George Kontopidis

The core lipocalin, bovine β-lactoglobulin.

Abstract The lipocalin family became established shortly after the structural similarity was noted between plasma retinol binding protein and the bovine milk protein, β-lactoglobulin. During the past 60 years, β-lactoglobulin has been studied by essentially every biochemical technique available and so there is a huge literature upon its properties. Despite all of these studies, no specific biological function has been ascribed definitively to the protein, although several possibilities have been suggested. During the processing of milk on an industrial scale, the unpredictable nature of the process has been put down to the presence of β-lactoglobulin and certainly the whey protein has been implicated in the initiation of aggregation that leads to the fouling of heat exchangers. This short review of the properties of the protein will concentrate mainly on studies carried out under essentially physiological conditions and will review briefly some of the possible functions for the protein that have been described.

The Ligand-binding Site of Bovine β-Lactoglobulin: Evidence for a Function?

Ever since the fortuitous observation that beta-lactoglobulin (beta-Lg), the major whey protein in the milk of ruminants, bound retinol, the details of the binding have been controversial. beta-Lg is a lipocalin, like plasma retinol-binding protein, so that ligand association was expected to make use of the central cavity in the protein. However, an early crystallographic analysis and some of the more recent solution studies indicated binding elsewhere. We have now determined the crystal structures of the complexes of the trigonal form of beta-Lg at pH 7.5 with bound retinol (R=21.4% for 7329 reflections between 20 and 2.4 A resolution, R(free)=30.6%) and with bound retinoic acid (R=22.7% for 7813 reflections between 20 and 2.34 A resolution, R(free)=29.8%). Both ligands are found to occupy the central calyx in a manner similar to retinol binding in retinol-binding protein. We find no evidence of binding at the putative external binding site in either of these structural analyses. Further, competition between palmitic acid and retinol reveals only palmitate bound to the protein. An explanation is provided for the lack of ligand binding to the orthorhombic crystal form also obtained at pH 7.5. Finally, the possible function of beta-Lg is discussed in the light of its species distribution and similarity to other lipocalins.

Discovery of a novel family of CDK inhibitors with the program LIDAEUS: structural basis for ligand-induced disordering of the activation loop

A family of 4-heteroaryl-2-amino-pyrimidine CDK2 inhibitor lead compounds was discovered with the new database-mining program LIDAEUS through in silico screening. Four compounds with IC(50) values ranging from 17 to 0.9 microM were selected for X-ray crystal analysis. Two distinct binding modes are observed, one of which resembles the hydrogen bonding pattern of bound ATP. In the second binding mode, the ligands trigger a conformational change in the activation T loop by inducing movement of Lys(33) and Asp(145) side chains. The family of molecules discovered provides an excellent starting point for the design and synthesis of tight binding inhibitors, which may lead to a new class of antiproliferative drugs.

Discovery of N-Phenyl-4-(thiazol-5-yl)pyrimidin-2-amine Aurora Kinase Inhibitors

Through cell-based screening of our kinase-directed compound collection, we discovered that a subset of N-phenyl-4-(thiazol-5-yl)pyrimidin-2-amines were potent cytotoxic agents against cancer cell lines, suppressed mitotic histone H3 phosphorylation, and caused aberrant mitotic phenotypes. It was subsequently established that these compounds were in fact potent inhibitors of aurora A and B kinases. It was shown that potency and selectivity of aurora kinase inhibition correlated with the presence of a substituent at the aniline para-position in these compounds. The anticancer effects of lead compound 4-methyl-5-(2-(4-morpholinophenylamino)pyrimidin-4-yl)thiazol-2-amine (18; K(i) values of 8.0 and 9.2 nM for aurora A and B, respectively) were shown to emanate from cell death following mitotic failure and increased polyploidy as a consequence of cellular inhibition of aurora A and B kinases. Preliminary in vivo assessment showed that compound 18 was orally bioavailable and possessed anticancer activity. Compound 18 (CYC116) is currently undergoing phase I clinical evaluation in cancer patients.

Design, synthesis, biological activity and structural analysis of cyclic peptide inhibitors targeting the substrate recruitment site of cyclin-dependent kinase complexes.

Inhibition of cyclin A- and cyclin E-associated cyclin-dependent kinase-2 (CDK2) activities is an effective way of selective induction of apoptotic cell death via the E2F pathway in tumour cells. The cyclin groove recognition motif (CRM) in the natural CDK-inhibitory (CDKI) tumour suppressor protein p27KIP1 was used as the basis for the design and synthesis of a series of cyclic peptides whose biological activity and structural characterisation by NMR and X-ray crystallography is reported. Whereas linear p27KIP1 sequence peptides were comparatively ineffective, introduction of side chain-to-tail constraints was found to be productive. An optimal macrocyclic ring size for the conformational constraint was determined, mimicking the intramolecular H-bonding system of p27. Molecular dynamics calculations of various macrocycles suggested a close correlation between ring flexibility and biological activity. Truncated inhibitor peptide analogues also confirmed the hypothesis that introduction of a cyclic conformational constraint is favourable in terms of affinity and potency. The structural basis for the potency increase in cyclic versus linear peptides was demonstrated through the determination and interpretation of X-ray crystal structures of complexes between CDK2/cylin A (CDK2A) and a constrained pentapeptide.

Performance and antioxidant status of broiler chickens supplemented with dried mushrooms (Agaricus bisporus) in their diet

In this study, we evaluated the growth performance and antioxidant status of broiler chicken supplemented with the edible mushroom Agaricus bisporus. Ninety 1-d-old female broiler chickens randomly allotted to 3 dietary treatments were given either a nutritionally balanced basal diet or the basal diet supplemented with 10 or 20 g of dried mushroom/kg of feed for 6 wk on an ad libitum basis. Body weight, feed intake, and feed conversion ratio values were monitored weekly. To evaluate the antioxidant status of broiler chicken, refrigerated liver, breast, and thigh tissues were assayed for levels of glutathione, reduced glutathione, glutathione reductase, glutathione peroxidase, and glutathione S-transferase, as well as malondialdehyde at 6 wk of age. Results showed that dietary mushroom supplementation at both inclusion levels was accepted well by the broiler chicken and improved feed efficiency compared with the control diet. Dietary mushroom inclusion at 20 g/kg improved both growth performance and feed efficiency compared with control diet at 42 d of age. Dietary mushroom at both inclusion levels reduced malondialdehyde production in liver, breast, and thigh tissues and elevated glutathione peroxidase, reduced glutathione, glutathione reductase, and glutathione S-transferase compared with the control treatment, the effects being dose-dependent. These results suggest that A. bisporus mushroom exerts both a growth-promoting and tissue antioxidant-protective activity when supplemented in broiler chicken diets.

Variation in trace element contents among chicken, turkey, duck, goose, and pigeon eggs analyzed by inductively coupled plasma mass spectrometry (ICP-MS).

Despite substantial interest in the trace element content of eggs by poultry breeders, nutritionists, and environmental scientists, available data about trace elements levels in eggs are scarce. Trace element contents in yolk and albumen of chicken, turkey, duck, goose, and pigeon eggs were analyzed to establish a baseline dataset and assess differences among trace element content in avian species. We measured the selenium (Se), zinc (Zn), manganese (Mn), cobalt (Co), copper (Cu), molybdenum (Mo), vanadium (V), chromium (Cr), nickel (Ni), arsenic (As), cadmium (Cd), and thallium (Tl) contents in both yolk and albumen by inductively coupled plasma mass spectrometry. One hundred twenty eggs deriving from 24 birds of each species, reared in the same poultry farm in northern Greece, were used; bird feed was common and based on cereals and legumes and contained no added vitamins or microminerals. Trace element contents in yolks were far higher than those in albumen, except for V and Ni. In yolks, the highest content for Se, Mo, and Tl were in pigeon eggs, for Zn, Mn, Cu, and Cr in turkey eggs, and for Co and Ni in goose eggs. In albumen, Se was highest in duck eggs, while Zn, Mn, and Co in pigeon ones. It is concluded that there is a substantial, up to threefold, variation for trace element contents in eggs among different domestic avian species offered the same feed.

REPLACE: a strategy for iterative design of cyclin-binding groove inhibitors.

We describe a drug‐design strategy termed REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) in which nonpeptidic surrogates for specific determinants of known peptide ligands are identified in silico by using a core peptide‐bound protein structure as a design anchor. In the REPLACE application example, we present the effective replacement of two critical binding motifs in a lead protein–protein interaction inhibitor pentapeptide with more druglike phenyltriazole and diphenyl ether groups. These were identified through docking of fragment libraries into the volume of the cyclin‐binding groove of CDK2/cyclin A vacated through truncation of the inhibitor peptide‐binding determinants. Proof of concept for this strategy was obtained through the generation of potent peptide–small‐molecule hybrids and by the confirmation of inhibitor‐binding modes in X‐ray crystal structures. This method therefore allows nonpeptide fragments to be identified without the requirement for a high‐sensitivity binding assay and should be generally applicable in replacing amino acids as individual residues or groups in peptide inhibitors to generate pharmaceutically acceptable lead molecules.

Dietary Supplementation of Benzoic Acid and Essential Oil Compounds Affects Buffering Capacity of the Feeds, Performance of Turkey Poults and Their Antioxidant Status, pH in the Digestive Tract, Intestinal Microbiota and Morphology

Three trials were conducted to evaluate the effect of supplementation of a basal diet with benzoic acid or thymol or a mixture of essential oil blends (MEO) or a combination of benzoic acid with MEO (BMEO) on growth performance of turkey poults. Control groups were fed a basal diet. In trial 1, benzoic acid was supplied at levels of 300 and 1,000 mg/kg. In trial 2, thymol or the MEO were supplied at levels of 30 mg/kg. In trial 3, the combination of benzoic acid with MEO was evaluated. Benzoic acid, MEO and BMEO improved performance, increased lactic acid bacteria populations and decreased coliform bacteria in the caeca. Thymol, MEO and BMEO improved antioxidant status of turkeys. Benzoic acid and BMEO reduced the buffering capacity compared to control feed and the pH values of the caecal content. Benzoic acid and EOs may be suggested as an effective alternative to AGP in turkeys.

Milk protein structure - what can it tell the dairy industry?

This paper describes the possible usefulness of knowledge of the three-dimensional structure of milk proteins to dairy scientists. After a brief introduction of the available methodology and the structures of milk proteins that are already available, the structure of bovine β-lactoglobulin is used to illustrate the possible applications of the structure to understanding the problems to which the protein contributes during milk processing.