RESUMEN
Bicyclic peptides assembled around small organic scaffolds are gaining an increasing interest as new potent, stable and highly selective therapeutics because of their uncommon ability to specifically recognize protein targets, of their small size that favor tissue penetration and of the versatility and easiness of the synthesis. We have here rationally designed bicyclic peptides assembled around a common tri-bromo-methylbenzene moiety in order to mimic the structure of the CFC domain of the oncogene Cripto-1 and, more specifically, to orient in the most fruitful way the hot spot residues H120 and W123. Through the CFC domain, Cripto-1 binds the ALK4 receptor and other protein partners supporting uncontrolled cell growth and proliferation. Soluble variants of CFC have the potential to inhibit these interactions suppressing the protein activity. A CFC analog named B3 binds ALK4 in vitro with an affinity in the nanomolar range. Structural analyses in solution via NMR and CD show that B3 has rather flexible conformations, like the parent CFC domain. The functional effects of B3 on the Cripto-1-positive NTERA cancer cell line have been evaluated showing that both CFC and B3 are cytotoxic for the cells and block the Cripto-1 intracellular signaling. Altogether, the data suggest that the administration of the soluble CFC and of the structurally related analog has the potential to inhibit tumor growth.
Asunto(s)
Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Péptidos/química , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Secuencias de Aminoácidos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Espectroscopía de Resonancia Magnética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Péptidos/farmacologíaRESUMEN
BACKGROUND: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb's epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. METHODS: We generated a mAb against PRAME immunizing mice with PRAME fragment 161-415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. RESULTS: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202-212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. CONCLUSIONS: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody's epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/farmacología , Desarrollo de Medicamentos , Epítopos/inmunología , Interferometría , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/inmunología , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Citometría de Flujo , Humanos , Cinética , Melanoma , Ratones , Terapia Molecular Dirigida , Unión Proteica/inmunología , Proteínas Recombinantes , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.