Current Enzyme Inhibition (v.11, #1)

Meet Our Editor: by Claudiu T. Supuran (1-2).

Preface: by Claudiu T. Supuran (3-3).

The central nervous system (CNS) is composed of numerous neural cells such as neurons and glias that have wide cell surface and numerous processes covered with plasma membranes. As the plasma membrane is enriched in cholesterol, the CNS composed of numerous neural cells with wide plasma membrane is a cholesterol- rich organ. Because inhibition of cholesterol supply induces cell death into neurons, the CNS is also an organ to highly require cholesterol. It is known that intercellular cholesterol transport is mainly maintained by generation of apolipoprotein- mediated high-density lipoproteins (HDL) in astrocytes and its supply to neuronal cells. Therefore, a study to elucidate a mechanism underlying the generation of HDL in astrocytes is one of important research subjects to understand cholesterol homeostasis in the brain.
In this context, it is of note that we have found cytosolic lipid-protein particles (CLPPs) to mediate intracellular cholesterol transport leading to HDL generation in rat astrocytes. The CLPPs are lipoprotein- like complexes composed of proteins such as caveolin-1 and cyclophilin A and lipids such as cholesterol, sphingomyelin, and phosphatidylcholine in the cytosol. It was shown by using specific enzyme inhibitors that exogenous apolipoprotein A-I (apoA-I) interacts with the membraneassociated ATP-binding cassette transporter A1 (ABCA1) to promote the CLPP-mediated intracellular transport of newly synthesized cholesterol through activation of ABCA1-binding phospholipase Cγ and protein kinase Cα. An inhibitor of phosphatidylcholine-specific phospholipase C, D609, reduces sphingomyelin synthesis and apoA-I-mediated HDL generation. These findings suggest that sphingomyelin synthesis is important for intracellular cholesterol transport to the plasma membrane and cholesterol efflux to generate apoA-I-containing HDL (apoA-I/HDL). A cyclophilin A inhibitor, cyclosporin A (Cs-A), suppresses not only exogenous apoA-I-mediated cholesterol efflux but also intracellular cholesterol transport through the inhibition of assembly of caveolin-1 and cholesterol to the CLPPs. Based on these findings, it is suggested that the CLPPs contribute to the intracellular cholesterol transport and cholesterol efflux to generate apoA-I/HDL. The mechanism underlying intracellular cholesterol transport, by which HDL generation and cholesterol homeostasis are regulated in the brain, is discussed focusing on our findings in this review.

Quantitative Enzymology by David J. Timson (12-31).
Accurate measurement of the quantitative aspects of enzyme-catalysed reactions is critical for a deeper understanding of their mechanisms, for their exploitation in biotechnology and for targeting enzymes by drug-like molecules. It is important to move beyond basic enzyme kinetics as encapsulated in the Michaelis-Menten equation. The type and magnitude of inhibition should be determined. Since the majority of enzyme-catalysed reactions involve more than one substrate, it is critical to understand how to treat these reactions quantitatively and how their kinetic behaviour depends on the type of mechanism occurring. Some reactions do not conform to 'standard' Michaelis-Menten treatment and exhibit phenomena such as cooperativity. Again it is important to put these phenomena onto a quantitative basis. Similarly the treatment of the effects of pH on enzymes is often vague and uninformative without a proper quantitative treatment. This review brings together tools and approaches for dealing with enzymes quantitatively together with original references for these approaches.

The new 3,4-dimethoxybenzoate complex of Cu(II) with nicotinamide was synthesized and characterized by elemental analysis, FT-IR spectroscopic study, UV-Vis spectrophotometry and mass spectrometry. The thermal behavior of the complex was studied by simultaneous TG, DTG and DTA methods in static air atmosphere. The infrared spectral characteristics of the complex is also discussed. In this complex, all ligands are coordinated to the metal ion as monodendate ligands. The final decomposition products were found to be the respective metal oxide. As it is well known, metal complexes of biologically important ligands are sometimes more effective than the free ligands. In this study the new compound was synthesized due to its functional groups such as veratric acid and nikotin amide, which have known biological characteristics and effective in terms of both enzyme and antifungal properties from recent studies. Therefore, antifulgal activity and enzyme inhibition properties of this complex have been examined.

Inhibition of Microbial Alkaline Phosphatase by Cimetidine; Kinetics and Molecular Model of Binding by Dariush Minai-Tehrani, Zohreh Soheili, Elnaz Yahyavi (39-45).
Cimetidine is a histamine H2-receptor antagonist which is used in gastrointestinal diseases. Although this drug is designed for H2 receptor, it can also attach to other enzymes. Alkaline phosphatase (ALP) is an important enzyme used for obtaining inorganic phosphate from organic phosphorylated substrate in microorganisms.
In this study, effect of cimetidine on the ALP of Escherichia coli and Saccharomyces cerevisiae was investigated and compared. Cultured cells were disrupted by ultrasound and the cell free extract was used for enzyme assay. The results showed that cimetidine inhibited ALP with un-competitive and competitive inhibition in E. coli and S. cerevisiae, respectively. Lower IC50 and Ki of the drug in E. coli determined that the drug bound to ALP of E. coli with higher affinity than the ALP of S. cerevisiae. The proposed molecular modeling suggested that, in E. coli, cimetidine bound to Mg2+ ion of active site, while in S. cerevisiae, the drug bound to Zn2+ ion. In conclusion, different patterns of pH, temperature profile, kinetic parameter, and type of inhibition demonstrated that the ALP of E. coli and S. cerevisiae were isozymes and cimetidine bound to these isozymes with different mechanisms.

Cysteine and Its Derivatives as New Delhi Metallo-beta-lactamase-1 Inhibitors by Cui-Gai Bai, Yin-Tong Xu, Ning-Ning Li, Jing-Han Wang, Cheng Yang, Yue Chen, Hong-Gang Zhou (46-57).
Nearly all antibiotics are ineffective to the antibiotic-resistant bacteria New Delhi metallo-beta -lactamase-1(NDM-1), one of the most important reasons is that antibiotics can be hydrolyzed by NDM-1 in these bacteria. Up to date, Many compounds, including captopril, are found to possess NDM-1 inhibition activity. Herein we report that some cysteine derivatives or homocysteine derivatives can inhibit the NDM-1 protein, and the lead compound 9 has the inhibition of the NDM-1 protein with IC50 of 1 µM, which is about 8 times potency of captopril.

Screening and Partial Purification of Cholinesterase Inhibitor from Microalgae by T. Vinoth Kumar, Rincy Yesudas, D. Geetharamani, S. Lakshmanasenthil, G Suja, B. V. Amritha Krishna, Amrutha Chacko (58-64).
The present study was carried out to screen microalgae for anticholinesterases. Acetylcholinesterase and butyrylcholinesterase were collected from human RBC ghost and sera respectively. Different algal species were collected from different study area and they were screened, isolated and cultivated with essential media. Crude extracts of the selected microalgae were fractionated by sequential extraction using various organic solvents (petroleum ether, chloroform, acetone and methanol). Cholinesterase enzyme activity and inhibitory activity were performed. Chloroform extract of Oscillatoria sp. showed maximum inhibition of 87.53% against acetylcholinesterase and acetone extract of Phormidium sp. showed maximum inhibition of 36.11% to butyrylcholinesterase. Eight fractions were isolated by TLC and the maximum inhibitory activity towards acetylcholinesterase was shown by the fraction 3 with the inhibition of 70.93 %, followed by fraction 8 with the inhibition of 60.12 %. The minimum inhibitory activity towards acetylcholinesterase was shown by fraction 1 with the inhibition of 43.03%. Functional groups of fraction no. 3 were determined and identified by FTIR.

The steroid derivative EM-1913 is a non-estrogenic inhibitor of steroid sulfatase (STS), an enzyme involved in the biosynthesis of estrogens and androgens. As an approach to treat estrogen-dependent diseases such as breast cancer, we want to test EM-1913 in an estrogen-dependent breast cancer tumor model in vivo. Ovariectomized nude mice were inoculated with estrogen-sensitive human breast cancer MCF-7 cells and stimulated with sulfated estradiol (E2S), which is transformed into the potent estrogen estradiol by STS. Tumors generated after 28 days were treated subcutaneously with EM-1913 administered in a mixture of ethanol (8%) and propylene glycol (92%). The three doses tested (100 µg, 4 mg/kg; 200 µg, 8 mg/kg; 500 µg, 20 mg/kg) blocked tumor growth induced by E2S. No apparent signs of toxicity in animals were observed at the different doses during the 30-day treatment period and at the end of treatment by measuring the body and liver weights. On a panel of cancer cell lines, we observed only very low cytotoxicity at high concentrations (over 10 µM). These in vivo and in vitro results confirm the potential of EM-1913 as an anticancer agent to treat estrogen-dependent diseases, at least in a xenograft model of breast cancer, but its use can also be extended to androgen-dependent diseases such as prostate cancer.