Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1

We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to preinhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties

Apoptosis detection in retinal ganglion cells using quantitative changes in multichannel fluorescence colocalization

KcapTR488 is a dual-fluorophore peptide sensor for the real-time reporting of programmed cell death by fluorescence imaging. KcapTR488 contains a nuclear localization sequence (NLS) conjugated with Texas Red, a caspase-cleavable sequence (DEVD), and a C-terminus conjugated to Alexa Fluor 488 (AF488). The synthesis and preliminary evaluation in cellulo of KcapTR488 for monitoring cell death by fluorescence imaging has been previously reported, but its utility in vivo has yet to be tested or validated. Herein, in vitro solution experiments verified the intramolecular fluorescence resonance energy transfer (FRET) between the two fluorophores and enabled a quantitative analysis of enzyme rates and selectivity. The sensor delivery kinetics in live rat models were quantified by ex vivo fluorescence microscopy. Studies in healthy control retinas demonstrated that KcapTR488 concentrated in the nucleus of retinal ganglion cells (RGC), with a strong colocalization of red and green fluorescence signals producing robust FRET signals, indicating an intact reporter. By contrast, using an acute but mild NMDA-induced retinal injury model, dual-color confocal ex vivo microscopy of cleaved KcapTR488 identified sensor activation as early as 2 h after injection. Quantitative changes in fluorescence colocalization were superior to changes in FRET for monitoring injury progression. Longitudinal monitoring revealed that the NLS-Texas Red fragment of the cleaved sensor moved out of the cell body, down the axon, and exited the retina, consistent with anterograde axonal transport. Thus, KcapTR488 may be a powerful tool to study RGC death pathways in live preclinical models of glaucoma

Acute Phencyclidine Alters Neural Oscillations Evoked by Tones in the Auditory Cortex of Rats

BACKGROUND/AIMS: The onset response to a single tone as measured by electroencephalography (EEG) is diminished in power and synchrony in schizophrenia. Because neural synchrony, particularly at gamma frequencies (30-80 Hz), is hypothesized to be supported by the N-methyl-D-aspartate receptor (NMDAr) system, we tested whether phencyclidine (PCP), an NMDAr antagonist, produced similar deficits to tone stimuli in rats. METHODS: Experiment 1 tested the effect of a PCP dose (1.0, 2.5, and 4.5 mg/kg) on response to single tones on intracranial EEG recorded over the auditory cortex in rats. Experiment 2 evaluated the effect of PCP after acute administration of saline or PCP (5 mg/kg), after continuous subchronic administration of saline or PCP (5 mg/kg/day), and after a week of drug cessation. In both experiments, a time-frequency analysis quantified mean power (MP) and phase locking factor (PLF) between 1 and 80 Hz. Event-related potentials (ERPs) were also measured to tones, and EEG spectral power in the absence of auditory stimuli. RESULTS: Acute PCP increased PLF and MP between 10 and 30 Hz, while decreasing MP and PLF between approximately 50 and 70 Hz. Acute PCP produced a dose-dependent broad-band increase in EEG power that extended into gamma range frequencies. There were no consistent effects of subchronic administration on gamma range activity. Acute PCP increased ERP amplitudes for the P16 and N70 components. CONCLUSIONS: Findings suggest that acute PCP-induced NMDAr hypofunction has differential effects on neural power and synchrony which vary with dose, time course of administration and EEG frequency. EEG synchrony and power appear to be sensitive translational biomarkers for disrupted NMDAr function, which may contribute to the pathophysiology of schizophrenia and other neuropsychiatric disorders

A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate

A Chemical Epitope-Targeting Strategy for Protein Capture Agents: The Serine 474 Epitope of the Kinase Akt2

Target and click: Peptide ligands targeted to the C-terminal motif of the kinase Akt2 were obtained by combining phosphate recognition of a dinuclear zinc(II) complex with in situ click chemistry to target this epitope. The peptide ligands (shown as XXXXX) selectively bind the C-terminal polypeptide of Akt2, and are selective for Akt2 relative to the Akt1 and Akt3 isoforms. The ligands differentially modulate Akt2 activity

Iterative in situ click chemistry creates antibody-like protein-capture agents

Iterative in situ click chemistry (see scheme for the tertiary ligand screen) and the one-bead-one-compound method for the creation of a peptide library enable the fragment-based assembly of selective high-affinity protein-capture agents. The resulting ligands are water-soluble and stable chemically, biochemically, and thermally. They can be produced in gram quantities through copper (I)-catalyzed cycloaddition

vProtein: Identifying Optimal Amino Acid Complements from Plant-Based Foods

Background: Indispensible amino acids (IAAs) are used by the body in different proportions. Most animal-based foods provide these IAAs in roughly the needed proportions, but many plant-based foods provide different proportions of IAAs. To explore how these plant-based foods can be better used in human nutrition, we have created the computational tool vProtein to identify optimal food complements to satisfy human protein needs. Methods: vProtein uses 1251 plant-based foods listed in the United States Department of Agriculture standard release 22 database to determine the quantity of each food or pair of foods required to satisfy human IAA needs as determined by the 2005 daily recommended intake. The quantity of food in a pair is found using a linear programming approach that minimizes total calories, total excess IAAs, or the total weight of the combination. Results: For single foods, vProtein identifies foods with particularly balanced IAA patterns such as wheat germ, quinoa, and cauliflower. vProtein also identifies foods with particularly unbalanced IAA patterns such as macadamia nuts, degermed corn products, and wakame seaweed. Although less useful alone, some unbalanced foods provide unusually good complements, such as Brazil nuts to legumes. Interestingly, vProtein finds no statistically significant bias toward grain/ legume pairings for protein complementation. These analyses suggest that pairings of plant-based foods should be based on the individual foods themselves instead of based on broader food group-food group pairings. Overall, the most efficien

Isolation and detection of circulating tumour cells from metastatic melanoma patients using a slanted spiral microfluidic device

Circulating Tumour Cells (CTCs) are promising cancer biomarkers. Several methods have been developed to isolate CTCs from blood samples. However, the isolation of melanoma CTCs is very challenging as a result of their extraordinary heterogeneity, which has hindered their biological and clinical study. Thus, methods that isolate CTCs based on their physical properties, rather than surface marker expression, such as microfluidic devices, are greatly needed in melanoma. Here, we assessed the ability of the slanted spiral microfluidic device to isolate melanoma CTCs via label-free enrichment. We demonstrated that this device yields recovery rates of spiked melanoma cells of over 80% and 55%, after one or two rounds of enrichment, respectively. Concurrently, a two to three log reduction of white blood cells was achieved with one or two rounds of enrichment, respectively. We characterised the isolated CTCs using multimarker flow cytometry, immunocytochemistry and gene expression. The results demonstrated that CTCs from metastatic melanoma patients were highly heterogeneous and commonly expressed stem-like markers such as PAX3 and ABCB5. The implementation of the slanted microfluidic device for melanoma CTC isolation enables further understanding of the biology of melanoma metastasis for biomarker development and to inform future treatment approaches

Induction of Tachykinin Production in Airway Epithelia in Response to Viral Infection

The tachykinins are implicated in neurogenic inflammation and the neuropeptide substance P in particular has been shown to be a proinflammatory mediator. A role for the tachykinins in host response to lung challenge has been previously demonstrated but has been focused predominantly on the release of the tachykinins from nerves innervating the lung. We have previously demonstrated the most dramatic phenotype described for the substance P encoding gene preprotachykinin-A (PPT-A) to date in controlling the host immune response to the murine gammaherpesvirus 68, in the lung.In this study we have utilised transgenic mice engineered to co-ordinately express the beta-galactosidase marker gene along with PPT-A to facilitate the tracking of PPT-A expression. Using a combination of these mice and conventional immunohistology we now demonstrate that PPT-A gene expression and substance P peptide are induced in cells of the respiratory tract including tracheal, bronchiolar and alveolar epithelial cells and macrophages after viral infection. This induction was observed 24h post infection, prior to observable inflammation and the expression of pro-inflammatory chemokines in this model. Induced expression of the PPT-A gene and peptide persisted in the lower respiratory tract through day 7 post infection.Non-neuronal PPT-A expression early after infection may have important clinical implications for the progression or management of lung disease or infection aside from the well characterised later involvement of the tachykinins during the inflammatory response