Parallel in vivo and in vitro selection using phage display identifies protease-dependent tumor-targeting peptides.

We recently developed activatable cell-penetrating peptides (ACPPs) that target contrast agents to in vivo sites of matrix metalloproteinase activity, such as tumors. Here we use parallel in vivo and in vitro selection with phage display to identify novel tumor-homing ACPPs with no bias for primary sequence or target protease. Specifically, phage displaying a library of ACPPs were either injected into tumor-bearing mice, followed by isolation of cleaved phage from dissected tumor, or isolated based on selective cleavage by extracts of tumor versus normal tissue. Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts. The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast. This uniquely identified ACPP was not cleaved by matrix metalloproteinases or various coagulation factors but was efficiently cleaved by plasmin and elastases, both of which have been shown to be aberrantly overexpressed in tumors. The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.

Treatment of Dementia With Bosutinib: An Open-Label Study of a Tyrosine Kinase Inhibitor.

OBJECTIVE: The pursuit of an effective therapeutic intervention for dementia has inspired interest in the class of medications known as tyrosine kinase inhibitors such as bosutinib. METHODS: Thirty-one patients with probable Alzheimer dementia or Parkinson spectrum disorder with dementia completed 12 months of bosutinib therapy and an additional 12 months of follow-up. The Clinical Dementia Rating scale (as estimated by the Quick Dementia Rating System [QDRS]) was the primary cognitive status outcome measure. Secondary outcome measures included the Repeatable Battery Assessment of Neuropsychological Status (RBANS) and the Montreal Cognitive Assessment. Cox regression methods were used to compare results with population-based estimates of cognitive decline. RESULTS: The present article reports on cognitive outcomes obtained at 12 months for 31 participants and up to 24 months for a 16-participant subset. Safety and tolerability of bosutinib were confirmed among the study population (Mage = 73.7 years, SDage = 14 years). Bosutinib was associated with less worsening in Clinical Dementia Rating (CDR) scores (hazard ratio = -0.62, p < 0.001, 95% confidence interval [CI]: -1.02 to -0.30) and less decline in RBANS performance (hazard ratio = -3.42, p < 0.001, 95% CI: -3.59 to -3.72) during the year of treatment than population-based estimates of decline. In the 24-month follow-up, wherein 16 patients were observed after 1 year postintervention, 31.2% of participants exhibited worsened CDR levels compared with their 12-month performances. CONCLUSIONS: Results support an overall positive outcome after 1 year of bosutinib. Future studies should explore the relationship between tyrosine kinases and neurodegenerative pathology as well as related avenues of treatment.

Focused transcranial ultrasound for treatment of neurodegenerative dementia.

INTRODUCTION: Preclinical studies support investigation of focused ultrasound for breakdown of cerebral pathologies in neurodegenerative conditions including Alzheimer's disease (AD) and Parkinson's disease (PD). METHODS: A focused transcranial Doppler device with probes (2 MHz, 520 mW/cm2) affixed bilaterally was used to target the hippocampus (AD) or substantia nigra (PD) with functional magnetic resonance imaging navigation for enhanced plaque removal. A total of 22 patients (n = 11 AD, n = 11 PD) underwent 8 consecutive, weekly, 1-hour treatments wherein sleep was encouraged naturally or pharmacologically. Cognitive and motor functioning assessment was carried out using standardized evaluations at baseline and conclusion. RESULTS: Of all, 62.5% of patients had one or more improved cognitive scores without data incongruence, 87% had stable or improved fine motor scores, and 87.5% had stable or improved gross motor scores. No adverse events were reported. DISCUSSION: The safety of focused transcranial Doppler and possible enhancement in patient functioning were suggested by outcome data.

A collaborative screening program for the discovery of inhibitors of HCV NS2/3 cis-cleaving protease activity.

This report describes the development of a cell-based assay for high-throughput screening and detection of small-molecule inhibitors for hepatitis C virus (HCV) NS2/3 protease. The HCV NS2/3 protease is essential for the normal infectious cycle of HCV. Generation of a cell-based assay for this cis-acting viral protease involved reporter constructs in which the NS2/3 protease sequence was inserted between the ,B-lactamase (BLA) reporter and a ubiquitin-based destabilization domain. In stable cell lines, NS2/3 cis cleavage of the NS2/3-BLA fusion protein resulted in differential stability of the cleaved versus uncleaved BLA reporter, providing a robust readout for protease activity. BLA reporter activity was shown to be a function of NS2/3-specific protease activity, by using genetic mutants of the NS2/3 sequence. In addition, the cell-based assay was validated and screened in a 384-well format on a fully automated robotic platform where small-molecule inhibitors of NS2/3 protease activity were identified.

A one-arm homologous recombination approach for developing nuclear receptor assays in somatic cells.

Drug discovery is in need of technologies that enable investigators to develop cell-based assays that accurately reflect the functional consequence of small molecule intervention on biological processes. Here, we describe a strategy that uses both one-arm homologous recombination and the beta-lactamase (BLA) reporter system, a sensitive and robust transcriptional reporter for gene activation. We demonstrate that this powerful approach can be utilized for developing cell-based assays for the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in HEK293 somatic cells. Specifically, one-arm homologous recombination was used to introduce the GAL4 DNA-binding domain (GAL4DBD) in the GR and MR genomic loci such that a chimeric GAL4DBD-GR (ligand-binding domain) [GAL4DBD-GR(LBD)] and GAL4DBD-MR(LBD) transcript is produced from the strong CMV promoter in HEK293 cells previously stably transfected with the UAS(GAL4)-BLA reporter construct. Dexamethasone- and aldosterone-responding BLA-positive cells were isolated by fluorescence-activated cell sorting, and then further expanded into separate cell lines. The sensitivity and robustness of the resulting GR and MR assays are demonstrated by the fact that the addition of dexamethasone and aldosterone to the two transgenic clonal cell lines for 16 h results in high Z' values (>0.8) and EC(50) values of 1 and 0.3 nM, respectively. These assays illustrate the flexibility of this technology to generate high-performance cellular assays for nuclear receptor targets without the need for target-specific cDNA.

Integration of G-protein coupled receptor signaling pathways for activation of a transcription factor (EGR-3).

We recently reported the use of a gene-trapping approach to isolate cell clones in which a reporter gene had integrated into genes modulated by T-cell activation. We have now tested a panel of clones from that report and identified the one that responds to a variety of G-protein coupled receptors (GPCR). The beta-lactamase tagged EGR-3 Jurkat cell was used to dissect specific GPCR signaling in vivo. Three GPCRs were studied, including the chemokine receptor CXCR4 (Gi-coupled) that was endogenously expressed, the platelet activation factor (PAF) receptor (Gq-coupled), and beta2 adrenergic receptor (Gs-coupled) that was both stably transfected. Agonists for each receptor activated transcription of the beta-lactamase tagged EGR-3 gene. Induction of EGR-3 through CXCR4 was blocked by pertussis toxin and PD58059, a specific inhibitor of MEK (MAPK/ERK kinase). Neither of these inhibitors blocked isoproterenol or PAF-mediated activation of EGR-3. Conversely, beta2- and PAF-mediated EGR-3 activation was blocked by the p38, specific inhibitor SB580. In addition, both beta2- and PAF-mediated EGR-3 activation could be synergistically activated by CXCR4 activation. This combined result indicates that EGR-3 can be activated through distinct signal transduction pathways by different GPCRs and that signals can be integrated and amplified to efficiently tune the level of activation.

The reliability of environmental measures of the college alcohol environment.

Much of what we know about students' drinking patterns and problems related to alcohol use is based on survey research. Although local and national survey data are important to alcohol-prevention projects, they do not sufficiently capture the complexity of the alcohol environment. Environmental prevention approaches to alcohol-related problems have been shown to be effective in community settings and researchers have begun to study and adapt such approaches for use on college campuses. Many environmental approaches require systematic scanning of the campus alcohol environment. This study assessed the inter-rater reliability of two environmental scanning tools (a newspaper content analysis form and a bulletin analysis form) designed to identify alcohol-related advertisements targeting college students. Inter-rater reliability for these forms varied across different rating categories and ranged from poor to excellent. Suggestions for future research are addressed.