Synthesis of novel phosphorothioates and phosphorodithioates and their differential inhibition of cholinesterases

The anticholinesterase activities of newly synthesized phosphorothioates and phosphorodithioates were investigated. The compounds were evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition potency through IC₅₀ determination. The selectivities of the synthesized compounds toward both enzymes were determined and compared in terms of their molecular structures.     

Biophysical study on the interaction of cartap hydrochloride and hemoglobin: Heme degradation and functional changes of protein

Cartap hydrochloride is a mildly perilous insecticide known as “Padan” which is used largely in agricultural farms to control weevil and caterpillars. The over use of cartap causes harmful effects on human health. Since the blood may acts as a target and carrier for insecticides, the effect of these compounds on blood in mammalian toxicology is very important. Hemoglobin is a tetramer protein that play critical role in oxygen transport. The aim of this study was to analyze and compare the function and structural changes of hemoglobin in the presence of different concentrations of cartap by employing different spectroscopic techniques. The obtained results show that cartap has a high hemolytic effect which is increased with cartap concentration and reduces the thermal midpoint of hemoglobin. Fluorescence measurements reveal heme degradation at different concentrations of cartap. In consequence of theoretical and experimental results, cartap has an undesirable effect on hemoglobin structure and function.     

Mechanisms of the self-assembly of EAK16-family peptides into fibrillar and globular structures: molecular dynamics simulations from nano- to micro-seconds

The self-assembly of EAK16-family peptides in a bulk solution was studied using a combination of all-atom and coarse-grained molecular dynamics simulations. In addition, specified concentrations of EAK16 peptides were induced to form fibrillary or globular assemblies in vitro. The results show that the combination of all-atom molecular dynamics simulations on the single- and double-chain levels and coarse-grained simulations on the many-chain level predicts the experimental observations reasonably well. At neutral pH conditions, EAK16-I and EAK16-II assemble into fibrillary structures, whereas EAK16-IV aggregates into globular assemblies. Mechanisms of the formation of fibrillar and globular assemblies are described using the simulation results.     

Detecting Morphologically Distinct Oligomeric Forms of ��-Synuclein

Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of α-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of α-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of α-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of α-synuclein in vitro, blocks extracellular α-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue.Parkinson disease (PD)² is the second most common neurodegenerative disorder of the elderly, affecting more than 500,000 people in the United States (1), with 50,000 new cases reported each year at an annual cost estimated at 10 billion dollars per year. Pathologically, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and formation of fibrillar cytoplasmic inclusions known as Lewy bodies and Lewy neurites (2, 3). The protein α-synuclein has been strongly linked to PD (4, 5) and other related neurodegenerative disorders (6, 7) by several lines of evidence. 1) It is the major component of the hallmark Lewy body aggregates associated with PD. 2) Mutations (A53T, A30P, and E46K, where A30P is human A30P α-synuclein; A53T is human A53T α-synuclein; E46K is human E46K α-synuclein) or multiplication in the α-synuclein gene have been linked to familial PD (8–10). 3) Overexpression of α-synuclein in transgenic mice and Drosophila has been shown to induce the formation of PD-like pathological phenotypes and behavior, although the animal models do not in general replicate neuronal loss patterns (11, 12).α-Synuclein is a small protein (14 kDa) expressed mainly in brain tissues and is primarily localized at the presynaptic terminals of neurons (13). The primary structure of α-synuclein consists of three distinct regions. The N-terminal region of α-synuclein includes the mutation sites associated with familial PD (A53T, A30P, and E46K) and contains six imperfectly conserved repeats (KTKEGV) that may facilitate protein-protein binding. This repeat section is predicted to form amphipathic α-helices, typical of the lipid-binding domain of apolipoproteins (14). The central region, non-amyloid component, is extremely hydrophobic and includes a 12-residue stretch (VTGVTAVAQKTV) that is essential for aggregation (15). The C-terminal region is enriched with acidic glutamate and aspartate residues and is responsible for the chaperone function of α-synuclein (16).α-Synuclein normally exists as an unfolded protein, but it can adopt several different folded conformations depending on the environment, including small aggregates or oligomers, spherical and linear protofibrils, as well as the fibrillar structure found in Lewy bodies (14, 15). A growing body of evidence implicates the oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death (16–18). Several different oligomeric forms of α-synuclein including spherical, annular (19), pore-like (20), and dopamine-stabilized structures have been identified in vitro (21).α-Synuclein is considered a cytosolic protein, and consequently its pathogenic effect was assumed to be limited to the cytoplasm of single cells. However, recent studies have suggested that α-synuclein also has extracellular pathogenic effects (22–25). α-Synuclein was detected in blood plasma and cerebrospinal fluid in both monomeric and oligomeric forms (22–25), and the presence of significantly elevated levels of oligomeric species of α-synuclein has been reported extracellularly in plasma and cerebrospinal fluid samples from patients with PD (23). Furthermore, various studies have shown that aggregated α-synuclein added extracellularly to the culture medium is cytotoxic (26–32).Despite all these studies, it is still not clear how the various aggregate forms of α-synuclein are involved in the progression of PD. Therefore, reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only for fundamental studies of how α-synuclein aggregates affect cell function but also as potential diagnostic and therapeutic agents for PD.Recently, we reported inhibition of both aggregation and extracellular toxicity of α-synuclein in vitro by a single chain variable domain antibody fragment (scFv) that specifically recognized an oligomeric form of α-synuclein (32). In this study, we describe a second scFv (syn-10H) that binds a larger later stage oligomeric form of α-synuclein than the previously reported scFv. The syn-10H scFv neutralizes α-synuclein-induced toxicity in both undifferentiated and differentiated SH-SY5Y human neuroblastoma cell line and inhibits α-synuclein aggregation in vitro. The syn-10H scFv reacts specifically with homogenized PD brain tissue but does not cross-react with similarly treated samples taken from Alzheimer disease (AD) or healthy brain samples. Such scFvs therefore have potential value as diagnostic reagents to identify the presence of specific oligomeric species in PD tissue and fluid samples. The scFvs also have value as therapeutic agents as they can be used either extracellularly or expressed intracellularly (intrabodies) to prevent formation of toxic aggregates in vivo whether inside or outside of cells. Intrabodies have been used efficiently to neutralize toxic effects of different pathogenic agents, including α-synuclein (33–36). Moreover, immunization studies in mouse models of PD have shown that extracellular antibodies can reduce accumulation of intracellular aggregates of α-synuclein (37), thereby providing precedent for the use of scFvs in potential passive vaccination strategies for treating PD.     

Regiocontrolled synthesis and HIV inhibitory activity of unsymmetrical binaphthoquinone and trimeric naphthoquinone derivatives of conocurvone

Unsymmetrical biquinone and trimeric quinone derivatives were synthesized using halotriflate-biselectrophilic naphthoquinones through stepwise regioselective quinone substitution chemistry and evaluated for their ability to inhibit the cytopathogenic effects of HIV-1 using an MTT colorimetric assay. Compounds were also screened for their ability to inhibit the activity of HIV-1 integrase in vitro. Pyranylated trimeric quinones and biquinones exhibited both antiviral activity and integrase inhibitory activity. Conocurvone 1 and trimeric quinone 21 were the most potent HIV integrase inhibitors in the series. All of the biquinones showed HIV inhibitory activity. Simple methoxy substituted biquinones did not inhibit HIV-1 integrase.     

Single chain variable fragments against beta-amyloid (Abeta) can inhibit Abeta aggregation and prevent abeta-induced neurotoxicity

Beta-amyloid (Abeta) is a major pathological determinant of Alzheimer's disease (AD). Both active and passive immunization studies have shown that antibodies against Abeta are effective in decreasing cerebral Abeta levels, reducing Abeta accumulation, and attenuating cognitive deficits in animal models of AD. However, the therapeutic potential of these antibodies in human AD patients is limited because of adverse inflammatory reactions and cerebral hemorrhaging associated with the treatments. Here we show that single chain variable fragments (scFv's) represent an attractive alternative to more conventional antibody-based therapeutics to reduce Abeta toxicity. The binding affinities and binding epitopes of two different scFv's to Abeta were characterized using a surface plasmon resonance (SPR) biosensor. An scFv binding the 17-28 region of Abeta effectively inhibited in vitro aggregation of Abeta as determined by thioflavin T (ThT) fluorescence staining and atomic force microscopy (AFM) analysis, while an scFv binding the carboxyl-terminal region of Abeta (residues 29-40) did not inhibit aggregation. The scFv to the 17-28 region when co-incubated with Abeta not only decreased aggregation but also eliminated any toxic effects of aggregated Abeta on the human neuroblastoma cell line, SH-SY5Y. The ability of scFv's to inhibit both aggregation and cytotoxicity of Abeta indicates that scFv's have potential therapeutic value for treating AD.     

Effect of metal chelators on the aggregation of beta-amyloid peptides in the presence of copper and iron

Amyloid β (Aβ) fibrils and amorphous aggregates are found in the brain of patients with Alzheimer’s disease (AD), and are implicated in the etiology of AD. The metal imbalance is also among leading causes of AD, owing to the fact that Aβ aggregation takes place in the synaptic cleft where Aβ, Cu(II) and Fe(III) are found in abnormally high concentrations. Aβ40 and Aβ42 are the main components of plaques found in afflicted brains. Coordination of Cu(II) and Fe(III) ions to Aβ peptides have been linked to Aβ aggregation and production of reactive oxygen species, two key events in the development of AD pathology. Metal chelation was proposed as a therapy for AD on the basis that it might prevent Aβ aggregation. In this work, we first examined the formation of Aβ40 and Aβ42 aggregates in the presence of metal ions, i.e. Fe(III) and Cu(II), which were detected by fluorescence spectroscopy and atomic force microscopy. Second, we studied the ability of the two chelators, ethylenediaminetetraacetic acid and 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol), to investigate their effect on the availability of these metal ions to interact with Aβ and thereby their effect on Aβ accumulation. Our findings show that Fe(III), but not Cu(II), promote aggregation of both Aβ40 and Aβ42. We also found that only clioquinol decreased significantly iron ion-induced aggregation of Aβ42. The presence of ions and/or chelators also affected the morphology of Aβ aggregates.     

Synthesis,characterization and antineoplastic activity of bis-aziridinyl dimeric naphthoquinone – A novel class of compounds with potent activity against acute myeloid leukemia cells

The synthesis, characterization and antileukemic activity of rationally designed amino dimeric naphthoquinone (BiQ) possessing aziridine as alkylating moiety is described. Bis-aziridinyl BiQ decreased proliferation of acute myeloid leukemia (AML) cell lines and primary cells from patients, and exhibited potent (nanomolar) inhibition of colony formation and overall cell survival in AML cells. Effective production of reactive oxygen species (ROS) and double stranded DNA breaks (DSB) induced by bis-aziridinyl BiQ is reported. Bis-dimethylamine BiQ, as the isostere of bis-aziridinyl BiQ but without the alkylating moiety did not show as potent anti-AML activity. Systemic administration of bis-aziridinyl BiQ was well tolerated in NSG mice.     

Isolation of a human single chain antibody fragment against oligomeric alpha-synuclein that inhibits aggregation and prevents alpha-synuclein-induced toxicity

Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of alpha-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson's Disease (PD). A natively unfolded protein, alpha-synuclein can adopt different aggregated morphologies, including oligomers, protofibrils and fibrils. The small oligomeric aggregates have been shown to be particularly toxic. Antibodies that neutralize the neurotoxic aggregates without interfering with beneficial functions of monomeric alpha-synuclein can be useful therapeutics. We were able to isolate single chain antibody fragments (scFvs) from a phage displayed antibody library against the target antigen morphology using a novel biopanning technique that utilizes atomic force microscopy (AFM) to image and immobilize specific morphologies of alpha-synuclein. The scFv described here binds only to an oligomeric form of alpha-synuclein and inhibits both aggregation and toxicity of alpha-synuclein in vitro. This scFv can have potential therapeutic value in controlling misfolding and aggregation of alpha-synuclein in vivo when expressed intracellularly in dopaminergic neurons as an intrabody.