Excellent healing rates and patient satisfaction after arthroscopic repair of medium to large rotator cuff tears with a single-row technique augmented with bone marrow vents.

PURPOSE: This study evaluated the repair integrity and patient clinical outcomes following arthroscopic rotator cuff repair of medium to large rotator cuff tears using a single-row technique consisting of medially based, triple-loaded anchors augmented with bone marrow vents in the rotator cuff footprint lateral to the repair. METHODS: This is a retrospective study of 52 patients (53 shoulders) comprising 36 males and 16 females with a median age of 62 (range 44-82) with more than 24-month follow-up, tears between 2 and 4 cm in the anterior-posterior dimension and utilizing triple-loaded anchors. Mann-Whitney test compared Western Ontario Rotator Cuff (WORC) outcome scores between patients with healed and re-torn cuff repairs. Multivariate logistic regression analysed association of variables with healing status and WORC score. Cuff integrity was assessed on MRI, read by a musculoskeletal fellowship-trained radiologist. RESULTS: Magnetic resonance imaging (MRI) demonstrated an intact repair in 48 of 53 shoulders (91%). The overall median WORC score was 95.7 (range 27.6-100.0). A significant difference in WORC scores were seen between patients with healed repairs 96.7 (range 56.7-100.0) compared with a re-tear 64.6 (27.6-73.8), p < 0.00056. CONCLUSIONS: Arthroscopic repair of medium to large rotator cuff tears using a triple-loaded single-row repair augmented with bone marrow vents resulted in a 91% healing rate by MRI and excellent patient reported clinical outcomes comparable to similar reported results in the literature. LEVEL OF EVIDENCE: IV.

Does duration of symptoms affect clinical outcome after hip arthroscopy for labral tears? Analysis of prospectively collected outcomes with minimum 2-year follow-up.

Limited research exists on the possible association between duration of symptoms and clinical outcomes following hip arthroscopy for labral tears. The purpose of this study was to evaluate whether duration of symptoms affected clinical and patient-reported outcome (PRO) scores following hip arthroscopy for labral tears. From 2008 to 2011, data were collected prospectively on all patients undergoing primary hip arthroscopy for labral tears. Workers' compensation cases, dysplasia cases and patients with previous ipsilateral hip surgeries were excluded. A total of 738 patients were identified with a minimum of 2-year follow-up, and clinical and PRO data were available for 680 patients. Uni- and multivariate analyses were performed to determine the relationship between duration of symptoms along with other variables and PROs. Overall, patients experienced significant improvements in all clinical and PRO scores. Results of univariate analysis revealed that all PROs were negatively associated with increasing Log10 months of symptoms as were pain and satisfaction scores. During multivariate analyses, increasing Log10 months of symptoms, age, body mass index and trauma were all negatively associated with PROs (P < 0.05). Our study demonstrates that clinical and PRO scores were negatively associated with increasing duration of symptoms prior to hip arthroscopy for treatment of labral tears. Although this implies that delay in treatment may adversely affect outcome, conservative treatment remains the gold standard first line of treatment. Surgeons should incorporate this information into their treatment algorithm to maximize patient outcomes following treatment for labral tears. Level of evidence: Level IV, prospective case series.

A high-throughput small-molecule ligand screen targeted to agonists and antagonists of the G-protein-coupled receptor GPR54.

Recent data have shown that the G-protein-coupled receptor GPR54 (also known as KiSS-1 receptor) regulates GnRH release from the hypothalamus. This essential role of GPR54 in controlling the hypothalamic-pituitary-gonadal axis makes it an attractive target for therapeutic intervention in reproductive and cancer medicine. Currently, there are no small-molecule modulators of GPR54 function for experimental or clinical use. To identify small-molecule compounds that modify GPR54 signal transduction, the authors have adapted a cell-based functional assay for high-throughput screening (HTS) using a commercially available homogeneous time-resolved fluorescence assay for inositol phosphate accumulation. They generated stable Chinese hamster ovary cell transfectants that express human GPR54 for use in this assay. After optimization in an automated HTS environment, they screened a library of 110,000 small-molecule compounds using 2 protocols, one to identify agonists and one to identify antagonists. Hits obtained in the primary screen were confirmed to be active in secondary in vitro assays. Compounds identified as agonists or antagonists from HTS and secondary screening will be characterized to identify agents with the potential to be developed as novel orally active agents to treat hormone-dependent disorders such as abnormal puberty, infertility, endometriosis, and sex steroid-dependent tumors.

Small-molecule activators of insulin-degrading enzyme discovered through high-throughput compound screening.

BACKGROUND: Hypocatabolism of the amyloid beta-protein (Abeta) by insulin-degrading enzyme (IDE) is implicated in the pathogenesis of Alzheimer disease (AD), making pharmacological activation of IDE an attractive therapeutic strategy. However, it has not been established whether the proteolytic activity of IDE can be enhanced by drug-like compounds. METHODOLOGY/PRINCIPAL FINDINGS: Based on the finding that ATP and other nucleotide polyphosphates modulate IDE activity at physiological concentrations, we conducted parallel high-throughput screening campaigns in the absence or presence of ATP and identified two compounds--designated Ia1 and Ia2--that significantly stimulate IDE proteolytic activity. Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE. Unexpectedly, we observed highly synergistic activation effects when the activity of Ia1 or Ia2 was tested in the presence of ATP, a finding that has implications for the mechanisms underlying ATP-mediated activation of IDE. Notably, Ia1 and Ia2 activated the degradation of Abeta by approximately 700% and approximately 400%, respectively, albeit only when Abeta was presented in a mixture also containing shorter substrates. CONCLUSIONS/SIGNIFICANCE: This study describes the first examples of synthetic small-molecule activators of IDE, showing that pharmacological activation of this important protease with drug-like compounds is achievable. These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP. Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility.

Two approaches to drug discovery in SOD1-mediated ALS.

Familial amyotrophic lateral sclerosis (ALS) accounts for 10% of all ALS cases; approximately 25% of these cases are due to mutations in the Cu/Zn superoxide dismutase gene (SOD1). To date, 105 different mutations spanning all 5 exons have been identified in the SOD1 gene. Mutant SOD1-associated ALS is caused by a toxic gain of function of the mutated protein. Therefore, regardless of the specific mechanism whereby mutant SOD1 initiates motor neuron death, the authors hypothesize that measures that decrease levels of mutant SOD1 protein should ameliorate the phenotype in transgenic mice and potentially in patients with SOD1-mediated disease. They have designed 2 cell-based screening assays to identify small, brain-permeant molecules that inactivate expression of the SOD1 gene or increase the degradation of the SOD1 protein. Here they describe the development and optimization of these assays and the results of high-throughput screening using a variety of compound libraries, including a total of more than 116,000 compounds. The majority of the hit compounds identified that down-regulated SOD1 were shown to be toxic in a cell-based viability assay or were nonselective transcription inhibitors, but work is continuing on a number of nonspecific inhibitors of SOD1 expression. Ultimately, the authors believe that these 2 cell-based assays will provide powerful strategies to identify novel therapies for the treatment of inherited SOD1-associated forms of ALS.

A high-throughput drug screen targeted to the 5'untranslated region of Alzheimer amyloid precursor protein mRNA.

The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5'untranslated region (5'UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Abeta) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5'UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5'UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5'UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5'UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5'UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Abeta production for AD.

Development of an assay to screen for inhibitors of tau phosphorylation by cdk5.

A high-throughput assay for tau phosphorylation by cdk5/p25 is described. Full-length recombinant tau was used as a substrate in the presence of saturating adenosine triphosphate (ATP). Using PHF-1, an antibody directed specifically against 2 tau phosphorylation epitopes (serine 396 and serine 404), an enzyme-linked immunosorbent assay (ELISA)-based colorimetric assay was formatted in 384-well plates. The assay was validated by measuring kinetic parameters for cdk5/p25 catalysis and known inhibitors. Rate constants for the site-specific phosphorylations at the PHF-1 epitopes were determined and suggested preferential phosphorylation at these sites. The performance of this assay in a high-throughput format was demonstrated and used to identify inhibitors of tau phosphorylation at specific epitopes phosphorylated by cdk5/p25.

Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line.

Neuronal ubiquitin C-terminal hydrolase (UCH-L1) has been linked to Parkinson's disease (PD), the progression of certain nonneuronal tumors, and neuropathic pain. Certain lung tumor-derived cell lines express UCH-L1 but it is not expressed in normal lung tissue, suggesting that this enzyme plays a role in tumor progression, either as a trigger or as a response. Small-molecule inhibitors of UCH-L1 would be helpful in distinguishing between these scenarios. By utilizing high-throughput screening (HTS) to find inhibitors and traditional medicinal chemistry to optimize their affinity and specificity, we have identified a class of isatin O-acyl oximes that selectively inhibit UCH-L1 as compared to its systemic isoform, UCH-L3. Three representatives of this class (30, 50, 51) have IC(50) values of 0.80-0.94 micro M for UCH-L1 and 17-25 micro M for UCH-L3. The K(i) of 30 toward UCH-L1 is 0.40 micro M and inhibition is reversible, competitive, and active site directed. Two isatin oxime inhibitors increased proliferation of the H1299 lung tumor cell line but had no effect on a lung tumor line that does not express UCH-L1. Inhibition of UCH-L1 expression in the H1299 cell line using RNAi had a similar proproliferative effect, suggesting that the UCH-L1 enzymatic activity is antiproliferative and that UCH-L1 expression may be a response to tumor growth. The molecular mechanism of this response remains to be determined.