The human Myt1 kinase (PKMYT1) is an important regulator of the G2/M transition in the cell cycle. Presently, limited knowledge about its substrate recognition is available. Here, various potential ...substrates were investigated by different antibody based techniques including fluorescence polarization immunoassays and immunoblotting. Regarding both Thr and Tyr kinase activity, only protein substrates were found to be phosphorylated by Myt1, whereas any tested peptide was not recognized. In silico molecular dynamics studies were used to compare the stability of the Myt1 peptide complex with Wee1 peptide complex and support the biochemical findings. Furthermore, a Myt1 kinase binding assay suggests Myt1 being insensitive to staurosporine.
One hundred and seventy-four pyrrolo3,4-ccarbazole-1,3(2H,6H)-dione derivatives reported as inhibitors of the kinase Wee1 were used for a molecular docking and three-dimensional quantitative ...structure-activity relationship (3D-QSAR) study. Due to the availability of the three-dimensional structure of the Wee1 kinase a receptor-based alignment strategy was applied. Six available Wee1-inhibitor crystal structures were analyzed using the docking program GOLD resulting in a good reproduction of the experimentally derived position and interaction of the cocrystallized inhibitors. Since only a low correlation between docking scores and inhibitory activities was obtained for the series of 174 inhibitors a receptor-based 3D-QSAR study was performed, dividing the data set into 144 training set molecules and an external test set of 30 compounds. Besides the ligand alignment derived from the docking study we tested several other alignment procedures as basis for the 3D-QSAR analysis. The most predictive model was obtained using the alignment from the GOLD docking study. The CoMFA model was found to be robust (q(LOO)(2)=0.764 and r(2)=0.870). The predictive ability of the model was further examined by carrying out leave-20%-out and leave-50%-out cross-validation (q(2)=0.747 for leave-20%-out and 0.737 for leave-50%-out) and predicting the activities of 30 inhibitors used as external test set (r(pred)(2)=0.790). The graphical analysis of the CoMFA contour plot together with the key residues of the binding pocket provided important insight into the relevant interactions of the inhibitors. The results not only provide information about the essential features of potent Wee1 inhibitors but also show the advantage of using receptor-based alignment for 3D-QSAR analysis.
In the human cell cycle, the Myt1 kinase is a crucial regulator of the G2/M transition. Because this membrane-associated kinase is hard to obtain and assay, there is a distinct lack of data so far. ...Here we report the derivatization of a glycoglycerolipid which was shown previously to be active in a Myt1 activity assay. These compounds were tested in a binding assay together with a set of common kinase inhibitors against a full-length Myt1 expressed in a human cell line. Dasatinib exhibited nanomolar affinity whereas broad coverage inhibitors such as sunitinib and staurosporine derivatives did not show any effect. We also carried out docking studies for the most potent compounds allowing further insights into the inhibitor interaction of this kinase. The glycoglycerolipids showed no significant effects in the binding assay, endorsing the idea of a mechanism of action distant from the active site.
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► Full-length human Myt1 kinase was used in a binding assay. ► Glycoglycerolipids were synthesized and tested but did not displace the tracer. ► A set of common kinase inhibitors was tested. ► PD0166285 and dasatinib had nanomolar affinities. ► Binding modes for these compounds were predicted.
Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, ...however, the cross-linking pattern is still unknown.
Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry.
MS2 data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations.
The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials.
The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin.
► In vitro cross-linking of short elastin peptides yields elastin-like biomaterials. ► Software was developed to allow identification of native polyfunctional cross-links. ► Intra- and intermolecular cross-links characteristic of elastin were identified. ► In vitro formation of desmosine was proven by means of mass spectrometry.
Within the last decade, interest in the development of new anticancer drugs increased mainly from emerging resistance against established drugs, which were found to be limited by the multidrug ...resistance (MDR) phenomenon. Several anticancer targets have been investigated for the development of structurally new drugs which were thought to be unaffected by the MDR phenomenon. Receptor tyrosine kinases (RTKs) make up one interesting group of anticancer targets. The overexpression and mutation of RTKs lead to an ongoing stimulus of cell growth and cancer progression. Early approaches to selective inhibition of single RTKs were generally disappointing in clinical studies, due in part to occurring resistance. Therefore, a new strategy involves the identification of multi-kinase inhibitors to slow the development of potential resistance. Moreover, the expected side effects of the first nonselective inhibitors were less dramatic than had been expected. We have discovered novel 4-benzylamino-α-carbolines as a new class of RTK inhibitors. Docking studies suggest a binding mode to the addressed target structures of the epidermal growth factor receptor (EGFR) and to the vascular endothelial growth factor receptor 2 (VEGFR2). Selectivity profiling against a panel of kinases and antiproliferative studies have highlighted one inhibitor, active in the nanomolar range, as a highly interesting candidate for further clinical studies.
The use of multiple target conformers has been applied successfully in virtual screening campaigns; however, a study on how to best combine scores for multiple targets in a hierarchic method that ...combines rigid and flexible docking is not available. In this study, we used a data set of 59 479 compounds to screen multiple conformers of four distinct protein targets to obtain an adapted and optimized combination of an established hierarchic method that employs the programs FRED and Surflex. Our study was extended and verified by application of our protocol to ten different data sets from the directory of useful decoys (DUD). We quantitated overall method performance in ensemble docking and compared several consensus scoring methods to improve the enrichment during virtual ligand screening. We conclude that one of the methods used, which employs a consensus weighted scoring of multiple target conformers, performs consistently better than methods that do not include such consensus scoring. For optimal overall performance in ensemble docking, it is advisable to first calculate a consensus of FRED results and use this consensus as a sub‐data set for Surflex screening. Furthermore, we identified an optimal method for each of the chosen targets and propose how to optimize the enrichment for any target.
A systematic study into the optimal combination of ranked score lists from ensemble docking when applied to a hierarchic docking protocol that combines rigid and flexible docking with FRED and Surflex, respectively, is presented. The results provide information and guidelines on how to optimally perform such hierarchic ensemble docking for use in structure‐based virtual screening.
