RESUMEN
Loss of WRN, a DNA repair helicase, was identified as a strong vulnerability of microsatellite instable (MSI) cancers, making WRN a promising drug target. We show that ATP binding and hydrolysis are required for genome integrity and viability of MSI cancer cells. We report a 2.2-Å crystal structure of the WRN helicase core (517-1,093), comprising the two helicase subdomains and winged helix domain but not the HRDC domain or nuclease domains. The structure highlights unusual features. First, an atypical mode of nucleotide binding that results in unusual relative positioning of the two helicase subdomains. Second, an additional ß-hairpin in the second helicase subdomain and an unusual helical hairpin in the Zn2+ binding domain. Modelling of the WRN helicase in complex with DNA suggests roles for these features in the binding of alternative DNA structures. NMR analysis shows a weak interaction between the HRDC domain and the helicase core, indicating a possible biological role for this association. Together, this study will facilitate the structure-based development of inhibitors against WRN helicase.
Asunto(s)
Dominio Catalítico , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Inestabilidad de Microsatélites , Helicasa del Síndrome de Werner/química , Helicasa del Síndrome de Werner/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Ciclo Celular/genética , Supervivencia Celular/genética , Cristalización , ADN/metabolismo , Daño del ADN/genética , Silenciador del Gen , Células HCT116 , Humanos , Hidrólisis , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Transfección , Zinc/metabolismo , Quinasa Tipo Polo 1RESUMEN
Aberrant WNT pathway activation, leading to nuclear accumulation of ß-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of ß-catenin and subsequent nuclear translocation. Restoring cellular degradation of ß-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to ß-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging ß-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein-protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards ß-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.
Asunto(s)
Bibliotecas de Moléculas Pequeñas/química , beta Catenina/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Mapas de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , beta Catenina/metabolismoRESUMEN
KRAS, the most common oncogenic driver in human cancers, is controlled and signals primarily through protein-protein interactions (PPIs). The interaction between KRAS and SOS1, crucial for the activation of KRAS, is a typical, challenging PPI with a large contact surface area and high affinity. Here, we report that the addition of only one atom placed between Y884SOS1 and A73KRAS is sufficient to convert SOS1 activators into SOS1 inhibitors. We also disclose the discovery of BI-3406. Combination with the upstream EGFR inhibitor afatinib shows in vivo efficacy against KRASG13D mutant colorectal tumor cells, demonstrating the utility of BI-3406 to probe SOS1 biology. These findings challenge the dogma that large molecules are required to disrupt challenging PPIs. Instead, a "foot in the door" approach, whereby single atoms or small functional groups placed between key PPI interactions, can lead to potent inhibitors even for challenging PPIs such as SOS1-KRAS.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína SOS1/metabolismo , Afatinib/química , Afatinib/metabolismo , Afatinib/uso terapéutico , Regulación Alostérica/efectos de los fármacos , Sitios de Unión , Dominio Catalítico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Humanos , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Proteína SOS1/agonistas , Proteína SOS1/antagonistas & inhibidores , Proteína SOS1/genéticaRESUMEN
Mouse double minuteâ 2 (MDM2) is a main and direct inhibitor of the crucial tumor suppressor p53. Reports from initial clinical trials showed that blocking this interaction with a small-molecule inhibitor can have great value in the treatment of cancer for patients with p53 wild-type tumors; however, it also revealed dose-limiting hematological toxicities and drug-induced resistance as main issues. To overcome the former, an inhibitor with superior potency and pharmacokinetic properties to ultimately achieve full efficacy with less-frequent dosing schedules is required. Toward this aim, we optimized our recently reported spiro-oxindole inhibitors by focusing on the crucial interaction with the amino acid side chain of His96MDM2 . The designed molecules required the targeted synthesis of structurally complex spiro[indole-3,2'-pyrrolo[2,3-c]pyrrole]-2,4'-diones for which we developed an unprecedented intramolecular azomethine ylide cycloaddition and investigated the results by computational methods. One of the new compounds showed superior cellular potency over previously reported BI-0252. This finding is a significant step toward an inhibitor suitable to potentially mitigate hematological on-target adverse effects.