INHIBIDORES DE LA TOXINA SHIGA TIPO 2, DESCUBRIMIENTO, SÍNTESIS Y ENSAYO DE MOLÉCULAS CANDIDATAS

Hemolytic uremic syndrome (HUS) is an endemic disease in Argentina, primarily triggered by infection with Shiga toxin–producing Escherichia coli (STEC). Shiga toxin type 2 (Stx2) inhibits protein synthesis, activating pro-apoptotic pathways. Currently, there is no specific treatment available to halt the progression of HUS. The objectives of this study were: (i) to identify molecules with anti-Stx2 activity that could be developed into therapeutic drugs, and (ii) to evaluate their ability to reduce cytotoxicity in HK-2 and VERO cells. Starting from the crystallographic structure of Stx2 (PDB ID: 1R4P) and using a structure-based drug design (SBDD) approach, molecules from different databases were selected. By analyzing molecular docking and molecular dynamics simulations of Stx2–drug complexes, a total of 20,105 compounds were screened and classified into three groups: (A) in-house compounds, (B) FDA-approved drugs, and (C) the Maybridge database. Binding free energies (ΔGᵤ) were calculated using the PBSA method, and the best candidates were selected. Four compounds from Group A were synthesized and three from Group B were acquired. For the selected molecules, the ΔGᵤ values (kcal/mol) were: A1, −14.62; A3, −12.60; A16, −16.67; A24, −19.45; B3, −7.96; and B18, −18.13. In vitro cytotoxicity assays of Stx2 on renal HK-2 and VERO cells showed significant differences in cell viability between monolayers treated with Stx2 alone (IC50 = 2.5–5 ng/mL) and those treated with Stx2 plus drug, as follows: (i) A3 at 10⁻² μM in HK-2 cells (p < 0.05, n = 8), and (ii) B18 at 10⁻³ μM in HK-2 cells (p < 0.05, n = 6) and at 10⁻¹ μM in VERO cells (p < 0.0001, n = 8). To improve the aqueous solubility of A3, eight derivatives sharing the same core structure were designed and subjected to docking analysis. The resulting binding free energies were −9.4 ± 0.5 and −9.3 kcal/mol for the mean ± SD and median, respectively. Overall, our results demonstrate that at least two of the compounds selected through SBDD exhibit anti-Stx2 activity. El Síndrome Urémico Hemolítico (SUH), enfermedad endémica en Argentina, cuyo principal desencadenante es la infección por E.coli productora de toxina Shiga (STEC). La toxina Shiga tipo 2 (Stx2), inhibe la síntesis de proteínas, desencadenando mecanismos proapoptóticos. No se dispone de un tratamiento específico para detener la progresión del SUH. Nuestros objetivos fueron: i) obtener moléculas con actividad anti-Stx2 transformables en fármacos y ii) ensayar su capacidad para disminuir la citotoxicidad sobre células HK2 y VERO. Partiendo de la estructura cristalizada de Stx2 (id:1R4P-PDB) y utilizando diseño de fármacos basado en la estructura (SBD), seleccionamos moléculas de diferentes bases de datos. Estudiando el acoplamiento molecular y la dinámica molecular de los complejos Stx2-droga, analizamos 20105 moléculas que clasificamos en tres grupos: A)“in house”, B)aprobadas por FDA y C)base Maybridge. Calculamos su energía libre de unión (ΔGu) con el método PBSA y elegimos las mejores candidatas. Sintetizamos cuatro del Grupo A y adquirimos tres del Grupo B. Para las moléculas seleccionadas, los valores de ΔGu (kcal/mol) fueron A1:-14.62;A3:-12.60;A16:-16.67; A24:-19.45;B3:-7.96 y B18:-18.13. Los resultados de ensayos in vitro de citotoxicidad de Stx2 sobre células renales HK2 y VERO, mostraron diferencias significativas entre la viabilidad de las monocapas tratadas solo con Stx2 (IC50= 2.5-5ng/ml) vs Stx2+droga, con el siguiente detalle: i) con A3:10-2μM en HK2, p