Discovery of a highly selective chemical inhibitor of matrix metalloproteinase-9 (MMP-9) that allosterically inhibits zymogen activation.

Aberrant activation of matrix metalloproteinases (MMPs) is a common feature of pathological cascades observed in diverse disorders, such as cancer, fibrosis, immune dysregulation, and neurodegenerative diseases. MMP-9, in particular, is highly dynamically regulated in several pathological processes. Development of MMP inhibitors has therefore been an attractive strategy for therapeutic intervention. However, a long history of failed clinical trials has demonstrated that broad-spectrum MMP inhibitors have limited clinical utility, which has spurred the development of inhibitors selective for individual MMPs. Attaining selectivity has been technically challenging because of sequence and structural conservation across the various MMPs. Here, through a biochemical and structural screening paradigm, we have identified JNJ0966, a highly selective compound that inhibited activation of MMP-9 zymogen and subsequent generation of catalytically active enzyme. JNJ0966 had no effect on MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14 catalytic activity and did not inhibit activation of the highly related MMP-2 zymogen. The molecular basis for this activity was characterized as an interaction of JNJ0966 with a structural pocket in proximity to the MMP-9 zymogen cleavage site near Arg-106, which is distinct from the catalytic domain. JNJ0966 was efficacious in reducing disease severity in a mouse experimental autoimmune encephalomyelitis model, demonstrating the viability of this therapeutic approach. This discovery reveals an unprecedented pharmacological approach to MMP inhibition, providing an opportunity to improve selectivity of future clinical drug candidates. Targeting zymogen activation in this manner may also allow for pharmaceutical exploration of other enzymes previously viewed as intractable drug targets.

A novel fluorogenic substrate for the measurement of endothelial lipase activity.

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.

Discovery of novel Cobactin-T based matrix metalloproteinase inhibitors via a ring closing metathesis strategy.

The discovery of potent N-hydroxyl caprolactam matrix metalloproteinase (MMP) inhibitors (6) based on the natural product Cobactin-T (2) is described. The synthetic method, which utilizes the ring closing metathesis reaction, is compatible to provide complementary (R) and (S) enantiomers. These compounds tested against MMP-2 and 9, show that the R stereochemistry (i.e., 16), which is opposite for that found in the natural product Cobactin-T is >1000-fold more active with IC(50) values of 0.2-0.6nM against both enzymes. The variation in the incorporation of the sulfonamide enzyme recognition element (Ar(2)XAr(1)SO(2)N(R(1)), 6), along with alterations in the RCM/double bond chemistry (R(2)) provided a series of sub nanomolar MMP inhibitors. For example, compounds 16 and 34 were found to be the most potent with IC(50) values against MMP-2 and MMP-9 found to be between 0.2 and 0.6nM with 34 being the most potent compound discovered (MMP-2 IC(50)=0.39nM and MMP-9 IC(50)=0.22nM). Compounds 16 and 34 showed acceptable drug-like properties in vivo with compound 34 showing oral bioavailability.

Evolutionary sequence modeling for discovery of peptide hormones.

There are currently a large number of "orphan" G-protein-coupled receptors (GPCRs) whose endogenous ligands (peptide hormones) are unknown. Identification of these peptide hormones is a difficult and important problem. We describe a computational framework that models spatial structure along the genomic sequence simultaneously with the temporal evolutionary path structure across species and show how such models can be used to discover new functional molecules, in particular peptide hormones, via cross-genomic sequence comparisons. The computational framework incorporates a priori high-level knowledge of structural and evolutionary constraints into a hierarchical grammar of evolutionary probabilistic models. This computational method was used for identifying novel prohormones and the processed peptide sites by producing sequence alignments across many species at the functional-element level. Experimental results with an initial implementation of the algorithm were used to identify potential prohormones by comparing the human and non-human proteins in the Swiss-Prot database of known annotated proteins. In this proof of concept, we identified 45 out of 54 prohormones with only 44 false positives. The comparison of known and hypothetical human and mouse proteins resulted in the identification of a novel putative prohormone with at least four potential neuropeptides. Finally, in order to validate the computational methodology, we present the basic molecular biological characterization of the novel putative peptide hormone, including its identification and regional localization in the brain. This species comparison, HMM-based computational approach succeeded in identifying a previously undiscovered neuropeptide from whole genome protein sequences. This novel putative peptide hormone is found in discreet brain regions as well as other organs. The success of this approach will have a great impact on our understanding of GPCRs and associated pathways and help to identify new targets for drug development.

Inter-agency indicators of out-of-home-care placement by age 13-14 years: A population record linkage study.

BACKGROUND: Cross-agency administrative data can improve cost-effective triage systems for child protection and other human service delivery. OBJECTIVE: To determine the minimum set of cross-agency indicators that could accurately classify placement in out-of-home-care (OOHC) before age 13-14 years. PARTICIPANTS AND SETTING: Participants were 72,079 Australian children (mean age = 13.16 years; SD = 0.37; 51.4% male) and their parents, for whom linked administrative records spanning the years 1994-2016 were available for analysis within the 'New South Wales Child Development Study'. METHODS: First, a series of logistic regression analyses were conducted to examine associations between cross-agency (health, justice, education) risk indicators and membership of the sub-cohort of 1239 children who had an OOHC placement prior to age 13-14 years, relative to (1) the sub-cohort of 55,473 children who had no previous contact with child protection services, and (2) the sub-cohort of 15,367 children who had been reported to child protection services but had no record of OOHC placement. We then explored the classification characteristics associated with a smaller combination of risk factors, and the utility of specific familial risk factors, for classifying membership of the OOHC subgroup. RESULTS: A combination of six risk indicators evident before OOHC placement can classify children placed in OOHC with approximately 95% accuracy, and the presence of at least four of these risk indicators provides excellent specificity (99.6%). CONCLUSIONS: A combination of risk factors observable in administrative datasets held by multiple government agencies may be used to target support services to prevent entry into OOHC for children from vulnerable families.

beta-N-Biaryl ether sulfonamide hydroxamates as potent gelatinase inhibitors: part 1. Design, synthesis, and lead identification.

A new series of beta-N-biaryl ether sulfonamide hydroxamates as novel gelatinase inhibitors is described. These compounds exhibit good potency for MMP-2 and MMP-9 without inhibiting MMP-1. The structure-activity relationships (SAR) reveal the biaryl ether type P1' moiety together with methanesulfonamide is the optimal combination that provides inhibitory activity of MMP-9 in the single-digit nanomolar range.

beta-N-Biaryl ether sulfonamide hydroxamates as potent gelatinase inhibitors: part 2. Optimization of alpha-amino substituents.

The introduction and the optimization of an alpha-amino substituent based on a series of alpha-hydroxy-beta-N-biaryl ether sulfonamide hydroxamates is described. The modification leads to a new series of MMP-2/MMP-9 inhibitors with enhanced inhibitory activities and improved ADME properties. An efficacy study on reducing the ischemia-induced brain edema in the rat transient middle cerebral artery occlusion (tMCAo) model is also demonstrated.

Syntheses and in vitro evaluation of arylsulfone-based MMP inhibitors with heterocycle-derived zinc-binding groups (ZBGs).

Several classes of arylsulfone-based MMP-2/-9 inhibitors utilizing 6- to 8-membered heterocyclic rings as zinc-binding groups (ZBGs) have been synthesized and their enzyme inhibitory activities were evaluated. Although a number of 6- and 7-membered heterocycles were effective, the most potent arylsulfone inhibitors are based on the rigid 1- or 3-hydroxypyridone ZBG.

1-Hydroxy-2-pyridinone-based MMP inhibitors: synthesis and biological evaluation for the treatment of ischemic stroke.

Matrix metalloproteinase-9 (MMP-9) has been implicated in the breakdown of the blood-brain barrier during cerebral ischemia. As a result, inhibition of MMP-9 may have utility as a therapeutic intervention in stroke. Towards this end, we have synthesized a series of 1-hydroxy-2-pyridinones that have excellent in vitro potency in inhibiting MMP-9 in addition to MMP-2. Representative compounds also demonstrate good efficacy in the mouse transient mid-cerebral artery occlusion (tMCAO) model of cerebral ischemia.

Ventilator weaning outcomes in chronic respiratory failure in children.

The purpose of this study was to describe mechanical ventilation weaning outcomes for children with chronic respiratory failure discharged from one of six post-acute rehabilitation facilities. Demographic, clinical and outcome data were collected from the medical record. Forty-four children were included in this prospective series; 20 (45%) were weaned off the ventilator at discharge. Children required significantly lower levels of ventilatory support at discharge than admission. Hourly use on the ventilator decreased from admission to discharge for the full cohort and for the subgroup who required a ventilator at discharge. Seventy-five percent of the children discharged with a ventilator had a portable unit. We conclude that nearly half of the children using mechanical ventilation achieve weaning during a postacute rehabilitation admission, whereas others have positive outcomes in severity, hours off the ventilator or portability of equipment.

Morphine Versus Methadone Treatment for Neonatal Withdrawal and Impact on Early Infant Development.

Objective. Compare developmental outcomes in infants treated with morphine versus methadone. Method. Retrospective chart review of newborns identified through use of ICD-9 code for neonatal abstinence syndrome (NAS). Thirty-six infants were evaluated-17 treated with methadone and 19 treated with morphine. Assessment was completed following treatment using the Bayley Scales of Infant and Toddler Development-Third Edition (Bayley-III). Scores in Cognitive, Language, and Motor domains were compared. Results. Comparison of scores between morphine- and methadone-treated groups revealed differences in mean Cognitive Composite (91.3 vs 83.0; P = .03410) and mean Total Motor Composite Scores (96.3 vs 89.6; P = .0149). Conclusion. Newborns with NAS treated with morphine had significantly higher scores in Cognitive and Gross Motor domains compared to infants treated with methadone. Development screening should be pursued to determine if this difference persists throughout early childhood. Results may influence accepted treatment protocols for NAS.

KChIPs and Kv4 alpha subunits as integral components of A-type potassium channels in mammalian brain.

Voltage-gated potassium (Kv) channels from the Kv4, or Shal-related, gene family underlie a major component of the A-type potassium current in mammalian central neurons. We recently identified a family of calcium-binding proteins, termed KChIPs (Kv channel interacting proteins), that bind to the cytoplasmic N termini of Kv4 family alpha subunits and modulate their surface density, inactivation kinetics, and rate of recovery from inactivation (An et al., 2000). Here, we used single and double-label immunohistochemistry, together with circumscribed lesions and coimmunoprecipitation analyses, to examine the regional and subcellular distribution of KChIPs1-4 and Kv4 family alpha subunits in adult rat brain. Immunohistochemical staining using KChIP-specific monoclonal antibodies revealed that the KChIP polypeptides are concentrated in neuronal somata and dendrites where their cellular and subcellular distribution overlaps, in an isoform-specific manner, with that of Kv4.2 and Kv4.3. For example, immunoreactivity for KChIP1 and Kv4.3 is concentrated in the somata and dendrites of hippocampal, striatal, and neocortical interneurons. Immunoreactivity for KChIP2, KChIP4, and Kv4.2 is concentrated in the apical and basal dendrites of hippocampal and neocortical pyramidal cells. Double-label immunofluorescence labeling revealed that throughout the forebrain, KChIP2 and KChIP4 are frequently colocalized with Kv4.2, whereas in cortical, hippocampal, and striatal interneurons, KChIP1 is frequently colocalized with Kv4.3. Coimmunoprecipitation analyses confirmed that all KChIPs coassociate with Kv4 alpha subunits in brain membranes, indicating that KChIPs 1-4 are integral components of native A-type Kv channel complexes and are likely to play a major role as modulators of somatodendritic excitability.

Identification of early developmental deficits in infants with prenatal heroin, methadone, and other opioid exposure.

OBJECTIVE: Study aims to examine development in infants following prenatal heroin, methadone, and opioid exposure, which adversely affects central and autonomic nervous systems. Abrupt discontinuation results in neurologic and behavioral findings as Neonatal Abstinence Syndrome (NAS). METHOD: Following NAS treatment, 28 infants (mean age 55 days [range 21-98 days], 57% male) were assessed using Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) Cognitive, Language, and Motor subscales. Outcomes were compared with a historical control. RESULTS: Mean Language and Cognition scores were significantly lower (P < .001) in the NAS group. Distributions of scores for Language (P < .001) and Cognition (P = .022) were also significantly different between NAS and historical control groups. CONCLUSION: Prenatal heroin, methadone, and other opioid exposure is associated with weaknesses in language and cognition. This information has important public health implications, drawing attention to an otherwise healthy infant population which may benefit from early intervention services.

Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland.

BACKGROUND: Regulation of raphe serotonergic cells is fundamental to the prevailing hypothesis of major depression pathophysiology. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis, but brainstem TPH mRNA expression has been difficult to measure and study. Recently, a novel paralog of TPH, TPH2 (or neuronal TPH), was described, but its anatomic expression is unknown. METHODS: In situ hybridization histochemical survey was conducted across Sprague-Dawley rat brain for TPH1 and TPH2 mRNA. Semiquantitative techniques were used to estimate relative mRNA levels in individual cells. RESULTS: Almost exclusively, TPH2 mRNA is expressed in raphe, in a pattern overlapping the histologically defined raphe nuclei. In sharp contrast, TPH1 (the previously known TPH) is expressed predominantly in pineal gland. There is no appreciable overlap in the expression of these paralogs. The level of TPH2 mRNA expression in individual raphe cells is approximately 2.5-fold greater than the level of TPH1 expression in pinealocytes. CONCLUSIONS: TPH2 mRNA has an anatomic expression pattern consistent with brainstem raphe nuclei and is likely to be the gene giving rise to the majority of TPH activity in these cells. The robust expression of TPH2 in brainstem should facilitate studies on the transcriptional regulation of raphe serotonin biosynthesis.

Serotonin 5-HT1A, 5-HT1B, and 5-HT2A receptor mRNA expression in subjects with major depression, bipolar disorder, and schizophrenia.

BACKGROUND: Alterations of serotonin neurotransmission are implicated in both mood disorders and schizophrenia. Specific serotonin-receptor-based abnormalities in these psychiatric illnesses have been intensively studied; however, it has been difficult to draw any conclusions because of a lack of consensus. These inconsistencies have most likely arisen from the unavailability of selective ligands. METHODS: Our study used in situ hybridization to quantify 5-HT(1A), 5-HT(1B), and 5-HT(2A) mRNA levels in the hippocampus (HC) and 5-HT(1A) and 5-HT(2A) mRNA levels in the dorsolateral prefrontal cortex (DLPFC) of subjects with a history of major depression disorder (MDD), bipolar disorder (BPD), schizophrenia, and a normal comparison group (15 subjects per group). RESULTS: In the DLPFC, there is a significant decrease in 5-HT(1A) mRNA of subjects with MDD and in 5-HT(2A) mRNA of subjects with BPD. Subjects with MDD have a significant decrease in 5-HT(1A) mRNA in the HC; subjects with BPD and schizophrenia had increased 5-HT(1B) mRNA levels and a significant decrease in 5-HT(2A) mRNA levels in the hippocampal formation. CONCLUSIONS: Alterations in 5-HT(1A,) 5-HT(1B), and 5-HT(2A) mRNA levels in the brains of subjects with both mood disorders and schizophrenia add further support for hypothesis of dysregulation of the serotonergic system in these psychiatric disorders.

Socially-induced brain 'fertilization': play promotes brain derived neurotrophic factor transcription in the amygdala and dorsolateral frontal cortex in juvenile rats.

Rough and tumble (R&T) play is assumed to have beneficial effects in developing organisms. To evaluate this idea, brain derived neurotrophic factor (BDNF) gene expression was evaluated in 32-day-old juvenile rats that were allowed to play for 30 min prior to sacrifice. In situ hybridization for BDNF mRNA revealed that the amygdala and dorsolateral frontal cortex had significantly elevated BDNF mRNA expression as a result of play. These effects suggest that play may help program higher brain regions involved in emotional behaviors.

Understanding venous leg ulcer pain: results of a longitudinal study.

Venous leg ulcer pain experienced during compression bandaging is poorly understood. A prospective, pilot cohort study was initiated to determine the feasibility of conducting a large-scale, repeated measures cohort study of venous leg ulcer pain and to document and describe the venous leg ulcer pain experience during the first 5 weeks of treatment with compression bandages. Eligible individuals admitted to a nurse-led community leg ulcer service in one Canadian community were recruited for the 5-week study. Pain assessment tools (ie, numerical rating scale and short form McGill Pain Questionnaire) were evaluated by 20 venous ulcer patients (mean age = 73.7 years) and their nurses for ease of use during one baseline and five weekly follow-up visits. Health-related quality of life (HRQL) information was obtained. Nurses reported on ease of integrating pain data collection into regular clinical care. Each pain assessment tool was audited for completion. Most participants found the pain assessment tools easy to use, but nurses reported lengthened visit times with some participants as a result of tool administration difficulties, particularly the visual analogue scale (VAS). Overall completeness of pain assessment tools ranged from 85.0% (visual analogue scale) to 96.3% (present pain intensity and word descriptor list). The vast majority of patients (18) reported ulcer pain at baseline. Total mean scores for all pain assessment tools used decreased over time, but most patients reported pain throughout the study. The most common pain descriptors used were "aching," "stabbing," "sharp," "tender," and "tiring." Health-related quality of life was low and did not change during the 5-week study. The results of this study suggest that the vast majority of venous ulcer patients experience pain and that it is feasible to examine this pain in individuals receiving care in the community over time.

Podcasting in undergraduate nursing programs.

Little information is available regarding the value of podcasting in nursing education. This mixed-methods study described nursing students' (n=101) perceptions of podcasted materials, the benefits of podcasting, and when and where students used podcasted materials. Students (86%) believed podcasts enriched their learning, and 95% reported podcasts as valuable tools in the learning environment. Most students (94%) would recommend podcasting in other courses and accessed podcast materials 3 times per week. More than half of the students (55%) accessed podcast materials in multiple places (ie, in the car, in the home, and at school).

Pediatric post-acute care hospital transitions: an evaluation of current practice.

OBJECTIVES: After discharge from an acute care hospital, some children require ongoing care at a post-acute care hospital. Care transitions occur at both admission to the post-acute care hospital and again at discharge to the home/community. Our objective was to report the current practices used during the admission to and discharge from 7 pediatric post-acute care hospitals in the United States. METHODS: Participants from 7 pediatric post-acute care hospitals completed a survey and rated the frequency of use of 20 practices to prepare and support children and their families during both admission to the hospital and at time of discharge to the home/community. For consistency with existing literature, practices were grouped into 4 previously reported categories: assessment, communication, education, and logistics. Descriptive statistics were used to report the frequency of use within practices and between hospitals. RESULTS: Only 2 of 10 admission practices and 3 of 10 discharge practices were reportedly "always" used by all hospitals. Assessment and communication practices were reported to be more frequently used (57%-100% of the time) than education and logistic procedures. Between hospitals, only the reported frequency of use of the discharge practices was statistically significantly different (P = .03). CONCLUSIONS: Variability exists in transition practices among 7 post-acute care pediatric hospitals. This report is the first known to detail the frequency of use of admission and discharge practices for pediatric post-acute care hospitals in the United States.

Glucocorticoid and mineralocorticoid receptor expression in the human hippocampus in major depressive disorder.

Approximately 50% of mood disorder patients exhibit hypercortisolism. Cortisol normally exerts its functions in the CNS via binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Both MR and GR are highly expressed in human hippocampus and several studies have suggested that alterations in the levels of MR or GR within this region may contribute to the dysregulation in major depressive disorder (MDD). Studies have also shown functional heterogeneity across the hippocampus, with posterior hippocampus preferentially involved in cognitive processes and anterior hippocampus involved in stress, emotion and affect. We therefore hypothesize that GR and MR expression in hippocampus of control and MDD patients may vary not only with disease, but also with regional specificity along the anterior/posterior axis. Student's t-test analysis showed decreased expression of MR in the MDD group compared to controls in the anterior, but not the posterior hippocampus, with no significant changes in GR. Linear regression analysis showed a marked difference in MR:GR correlation between suicide and non-suicide patients in the posterior hippocampus. Our findings are consistent with previous reports of hippocampal corticosteroid receptor dysregulation in mood disorders, but extend those findings by analysis across the anterior/posterior axis of the hippocampus. A decrease in MR in the anterior but not posterior hippocampus of MDD patients emphasizes the important functional role of the anterior hippocampus in neuroendocrine regulation in humans.