Search for small molecules with antipsychotic activity based on neurotensin

A series of systemically active N-acylprolyltyrosines was constructed based on the structure of endogenous neuroleptic-like tridecapeptide neurotensin that exhibits activity on intracerebral administration. Among these dipeptides, N-caproyl-L-prolyl-L-tyrosine methyl ester (Dilept) was chosen for further development. The paper briefly describes the main concepts of Dilept design and presents new experimental data of the Dilept effects in a ‘translational’ model of schizophrenia, i.e., the deficit of prepulse inhibition caused by dopamine-mimetic apomorphine or ketamine, a noncompetitive blocker of N-methyl-D-aspartic acid. Dilept demonstrated effectiveness in this model in both intraperitoneal and oral administration, which makes it possible to predict its application as an antipsychotic agent.     

Kinetic characterization of small DNA-binding molecules interacting with a DNA strand on a quartz crystal microbalance

Quantitative studies of the binding of various DNA-binding antibiotics with dsDNA are useful for drug design, not only for effective antibiotics, but also for antitumor drugs. We studied the binding kinetics, association and dissociation rate constants, and association constants (k_on, k_off, and K_a, respectively) of intercalators and groove binders, including various antibiotics, to double-stranded DNA (dA₃₀·dT₃₀ and dG₃₀·dC₃₀) immobilized on a highly sensitive 27 MHz quartz-crystal microbalance (QCM) in aqueous solution. Although a simple ethidium bromide intercalator bound to both dA₃₀·dT₃₀ and dG₃₀·dC₃₀, antibiotics that are side-binding intercalators, such as daunomycin, aclacinomycin A, and actinomycin D, with sugar or peptide moieties on the intercalator parts selectively bound to dG₃₀·dC₃₀ with high K_a and small k_off values. Nogalamycin, a dumbbell-shaped penetrating intercalator, showed low k_on and k_off values owing to slow duplex unwinding during the penetration process. Groove binders (Hoechst 33258, distamycin A, and mithramycin) had high K_a values owing to the high k_on values. Kinetic parameters depended largely on molecular shapes and DNA-binding molecule binding modes.     

Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts

The expression of 4 pluripotency genes (Oct4, Sox2, c-Myc and Klf4) in mouse embryonic fibroblasts can reprogramme them to a pluripotent state. We have investigated the expression of these pluripotency genes when human somatic 293T cells are permeabilized and incubated in extracts of mouse embryonic stem (ES) cells. Expression of all 4 genes was induced over 1–8 h. Gene expression was associated with loss of repressive histone H3 modifications and increased recruitment of RNA polymerase II at the promoters. Lamin A/C, which is typically found only in differentiated cells, was also removed from the nuclei. When 293T cells were returned to culture after exposure to ES cell extract, the expression of the pluripotency genes continued to rise over the following 48 h of culture, suggesting that long-term reprogramming of gene expression had been induced. This provides a methodology for studying the de-differentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells to a pluripotent state without genetically altering them.     

Development of novel small molecules for the treatment of ALS

Amyotrophic lateral sclerosis (ALS) is a rare and progressive neurodegenerative disease with unknown etiology. It is caused by the degeneration of motor neurons responsible for controlling voluntary muscles. It has been reported that mutations in the superoxide dismutase (SOD) 1 gene can lead to ALS. SOD1 abnormalities have been identified in both familial, as well as sporadic ALS cases. SOD2 is a highly inducible SOD that works in conjunction with SOD1. SOD2 can be induced through activation of NF-κBs. We previously reported that the novel small molecule, SRI-22818, increases NF-κB expression and activation and SOD2 levels in vitro and has activity in vivo in the SOD1-G93A reference model of ALS. We report herein the synthesis and biological evaluation of SRI-22818 analogs.     

Kinetic instability of p53 core domain mutants: implications for rescue by small molecules

Oncogenic mutations in the tumor suppressor protein p53 are found mainly in its DNA-binding core domain. Many of these mutants are thermodynamically unstable at body temperature. Here we show that these mutants also denature within minutes at 37 degrees C. The half-life (t(1/2)) of the unfolding of wild-type p53 core domain was 9 min. Hot spot mutants denatured more rapidly with increasing thermodynamic instability. The highly destabilized mutant I195T had a t(1/2) of less than 1 min. The wild-type p53-(94-360) construct, containing the core and tetramerization domains, was more stable, with t(1/2) = 37 min at 37 degrees C, similar to full-length p53. After unfolding, the denatured proteins aggregated, the rate increasing with higher concentrations of protein. A derivative of the p53-stabilizing peptide CDB3 significantly slowed down the unfolding rate of the p53 core domain. Drugs such as CDB3, which rescue the conformation of unstable mutants of p53, have to act during or immediately after biosynthesis. They should maintain the mutant protein in a folded conformation and prevent its aggregation, allowing it enough time to reach the nucleus and bind its sequence-specific target DNA or the p53 binding proteins that will stabilize it.     

Principles of signaling pathway modulation for enhancing human naive pluripotency induction

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Expanding chemical space of DNA-binding molecules with three base-binding units

A new molecule NC3-3 designed to expand chemical space of parent molecule NCD by adding the third base-binding unit was reported. NC3-3 bound to the G-G mismatch in the 5′-CGG-3′/5′-CGG-3′ motif but not to that in 5′-GGC-3′/5′-GGC-3′. This binding selectivity is similar to that reported for NCD. Fluorimetric screening of NCD and NC3-3 to dsDNA library containing yGw/xGz motifs showed that NC3-3 still kept the sequence selectivity as we observed for NCD-binding. The third naphthyridine heterocycle in NC3-3 affected the mode of the binding, but a little effect on the sequence selectivity.     

The search of small molecules with antipsychotic activity on the background of neurotensin

Tridecapeptide neurotensin (NT) is known to exert the neuroleptic-like effects in case of its intracerebral administration. The group of systemically active dipeptides , acylprolyltyrosines, was constructed on the background of NT. Methyl ester of N-caproyl-L-prolyl-L-tyrosine (Dilept) was chosen for further development. The paper is dealing with main principles of Dilept'design and with analysis of the experimental data concerning its effect on the "translational" model of schizophrenia--the deficit of prepulse inhibition of the acoustic startle-reaction caused by either dopamine-mimetic, apomorphine, or by the uncompetitive NMDA-blocker, ketamine. Dilept was shown to attenuate these deficits both in case ofintraperitoneal and peroral administration. Dilept is considered as a potential antipsychotic.     

Analysis of epidermal proteins for DNA-binding activity

A group of DNA-binding proteins from the soluble extract of newborn rat epidermis have been separated by chromatography using DNA-cellulose columns. The electrophoretogram of the DNA-binding proteins eluted from a single stranded DNA-cellulose column shows five major proteins of molecular weights ranging between 25K to 40K. Both the epidermal protein filaggrin and most keratins, except two high molecular weight keratins, do not show in vitro DNA-binding activity.     

DNMT3B inhibits the re-expression of genes associated with induced pluripotency

DNMT3B is a de novo DNA methyltransferase that is highly expressed in mouse and human embryonic stem (ES) cells and has been shown to be essential for differentiation of mouse ES cells toward different lineages. In the present study, we found that DNMT3B is rapidly down-regulated in human ES cells during retinoic acid (RA)-induced differentiation compared with DNMT3A2, which is also highly expressed in ES cells. Silencing of DNMT3B in human ES cells by an inducible shRNAi system leads to a reduction of clonal ability of the stem cells, while expression of OCT4 and NANOG is unchanged. By contrast, the germline-specific genes VASA and SCP3 and the surface antigen BE12 are down regulated following DNMT3B knockdown. Upon retinoic acid-induced differentiation, we found that depletion of DNMT3B leads to a decrease in expression of the surface antigen A2B5 and of neural tube-associated genes PAX7 and BRN3A. Consistent with its importance in stem cell differentiation, we observed that silencing of DNMT3B facilitates the generation of cells that bear the hallmarks of pluripotency. Our findings suggest a role of DNMT3B in controlling the differentiation of human ES cells and in the generation of iPS cells.