Emmanuel Mikros

Roscovitine-derived, dual-specificity inhibitors of cyclin-dependent kinases and casein kinases 1.

Cyclin-dependent kinases (CDKs) and casein kinases 1 (CK1) are involved in the two key molecular features of Alzheimers disease, production of amyloid-beta peptides (extracellular plaques) and hyper-phosphorylation of Tau (intracellular neurofibrillary tangles). A series of 2,6,9-trisubstituted purines, structurally related to the CDK inhibitor roscovitine, have been synthesized. They mainly differ by the substituent on the C-6 position. These compounds were screened for kinase inhibitory activities and antiproliferative effects. Several biaryl derivatives displayed potent inhibition of both CDKs and CK1. In particular, derivative 13a was a potent inhibitor of CDK1/cyclin B (IC 50: 220 nM), CDK5/p25 (IC 50: 80 nM), and CK1 (IC 50: 14 nM). Modeling of these molecules into the ATP-binding pocket of CK1delta provided a rationale for the increased selectivity toward this kinase. 13a was able to prevent the CK1-dependent production of amyloid-beta in a cell model. CDK/CK1 dual-specificity inhibitors may have important applications in Alzheimers disease and cancers.

1H NMR-Based Metabonomics for the Classification of Greek Wines According to Variety, Region, and Vintage. Comparison with HPLC Data

A sensitive and simple method was developed for the classification of wines according to variety, geographical origin, and vintage using NMR-based metabonomics. Polyphenol-rich extracts were prepared from 67 varietal wines from the principal wine-producing regions of Greece, using adsorption resin XAD-4. 1D (1)H NMR spectra obtained from the corresponding extracts were segmented, integrated, and normalized, and the data were subjected to principal component analysis. The chemometric classification of wines according to their phenolic profile allows discrimination between wines from different wineries of the same wine-producing zone and between different vintages for wines of the same variety.

(1)H NMR-based metabonomic investigation of the effect of two different exercise sessions on the metabolic fingerprint of human urine.

Physical exercise modifies animal metabolism profoundly. Until recently, biochemical investigations related to exercise focused on a small number of biomolecules. In the present study, we used a holistic analytical approach to investigate changes in the human urine metabolome elicited by two exercise sessions differing in the duration of the rest interval between repeated efforts. Twelve men performed three sets of two 80 m maximal runs separated by either 10 s or 1 min of rest. Analysis of pre- and postexercise urine samples by (1)H NMR spectroscopy and subsequent multivariate statistical analysis revealed alterations in the levels of 22 metabolites. Urine samples were safely classified according to exercise protocol even when applying unsupervised methods of statistical analysis. Separation of pre- from postexercise samples was mainly due to lactate, pyruvate, hypoxanthine, compounds of the Krebs cycle, amino acids, and products of branched-chain amino acid (BCAA) catabolism. Separation of the two rest intervals was mainly due to lactate, pyruvate, alanine, compounds of the Krebs cycle, and 2-oxoacids of BCAA, all of which increased more with the shorter interval. Metabonomics provides a powerful methodology to gain insight in metabolic changes induced by specific training protocols and may thus advance our knowledge of exercise biochemistry.

Recent advances and new strategies in the NMR-based identification of natural products.

Nature comprises an untapped pool of unique compounds with high structural uniqueness and exceptional properties. At the core of natural products (NPs) discovery is the identification procedure and NMR remains the most efficient method. Technical improvements such as miniaturized and crycogenic NMR probes along with hyphenation capabilities and computational support are at the center of evolution. Concepts such as dereplication and metabolomics are increasingly adopted in NPs using the power of databases, currently fragmented. The introduction and utilization of these technical and computational implements could lead NPs research to more comprehensive structure identification and new holistic perspectives.

Metabonomic identification of novel biomarkers in doxorubicin cardiotoxicity and protective effect of the natural antioxidant oleuropein.

Doxorubicin (DXR) is a commonly used antineoplastic agent; however, its use is limited due to cardiotoxicity. Oxidative stress and consequent alterations of cardiac energetics are involved in the development of DXR toxicity. Oleuropein (Oleu) is a phenolic antioxidant, present in olive tree, reported to confer protection against DXR cardiotoxicity. In this study, NMR based‐metabonomics was applied to characterize the metabolic profile of the acute DXR cardiotoxicity in rats and to evaluate the metabolic alterations conferred by co‐treatment with Oleu. Wistar rats were divided into six groups and treated as follows: control group with a single injection of 2 mL normal saline intraperitoneally (i.p.), DXR group with a single dose of 20 mg/kg, i.p and DXR plus Oleu groups with 20mg/kg DXR i.p., and 100 or 200 mg/kg/BW of Oleu i.p. for 5 or 3 consecutive days starting either 2 days before or on the day of DXR administration. Hearts were excised 72 h after DXR treatment and 1H‐NMR spectra of aqueous myocardium extracts were recorded. Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis (PLS‐DA) revealed differences in the metabolic profile between control and DXR attributed to several metabolites. A number of them were quantified by integration of the NMR spectra. Myocardial levels of acetate and succinate were increased in DXR compared to controls, while branched amino acids were decreased. These results correlate with nonenzymatic conversion of pyruvate to acetate and of α‐ketoglutarate to succinate by DXR free radicals. Oleu completely restored the changes of metabolites to the normal levels. Acetate and succinate constitute novel biomarkers related to DXR, and Oleu treatment aids the compensation of distressed energy metabolic pathways. Copyright

1H NMR Metabonomic Analysis in Renal Cell Carcinoma: a Possible Diagnostic Tool

(1)H NMR based metabonomic approach was applied in order to monitor the alterations of plasma metabolic profile in Renal Cell Carcinoma (RCC) patients and controls. (1)H NMR spectra of plasma samples from 32 RCC patients and 13 controls (patients exhibiting benign urologic disease) were recorded and analyzed using multivariate statistical techniques. Alterations in the levels of LDL/VLDL, NAC, lactate, and choline were observed between RCC patients and controls discriminating these groups in Principal Component Analysis (PCA) plots. Post OSC PLS-DA presented a satisfactory clustering between T1 with T3 RCC patients. Decrease in plasma lipid concentrations in RCC patients was verified using conventional clinical chemistry analysis. The results suggest that combination of (1)H NMR spectroscopy with PCA has potential in cancer diagnosis; however, a limitation of the method to monitor RCC is that major biomarkers revealed (lipoproteins and choline) in this metabolic profile are not unique to RCC but may be the result of the presence of any malignancy.

Oleuropein prevents doxorubicin-induced cardiomyopathy interfering with signaling molecules and cardiomyocyte metabolism

Oleuropein, a natural phenolic compound, prevents acute doxorubicin (DXR)-induced cardiotoxicity but there is no evidence regarding its role in chronic DXR-induced cardiomyopathy (DXR-CM). In the present study, we investigated the role of oleuropein in DXR-CM by addressing cardiac geometry and function (transthoracic echocardiography), cardiac histopathology, nitro-oxidative stress (MDA, PCs, NT), inflammatory cytokines (IL-6, Big ET-1), NO homeostasis (iNOS and eNOS expressions), kinases involved in apoptosis and metabolism (Akt, AMPK) and myocardial metabonomics. Rats were randomly divided into 6 groups: Control, OLEU-1 and OLEU-2 [oleuropein at 1000 and 2000 mg/kg in total, respectively, intraperitoneally (i.p.) for 14 days], DXR (18 mg/kg, i.p. divided into 6 equal doses for 2 weeks), DXR-OLEU-1 and DXR-OLEU-2 (both oleuropein and DXR as previously described). Impaired left ventricular contractility and inflammatory and degenerative pathology lesions were encountered only in the DXR group. The DXR group also had higher MDA, PCs, NT, IL-6 and Big ET-1 levels, higher iNOS and lower eNOS, Akt and AMPK activation compared to controls and the oleuropein-treated groups. Metabonomics depicted significant metabolite alterations in the DXR group suggesting perturbed energy metabolism and protein biosynthesis. The effectiveness of DXR in inhibiting cell proliferation is not compromised when oleuropein is present. We documented an imbalance between iNOS and eNOS expressions and a disturbed protein biosynthesis and metabolism in DXR-CM; these newly recognized pathways in DXR cardiotoxicity may help identifying novel therapeutic targets. Activation of AMPK and suppression of iNOS by oleuropein seem to prevent the structural, functional and histopathological cardiac effects of chronic DXR toxicity.

1H NMR Study on the Short- and Long-Term Impact of Two Training Programs of Sprint Running on the Metabolic Fingerprint of Human Serum

Metabonomics is an established strategy in the exploration of the effects of various stimuli on the metabolic fingerprint of biofluids. Here, we present an application of (1)H NMR-based metabonomics on the field of exercise biochemistry. Fourteen men were assigned to either of two training programs, which lasted 8 weeks and involved sets of 80-m maximal runs separated by either 10 s or 1 min of rest. Analysis of pre- and postexercise serum samples, both at the beginning and end of training, by (1)H NMR spectroscopy and subsequent multivariate statistical techniques revealed alterations in the levels of 18 metabolites. Validated O-PLS models could classify the samples in regard to exercise, the separation being mainly due to lactate, pyruvate, alanine, leucine, valine, isoleucine, arginine/lysine, glycoprotein acetyls, and an unidentified metabolite resonating at 8.17 ppm. Samples were also classified safely with respect to training, the separation being mainly due to lactate, pyruvate, methylguanidine, citrate, glucose, valine, taurine, trimethylamine N-oxide, choline-containing compounds, histidines, acetoacetate/acetone, glycoprotein acetyls, and lipids. Samples could not be classified according to the duration of the rest interval between sprints. Our findings underline the power of metabonomics to offer new insights into the short- and long-term impact of exercise on metabolism.

Novel Inverse Binding Mode of Indirubin Derivatives Yields Improved Selectivity for DYRK Kinases

DYRK kinases are involved in alternative pre-mRNA splicing as well as in neuropathological states such as Alzheimers disease and Down syndrome. In this study, we present the design, synthesis, and biological evaluation of indirubins as DYRK inhibitors with enhanced selectivity. Modifications of the bis-indole included polar or acidic functionalities at positions 5′ and 6′ and a bromine or a trifluoromethyl group at position 7, affording analogues that possess high activity and pronounced specificity. Compound 6i carrying a 5′-carboxylate moiety demonstrated the best inhibitory profile. A novel inverse binding mode, which forms the basis for the improved selectivity, was suggested by molecular modeling and confirmed by determining the crystal structure of DYRK2 in complex with 6i. Structure–activity relationships were further established, including a thermodynamic analysis of binding site water molecules, offering a structural explanation for the selective DYRK inhibition.

Identification of the substrate recognition and transport pathway in a eukaryotic member of the nucleobase-ascorbate transporter (NAT) family.

Using the crystal structure of the uracil transporter UraA of Escherichia coli, we constructed a 3D model of the Aspergillus nidulans uric acid-xanthine/H+ symporter UapA, which is a prototype member of the Nucleobase-Ascorbate Transporter (NAT) family. The model consists of 14 transmembrane segments (TMSs) divided into a core and a gate domain, the later being distinctly different from that of UraA. By implementing Molecular Mechanics (MM) simulations and quantitative structure-activity relationship (SAR) approaches, we propose a model for the xanthine-UapA complex where the substrate binding site is formed by the polar side chains of residues E356 (TMS8) and Q408 (TMS10) and the backbones of A407 (TMS10) and F155 (TMS3). In addition, our model shows several polar interactions between TMS1-TMS10, TMS1-TMS3, TMS8-TMS10, which seem critical for UapA transport activity. Using extensive docking calculations we identify a cytoplasm-facing substrate trajectory (D360, A363, G411, T416, R417, V463 and A469) connecting the proposed substrate binding site with the cytoplasm, as well as, a possible outward-facing gate leading towards the substrate major binding site. Most importantly, re-evaluation of the plethora of available and analysis of a number of herein constructed UapA mutations strongly supports the UapA structural model. Furthermore, modeling and docking approaches with mammalian NAT homologues provided a molecular rationale on how specificity in this family of carriers might be determined, and further support the importance of selectivity gates acting independently from the major central substrate binding site.