Discovery of highly potent and selective D4 ligands by interactive SAR study

A series of thienylmethylphenylpiperazins was synthesized and tested for affinity towards the five subtypes of dopaminergic receptors. Compound 5f showed more than 1000 folds selectivity to D4 receptors; analogue 5e showed the highest affinity to D4 receptors with K_i 3.9 nM. An interactive SAR approach was adopted and lead to compound 14a with K_i (D4) as low as 0.03 nM. Molecular docking studies showed a potential, first to report arene cation interaction between the D4 unique residue Arg-186 and the ligands’ arene moiety, explaining the importance of having a strong negative electrostatic potential at this area of the compound structure.     

The ameliorating effects of 2,3-dihydroxy-4-methoxyacetophenone on scopolamine-induced memory impairment in mice and its neuroprotective activity

We isolated 2,3-dihydroxy-4-methoxyacetophenone, a neuroprotective compound from Cynenchum paniculatum in our previous study.The present study was conducted to investigate the possible neuroprotective effect of 2,3-dihydroxy-4-methoxyacetophenone that has been previously isolated from Cynenchum paniculatum on hippocampal neuronal cell line, HT22 cells and its possible cognitive-enhancing effect on scopolamine-induced amnesia in mice.Neuroprotective effect against glutamate-induced neurotoxicity in HT22 cells was evaluated by MTT assay. Also, cognitive enhancing effect against scopolamine (1 mg/kg, ip) induced learning and memory deficit was measured by Morris water maze test. Oral administered of 2,3-dihydroxy-4-methoxyacetophenone (1, 10, 20, 40 and 50 mg/kg) to amnesic mice induced by scopolamine. In Morris water maze test, 2,3-dihydroxy-4-methoxyacetophenone (50 mg/kg) improved the impairment of spatial memory induced by scopolamine. 2,3-Dihydroxy-4-methoxyacetophenone protect HT22 cells on glutamate induced cell-death in a dose-dependent manner (EC₅₀ value: 10.94 μM). Furthermore, 2,3-dihydroxy-4-methoxyacetophenone was found to inhibit [Ca²⁺] accumulation in HT22 cells and had antioxidantive activity. The results showed that 2,3-dihydroxy-4-methoxyacetophenone exert neuroprotective and cognitive-enhancing activities through its antioxidant activity. We suggest that 2,3-dihydroxy-4-methoxyacetophenone improves cognitive function and may be helpful for the treatment of Alzheimer’s disease.     

Phosphodiesterase inhibitors. Part 6: Design,synthesis, and structure–activity relationships of PDE4-inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory activity

We previously identified KCA-1490 [(?)-6-(7-methoxy-2-trifluoromethyl-pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone], a dual PDE3/4 inhibitor. In the present study, we found highly potent selective PDE4 inhibitors derived from the structure of KCA-1490. Among them, N-(3,5-dichloropyridin-4-yl)-7-methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridine-4-carboxamide (2a) had good anti-inflammatory effects in an animal model.     

PEG conjugates of potent α4 integrin inhibitors,maintaining sustained levels and bioactivity in vivo,following subcutaneous administration

Mitsunobu reactions were employed to link t-butyl esters of α4 integrin inhibitors at each of the termini of a three-arm, 40 kDa, branched PEG. Cleavage of the t-butyl esters using HCO₂H provided easily isolated PEG derivatives, which are potent α4 integrin inhibitors, and which achieve sustained levels and bioactivity in vivo, following subcutaneous administration to rats.     

Synthesis and optimization of a novel series of HCV NS3 protease inhibitors: 4-Arylproline analogs

In this report we describe the synthesis and evaluation of diverse 4-arylproline analogs as HCV NS3 protease inhibitors. Introduction of this novel P2 moiety opened up new SAR and, in combination with a synthetic approach providing a versatile handle, allowed for efficient exploitation of this novel series of NS3 protease inhibitors. Multiple structural modifications of the aryl group at the 4-proline, guided by structural analysis, led to the identification of analogs which were very potent in both enzymatic and cell based assays. The impact of this systematic SAR on different drug properties is reported.     

Design,synthesis and biological evaluation of novel aminothiazoles as antiviral compounds acting against human rhinovirus

We describe here the design, synthesis and biological evaluation of antiviral compounds acting against human rhinovirus (HRV). A series of aminothiazoles demonstrated pan-activity against the HRV genotypes screened and productive structure–activity relationships. A comprehensive investigational library was designed and performed allowing the identification of potent compounds with lower molecular weight and improved ADME profile. 31d-1, 31d-2, 31f showed good exposures in CD-1 mice. The mechanism of action was discovered to be a host target: the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). The identification of the pan-HRV active compound 31f combined with a structurally distinct literature compound T-00127-HEV1 allowed the assessment of target related tolerability of inhibiting this kinase for a short period of time in order to prevent HRV replication.     

Characterization of Tudor-sn-containing granules in the silkworm,Bombyx mori

The Tudor-sn protein, which contains four staphylococcal nuclease domains and a Tudor domain, is a ubiquitous protein found in almost all organisms. It has been reported that Tudor-sn in mammals participates in various cellular pathways involved in gene regulation, cell growth, and development. In insects, we have previously identified a Tudor-sn ortholog in the silkworm, Bombyx mori, and detected its interactions between with Argonaute proteins. The role of Tudor-sn in silkworm, however, still remains largely unknown. In this study, we demonstrated that silkworm Tudor-sn is a stress granule (SG) protein, and determined its interactions with other SG proteins using Bimolecular Fluorescence Complementation assay and Insect Two-Hybrid method. Depletions of Argonaute proteins and SG-marker protein Tia1 by RNAi impaired the involvement of Tudor-sn in the SG formation. Protein domain deletion analysis of Tudor-sn demonstrated that SN2 is the key domain required for the aggregation of Tudor-sn in SGs.     

In vitro characterization of the inhibitor resistance of glutamate-1-semialdehyde aminotransferase from the cyanobacterium Synechococcus PCC630l GR6

Glutamate-l-semialdehyde (GSA) aminotransferase catalyses the final step in the C5 pathway converting glutamate to the tetrapyrrole precursor δ-aminolaevulinic acid. This enzyme is sensitive to gabaculine (2,3-dihydro-3-amino benzoic acid) and to 4-amino-5-fluoropentanoic acid (AFPA), which are irreversible, mechanism-based inhibitors of pyridoxal phosphatedependent enzymes. Spontaneous mutants of Synechococcus PCC6301 resistant to these inhibitors contain altered enzyme that displays corresponding resistance to high concentrations of the inhibitor. The enzyme from strain GR6, resistant to both inhibitors, contains a three-amino-acid deletion at positions 5–7 and a Met²⁴⁸ → Ile substitution. The enzyme from strain K40 resistant to AFPA but not to gabaculine, contains a Ser¹⁶³ → Thr substitution. GSA aminotransferases containing either the deletion or the substitution that are characteristic of the GR6 mutant were produced in Escherichia coli using the expression vector pMalc2. These engineered mutant enzymes were characterized in terms of their catalytic parameters and sensitivities to gabaculine and AFPA. Furthermore, maltose binding protein/aminotransferase fusion proteins were characterized spectrophotometrically to monitor the interaction of bound cofactor with diamino- and dioxocompounds related to the substrate and both inhibitors. Results were compared with those for similarly produced recombinant wild-type, K40 and GR6 GSA aminotransferases. The engineered products with either the N-terminal deletion or the Met²⁴⁸ → Ile substitution displayed catalytic efficiencies that were intermediate between the wild-type and GR6 or K40 enzymes. However, with respect to their absorption spectra, sensitivity to inhibitors and the reactivity of bound cofactor, they were essentially wild-type. These in vitro studies demonstrate that both changes in enzyme structure are necessary to obtain the distinctive properties of the GR6 aminotransferase, including resistance to high concentrations of gabaculine and AFPA.     

CXCR4-derived synthetic peptides inducing anti-HIV-1 antibodies

Despite almost 30 years since the identification of the human immunodeficiency virus type I (HIV-1), development of effective AIDS vaccines has been hindered by the high mutability of HIV-1. The HIV-1 co-receptors CCR5 and CXCR4 are genetically stable, but viral proteins may mutate rapidly during the course of infection. CXCR4 is a seven transmembrane G protein-coupled receptor, possessing an N-terminal region (NT) and three extracellular loops (ECL1-3). Previous studies have shown that the CXCR4-ED-derived peptides inhibit the entry of HIV-1 by interacting with gp120, an HIV-1 envelope glycoprotein. In the present study, antigenicity of CXCR4-derived peptides has been investigated and the anti-HIV-1 effects of induced antisera have been assessed. It was found that CXCR4-ED-derived antigen molecules immunize mice, showing that the linear peptides have higher antigenicity than the cyclic peptides. The L1- and L2-induced antisera inhibited the HIV-1 entry significantly, while anti-N1 antibodies have no inhibitory activity. This study produced promising examples for the design of AIDS vaccines which target the human protein and can overcome mutability of HIV-1.