Multimeric Rhodamine Dye-Induced Aggregation of Silver Nanoparticles for Surface-Enhanced Raman Scattering.

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.

Discovery of Inhibitors for Proliferating Cell Nuclear Antigen Using a Computational-Based Linked-Multiple-Fragment Screen.

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.

Selective Inhibition of STAT3 Phosphorylation Using a Nuclear-Targeted Kinase Inhibitor.

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.

Acm1 contributes to nuclear positioning by inhibiting Cdh1-substrate interactions.

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.