction adverse ventricular remodeling by increasing angiogenesis and reducing fibrosis. Encapsulation of HGFdf in SHIELD improves clinical translatability by enabling minimally-invasive delivery and subsequent retention and sustained administration of this novel, potent angiogenic protein analog. Biotechnol. Bioeng. 2017;114: 2379-2389. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Preparaciones de Acción Retardada/administración & dosificación , Factor de Crecimiento de Hepatocito/administración & dosificación , Hidrogeles/química , Infarto del Miocardio/tratamiento farmacológico , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/administración & dosificación , Disfunción Ventricular Izquierda/prevención & control , Proteínas Angiogénicas/administración & dosificación , Proteínas Angiogénicas/química , Proteínas Angiogénicas/genética , Animales , Preparaciones de Acción Retardada/química , Difusión , Factor de Crecimiento de Hepatocito/análogos & derivados , Factor de Crecimiento de Hepatocito/genética , Inyecciones , Masculino , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/patología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Resistencia al Corte , Resultado del Tratamiento , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/patología , ViscosidadRESUMEN
Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address this need, we developed a matriptase-sensitive protein biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) reporter system. In this platform, two adjoining protein domains, connected by a protease-labile linker, produce fluorescence when assembled and are nonfluorescent when the linker is cleaved by matriptase. A panel of ddRFP-based matriptase biosensor designs was created that contained different linker lengths between the protein domains. These constructs were characterized for linker-specific cleavage, matriptase activity, and matriptase selectivity; a biosensor containing a RSKLRVGGH linker (termed B4) was expressed at high yields and displayed both high catalytic efficiency and matriptase specificity. This biosensor detects matriptase inhibition by soluble and yeast cell surface expressed inhibitor domains with up to a 5-fold dynamic range and also detects matriptase activity expressed by human cancer cell lines. In addition to matriptase, we highlight a strategy that can be used to create effective biosensors for quantifying activity and inhibition of other proteases of interest.
Asunto(s)
Técnicas Biosensibles/métodos , Proteínas Luminiscentes/metabolismo , Péptido Hidrolasas/análisis , Serina Endopeptidasas/metabolismo , Western Blotting , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Escherichia coli/genética , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Péptido Hidrolasas/metabolismo , Multimerización de Proteína , Serina Endopeptidasas/análisis , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-Actividad , Proteína Fluorescente RojaRESUMEN
Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell trafficking behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications. STATEMENT OF SIGNIFICANCE: Growth factors are promising therapeutic proteins that have the ability to modulate morphogenetic behaviors, including cell survival, proliferation, migration and differentiation. However, the translation of growth factors into clinical therapies has been hindered by properties such as poor protein stability, low recombinant expression yield, and non-physiological delivery, which lead to suboptimal efficacy and adverse side effects. To address these needs, researchers are employing clever molecular and material engineering and design strategies to both improve the intrinsic properties of growth factors and effectively control their delivery into tissue. This review highlights examples of interdisciplinary tools and technologies used to augment the therapeutic potential of growth factors for clinical applications in regenerative medicine.