Studies on non-steroidal inhibitors of aromatase enzyme; 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives

Steroidal and non-steroidal aromatase inhibitors target the suppression of estrogen biosynthesis in the treatment of breast cancer. Researchers have increasingly focused on developing non-steroidal derivatives for their potential clinical use avoiding steroidal side-effects.Non-steroidal derivatives generally have planar aromatic structures attached to the azole ring system. One part of this ring system comprises functional groups that inhibit aromatization through the coordination of the haem group of the aromatase enzyme. Replacement of the triazole ring system and development of aromatic/cyclic structures of the side chain can increase selectivity over aromatase enzyme inhibition.In this study, 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives were synthesized and physical analyses and structural determination studies were performed. The IC₅₀ values were determined by a fluorescence-based aromatase inhibition assay and compound 1 (4-(2-hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole) were found potent inhibitor of enzyme (IC₅₀:0.42?nM). Then, their antiproliferative activity over MCF-7 and HEK-293 cell lines was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds 1, 7, 8, 13, 15, 18, 21 were active against MCF-7 breast cancer cells. Lastly, a series of docking experiments were undertaken to analyze the crystal structure of human placental aromatase and identify the possible interactions between the most active structure and the active site.     

Characterization of a Highly Conserved Binding Site of Mlh1 Required for Exonuclease I-Dependent Mismatch Repair

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K_d values ranging from 8.1 to 17.4 μM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.     

Crystal Structure of LipL32, the Most Abundant Surface Protein of Pathogenic Leptospira spp.

Spirochetes of the genus Leptospira cause leptospirosis in humans and animals worldwide. Proteins exposed on the bacterial cell surface are implicated in the pathogenesis of leptospirosis. However, the biological role of the majority of these proteins is unknown; this is principally due to the lack of genetic systems for investigating Leptospira and the absence of any structural information on leptospiral antigens. To address this, we have determined the 2.0-Å-resolution structure of the lipoprotein LipL32, the most abundant outer-membrane and surface protein present exclusively in pathogenic Leptospira species. The extracellular domain of LipL32 revealed a compact, globular, “jelly-roll” fold from which projected an unusual extended β-hairpin that served as a principal mediator of the observed crystallographic dimer. Two acid-rich patches were also identified as potential binding sites for positively charged ligands, such as laminin, to which LipL32 has a propensity to bind. Although LipL32 shared no significant sequence identity to any known protein, it possessed structural homology to the adhesins that bind components of the extracellular matrix, suggesting that LipL32 functions in an analogous manner. Moreover, the structure provides a framework for understanding the immunological role of this major surface lipoprotein.     

Hijacking of a substrate-binding protein scaffold for use in mycobacterial cell wall biosynthesis

The waxy cell wall is crucial to the survival of mycobacteria within the infected host. The cell wall is a complex structure rich in unusual molecules that includes two related lipoglycans, the phosphatidylinositol mannosides (PIMs) and lipoarabinomannans (LAMs). Many proteins implicated in the PIM/LAM biosynthetic pathway, while attractive therapeutic targets, are poorly defined. The 2.4A resolution crystal structure of an essential lipoprotein, LpqW, implicated in LAM biosynthesis is reported here. LpqW adopts a scaffold reminiscent of the distantly related, promiscuous substrate-binding proteins of the ATP-binding cassette import system. Nevertheless, the unique closed conformation of LpqW suggests that mycobacteria and other closely related pathogens have hijacked this scaffold for use in key processes of cell wall biosynthesis. In silico docking provided a plausible model in which the candidate PIM ligand binds within a marked electronegative region located on the surface of LpqW. We suggest that LpqW represents an archetypal lipoprotein that channels intermediates from a pathway for mature PIM production into a pathway for LAM biosynthesis, thus controlling the relative abundance of these two important components of the cell wall.     

Melatonin prevents oxidant damage in various tissues of rats with hyperthyroidism

Impairment of thyroid functions brings about pathological changes in different organs of body. Findings of in vivo and in vitro studies indicate that thyroid hormones have a considerable impact on oxidative stress. Melatonin reduces oxidative damage through its free radical eliminating and direct anti-oxidant effects. The present study was undertaken to determine how a 3-week period of intraperitoneal melatonin administration affected oxidative damage caused in experimental hyperthyroidism in rat. The experimental animals were divided into 3 groups (control, hyperthyroidism, hyperthyroidism+melatonin). Malondialdehyde (MDA) and glutathione (GSH) levels were determined in different tissues. MDA levels in cerebral, liver and cardiac tissues in hyperthyroidism group were significantly higher than those in control and hyperthyroidism+melatonin supplemented groups (p<0.001). The highest GSH levels were observed in the group that was administered melatonin in addition to having hyperthyroidism (p<0.001). These results show that hyperthyroidism increased oxidative damage in cerebral, hepatic and cardiac tissues of rat. Melatonin supplementation may also suppress oxidative damage.     

Quantitative Magnetic Resonance (QMR) for Longitudinal Evaluation of Body Composition Changes With Two Dietary Regimens

We have recently reported a validation study of a prototype low‐field strength quantitative magnetic resonance (QMR) instrument for measurement of human body composition (EchoMRI‐AH). QMR was very precise, but underreported fat mass (FM) by 2–4 kg when compared to a 4‐compartment (4C) model in this cross‐sectional study. Here, we report the performance of an updated instrument in two longitudinal studies where FM was decreasing. Healthy obese volunteers were given a modest energy deficit diet for 8 weeks (study A) and obese patients with heart failure and/or at high cardiovascular risk were prescribed a low energy liquid diet for 6 weeks (study B). FM was measured at the start and end of these periods by QMR, dual‐energy X‐ray absorptiometry (DXA) and 4C. A higher proportion of the weight lost came from fat in study A compared with study B, where loss of total body water (TBW) played a greater part. The intraclass correlation between QMR and 4C estimates of FM loss (ΔFat) was 0.95, but 20 of 22 estimates of ΔFat by QMR were lower than the corresponding estimate by the 4C model. Bland–Altman analysis demonstrated that estimates of FM loss by QMR were ～1.0 and 0.7 kg lower than those obtained with 4C (P = 0.0008) and DXA (P = 0.049), respectively. Measurement precision remained high. QMR measurement should prove valuable for quantifying modest changes of FM in small trials.     

Synthesis and biological evaluation of aminomethyl and alkoxymethyl derivatives as carbonic anhydrase,acetylcholinesterase and butyrylcholinesterase inhibitors

Compounds containing nitrogen and sulfur atoms can be widely used in various fields such as industry, medicine, biotechnology and chemical technology. Therefore, the reactions of aminomethylation and alkoxymethylation of mercaptobenzothiazole, mercaptobenzoxazole and 2-aminothiazole were developed. Additionally, the alkoxymethyl derivatives of mercaptobenzoxazole and 2-aminothiazole were synthesized by a reaction with hemiformals, which are prepared by the reaction of alcohols and formaldehyde. In this study, the inhibitory effects of these molecules were investigated against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) enzymes and carbonic anhydrase I, and II isoenzymes (hCA I and II). Both hCA isoenzymes were significantly inhibited by the recently synthesized molecules, with K_i values in the range of 58–157?nM for hCA I, and 81–215?nM for hCA II. Additionally, the K_i parameters of these molecules for BChE and AChE were calculated in the ranges 23–88 and 18–78?nM, respectively.     

The small CAB-like proteins of the cyanobacterium Synechocystis sp. PCC 6803: their involvement in chlorophyll biogenesis for Photosystem II

The five small CAB-like proteins (ScpA-E) of the cyanobacterium Synechocystis sp. PCC 6803 belong to the family of stress-induced light-harvesting-like proteins, but are constitutively expressed in a mutant deficient of Photosystem I (PSI). Using absorption, fluorescence and thermoluminescence measurements this PSI-less strain was compared with a mutant, in which all SCPs were additionally deleted. Depletion of SCPs led to structural rearrangements in Photosystem II (PSII): less photosystems were assembled; and in these, the Q(B) site was modified. Despite the lower amount of PSII, the SCP-deficient cells contained the same amount of phycobilisomes (PBS) as the control. Although the excess PBS were functionally disconnected, their fluorescence was quenched under high irradiance by the activated Orange Carotenoid Protein (OCP). Additionally the amount of OCP, but not of the iron-stress induced protein (isiA), was higher in this SCP-depleted mutant compared with the control. As previously described, the lack of SCPs affects the chlorophyll biosynthesis (Vavilin, D., Brune, D. C., Vermaas, W. (2005) Biochim Biophys Acta 1708, 91-101). We demonstrate that chlorophyll synthesis is required for efficient PSII repair and that it is partly impaired in the absence of SCPs. At the same time, the amount of chlorophyll also seems to influence the expression of ScpC and ScpD.