Isoform selectivities of novel 4-hydroxycoumarin imines as inhibitors of myosin II.

Muscle myosin inhibition could be used to treat many medical conditions involving hypercontractile states, including muscle spasticity, chronic musculoskeletal pain, and hypertrophic cardiomyopathy. A series of 13 advanced analogs of 3-(N-butylethanimidoyl)ethyl)-4-hydroxy-2H-chromen-2-one (BHC) were synthesized to explore extended imine nitrogen side chains and compare aldimines vs. ketimines. None of the new analogs inhibit nonmuscle myosin in a cytokinesis assay. ATPase structure-activity relationships reveal that selectivity for cardiac vs. skeletal myosin can be tuned with subtle structural changes. None of the compounds inhibited smooth muscle myosin II. Docking the compounds to homology models of cardiac and skeletal myosin II gave rationales for the effects of side arm length on inhibition selectivity and for cardiac vs. skeletal myosin. Properties including solubility, stability and toxicity, suggest that certain BHC analogs may be useful as candidates for preclinical studies or as lead compounds for advanced candidates for drugs with cardiac or skeletal muscle myosin selectivity.

X-ray and NMR crystallography in an enzyme active site: the indoline quinonoid intermediate in tryptophan synthase.

Chemical-level details such as protonation and hybridization state are critical for understanding enzyme mechanism and function. Even at high resolution, these details are difficult to determine by X-ray crystallography alone. The chemical shift in NMR spectroscopy, however, is an extremely sensitive probe of the chemical environment, making solid-state NMR spectroscopy and X-ray crystallography a powerful combination for defining chemically detailed three-dimensional structures. Here we adopted this combined approach to determine the chemically rich crystal structure of the indoline quinonoid intermediate in the pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase under conditions of active catalysis. Models of the active site were developed using a synergistic approach in which the structure of this reactive substrate analogue was optimized using ab initio computational chemistry in the presence of side-chain residues fixed at their crystallographically determined coordinates. Various models of charge and protonation state for the substrate and nearby catalytic residues could be uniquely distinguished by their calculated effects on the chemical shifts measured at specifically (13)C- and (15)N-labeled positions on the substrate. Our model suggests the importance of an equilibrium between tautomeric forms of the substrate, with the protonation state of the major isomer directing the next catalytic step.

Reduction of Progranulin-Induced Breast Cancer Stem Cell Propagation by Sortilin-Targeting Cyclotriazadisulfonamide (CADA) Compounds.

Cyclotriazadisulfonamide (CADA) compounds selectively down-modulate two human proteins of potential therapeutic interest, cluster of differentiation 4 (CD4) and sortilin. Progranulin is secreted from some breast cancer cells, causing dedifferentiation of receiving cancer cells and cancer stem cell proliferation. Inhibition of progranulin binding to sortilin, its main receptor, can block progranulin-induced metastatic breast cancer using a triple-negative in vivo xenograft model. In the current study, seven CADA compounds (CADA, VGD020, VGD071, TL020, TL023, LAL014, and DJ010) were examined for reduction of cellular sortilin expression and progranulin-induced breast cancer stem cell propagation. In addition, inhibition of progranulin-induced mammosphere formation was examined and found to be most significant for TL020, TL023, VGD071, and LAL014. Full experimental details are given for the synthesis and characterization of the four new compounds (TL020, TL023, VGD071, and DJ010). Comparison of solubilities, potencies, and cytotoxicities identified VGD071 as a promising candidate for future studies using mouse breast cancer models.

Tracheal colonization factor A (TcfA) is a biomarker for rapid and specific detection of Bordetella pertussis.

Pertussis is a highly contagious disease for which prompt, point-of-care diagnosis remains an unmet clinical need. Results from conventional test modalities (nucleic acid detection, serology, and culture) take hours to days. To overcome this challenge, we identified a new biomarker (tracheal colonization factor A, TcfA) for detection of Bordetella pertussis infection by lateral flow immunoassay (LFIA). We developed a library of 28 epitope-mapped monoclonal antibodies against TcfA and incorporated three antibodies into a LFIA. The LFIA did not cross-react with common bacterial or fungal organisms, but did react with nine distinct B. pertussis strains. The minimal linear epitope sequences targeted by the LFIA were conserved in 98% of 954 B. pertussis isolates collected across 12 countries from 1949-2017. The LFIA's limit of detection was 3.0 × 105 CFU/mL with B. pertussis cells in buffer, 6.2 × 105 CFU/mL with nasopharyngeal washes from a non-human primate model, and 2.3 ng/mL with recombinant TcfA. The LFIA reacted with patient nasopharyngeal swab specimens containing as few as 1.8 × 106 B. pertussis genomes/mL and showed no false-positives. Rapid (< 20 min) LFIA detection of TcfA as a biomarker for B. pertussis infection is feasible and may facilitate early detection of pertussis.

Asymmetries in global-local processing ability in elderly people with the apolipoprotein e-epsilon4 allele.

Previous studies have identified cognitive asymmetries in elderly people at increased risk for Alzheimer's disease (AD) by comparing standardized neuropsychological tests of verbal and spatial abilities in both preclinical AD and apolipoprotein epsilon4+ elderly groups. This prospective study investigated cognitive asymmetries within a single test by comparing cognitively intact elderly (with and without the epsilon4+ allele) on a learning and memory measure that uses global and local visuospatial stimuli. Both groups demonstrated comparable overall learning and recall. But the epsilon4+ group had a significantly larger discrepancy between their global and local learning scores and had a greater proportion of individuals with more than a one standard deviation difference between their immediate recall of the global and local elements, relative to the epsilon4- group. These findings build on previous studies identifying subgroups of elderly people at greater risk for AD who often demonstrate increased cognitive asymmetries relative to groups without significant risk factors.

Uniform-sign cross-peak double-quantum-filtered correlation spectroscopy.

We detail the uniform-sign cross-peak double-quantum-filtered correlation spectroscopy (UC2QF COSY) experiment, a new through-bond correlation method for disordered solids. This experiment is a refocused version of the popular double-quantum-filtered correlation spectroscopy experiment in liquids. Its key feature is that it provides in-phase and doubly absorptive line shapes, which renders it robust for chemical shift correlation in solids. Both theory and experiment point to distinct advantages of this protocol, which are illustrated by several experiments under challenging conditions, including fast magic-angle spinning (30kHz), anisotropic molecular motion, and (13)C correlation spectroscopy at the natural abundance isotope level.

Brain response correlates of decisional capacity in schizophrenia: a preliminary FMRI study.

The capacity of schizophrenia patients to make decisions regarding research consent relates to neurocognition, but the exact nature of the relationship is unclear. The authors examined the correlation of scores on the MacArthur Competence Assessment Tool for Clinical Research with functional magnetic resonance imaging brain response during a verbal learning task. Understanding of a consent form correlated with activation of the right hippocampus during verbal learning and with brain response in a large area that included the bilateral parahippocampus, cerebellum, and thalamus. Reasoning scores were not significantly related to brain activation. Understanding deficits during informed consent relates to particular brain abnormalities among schizophrenia patients.

Effect of protonation on the conformation of cinchonidine.

The protonation of cinchona alkaloids in solution leads to the severe restriction of their internal rotational degrees of freedom and to the locking of the molecule around a specific conformation held in place by a bridging counterion of the acid used for protonation. For HF, direct interactions were detected by NMR between the fluorine anion and not only with the acidic hydroxo group but also with non-acidic hydrogen atoms in the quinoline ring.

Constant-time through-bond 13C correlation spectroscopy for assigning protein resonances with solid-state NMR spectroscopy.

Even as available magnetic fields for NMR continue to increase, resolution remains one of the most critical limitations in assigning and solving structures of larger biomolecules. Here we present a novel constant-time through-bond correlation spectroscopy for solids that offers superior resolution for 13C chemical shift assignments in proteins. In this experiment, the indirect evolution and transfer periods are combined into a single constant time interval, offering increased resolution while not sacrificing sensitivity. In GB1, this allows us to resolve peaks that are otherwise unresolved and to make assignments in the absence of multibond transfers.

The amide rotational barrier in isonicotinamide: Dynamic NMR and ab initio studies.

We report use of dynamic nuclear magnetic resonance (NMR) to measure the amide rotational barrier in isonicotinamide. A significant challenge to obtaining good transition rates from dynamic NMR data is suppression of errors due to inherent line widths associated with transverse relaxation. We address this challenge with a fitting procedure that incorporates transverse relaxation over the temperature range of interest simply and reliably. The fitting model is nonlinear in only one of the fit parameters, namely, the activation enthalpy. This reduces parameter estimation to solution of a single transcendental equation, which avoids both a fine search over a multidimensional parameter space and extrapolation of a "limiting line width" solely from slow-exchange data. The activation enthalpy Delta H++ measured for isonicotinamide, +14.1 +/- 0.2 kcal/mol, falls between those of its regioisomers picolinamide and nicotinamide, which were reported in an earlier study. In that study, ab initio calculations of the rotational barriers helped to discern the relative importance of steric, electronic, and hydrogen-bonding effects in this biochemically significant combination of pyridine-ring and carboxamide moieties. A direct comparison between isonicotinamide and nicotinamide, where steric and hydrogen-bonding effects differ only slightly, permits a closer study of electronic considerations.

Through-bond 13C-13C correlation at the natural abundance level: refining dynamic regions in the crystal structure of vitamin-D3 with solid-state NMR.

Recently, we presented a novel nuclear magnetic resonance experiment for establishing through-bond connectivity in disordered solids using scalar coupling-driven correlation. This method, a variant of the popular double-quantum-filtered correlation spectroscopy experiment in liquids, is robust under fast magic-angle-spinning conditions and in the presence of dynamics. Here, we show that this new experiment, the UC2QF COSY, can be extended to 13C natural abundance correlation in moderately sized molecules, allowing the assignment of the 54 peaks of the solid-state NMR spectrum of microcrystalline vitamin-D3. In this case, comparison between the assigned peaks and ab initio calculations of the chemical shifts based on the crystal coordinates permits a refinement of the average structure in dynamic regions reported as disordered in the crystal structure.

The amide rotational barriers in picolinamide and nicotinamide: NMR and ab initio studies.

Pyridine carboxamides are a class of medicinal agents with activity that includes the reduction of iron-induced renal damage, the regulation of nicotinamidase activity, and radio- and chemosensitization. Such pharmacological activities, and the prevalence of the carboxamide moiety and the importance of amide rotations in biology, motivate detailed investigation of energetics in these systems. In this study, we report the use of dynamic nuclear magnetic resonance to measure the amide rotational barriers in the pyridine carboxamides picolinamide and nicotinamide. The activation enthalpies and entropies of DeltaH++ = 12.9 +/- 0.3 kcal/mol and DeltaS++ = -7.7 +/- 0.9 cal/mol K for nicotinamide and DeltaH++ = 18.3 +/- 0.4 kcal/mol and DeltaS++ = +1.3 +/- 1.0 cal/mol K for picolinamide report a substantial energetic difference for these regioisomers. Ab initio calculations of the rotational barriers are in good agreement with the experimentally determined values and help partition the 5.4 kcal/mol enthalpy difference into its major contributions. Of principal importance are the variations in steric interactions in the ground states of picolinamide and nicotinamide, superior pi electron donation from the pyridine ring in the transition state of nicotinamide, and an intramolecular hydrogen bond in the ground state of picolinamide.

Effects of task structure on category priming in patients with Parkinson's disease and in healthy individuals.

Lexical decision tasks have been used to study both shifts of attention and semantic processing in Parkinson's Disease (PD). Whereas other laboratories have reported normal levels of semantic priming among PD patients, our laboratory has reported abnormally large levels. In this study, two experiments were performed to determine the influence of task structure on the extent of semantic priming during lexical decision-making and pronunciation tasks among PD patients and neurologically healthy controls. In Experiment 1, the effect of Prime Dominance (the ratio of category to neutral trials) on lexical decision-making was studied. Although equal numbers of word and nonword trials were presented, half of the PD patients and controls were studied under Category Prime Dominance (category : neutral prime ratio of 2:1) and half were studied under Neutral Prime Dominance (category : neutral prime ratio of 1:2). In Experiment 2, PD and control participants were studied on lexical decision-making and pronunciation tasks where twice as many words as nonword trials were presented, consistent with other studies from our laboratory. In Experiment 1, we found no group differences in the magnitude of priming and no effect of Prime Dominance. Moreover, the findings were similar in pattern and magnitude to results published by Neely (1977). In Experiment 2, we observed larger priming effects among PD patients than among controls, but only on the lexical decision (LD) task. These results support the hypothesis that abnormally large category-priming effects appear in LD studies of PD patients when the number of word trials exceeds the number of nonword trials. Furthermore, increased lexical priming in PD appears to be due to processes operating during the decision-making period that follows presentation of the lexical target.