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.

Targeting the Hsp90 C-terminal domain to induce allosteric inhibition and selective client downregulation.

BACKGROUND: Inhibition of Hsp90 is desirable due to potential downregulation of oncogenic clients. Early generation inhibitors bind to the N-terminal domain (NTD) but C-terminal domain (CTD) inhibitors are a promising class because they do not induce a heat shock response. Here we present a new structural class of CTD binding molecules with a unique allosteric inhibition mechanism. METHODS: A hit molecule, NSC145366, and structurally similar probes were assessed for inhibition of Hsp90 activities. A ligand-binding model was proposed indicating a novel Hsp90 CTD binding site. Client protein downregulation was also determined. RESULTS: NSC145366 interacts with the Hsp90 CTD and has anti-proliferative activity in tumor cell lines (GI50=0.2-1.9µM). NSC145366 increases Hsp90 oligomerization resulting in allosteric inhibition of NTD ATPase activity (IC50=119µM) but does not compete with NTD or CTD-ATP binding. Treatment of LNCaP prostate tumor cells resulted in selective client protein downregulation including AR and BRCA1 but without a heat shock response. Analogs had similar potencies in ATPase and chaperone activity assays and variable effects on oligomerization. In silico modeling predicted a binding site at the CTD dimer interface distinct from the nucleotide-binding site. CONCLUSIONS: A set of symmetrical scaffold molecules with bisphenol A cores induced allosteric inhibition of Hsp90. Experimental evidence and molecular modeling suggest that the binding site is independent of the CTD-ATP site and consistent with unique induction of allosteric effects. GENERAL SIGNIFICANCE: Allosteric inhibition of Hsp90 via a mechanism used by the NSC145366-based probes is a promising avenue for selective oncogenic client downregulation.

Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors.

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.