Structural Basis for Isoform Selective Nitric Oxide Synthase Inhibition by Thiophene-2-Carboximidamides

The over production of nitric oxide in the brain by neuronal nitric oxide synthase (nNOS) is associated with a number of neurodegenerative diseases. Although inhibiting nNOS is an important therapeutic goal, it is important not to inhibit endothelial NOS (eNOS) owing to the critical role played by eNOS in maintaining vascular tone. While it has been possible to develop nNOS selective aminopyridine inhibitors, many of the most potent and selective inhibitors exhibit poor bioavailability properties. Our group and others have turned to more biocompatible thiophene-2-carboximidamides (T2C) inhibitors as potential nNOS selective inhibitors. We have used crystallography and computational methods to better understand how and why 2 commercially developed T2C inhibitors exhibit selectivity for human nNOS over human eNOS. As with many of the aminopyridine inhibitors, a critical active site Asp residue in nNOS vs Asn in eNOS is largely responsible for controlling selectivity. We also present thermodynamic integration results to better understand the change in pKa and thus charge of inhibitors once bound to the active site. In addition, relative free energy calculations underscore the importance of enhanced electrostatic stabilization of inhibitors bound to the nNOS active site compared to eNOS

Patent No. US 9,090,589 B2: Specific NNOS Inhibitors for the Therapy and Prevention of Human Melanoma

Methods for melanoma treatment and prevention with selective nitric oxide synthase inhibitor compounds and related pharmaceutical compositions, alone or in conjunction with one or more other melanoma therapies

Pediatric Feeding Disorder: Consensus Definition and Conceptual Framework

Pediatric feeding disorders (PFDs) lack a universally accepted definition. Feeding disorders require comprehensive assessment and treatment of 4 closely related, complementary domains (medical, psychosocial, and feeding skill-based systems and associated nutritional complications). Previous diagnostic paradigms have, however, typically defined feeding disorders using the lens of a single professional discipline and fail to characterize associated functional limitations that are critical to plan appropriate interventions and improve quality of life. Using the framework of the World Health Organization International Classification of Functioning, Disability, and Health, a unifying diagnostic term is proposed: “Pediatric Feeding Disorder” (PFD), defined as impaired oral intake that is not age-appropriate, and is associated with medical, nutritional, feeding skill, and/or psychosocial dysfunction. By incorporating associated functional limitations, the proposed diagnostic criteria for PFD should enable practitioners and researchers to better characterize the needs of heterogeneous patient populations, facilitate inclusion of all relevant disciplines in treatment planning, and promote the use of common, precise, terminology necessary to advance clinical practice, research, and health-care policy

Inhibition of Interferon-Gamma-Stimulated Melanoma Progression by Targeting Neuronal Nitric Oxide Synthase (nNOS)

Interferon-gamma (IFN-γ) is shown to stimulate melanoma development and progression. However, the underlying mechanism has not been completely defined. Our study aimed to determine the role of neuronal nitric oxide synthase (nNOS)-mediated signaling in IFN-γ-stimulated melanoma progression and the anti-melanoma effects of novel nNOS inhibitors. Our study shows that IFN-γ markedly induced the expression levels of nNOS in melanoma cells associated with increased intracellular nitric oxide (NO) levels. Co-treatment with novel nNOS inhibitors effectively alleviated IFN-γ-activated STAT1/3. Further, reverse phase protein array (RPPA) analysis demonstrated that IFN-γ induced the expression of HIF1α, c-Myc, and programmed death-ligand 1 (PD-L1), in contrast to IFN-α. Blocking the nNOS-mediated signaling pathway using nNOS-selective inhibitors was shown to effectively diminish IFN-γ-induced PD-L1 expression in melanoma cells. Using a human melanoma xenograft mouse model, the in vivo studies revealed that IFN-γ increased tumor growth compared to control, which was inhibited by the co-administration of nNOS inhibitor MAC-3-190. Another nNOS inhibitor, HH044, was shown to effectively inhibit in vivo tumor growth and was associated with reduced PD-L1 expression levels in melanoma xenografts. Our study demonstrates the important role of nNOS-mediated NO signaling in IFN-γ-stimulated melanoma progression. Targeting nNOS using highly selective small molecular inhibitors is a unique and effective strategy to improve melanoma treatment

Family history of severe cardiovascular disease in Marfan syndrome is associated with increased aortic diameter and decreased survival

Objectives.We attempted to determine whether a family history of severe cardiovascular disease in patients with the Marfan syndrome is associated with increased aortic dilation or decreased survival, or both.Background.The prognostic importance of a family history of severe cardiovascular disease in patients with the Marfan syndrome has been incompletely examined. We hypothesized that such a family history would correlate with increased aortic dilation and would be associated with decreased survival.Methods.One hundred eight affected patients and 48 unaffected family members from 33 multigenerational families with the Marfan syndrome underwent echocardiographic measurement of the aortic root, arch and mid-abdominal aorta. Date of birth and age at death ascertained from family pedigrees were used to perform life table analysis and estimate survival.Results.Aortic root and arch diameters were significantly greater in patients with a family history of severe cardiovascular disease than in patients without such a family history. Of subjects in the highest quartile for aortic size, >80% had such a family history in contrast to <10% of those in the lowest quartile (chisquare 57.37, p < 0.00001). Mean age at death and cumulative probability of survival were significantly lower in patients with such a family history.Conclusions.Among patients with the Marfan syndrome, aortic dilation is greater and life expectancy shorter in those with a family history of severe cardiovascular manifestations. These data suggest that such a family history is an important risk factor for cardiovascular events in patients with the Marfan syndrome

A Small Peptide Increases Drug Delivery in Human Melanoma Cells

Melanoma is the most fatal type of skin cancer and is notoriously resistant to chemotherapies. The response of melanoma to current treatments is difficult to predict. To combat these challenges, in this study, we utilize a small peptide to increase drug delivery to melanoma cells. A peptide library array was designed and screened using a peptide array-whole cell binding assay, which identified KK-11 as a novel human melanoma-targeting peptide. The peptide and its D-amino acid substituted analogue (VPWxEPAYQrFL or D-aa KK-11) were synthesized via a solid-phase strategy. Further studies using FITC-labeled KK-11 demonstrated dose-dependent uptake in human melanoma cells. D-aa KK-11 significantly increased the stability of the peptide, with 45.3% remaining detectable after 24 h with human serum incubation. Co-treatment of KK-11 with doxorubicin was found to significantly enhance the cytotoxicity of doxorubicin compared to doxorubicin alone, or sequential KK-11 and doxorubicin treatment. In vivo and ex vivo imaging revealed that D-aa KK-11 distributed to xenografted A375 melanoma tumors as early as 5 min and persisted up to 24 h post tail vein injection. When co-administered, D-aa KK-11 significantly enhanced the anti-tumor activity of a novel nNOS inhibitor (MAC-3-190) in an A375 human melanoma xenograft mouse model compared to MAC-3-190 treatment alone. No apparent systemic toxicities were observed. Taken together, these results suggest that KK-11 may be a promising human melanoma-targeted delivery vector for anti-melanoma cargo

Structural Basis for Isoform Selective Nitric Oxide Synthase Inhibition by Thiophene-2-Carboximidamides

The over production of nitric oxide in the brain by neuronal nitric oxide synthase (nNOS) is associated with a number of neurodegenerative diseases. Although inhibiting nNOS is an important therapeutic goal, it is important not to inhibit endothelial NOS (eNOS) owing to the critical role played by eNOS in maintaining vascular tone. While it has been possible to develop nNOS selective aminopyridine inhibitors, many of the most potent and selective inhibitors exhibit poor bioavailability properties. Our group and others have turned to more biocompatible thiophene-2-carboximidamides (T2C) inhibitors as potential nNOS selective inhibitors. We have used crystallography and computational methods to better understand how and why 2 commercially developed T2C inhibitors exhibit selectivity for human nNOS over human eNOS. As with many of the aminopyridine inhibitors, a critical active site Asp residue in nNOS vs Asn in eNOS is largely responsible for controlling selectivity. We also present thermodynamic integration results to better understand the change in pKa and thus charge of inhibitors once bound to the active site. In addition, relative free energy calculations underscore the importance of enhanced electrostatic stabilization of inhibitors bound to the nNOS active site compared to eNOS

PLP and GABA Trigger GabR-mediated Transcription Regulation in Bacillus Subtilis via External Aldimine Formation

The Bacillus subtilis protein regulator of the gabTD operon and its own gene (GabR) is a transcriptional activator that regulates transcription of γ-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5′-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the “active site” in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic γ-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, (S)-4-amino-5-fluoropentanoic acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) from Escherichia coli as a control. A comparison between the structures of the Eb/O–PLP–AFPA complex and Asp-AT–PLP–AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP

Parent and child agreement for acute stress disorder, post-traumatic stress disorder and other psychopathology in a prospective study of children and adolescents exposed to single-event trauma

Examining parent-child agreement for Acute Stress Disorder (ASD) and Post-Traumatic Stress Disorder (PTSD) in children and adolescents is essential for informing the assessment of trauma-exposed children, yet no studies have examined this relationship using appropriate statistical techniques. Parent-child agreement for these disorders was examined by structured interview in a prospective study of assault and motor vehicle accident (MVA) child survivors, assessed at 2-4 weeks and 6 months post-trauma. Children were significantly more likely to meet criteria for ASD, as well as other ASD and PTSD symptom clusters, based on their own report than on their parent's report. Parent-child agreement for ASD was poor (Cohen's κ = -.04), but fair for PTSD (Cohen's κ = .21). Agreement ranged widely for other emotional disorders (Cohen's κ = -.07-.64), with generalised anxiety disorder found to have superior parent-child agreement (when assessed by phi coefficients) relative to ASD and PTSD. The findings support the need to directly interview children and adolescents, particularly for the early screening of posttraumatic stress, and suggest that other anxiety disorders may have a clearer presentation post-trauma