RESUMEN
Proliferating cell nuclear antigen (PCNA) is a central factor in DNA replication and repair pathways that plays an essential role in genome stability. The functional roles of PCNA are mediated through an extensive list of protein-protein interactions, each of which transmits specific information in protein assemblies. The flexibility at the PCNA-protein interaction interfaces offers opportunities for the discovery of functionally selective inhibitors of DNA repair pathways. Current fragment-based drug design methodologies can be limited by the flexibility of protein interfaces. These factors motivated an approach to defining compounds that could leverage previously identified subpockets on PCNA that are suitable for fragment-binding sites. Methodologies for screening multiple connected fragment-binding events in distinct subpockets are deployed to improve the selection of fragment combinations. A flexible backbone based on N-alkyl-glycine amides offers a scaffold to combinatorically link multiple fragments for in silico screening libraries that explore the diversity of subpockets at protein interfaces. This approach was applied to discover new potential inhibitors of DNA replication and repair that target PCNA in a multiprotein recognition site. The screens of the libraries were designed to computationally filter ligands based upon the fragments and positions to <1%, which were synthesized and tested for direct binding to PCNA. Molecular dynamics simulations also revealed distinct features of these novel molecules that block key PCNA-protein interactions. Furthermore, a Bayesian classifier predicted 15 of the 16 new inhibitors to be modulators of protein-protein interactions, demonstrating the method's utility as an effective screening filter. The cellular activities of example ligands with similar affinity for PCNA demonstrate unique properties for novel selective synergy with therapeutic DNA-damaging agents in drug-resistant contexts.
RESUMEN
Isotopic variants of Rhodamine 6G (R6G) have previously been used as a method of multiplexed detection for Surface Enhanced Raman Spectroscopy (SERS), including protein detection and quantification. Challenges exist, however, with producing long-term stable SERS signals with exposure to silver or gold metal surfaces without the use of additional protective coatings of nanomaterials. Here, novel rhodamine "dimers" and "trimers" have been created that demonstrate a higher avidity for metal nanoparticles and induce aggregation to create plasmonic "hotspots" as indicated by enhanced Raman scattering in situ. These aggregates can be formed in a colloid, on surfaces, or membrane substrates such as poly(vinylidene fluoride) for applications in biosciences. The integrity of the materials and Raman signals are maintained for months of time on different substrates. These dye materials should provide avenues for simplified in situ generation of sensors for Raman-based assays especially in settings requiring highly robust performance.
RESUMEN
The discovery of compounds that selectively modulate signaling and effector proteins downstream of EGFR could have important implications for understanding specific roles for pathway activation. A complicating factor for receptor tyrosine kinases is their capacity to be translocated to the nucleus upon ligand engagement. Once localized in subcellular compartments like the nucleus, the roles for EGFR take on additional features, many of which are still being revealed. Additionally, nuclear localization of EGFR has been implicated in downstream events that have significance for therapy resistance and disease progression. The challenges to addressing the differential roles for EGFR in the nucleus motivated experimental approaches that can selectively modulate its subcellular function. By adding modifications to the established EGFR kinase inhibitor gefitinib, an approach to small molecule conjugates with a unique nuclear-targeting peptoid sequence was tested in both human and murine breast tumor cell models for their capacity to inhibit EGF-stimulated activation of ERK1/2 and STAT3. While gefitinib alone inhibits both of these downstream effectors, data acquired here indicate that compartmentalization of the gefitinib conjugates allows for pathway specific inhibition of STAT3 while not affecting ERK1/2 signaling. The inhibitor conjugates offered a more direct route to evaluate the role of EGF-stimulated epithelial-to-mesenchymal transition in these breast cancer cell models. These conjugates revealed that STAT3 activation is not involved in EGF-induced EMT, and instead utilization of the cytoplasmic MAP kinase signaling pathway is critical to this process. This is the first example of a conjugate kinase inhibitor capable of partitioning to the nucleus and offers a new approach to enhancing kinase inhibitor specificity.
Asunto(s)
Descubrimiento de Drogas , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Brefeldino A/farmacología , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Humanos , Peptoides/administración & dosificación , Peptoides/química , Peptoides/farmacología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Inhibidores de la Síntesis de la Proteína/farmacología , Factor de Transcripción STAT3/metabolismoRESUMEN
The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6.
Asunto(s)
Benzopiranos/química , Benzopiranos/farmacología , Sondas Moleculares/química , Sondas Moleculares/farmacología , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Proteínas Oncogénicas Virales/química , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Represoras/químicaRESUMEN
While the fragment-based drug design approach continues to gain importance, gaps in the tools and methods available in the identification and accurate utilization of protein subpockets have limited the scope. The importance of these features of small molecule-protein recognition is highlighted with several examples. A generalized solution for the identification of subpockets and corresponding chemical fragments remains elusive, but there are numerous advancements in methods that can be used in combination to address subpockets. Finally, additional examples of approaches that consider the relative importance of small-molecule co-dependence of protein conformations are highlighted to emphasize an increased significance of subpockets, especially at protein interfaces.
Asunto(s)
Diseño de Fármacos , Proteínas/química , Adenosina Trifosfato/química , Sitios de Unión , LigandosRESUMEN
Expression and function of the human papillomavirus (HPV) early protein 6 (E6) is necessary for viral replication and oncogenesis in cervical cancers. HPV E6 targets the tumor suppressor protein p53 for degradation. To achieve this, "high-risk" HPV E6 proteins bind to and modify the target specificity of the ubiquitin ligase E6AP (E6 associated protein). This E6-dependent loss of p53 enables the virus to bypass host cell defenses and facilitates virally induced activation of the cell cycle progression during viral replication. Disruption of the interaction between E6 and E6AP and stabilization of p53 should decrease viability and proliferation of HPV positive cells. A new in vitro high-throughput binding assay was developed to assay binding between HPV-16 E6 and E6AP and to identify compounds that inhibit this interaction. The compound luteolin emerged from the screen and a library of novel flavones based on its structure was synthesized and characterized using this in vitro binding assay. The compounds identified in this study disrupt the E6/E6AP interaction, increase the levels of p53 and p21(Cip1/Waf1), and decrease proliferation of HPV positive cell lines. The new class of flavonoid E6 inhibitors displays a high degree of specificity for HPV positive cells. Docking analyses suggest that these compounds bind in a hydrophobic pocket at the interface between E6 and E6AP and mimic the leucines in the conserved α-helical motif of E6AP. The activity and specificity of these compounds represent a promising new lead for development as an antiviral therapy in the treatment of HPV infection and cervical cancer.
Asunto(s)
Flavonoides/química , Papillomavirus Humano 16/efectos de los fármacos , Modelos Moleculares , Proteínas Oncogénicas Virales/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Neoplasias del Cuello Uterino/virología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Descubrimiento de Drogas , Femenino , Flavonoides/antagonistas & inhibidores , Flavonoides/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Luteolina/farmacología , Estructura Molecular , Proteínas Oncogénicas Virales/química , Unión Proteica/fisiología , Proteínas Represoras/química , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
The anaphase-promoting complex (APC) is tightly regulated during cell division, often by pseudosubstrate binding to its coactivators Cdh1 and Cdc20. Budding yeast Acm1 is a Cdh1 pseudosubstrate inhibitor whose biological function is unknown. We show here that cells lacking Acm1 have defects in nuclear positioning and spindle morphology during mitosis. However, Cdh1 substrates are not destabilized in the absence of Acm1 and expression of inactive Cdh1 mutants that retain substrate binding is sufficient for the acm1 phenotype. We conclude that Acm1 is not required to inhibit APC(Cdh1) activity but rather prevents untimely Cdh1-substrate interactions. We further provide evidence suggesting that the substrate primarily responsible for the acm1 phenotype is the bud neck-localized kinase, Hsl1. Our results imply that at least some coactivator-substrate interactions require regulation. Several unrelated APC pseudosubstrates have been identified in diverse eukaryotes and their ability to simultaneously inhibit enzymatic activity and substrate binding may partly explain why this regulatory mechanism has been selected repeatedly during evolution.