Anticancer thiopyrano[2,3-d][1,3]thiazol-2-ones with norbornane moiety. Synthesis, cytotoxicity, physico-chemical properties, and computational studies

A series of novel 9-substituted-3,7-dithia-5-azatetracyclo[9.2.1.0(2,10).0(4,8)]tetradecen-4(8)-ones-6 have been synthesized by a stereoselective hetero-Diels-Alder reaction of 5-ylidene-4-thioxo-2-thiazolidone derivatives with norbornene-2. All the compounds have been evaluated for antitumor activity in in vitro human tumor cell lines, and 10 of them possessed significant and selective cytotoxicity (MGM logGI50 approximately -4.17 to -4.98, for individual cell lines logGI50 up to -8). COMPARE analyses of differential growth inhibition patterns of compounds at the GI50 level showed high correlations with some of the antitubulin agents. The lipophilicity of the compounds was studied by RP-TLC and found to correlate well with calculated logP values. Docking and structure-activity relationship studies produced seven QSAR models with 2 or 3 variables, with correlation coefficients r2>0.9 and leave-one-out cross-validation correlation coefficients, q2>0.8.     

The adhesion molecule CHL1 regulates uncoating of clathrin-coated synaptic vesicles

In searching for binding partners of the intracellular domain of the immunoglobulin superfamily adhesion molecule CHL1, we identified the clathrin-uncoating ATPase Hsc70. CHL1 gene ablation resulted in reduced targeting of Hsc70 to the synaptic plasma membrane and synaptic vesicles, suggesting CHL1 as a synapse-targeting cue for Hsc70. CHL1 accumulates in presynaptic membranes and, in response to synapse activation, is targeted to synaptic vesicles by endocytosis. CHL1 deficiency or disruption of the CHL1/Hsc70 complex results in accumulation of abnormally high levels of clathrin-coated synaptic vesicles with a reduced ability to release clathrin. Generation of new clathrin-coated synaptic vesicles in an activity-dependent manner is inhibited when the CHL1/Hsc70 complex is disrupted, resulting in impaired uptake and release of FM dyes in synaptic boutons. Abnormalities in clathrin-dependent synaptic vesicle recycling may thus underlie brain malfunctions in humans and mice that carry mutations in the CHL1 gene.     

A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte

We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore ( I(N*)/I(T*)) upon binding of Fab57P. When the peptide was labeled in the C terminus, the I(N*)/I(T*) ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats.     

Behavioral Characterization of A53T Mice Reveals Early and Late Stage Deficits Related to Parkinson’s Disease

Parkinson''s disease (PD) pathology is characterized by the formation of intra-neuronal inclusions called Lewy bodies, which are comprised of alpha-synuclein (α-syn). Duplication, triplication or genetic mutations in α-syn (A53T, A30P and E46K) are linked to autosomal dominant PD; thus implicating its role in the pathogenesis of PD. In both PD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of protein aggregates (i.e., α-syn) and neurodegeneration. Characterization of the timing and nature of symptomatic dysfunction is important for understanding the impact of α-syn on disease progression. Furthermore, this knowledge is essential for identifying pathways and molecular targets for therapeutic intervention. To this end, we examined various functional and morphological endpoints in the transgenic mouse model expressing the human A53T α-syn variant directed by the mouse prion promoter at specific ages relating to disease progression (2, 6 and 12 months of age). Our findings indicate A53T mice develop fine, sensorimotor, and synaptic deficits before the onset of age-related gross motor and cognitive dysfunction. Results from open field and rotarod tests show A53T mice develop age-dependent changes in locomotor activity and reduced anxiety-like behavior. Additionally, digigait analysis shows these mice develop an abnormal gait by 12 months of age. A53T mice also exhibit spatial memory deficits at 6 and 12 months, as demonstrated by Y-maze performance. In contrast to gross motor and cognitive changes, A53T mice display significant impairments in fine- and sensorimotor tasks such as grooming, nest building and acoustic startle as early as 1–2 months of age. These mice also show significant abnormalities in basal synaptic transmission, paired-pulse facilitation and long-term depression (LTD). Combined, these data indicate the A53T model exhibits early- and late-onset behavioral and synaptic impairments similar to PD patients and may provide useful endpoints for assessing novel therapeutic interventions for PD.     

Detection of multi-color fluorescent objects with single photon spectrometer

Single photon counting is the most sensitive and accurate method for detection of very weak fluorescent signals obtained in many applications such as DNA sequencing, detection of biological reporters on micro-beads, detection of droplets in micro-fluidic systems, etc. In this paper we describe the use of single photon spectrometer for detection and characterization of very weak multicolor fluorescence produced by mixtures of various fluorescent dyes and quantum dots.     

Investigating the competitive effects of cholesterol and melatonin in model lipid membranes

We have studied the impact of cholesterol and/or melatonin on the static and dynamical properties of bilayers made of DPPC or DOPC utilizing neutron scattering techniques, Raman spectroscopy and molecular dynamics simulations. While differing in the amplitude of the effect due to cholesterol or melatonin when comparing their interactions with the two lipids, their addition ensued recognizable changes to both types of bilayers. As expected, based on the two-component systems of lipid/cholesterol or lipid/melatonin studied previously, we show the impact of cholesterol and melatonin being opposite and competitive in the case of three-component systems of lipid/cholesterol/melatonin. The effect of cholesterol appears to prevail over that of melatonin in the case of structural properties of DPPC-based bilayers, which can be explained by its interactions targeting primarily the saturated lipid chains. The dynamics of hydrocarbon chains represented by the ratio of trans/gauche conformers reveals the competitive effect of cholesterol and melatonin being somewhat more balanced. The additive yet opposing effects of cholesterol and melatonin have been observed also in the case of structural properties of DOPC-based bilayers. We report that cholesterol induced an increase in bilayer thickness, while melatonin induced a decrease in bilayer thickness in the three-component systems of DOPC/cholesterol/melatonin. Commensurately, by evaluating the projected area of DOPC, we demonstrate a lipid area decrease with an increasing concentration of cholesterol, and a lipid area increase with an increasing concentration of melatonin. The demonstrated condensing effect of cholesterol and the fluidizing effect of melatonin appear in an additive manner upon their mutual presence.     

Heme-bound nitroxyl, hydroxylamine, and ammonia ligands as intermediates in the reaction cycle of cytochrome c nitrite reductase: a theoretical study

In this article, we consider, in detail, the second half-cycle of the six-electron nitrite reduction mechanism catalyzed by cytochrome c nitrite reductase. In total, three electrons and four protons must be provided to reach the final product, ammonia, starting from the HNO intermediate. According to our results, the first event in this half-cycle is the reduction of the HNO intermediate, which is accomplished by two PCET reactions. Two isomeric radical intermediates, HNOH^? and H₂NO^?, are formed. Both intermediates are readily transformed into hydroxylamine, most likely through intramolecular proton transfer from either Arg₁₁₄ or His₂₇₇. An extra proton must enter the active site of the enzyme to initiate heterolytic cleavage of the N–O bond. As a result of N–O bond cleavage, the H₂N⁺ intermediate is formed. The latter readily picks up an electron, forming H₂N^+?, which in turn reacts with Tyr₂₁₈. Interestingly, evidence for Tyr₂₁₈ activity was provided by the mutational studies of Lukat (Biochemistry 47:2080, 2008), but this has never been observed in the initial stages of the overall reduction process. According to our results, an intramolecular reaction with Tyr₂₁₈ in the final step of the nitrite reduction process leads directly to the final product, ammonia. Dissociation of the final product proceeds concomitantly with a change in spin state, which was also observed in the resonance Raman investigations of Martins et al. (J Phys Chem B 114:5563, 2010).