Electrocatalytic assay for monitoring methylglyoxal-mediated protein glycation.

Protein glycation is a complex process that plays an important role in diabetes mellitus, aging, and the regulation of protein function in general. As a result, current methodological research on proteins is focused on the development of novel approaches for investigating glycation and the possibility of monitoring its modulation and selective inhibition. In this paper, a first sensing strategy for protein glycation is proposed, based on protein electroactivity measurement. Concretely, the label-free method proposed is based on the application of a constant-current chronopotentiometric stripping (CPS) analysis at Hg-containing electrodes. The glycation process was monitored as the decrease in the electrocatalytic protein signal, peak H, observed at highly negative potentials at around -1.8 V (vs Ag/AgCl3 M KCl), which was previously ascribed to a catalytic hydrogen evolution reaction (CHER). Using this method, a model protein bovine serum albumin was investigated over 3 days of incubation with the glycation agent methylglyoxal in the absence or presence of the glycation inhibitor aminoguanidine (pimagedine). The electrochemical methodology presented here could open up new possibilities in research on protein glycation and oxidative modification. The methodology developed also provides a new option for the analysis of protein intermolecular interactions using electrochemical sensors, which was demonstrated by the application of a silver solid amalgam electrode (AgSAE) for monitoring the glycation process in samples of bovine serum albumin, human serum albumin, and lysozyme.

Caffeine inhibits acetylcholinesterase, but not butyrylcholinesterase.

Caffeine is an alkaloid with a stimulant effect in the body. It can interfere in transmissions based on acetylcholine, epinephrine, norepinephrine, serotonin, dopamine and glutamate. Clinical studies indicate that it can be involved in the slowing of Alzheimer disease pathology and some other effects. The effects are not well understood. In the present work, we focused on the question whether caffeine can inhibit acetylcholinesterase (AChE) and/or, butyrylcholinesterase (BChE), the two enzymes participating in cholinergic neurotransmission. A standard Ellman test with human AChE and BChE was done for altering concentrations of caffeine. The test was supported by an in silico examination as well. Donepezil and tacrine were used as standards. In compliance with Dixon's plot, caffeine was proved to be a non-competitive inhibitor of AChE and BChE. However, inhibition of BChE was quite weak, as the inhibition constant, Ki, was 13.9 ± 7.4 mol/L. Inhibition of AChE was more relevant, as Ki was found to be 175 ± 9 µmol/L. The predicted free energy of binding was -6.7 kcal/mol. The proposed binding orientation of caffeine can interact with Trp86, and it can be stabilize by Tyr337 in comparison to the smaller Ala328 in the case of human BChE; thus, it can explain the lower binding affinity of caffeine for BChE with reference to AChE. The biological relevance of the findings is discussed.

Transferable scoring function based on semiempirical quantum mechanical PM6-DH2 method: CDK2 with 15 structurally diverse inhibitors.

A semiempirical quantum mechanical PM6-DH2 method accurately covering the dispersion interaction and H-bonding was used to score fifteen structurally diverse CDK2 inhibitors. The geometries of all the complexes were taken from the X-ray structures and were reoptimised by the PM6-DH2 method in continuum water. The total scoring function was constructed as an estimate of the binding free energy, i.e., as a sum of the interaction enthalpy, interaction entropy and the corrections for the inhibitor desolvation and deformation energies. The applied scoring function contains a clear thermodynamical terms and does not involve any adjustable empirical parameter. The best correlations with the experimental inhibition constants (ln K (i)) were found for bare interaction enthalpy (r (2) = 0.87) and interaction enthalpy corrected for ligand desolvation and deformation energies (r (2) = 0.77); when the entropic term was considered, however, the correlation becomes worse but still acceptable (r (2) = 0.52). The resulting correlation based on the PM6-DH2 scoring function is better than previously published function based on various docking/scoring, SAR studies or advanced QM/MM approach, however, the robustness is limited by number of available experimental data used in the correlation. Since a very similar correlation between the experimental and theoretical results was found also for a different system of the HIV-1 protease, the suggested scoring function based on the PM6-DH2 method seems to be applicable in drug design, even if diverse protein-ligand complexes have to be ranked.

Anterior gradient protein 3 is associated with less aggressive tumors and better outcome of breast cancer patients.

Anterior gradient protein (AGR) 3 is a highly related homologue of pro-oncogenic AGR2 and belongs to the family of protein disulfide isomerases. Although AGR3 was found in breast, ovary, prostate, and liver cancer, it remains of yet poorly defined function in tumorigenesis. This study aimed to determine AGR3 expression in a cohort of 129 primary breast carcinomas and evaluate the clinical and prognostic significance of AGR3 in these tumors. The immunohistochemical analysis revealed the presence of AGR3 staining to varying degrees in 80% of analyzed specimens. The percentage of AGR3-positive cells significantly correlated with estrogen receptor, progesterone receptor (both P<0.0001) as well as low histological grade (P=0.003), and inversely correlated with the level of Ki-67 expression (P<0.0001). In the whole cohort, AGR3 expression was associated with longer progression-free survival (PFS), whereas AGR3-positive subgroup of low-histological grade tumors showed both significantly longer PFS and overall survival. In conclusion, AGR3 is associated with the level of differentiation, slowly proliferating tumors, and more favorable prognosis of breast cancer patients.

The role of AGR2 and AGR3 in cancer: similar but not identical.

In the past decades, highly related members of the protein disulphide isomerase family, anterior gradient protein AGR2 and AGR3, attracted researchers' attention due to their putative involvement in developmental processes and carcinogenesis. While AGR2 has been widely demonstrated as a metastasis-related protein whose elevated expression predicts worse patient outcome, little is known about AGR3's role in tumour biology. Thus, we aim to confront the issue of AGR3 function in physiology and pathology in the following review by comparing this protein with the better-described homologue AGR2. Relying on available data and in silico analyses, we show that AGR proteins are co-expressed or uncoupled in context-dependent manners in diverse carcinomas and healthy tissues. Further, we discuss plausible roles of both proteins in tumour-associated processes such as differentiation, proliferation, migration, invasion and metastasis. This work brings new hints and stimulates further thoughts on hitherto unresolved conundrum of anterior gradient protein function.

Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder.

Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein-ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.

Influence of mutation type on prognostic and predictive values of TP53 status in primary breast cancer patients.

High rates of mutation in the TP53 tumor suppressor gene have been found in many human cancers, including breast tumors, making p53 one of the most studied proteins in oncology. However, the prognostic and predictive value of alterations in this gene remains ambiguous. To analyze the clinical value of somatic TP53 mutations, we collected clinical and molecular data on 210 women with primary breast cancer. We found significant associations of p53 mutations with tumor grade, metastasis, molecular subtype, Her2 status and inverse correlations with estrogen and progesterone receptor status. Cox proportional hazard analysis confirmed a strong prognostic value of p53 mutation for overall survival rate and highlighted significant interactions with lymph node involvement and tumor size. In relation to treatment options, TP53 mutations were associated with poor response to anthracyclines and radiotherapy. Categorization of TP53 mutations according to their type and location revealed that patients with nonsense mutation have the poorest prognosis in comparison with wild-type cases and other types of mutations in this gene. Classification of TP53 mutations with respect to the degree of disturbance of protein structure showed association of disruptive mutations with poorer patients' outcome in contrast to wild-type and non-disruptive mutations. In conclusion, the present study confirms p53 as a potential predictive and prognostic factor in oncology practice and highlights the growing evidence that distinct types of mutations have different clinical impacts.

Quantum mechanical scoring: structural and energetic insights into cyclin-dependent kinase 2 inhibition by pyrazolo[1,5-a]pyrimidines.

A quantum mechanics (QM)-based scoring function has been applied to complexes of cyclin-dependent kinase 2 (CDK2) and thirty-one pyrazolo[1,5-a]pyrimidine-based inhibitors and their bioisosteres. A hybrid three-layer QM/MM setup (DFT-D/PM6-D3H4X/AMBER in generalized Born solvent) was used here for the first time as an extension of our previous full QM and SQM/MM (SQM means semiempirical QM) approaches. Two approaches to obtain the structures of the CDK2/inhibitor complexes were examined: i) building the modifications from one X-ray structure available coupled with a conformational search and ii) docking the compounds into CDK2. The QM-based scoring entailed a QM/SQM/MM optimization followed by calculations of the binding scores which were subsequently correlated with the experimental binding free energies. The correlation for the building protocol was good (r(2) = 0.64, predictive index = 0.81), whereas the docking approach failed. A decomposition of the interaction energies to ligand fragments enabled us to rationalize the differences in the binding affinities. In conclusion, we have developed and refined a QM-based scoring protocol and successfully applied it to reproduce the binding affinities in congeneric series of CDK2 inhibitors and to rationalize their potency. We thus propose that such a tool can be used in computer-aided rational drug design.

Semiempirical quantum mechanical method PM6-DH2X describes the geometry and energetics of CK2-inhibitor complexes involving halogen bonds well, while the empirical potential fails.

In the present study, we have investigated complexes of CK2 protein kinase with halogenated inhibitors by means of the advanced semiempirical quantum mechanical (SQM) PM6 method (called PM6-DH2X), which describes various types of noncovalent interactions including halogen bonding well. The PM6-DH2X method provides reliable geometries of those CK2 protein kinase-inhibitor complexes involving halogen bonds that agree well with the X-ray crystal structures. When the Amber empirical potential is applied, this agreement becomes considerably worse. Similarly, the binding free energies determined by the PM6-DH2X SQM method are much closer to the experimental inhibition constants than those based on the Amber empirical potential.

Missense mutations located in structural p53 DNA-binding motifs are associated with extremely poor survival in chronic lymphocytic leukemia.

PURPOSE: There is a distinct connection between TP53 defects and poor prognosis in chronic lymphocytic leukemia (CLL). It remains unclear whether patients harboring TP53 mutations represent a homogenous prognostic group. PATIENTS AND METHODS: We evaluated the survival of patients with CLL and p53 defects identified at our institution by p53 yeast functional assay and complementary interphase fluorescence in situ hybridization analysis detecting del(17p) from 2003 to 2010. RESULTS: A defect of the TP53 gene was identified in 100 of 550 patients. p53 mutations were strongly associated with the deletion of 17p and the unmutated IgVH locus (both P < .001). Survival assessed from the time of abnormality detection was significantly reduced in patients with both missense (P < .001) and nonmissense p53 mutations (P = .004). In addition, patients harboring missense mutation located in p53 DNA-binding motifs (DBMs), structurally well-defined parts of the DNA-binding domain, manifested a clearly shorter median survival (12 months) compared with patients having missense mutations outside DBMs (41 months; P = .002) or nonmissense alterations (36 months; P = .005). The difference in survival was similar in the analysis limited to patients harboring mutation accompanied by del(17p) and was also confirmed in a subgroup harboring TP53 defect at diagnosis. The patients with p53 DBMs mutation (at diagnosis) also manifested a short median time to first therapy (TTFT; 1 month). CONCLUSION: The substantially worse survival and the short TTFT suggest a strong mutated p53 gain-of-function phenotype in patients with CLL with DBMs mutations. The impact of p53 DBMs mutations on prognosis and response to therapy should be analyzed in investigative clinical trials.

Interaction energies for the purine inhibitor roscovitine with cyclin-dependent kinase 2: correlated ab initio quantum-chemical, DFT and empirical calculations.

The interaction between roscovitine and cyclin-dependent kinase 2 (cdk2) was investigated by performing correlated ab initio quantum-chemical calculations. The whole protein was fragmented into smaller systems consisting of one or a few amino acids, and the interaction energies of these fragments with roscovitine were determined by using the MP2 method with the extended aug-cc-pVDZ basis set. For selected complexes, the complete basis set limit MP2 interaction energies, as well as the coupled-cluster corrections with inclusion of single, double and noninteractive triples contributions [CCSD(T)], were also evaluated. The energies of interaction between roscovitine and small fragments and between roscovitine and substantial sections of protein (722 atoms) were also computed by using density-functional tight-binding methods covering dispersion energy (DFTB-D) and the Cornell empirical potential. Total stabilisation energy originates predominantly from dispersion energy and methods that do not account for the dispersion energy cannot, therefore, be recommended for the study of protein-inhibitor interactions. The Cornell empirical potential describes reasonably well the interaction between roscovitine and protein; therefore, this method can be applied in future thermodynamic calculations. A limited number of amino acid residues contribute significantly to the binding of roscovitine and cdk2, whereas a rather large number of amino acids make a negligible contribution.

Binding of fatty acids to beta-cryptogein: quantitative structure-activity relationships and design of selective protein mutants.

Binding of fatty acids to cryptogein, the proteinaceous elicitor from Phytophthora, was studied by using molecular docking and quantitative structure-activity relationships analysis. Fatty acids bind to the groove located inside the cavity of cryptogein. The structure-activity model was constructed for the set of 27 different saturated and unsaturated fatty acids explaining 87% (81% cross-validated) of the quantitative variance in their binding affinity. The difference in binding between saturated and unsaturated fatty acids was described in the model by three electronic descriptors: the energy of the lowest unoccupied molecular orbital, the energy of the highest occupied molecular orbital, and the heat of formation. The presence of double bonds in the ligand generally resulted in stronger binding. The difference in binding within the group of saturated fatty acids was explained by two steric descriptors, i.e., ellipsoidal volume and inertia moment of length, and one hydrophobicity descriptor, i.e., lipophility. The developed model predicted strong binding for two biologically important molecules, geranylgeranyol and farnesol playing an important role in plant signaling as lipid anchors of some membrane proteins. Elicitin mutants selectively binding only one type of ligand were designed for future experimental studies.