In Silico and In Vitro Evaluation of Bevacizumab Biosimilar MB02 as an Antitumor Agent in Canine Mammary Carcinoma

Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC.Fil: Cardama, Georgina Alexandra. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bucci, Paula Lorena. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lemos, Jesus. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Llavona, Candela. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Benavente, Micaela Andrea. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Hellmén, Eva. Gobierno de la Provincia de Buenos Aires. Hospital El Cruce Doctor Nestor Carlos Kirchner. Centro de Medicina Traslacional.; ArgentinaFil: Fara, María Laura. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; ArgentinaFil: Medrano, Eduardo. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; ArgentinaFil: Spitzer, Eduardo. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; ArgentinaFil: Demarco, Ignacio A.. No especifíca;Fil: Sabella, Patricia. No especifíca;Fil: Garona, Juan. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alonso, Daniel Fernando. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Desarrollo de nuevos inhibidores de Rho GTPasas con actividad antitumoral mediante un screening virtual basado en docking

Fil: Cardama, Georgina A. Universidad Nacional de Quilmes. Departamente de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina.La vía de señalización de las Rho GTPasas cumple un rol fundamental en la regulación de múltiples procesos celulares. La activación aberrante de Rac1, un miembro de la familia de las Rho GTPasas, está asociado con tumorigénesis, progresión tumoral y metástasis en diferentes tipos tumorales. Particularmente, la vía de Rac1 se encuentra sobreexpresada e hiperactivada en diferentes tipos de cáncer, incluyendo al carcinoma mamario agresivo y al glioblastoma. Es por esto que la vía de Rac1 es considerada como un blanco molecular interesante. Tomando como punto de partida la estructura tridimensional de la proteína Rac1 y un subset de 250.000 estructuras tridimensionales de compuestos tipo droga de la base de datos libre ZINC, se llevó adelante un screening virtual basado en docking. A partir de dicho experimento in silico, se identificaron una serie de compuestos que posiblemente presentaban afinidad por el sitio de interacción de Rac1 con sus activadores tipo GEF. Se seleccionaron 11 compuestos del screening in silico y se evaluaron in vitro, identificando al Hit1 (ZINC69391) como el candidato más interesante para posterior desarrollo. En primer lugar, se evaluó el compuesto en modelos de carcinoma mamarios agresivos. Se observó que ZINC69391 fue capaz de inhibir la interacción de Rac1 con su activador tipo GEF Tiam1, llevando a la inhibición de la activación de Rac1 mediada por EGF, uno de los factores de crecimiento que activan esta vía de señalización, sin interferir con la activación de la GTPasa íntimamente relacionada Cdc42. Se observó que ZINC69391 fue capaz de inhibir la proliferación, la migración celular y arrestar el ciclo celular en fase G1 de células de carcinoma mamario agresivas. Además, ZINC69391 mostró tener efecto antimetastásico in vivo en un modelo murino singénico. Con el objetivo de optimizar el compuesto, a partir de ZINC69391 se diseñaron racionalmente y se sintetizaron una serie de análogos. Se identificó a un análogo más activo denominado 1A-116. El compuesto 1A-116 fue capaz de inhibir la interacción de Rac1 con P-Rex1, un GEF clave para la progresión de tumores mamarios, y logró inhibir la activación de Rac1 a concentraciones menores que el compuesto parental sin afectar la activación de Cdc42 en el modelo de carcinoma mamario. Mostró una mayor actividad antitumoral y antimetastásica in vivo en un modelo murino singénico. Por otro lado, también se evaluó el compuesto ZINC69391 en células de glioblastoma, mostrando que es capaz de interferir la interacción de Rac1 con otro GEF específico relacionado directamente con la agresividad de glioblastomas en pacientes, denominado Dock180. ZINC69391 inhibió la activación de Rac1 en respuesta a EGF en células de glioblastoma humano y también la fosforilación de Pak1, un efector importante de Rac1. ZINC69391 mostró inhibir la proliferación celular, arrestar el ciclo celular en fase G1 y gatillar un efecto proapoptótico. ZINC69391 bloqueó la migración e invasión celular a través de la modulación del citoesqueleto de actina, eventos claves para la patogénesis de este tipo tumoral. En un modelo intracraneal xenogénico murino el tratamiento diario con 20 mg/kg/dia de ZINC69391 vía i.p logró un aumento significativo en la sobrevida de los animales en comparación con el grupo control. También evaluamos el efecto del análogo 1A-116 en células de glioblastoma humano y fue más potente que el compuesto parental, presentando una mayor actividad antiproliferativa y antiinvasiva. Estos nuevos inhibidores de Rac1 desarrollados son capaces de modular diferentes eventos celulares mediados por esta GTPasa y podrían ser utilizados como una estrategia novedosa para el tratamiento de tumores en los cuales Rac1 cumple un rol importante. Los compuestos muestran una gran potencialidad de aplicación en la clínica para el manejo terapéutico de diferentes tipos tumorales, tales como el carcinoma mamario agresivo y el glioblastoma

In Silico and In Vitro Evaluation of Bevacizumab Biosimilar MB02 as an Antitumor Agent in Canine Mammary Carcinoma

Simple Summary Canine mammary carcinomas (CMC) are associated with poor clinical outcomes and high mortality. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and tumor progression in many solid tumors, including mammary carcinomas. The goal of this work was to establish the therapeutic value of MB02 monoclonal antibody biosimilar to bevacizumab that targets VEGF in CMC. For this purpose, first, we were able to predict in silico that bevacizumab was able to recognize and bind canine VEGF. This was confirmed in vitro using an ELISA-based assay. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These results show a high potential for MB02 as a promising therapeutic agent for the management of CMC. Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC

Rho GTPases as molecular targets in cancer. Strategies and therapeutic opportunities

Rho GTPases are molecular switches that control the different cellular processes. Deregulation of these proteins is associated to transformation and malignant progression in several cancer types. Given the evidence available of the role of Rho GTPases in cancer it is suggested that these proteins can serve as potential therapeutic targets. This review focuses on the strategies used to develop Rho GTPases modulators and their potential use in therapeutic settings

A Functional Phenylacetic Acid Catabolic Pathway Is Required for Full Pathogenicity of Burkholderia cenocepacia in the Caenorhabditis elegans Host Model▿

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex, a group of metabolically versatile bacteria that have emerged as opportunistic pathogens in cystic fibrosis and immunocompromised patients. Previously a screen of transposon mutants in a rat pulmonary infection model identified an attenuated mutant with an insertion in paaE, a gene related to the phenylacetic acid (PA) catabolic pathway. In this study, we characterized gene clusters involved in the PA degradation pathway of B. cenocepacia K56-2 in relation to its pathogenicity in the Caenorhabditis elegans model of infection. We demonstrated that targeted-insertion mutagenesis of paaA and paaE, which encode part of the putative PA-coenzyme A (CoA) ring hydroxylation system, paaZ, coding for a putative ring opening enzyme, and paaF, encoding part of the putative beta-oxidation system, severely reduces growth on PA as a sole carbon source. paaA and paaE insertional mutants were attenuated for virulence, and expression of paaE in trans restored pathogenicity of the paaE mutant to wild-type levels. Interruption of paaZ and paaF slightly increased virulence. Using gene interference by ingested double-stranded RNA, we showed that the attenuated phenotype of the paaA and paaE mutants is dependent on a functional p38 mitogen-activated protein kinase pathway in C. elegans. Taken together, our results demonstrate that B. cenocepacia possesses a functional PA degradation pathway and that the putative PA-CoA ring hydroxylation system is required for full pathogenicity in C. elegans

Preclinical Efficacy and Toxicology Evaluation of RAC1 Inhibitor 1A-116 in Human Glioblastoma Models

Malignant gliomas are the most common primary central nervous system tumor in adults. Despite current therapeutics, these tumors are associated with poor prognosis and a median survival of 16 to 19 months. This highlights the need for innovative treatments for this incurable disease. Rac1 has long been associated with tumor progression and plays a key role in glioma’s infiltrative and invasive nature. The aim of this study is to evaluate the 1A-116 molecule, a Rac1 inhibitor, as targeted therapy for this aggressive disease. We found that targeting Rac1 inhibits cell proliferation and cell cycle progression using different in vitro human glioblastoma models. Additionally, we evaluated 1A-116 in vivo, showing a favorable toxicological profile. Using in silico tools, 1A-116 is also predicted to penetrate the blood–brain barrier and present a favorable metabolic fate. In line with these results, 1A-116 i.p daily treatment resulted in a dose-dependent antitumor effect in an orthotopic IDH-wt glioma model. Altogether, our study provides a strong potential for clinical translation of 1A-116 as a signal transduction-based precision therapy for glioma and also increases the evidence of Rac1 as a key molecular target

Antiviral effect of high-dose ivermectin in adults with COVID-19: A proof-of-concept randomized trial

Background: There are limited antiviral options for the treatment of patients with COVID-19. Ivermectin (IVM), a macrocyclic lactone with a wide anti-parasitary spectrum, has shown potent activity against SARS-CoV-2 in vitro. This study aimed at assessing the antiviral effect of IVM on viral load of respiratory secretions and its relationship with drug concentrations in plasma. Methods: Proof-of-concept, pilot, randomized, controlled, outcome-assessor blinded trial to evaluate antiviral activity of high-dose IVM in 45 COVID-19 hospitalized patients randomized in a 2:1 ratio to standard of care plus oral IVM at 0·6 mg/kg/day for 5 days versus standard of care in 4 hospitals in Argentina. Eligible patients were adults with RT-PCR confirmed SARS-CoV-2 infection within 5 days of symptoms onset. The primary endpoint was the difference in viral load in respiratory secretions between baseline and day-5, by quantitative RT-PCR. Concentrations of IVM in plasma were measured. Study registered at ClinicalTrials.gov: NCT04381884. Findings: 45 participants were recruited (30 to IVM and 15 controls) between May 18 and September 9, 2020. There was no difference in viral load reduction between groups but a significant difference was found in patients with higher median plasma IVM levels (72% IQR 59–77) versus untreated controls (42% IQR 31–73) (p = 0·004). Mean ivermectin plasma concentration levels correlated with viral decay rate (r: 0·47, p = 0·02). Adverse events were similar between groups. No differences in clinical evolution at day-7 and day-30 between groups were observed. Interpretation: A concentration dependent antiviral activity of oral high-dose IVM was identified at a dosing regimen that was well tolerated. Large trials with clinical endpoints are necessary to determine the clinical utility of IVM in COVID-19. Funding: This work was supported by grant IP-COVID-19-625, Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación, Argentina and Laboratorio ELEA/Phoenix, Argentina