Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity

Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme\u27s activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 μM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy. © 2013 Shen et al

Recoveries from screening phage displayed libraries against the soluble ectodomain of PSMA.

<p>Recoveries from screening phage displayed libraries against the soluble ectodomain of PSMA.</p

Structure of the Peptide 562 tetramer.

<p>Structure of the Peptide 562 tetramer.</p

Binding and internalization of 5FAM-562 and 5FAM-563 in LNCaP cells.

<p>5FAM-562 (1 µM, <i>A–C</i>, green) and 5FAM-563 (1 µM , <i>D–F</i>, green) bound and internalized in LNCaP cells and co-localized with PSMA in intracellular vesicles visualized with anti-PSMA-Mab (red, scale is 20 µm).</p

Immunofluorescence microscopy of phage clones from round 3 of panning.

<p>Randomly selected PSMA-targeted phage clones (C, E, G, H, J, L) from the third round of screening bound specifically to LNCaP cells (“L”), but not PC3 cells (“P”). While staining was preferentially bound at cell surface, there was evidence of internalization (scale 20 µm).</p

Sequences of phage clones isolated after 3-round of panning and IC₅₀ values of the peptides analyzed.

*<p>Cys(Acm) in peptides 564 and 565.</p>†<p>Ki value from the Cheng-Prussof equation.</p>a<p>, no inhibition observed at 14.5 µM.</p>b<p>, no inhibition observed at 55.5 µM.</p>c<p>, no inhibition observed at 1 mM.</p><p>nd, not determined.</p

Immunofluorescence microscopy of phage clones from round 2 of panning.

<p><i>(A)</i> PSMA (100 nM) inhibited the binding of Clone 1 (ARLSHRPSYLLVCA) and Clone 2 (GTAVASRVYSLHSLM) to PSMA-postive LNCaP cells, indicating that the uptake was receptor specific (scale is 30 µm). <i>(B)</i> Immunofluorescence microscopy of phage clones from round 2 of panning indicate that the panning process is biased toward PSMA binding (“*” denotes <i>P<0.05</i>).</p

Tunable Ultrasmall Visible-to-Extended Near-Infrared Emitting Silver Sulfide Quantum Dots for Integrin-Targeted Cancer Imaging

The large size of many near-infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines <i>in vivo</i> uptake to the peritumoral space and results in high liver retention. In this study, we developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from 500 to 1200 nm and a QD core diameter between 1.5 and 9 nm. Conjugation of a tumor-avid cyclic pentapeptide (Arg-Gly-Asp-DPhe-Lys) resulted in monodisperse, water-soluble QDs (hydrodynamic diameter < 10 nm) without loss of the peptide’s high binding affinity to tumor-associated integrins (<i>K</i>_I = 1.8 nM/peptide). Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and noncytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood