Tacrine-melatonin hybrids as multifunctional agents for Alzheimer's disease, with cholinergic, antioxidant, and neuroprotective properties.

Tacrine-melatonin hybrids were designed and synthesized as new multifunctional drug candidates for Alzheimer's disease. These compounds may simultaneously palliate intellectual deficits and protect the brain against both beta-amyloid (A beta) peptide and oxidative stress. They show improved cholinergic and antioxidant properties, and are more potent and selective inhibitors of human acetylcholinesterase (hAChE) than tacrine. They also capture free radicals better than melatonin. Molecular modeling studies show that these hybrids target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. At sub-micromolar concentrations they efficiently displace the binding of propidium iodide from the PAS and could thus inhibit A beta peptide aggregation promoted by AChE. Moreover, they also inhibit A beta self-aggregation and display neuroprotective properties in a human neuroblastoma line against cell death induced by various toxic insults, such as A beta(25-35), H(2)O(2), and rotenone. Finally, they exhibit low toxicity and may be able to penetrate the central nervous system according to an in vitro parallel artificial membrane permeability assay for the blood-brain barrier (PAMPA-BBB).

Dual binding site acetylcholinesterase inhibitors: potential new disease-modifying agents for AD.

The therapeutic potential of acetylcholinesterase (AChE) inhibitors has been strengthened recently by evidence showing that besides their role in cognitive function, they might contribute to slow down the neurodegeneration in Alzheimer's disease (AD) patients. It is known that AChE exerts secondary noncholinergic functions, related to its peripheral anionic site, in cell adhesion and differentiation, and recent findings also support its role in mediating the processing and deposition of beta-amyloid (Abeta) peptide. AChE is one of the proteins that colocalizes with Abeta peptide deposits in the brain of AD patients and promotes Abeta fibrillogenesis by forming stable AChEA beta complexes. Additionally, it has also been postulated that AChE binds through its peripheral site to the Abeta nonamyloidogenic form and acts as a pathological chaperone inducing a conformational transition to the amyloidogenic form (Inestrosa et al., 1996; Bartolini et al., 2003). Anew series of dual binding site AChE inhibitors has been designed and synthesized as new potent AChE inhibitors, which might simultaneously alleviate cognitive deficits and behave as disease-modifying agents by inhibiting Abeta peptide aggregation through binding to both catalytic and peripheral sites of the enzyme.

Manzamine B and E and ircinal A related alkaloids from an Indonesian Acanthostrongylophora sponge and their activity against infectious, tropical parasitic, and Alzheimer's diseases.

Four new manzamine-type alkaloids, 12,28-oxamanzamine E (2), 12,34-oxa-6-hydroxymanzamine E (3), 8-hydroxymanzamine B (5), and 12,28-oxaircinal A (11), were isolated from three collections of an Indonesian sponge of the genus Acanthostrongylophora together with 13 known manzamine alkaloids, ircinal A, ircinol A, xestomanzamine A, manzamines A, E, F, J, and Y, manadomanzamines A and B, neo-kauluamine, 8-hydroxymanzamine A, and manzamine A N-oxide. The structures of the new compounds were elucidated by means of 1D and 2D NMR spectroscopic methods. Three of these compounds (2, 3, and 11) possess a unique manzamine-type aminal ring system generated through an ether linkage between carbons 12-28 or between carbons 12-34. In the case of manzamine B and related metabolites, carbons 11 and 12 of the typical manzamine structure have an epoxide group and add to our growing understanding of manzamine structure-activity relationships (SAR) and metabolism. The bioactivity and SAR for a number of previously reported manzamine-related metabolites against malaria, leishmania, tuberculosis, and HIV-1 are also presented. Manzamine Y (9) showed significant inhibitory activity of GSK3, an enzyme implicated in Alzheimer's disease pathology. The toxicity of manzamine A and neo-kauluamine was evaluated against both medaka fry and eggs.

Design, synthesis, and biological evaluation of dual binding site acetylcholinesterase inhibitors: new disease-modifying agents for Alzheimer's disease.

New dual binding site acetylcholinesterase (AChE) inhibitors have been designed and synthesized as new potent drugs that may simultaneously alleviate cognitive deficits and behave as disease-modifying agents by inhibiting the beta-amyloid (A beta) peptide aggregation through binding to both catalytic and peripheral sites of the enzyme. Particularly, compounds 5 and 6 emerged as the most potent heterodimers reported so far, displaying IC50 values for AChE inhibition of 20 and 60 pM, respectively. More importantly, these dual AChE inhibitors inhibit the AChE-induced A beta peptide aggregation with IC50 values 1 order of magnitude lower than that of propidium, thus being the most potent derivatives with this activity reported up to date. We therefore conclude that these compounds are very promising disease-modifying agents for the treatment of Alzheimer's disease (AD).

Synthesis and biological evaluation of tacrine-thiadiazolidinone hybrids as dual acetylcholinesterase inhibitors.

The synthesis of tacrine-thiadiazolidinone hybrids is described. These compounds are designed as dual acetylcholinesterase inhibitors binding simultaneously to the peripheral and catalytic sites of the enzyme. All tested compounds exhibit significant AChE inhibitory activity. Competition assays using propidium as reference of selective ligand for the peripheral anionic site on acetylcholinesterase indicates the influence of the designed compounds over the peripheral site. They can be considered as new leads in the optimization of Alzheimer's disease modifying agents.

Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate.

DNA polymerase mu (Pol mu) is a DNA-dependent DNA polymerase closely related to terminal deoxynucleotidyl transferase (TdT), and prone to induce template/primer misalignments and misincorporation. In addition to a proposed general role in non-homologous end joining of double-strand breaks, its mutagenic potential and preferential expression in secondary lymphoid tissues support a role in somatic hypermutation (SHM) of immunoglobulin genes. Here, we show that human Pol mu protein is expressed in the nucleus of centroblasts obtained from human tonsils, forming a characteristic foci pattern resembling that of other DNA repair proteins in response to DNA damage. Overexpression of human Pol mu in Ramos cells, in which the SHM process is constitutive, augmented the somatic mutations specifically at the variable (V) region of the immunoglobulin genes. The nature of the mutations introduced, mostly base substitutions, supports the contribution of Pol mu to mutation of G and C residues during SHM. In vitro analysis of Pol mu misincorporation on specific templates, that mimic DNA repair intermediates and correspond to mutational hotspots, indicated that many of the mutations observed in vivo can be explained by the capacity of Pol mu to induce transient template/primer misalignments.

DNA polymerase lambda, a novel DNA repair enzyme in human cells.

DNA polymerase lambda (pol lambda) is a novel family X DNA polymerase that has been suggested to play a role in meiotic recombination and DNA repair. The recent demonstration of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in pol lambda supports a function of this enzyme in base excision repair. However, the biochemical properties of the polymerization activity of this enzyme are still largely unknown. We have cloned and purified human pol lambda to homogeneity in a soluble and active form, and we present here a biochemical description of its polymerization features. In support of a role in DNA repair, pol lambda inserts nucleotides in a DNA template-dependent manner and is processive in small gaps containing a 5'-phosphate group. These properties, together with its nucleotide insertion fidelity parameters and lack of proofreading activity, indicate that pol lambda is a novel beta-like DNA polymerase. However, the high affinity of pol lambda for dNTPs (37-fold over pol beta) is consistent with its possible involvement in DNA transactions occurring under low cellular levels of dNTPs. This suggests that, despite their similarities, pol beta and pol lambda have nonredundant in vivo functions.

Modulatory mechanism of the endogenous peptide catestatin on neuronal nicotinic acetylcholine receptors and exocytosis.

The catestatin fragment of chromogranin A is the first known endogenous compound able to inhibit catecholamine release elicited by the activation of neuronal nicotinic acetylcholine receptors (nAChRs) of different animal species and catecholaminergic cell types. However, how catestatin regulates the receptor activity, which subunit combination of the heteropentameric forms of receptor is better blocked by the peptide, or how it affects the different stages of the exocytotic process have not yet been evaluated. To address these questions, we have assayed the effects of catestatin: (first) on the inward currents elicited by ACh (I(ACh)) in voltage-clamped oocytes expressing different combinations of nAChR subunits; and (second) on the cytosolic Ca2+ concentration, [Ca2+]c, and quantal release of catecholamines simultaneously monitored in single adrenal chromaffin cells stimulated with ACh. Catestatin potently blocks all the subtypes of nAChRs studied. Furthermore, it inhibits the alpha3beta4 current in a reversible, noncompetitive, voltage-, and use-dependent manner, a behavior compatible with open-channel blockade. In fura-2-loaded single chromaffin cells, the peptide reduced the [Ca2+]c signal and the total release of catecholamines elicited by ACh; however, catestatin did not modify the kinetics or the last step of the exocytotic process. Our results suggest that catestatin might play an autocrine regulatory role in neuroendocrine secretion through its interaction with different native nAChR subtypes; the extent of receptor blockade by the peptide could be acutely regulated by the intensity and duration of the presynaptic stimulus.