Charles P. Scott

Anti-inflammatory compounds parthenolide and Bay 11-7082 are direct inhibitors of the inflammasome.

Activation of the inflammasome generates the pro-inflammatory cytokines interleukin-1β and -18, which are important mediators of inflammation. Abnormal activation of the inflammasome leads to many inflammatory diseases, including gout, silicosis, neurodegeneration, and genetically inherited periodic fever syndromes. Therefore, identification of small molecule inhibitors that target the inflammasome is an important step toward developing effective therapeutics for the treatment of inflammation. Here, we show that the herbal NF-κB inhibitory compound parthenolide inhibits the activity of multiple inflammasomes in macrophages by directly inhibiting the protease activity of caspase-1. Additional investigations of other NF-κB inhibitors revealed that the synthetic IκB kinase-β inhibitor Bay 11-7082 and structurally related vinyl sulfone compounds selectively inhibit NLRP3 inflammasome activity in macrophages independent of their inhibitory effect on NF-κB activity. In vitro assays of the effect of parthenolide and Bay 11-7082 on the ATPase activity of NLRP3 demonstrated that both compounds inhibit the ATPase activity of NLRP3, suggesting that the inhibitory effect of these compounds on inflammasome activity could be mediated in part through their effect on the ATPase activity of NLRP3. Our results thus elucidate the molecular mechanism for the therapeutic anti-inflammatory activity of parthenolide and identify vinyl sulfones as a new class of potential therapeutics that target the NLRP3 inflammasome.

Structural requirements for the biosynthesis of backbone cyclic peptide libraries

BACKGROUND Combinatorial methods for the production of molecular libraries are an important source of ligand diversity for chemical biology. Synthetic methods focus on the production of small molecules that must traverse the cell membrane to elicit a response. Genetic methods enable intracellular ligand production, but products must typically be large molecules in order to withstand cellular catabolism. Here we describe an intein-based approach to biosynthesis of backbone cyclic peptide libraries that combines the strengths of synthetic and genetic methods. RESULTS Through site-directed mutagenesis we show that the DnaE intein from Synechocystis sp. PCC6803 is very promiscuous with respect to peptide substrate composition, and can generate cyclic products ranging from four to nine amino acids. Libraries with five variable amino acids and either one or four fixed residues were prepared, yielding between 10(7) and 10(8) transformants. The majority of randomly selected clones from each library gave cyclic products. CONCLUSIONS We have developed a versatile method for producing intracellular libraries of small, stable cyclic peptides. Genetic encoding enables facile manipulation of vast numbers of compounds, while low molecular weight ensures ready pharmacophore identification. The demonstrated flexibility of the method towards both peptide length and composition makes it a valuable addition to existing methods for generating ligand diversity.

A modified Hammersmith functional motor scale for use in multi-center research on spinal muscular atrophy

The Hammersmith functional motor scale for children with spinal muscular atrophy was modified to establish a standard measure of functional ability in children with non-ambulant spinal muscular atrophy types 2 and 3 in a longitudinal multi-center clinical trial. This study assessed the intra- and interrater reliability and the test-retest stability of a modified version of the scale. Both intra- and interrater reliability were established. Results indicate that the scale is reliable and stable over a 6 month period. Reliability was maintained when patient sample criteria were expanded to include children younger than 30 months and children with popliteal angles greater than 20 degrees . These data establish the modified Hammersmith functional motor scale for children with spinal muscular atrophy as a reliable instrument for use in multi-center treatment trials in non-ambulant spinal muscular atrophy children. Our data provides additional support for the use of original scale items in terms of ease of administration, usefulness and reliability, while incorporating modifications to optimize its use in a multi-center clinical research setting.

Compound muscle action potential and motor function in children with spinal muscular atrophy.

Reliable outcome measures that reflect the underlying disease process and correlate with motor function in children with SMA are needed for clinical trials. Maximum ulnar compound muscle action potential (CMAP) data were collected at two visits over a 4–6‐week period in children with SMA types II and III, 2–17 years of age, at four academic centers. Primary functional outcome measures included the Modified Hammersmith Functional Motor Scale (MHFMS) and MHFMS‐Extend. CMAP negative peak amplitude and area showed excellent discrimination between the ambulatory and non‐ambulatory SMA cohorts (ROC = 0.88). CMAP had excellent test–retest reliability (ICC = 0.96–0.97, n = 64) and moderate to strong correlation with the MHFMS and MHFMS‐Extend (r = 0.61–0.73, n = 68, P < 0.001). Maximum ulnar CMAP amplitude and area is a feasible, valid, and reliable outcome measure for use in pediatric multicenter clinical trials in SMA. CMAP correlates well with motor function and has potential value as a relevant surrogate for disease status. Muscle Nerve, 2010

RASCH ANALYSIS OF CLINICAL OUTCOME MEASURES IN SPINAL MUSCULAR ATROPHY

Introduction: Trial design for SMA depends on meaningful rating scales to assess outcomes. In this study Rasch methodology was applied to 9 motor scales in spinal muscular atrophy (SMA). Methods: Data from all 3 SMA types were provided by research groups for 9 commonly used scales. Rasch methodology assessed the ordering of response option thresholds, tests of fit, spread of item locations, residual correlations, and person separation index. Results: Each scale had good reliability. However, several issues impacting scale validity were identified, including the extent that items defined clinically meaningful constructs and how well each scale measured performance across the SMA spectrum. Conclusions: The sensitivity and potential utility of each SMA scale as outcome measures for trials could be improved by establishing clear definitions of what is measured, reconsidering items that misfit and items whose response categories have reversed thresholds, and adding new items at the extremes of scale ranges. Muscle Nerve 49:422–430, 2014

Mycobacterium tuberculosis Type II NADH-Menaquinone Oxidoreductase Catalyzes Electron Transfer through a Two-Site Ping-Pong Mechanism and Has Two Quinone-Binding Sites

Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the quinone pool and plays an essential role in the oxidative phosphorylation system of Mycobacterium tuberculosis (Mtb). The absence of NDH-2 in the mammalian mitochondrial electron transport chain makes this enzyme an attractive target for antibiotic development. To fully establish the kinetic properties of this enzyme, we studied the interaction of Mtb NDH-2 with substrates, NADH, and various quinone analogues and their products in both membrane and soluble environments. These studies, and comparative analyses of the kinetics with thio-NAD+ and quinone electron acceptors, provided evidence that Mtb NDH-2 catalyzes the transfer electrons from NADH to quinone substrates by a nonclassical, two-site ping-pong kinetic mechanism whereby substrate quinones bind to a site that is distinct from the NADH-binding site. Furthermore, the effects of quinols on Mtb NDH-2 catalytic activity demonstrate the presence of two binding sites for quinone ligands, one favoring the reduced form and the other favoring the oxidized form.

A man who inherited his SRY gene and Leri-Weill dyschondrosteosis from his mother and neurofibromatosis type 1 from his father

We report on a man with neurofibromatosis type 1 (NF1) and Leri-Weill dyschondrosteosis (LWD). His father had NF1. His mother had LWD plus additional findings of Turner syndrome (TS): high arched palate, bicuspid aortic valve, aortic stenosis, and premature ovarian failure. The probands karyotype was 46,X,dic(X;Y)(p22.3;p11.32). Despite having almost the same genetic constitution as 47,XXY Klinefelter syndrome, he was normally virilized, although slight elevation of serum gonadotropins indicated gonadal dysfunction. His mothers karyotype was mosaic 45,X[17 cells]/46,X,dic(X;Y)(p22.3;p11.32)[3 cells].ish dic(X;Y)(DXZ1 +,DYZ1 + ). The dic(X;Y) chromosome was also positive for Y markers PABY, SRY, and DYZ5, but negative for SHOX. The dic(X;Y) chromosome was also positive for X markers DXZ1 and a sequence < 300 kb from PABX, suggesting that the deletion encompassed only pseudoautosomal sequences. Replication studies indicated that the normal X and the dic(X;Y) were randomly inactivated in the probands lymphocytes. LWD in the proband and his mother was explained by SHOX haploinsufficiency. The mothers female phenotype was most likely due to 45,X mosaicism. This family segregating Mendelian and chromosomal disorders illustrates extreme sex chromosome variation compatible with normal male and female sexual differentiation. The case also highlights the importance of karyotyping for differentiating LWD and TS, especially in patients with findings such as premature ovarian failure or aortic abnormalities not associated with isolated SHOX haploinsufficiency.

Reliability and validity of the TIMPSI for infants with spinal muscular atrophy type I.

Purpose: This study examined the reliability and validity of the Test of Infant Motor Performance Screening Items (TIMPSI) in infants with type I spinal muscular atrophy (SMA). Methods: After training, 12 evaluators scored 4 videos of infants with type I SMA to assess interrater reliability. Intrarater and test-retest reliability was further assessed for 9 evaluators during a SMA type I clinical trial, with 9 evaluators testing a total of 38 infants twice. Relatedness of the TIMPSI score to ability to reach and ventilatory support was also examined. Results: Excellent interrater video score reliability was noted (intraclass correlation coefficient, 0.97–0.98). Intrarater reliability was excellent (intraclass correlation coefficient, 0.91–0.98) and test-retest reliability ranged from r = 0.82 to r = 0.95. The TIMPSI score was related to the ability to reach (P ⩽ .05). Conclusion: The TIMPSI can reliably be used to assess motor function in infants with type I SMA. In addition, the TIMPSI scores are related to the ability to reach, an important functional skill in children with type I SMA.

High level expression of the large subunit of mouse ribonucleotide reductase in a baculovirus system

The large subunit of ribonucleotide reductase from mouse has been overexpressed in Spodoptera frugiperda cells infected with recombinant baculovirus. The expressed protein was purified by affinity chromatography to apparent homogeneity as determined by SDS‐PAGE. The homogeneous protein is recognized in Western blot analysis by a monoclonal antibody raised to the large subunit of ribonucleotide reductase from calf thymus, has the correct N‐terminal sequence, and, in the presence of the small subunit of mouse ribonucleotide reductase and nucleoside triphosphate effectors, catalyzes the reduction of both purine and pyrimidine nucleoside diphosphates.

Taking the chemical out of chemical genetics

Screening a library of expressed cyclic peptides identified clones that reverse the cytotoxicity of alpha-synuclein in yeast and Caenorhabditis elegans. The results suggest a new approach for intervention in Parkinsons disease, and perhaps a druggable target.