Design and synthesis of novel 1,2,3,4-tetrazines as new anti-leukemia cancer agents.

A series of novel 1,2,3,4-tetrazines were designed and synthesized. 1 H-NMR spectroscopy, 13 C NMR spectroscopy, and HRMS were used to determine the structures of this novel compounds. Computational approaches suggested that DHFR is a putative target for the newly synthesized 11 compounds. Extensive molecular dynamics simulations followed by molecular docking simulations were employed to evaluate DHFR as a potential target protein. The anticancer activities of the compounds were evaluated against five different types of leukemia cell lines (Jurkat, Nalm-6, Reh, K562, and Molt-4) and one non-leukemic cell line (Hek293T) by MTT test in vitro and imatinib was used as a control drug. Among these compounds, 3a exhibited the best activity against all the leukemic cell lines, except Reh cell line. For Nalm-6, K562, Jurkat, and Molt-4 cell lines, IC50 values were found to be 15.98, 19.12, 23.15, and 25.80 µM, respectively. Our work focuses on the synthesis of original and novel 1,2,3,4-tetrazine derivatives while contributing to the ongoing effort to discover more potent new antileukemia agents.

Synthesis, molecular modeling, and biological evaluation of novel imatinib derivatives as anticancer agents.

Different derivatives of imatinib were synthesized by a 3-step reaction method. The structures of the new compounds were characterized by spectroscopic methods. For quantitative evaluation of the biological activity of the compounds, MTT assays were performed, where four BCR-ABL negative leukemic cell lines (Jurkat, Reh, Nalm-6 and Molt-4), one BCR-ABL positive cell line (K562), and one non-leukemic cell line (Hek293T) were incubated with various concentrations of the derivatives. Although imatinib was specifically designed for the BCR-ABL protein, our results showed that it was also effective on BCR-ABL negative cell lines except for Reh cell line. Compound 9 showed lowest IC50 values against Nalm-6 cells as 1.639 µM, also the values of Compound 10 for each cell were very close to imatinib. Molecular docking simulations suggest that except for compound 6, the compounds prefer a DFG-out conformation of the ABL kinase domain. Among them, compound 10 has the highest affinity for ABL kinase domain that is close to the affinity of imatinib. The common rings between compound 10 and imatinib adopt exactly the same conformation and same type of interactions in the ATP binding site with the ABL kinase domain.

Side effect prediction based on drug-induced gene expression profiles and random forest with iterative feature selection.

One in every ten drug candidates fail in clinical trials mainly due to efficacy and safety related issues, despite in-depth preclinical testing. Even some of the approved drugs such as chemotherapeutics are notorious for their side effects that are burdensome on patients. In order to pave the way for new therapeutics with more tolerable side effects, the mechanisms underlying side effects need to be fully elucidated. In this work, we addressed the common side effects of chemotherapeutics, namely alopecia, diarrhea and edema. A strategy based on Random Forest algorithm unveiled an expression signature involving 40 genes that predicted these side effects with an accuracy of 89%. We further characterized the resulting signature and its association with the side effects using functional enrichment analysis and protein-protein interaction networks. This work contributes to the ongoing efforts in drug development for early identification of side effects to use the resources more effectively.

Methylation-targeted specificity of the DNA binding proteins R.DpnI and MeCP2 studied by molecular dynamics simulations.

DNA methylation plays a major role in organismal development and the regulation of gene expression. Methylation of cytosine bases and the cellular roles of methylated cytosine in eukaryotes are well established, as well as methylation of adenine bases in bacterial genomes. Still lacking, however, is a general mechanistic understanding, in structural and thermodynamic terms, of how proteins recognize methylated DNA. Toward this aim, we present the results of molecular dynamics simulations, alchemical free energy perturbation, and MM-PBSA calculations to explain the specificity of the R.DpnI enzyme from Streptococcus pneumonia in binding to adenine-methylated DNA with both its catalytic and winged-helix domains. We found that adenine-methylated DNA binds more favorably to the catalytic subunit of R.DpnI (-4 kcal mol-1) and to the winged-helix domain (-1.6 kcal mol-1) than non-methylated DNA. In particular, N6-adenine methylation is found to enthalpically stabilize binding to R.DpnI. In contrast, C5-cytosine methylation entropically favors complexation by the MBD domain of the human MeCP2 protein with almost no contribution of the binding enthalpy.

How Hydrophilic Proteins Form Nonspecific Complexes.

In the crowded environment of cells, proteins frequently encounter other proteins in many possible orientations. Most of these encounters are short-lived because the physicochemical properties of the two binding patches do not match. However, even for protein pairs that bind tightly, it is not an easy task to find the correct binding site on the partner protein and align with it. So far not well understood is the source of interaction specificity that favors a small set of specific "native" interactions over the multitude of alternative orientations. We used molecular dynamics simulations to study nonspecific complexes formed by barnase and barstar, cytochrome c and cytochrome c peroxidase, and the complex of the N-terminal domain of enzyme I with the histidine-containing phosphocarrier. We found that spontaneously forming nonspecific encounters involve interaction interfaces smaller than those of the specific complexes and are attracted by shorter-range direct interactions between the proteins.

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2α/CK2ß interaction.

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2ß in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α(1-335) and the fluorescent probe CF-Ahx-Pc as a CK2ß analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α(1-335). Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2ß. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α(1-335) complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α(1-335) were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a KD value of 240nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2ß, with an IC50 value of 92µM.

Energetics of Hydrophilic Protein-Protein Association and the Role of Water.

Hydrophilic protein-protein interfaces constitute a major part of all protein-protein interfaces and are thus of great importance. However, the quantitative characterization of their association is still an ongoing challenge and the driving force behind their association remains poorly characterized. Here, we have addressed the association of hydrophilic proteins and the role of water by means of extensive molecular dynamics simulations in explicit water using three well studied protein complexes; Barnase-Barstar, cytochrome c-cytochrome c peroxidase, and the N-terminal domain of enzyme I-histidine-containing phosphocarrier. The one-dimensional free energy profiles obtained from umbrella sampling simulations are downhill or, in other words, barrierless. Using these one-dimensional free energy profiles, the computed standard free energies of binding are -12.7 ± 1.1 kcal/mol, -9.4 ± 0.7 kcal/mol, and -8.4 ± 1.9 kcal/mol that are in reasonable to very good agreement with the experimental values of -19.6 kcal/mol, -8.8 kcal/mol, and -7.8 kcal/mol. As expected, analysis of the confined water between the hydrophilic complex partners shows that the density and the orientational order parameter deviate noticeably from the bulk values, especially at close separations of the confining proteins.

ERAD and protein import defects in a sec61 mutant lacking ER-lumenal loop 7.

BACKGROUND: The Sec61 channel mediates protein translocation across the endoplasmic reticulum (ER) membrane during secretory protein biogenesis, and likely also during export of misfolded proteins for ER-associated degradation (ERAD). The mechanisms of channel opening for the different modes of translocation are not understood so far, but the position of the large ER-lumenal loop 7 of Sec61p suggests a decisive role. RESULTS: We show here that the Y345H mutation in L7 which causes diabetes in the mouse displays no ER import defects in yeast, but a delay in misfolded protein export. A complete deletion of L7 in Sec61p resulted in viable, cold- and tunicamycin-hypersensitive yeast cells with strong defects in posttranslational protein import of soluble proteins into the ER, and in ERAD of soluble substrates. Membrane protein ERAD was only moderately slower in sec61∆L7 than in wildtype cells. Although Sec61∆L7 channels were unstable in detergent, co-translational protein integration into the ER membrane, proteasome binding to Sec61∆L7 channels, and formation of hetero-heptameric Sec complexes were not affected. CONCLUSIONS: We conclude that L7 of Sec61p is required for initiation of posttranslational soluble protein import into and misfolded soluble protein export from the ER, suggesting a key role for L7 in transverse gating of the Sec61 channel.

BiP-mediated closing of the Sec61 channel limits Ca2+ leakage from the ER.

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic protein-conducting channel, the Sec61 complex. Previous work has characterized the Sec61 channel as a potential ER Ca(2+) leak channel and identified calmodulin as limiting Ca(2+) leakage in a Ca(2+)-dependent manner by binding to an IQ motif in the cytosolic aminoterminus of Sec61α. Here, we manipulated the concentration of the ER lumenal chaperone BiP in cells in different ways and used live cell Ca(2+) imaging to monitor the effects of reduced levels of BiP on ER Ca(2+) leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca(2+) leakage via the Sec61 complex. When we replaced wild-type Sec61α with mutant Sec61αY344H in the same model cell, however, Ca(2+) leakage from the ER increased and was no longer affected by manipulation of the BiP concentration. Thus, BiP limits ER Ca(2+) leakage through the Sec61 complex by binding to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344.

Druggability of dynamic protein-protein interfaces.

The conformational flexibility of protein targets is being increasingly recognized in the drug discovery and design processes. When working on a particular disease-related biochemical pathway, it is of crucial importance to carefully select druggable protein binding pockets among all those cavities that may appear transiently or permanently on the respective protein surface. In this review, we will focus on the conformational dynamics of proteins that governs the formation and disappearance of such transient pockets on protein surfaces. We will also touch on the issue of druggability of transiently formed pockets. For example, protein cavities suitable to bind small drug-like molecules show an increased pocket size and buriedness when compared to empty sites. Interestingly, we observed in molecular dynamics simulations of five different protein systems that the conformational transitions on the protein surface occur almost barrierless and large pockets are found at similar frequencies as small pockets, see below. Thus, the dynamical processes at protein surfaces are better visualized as fluid-like motion than as energetically activated events. We conclude by comparing two computational tools, EPOS and MDpocket, for identifying transient pockets in PDK1 kinase. We illustrate how the obtained results depend on the way in which corresponding pockets in different molecular dynamics snapshots are connected to each other.

A comparative molecular dynamics study of methylation state specificity of JMJD2A.

Histone modifications have great importance in epigenetic regulation. JMJD2A is a histone demethylase which is selective for di- and trimethyl forms of residues Lys9 and Lys36 of Histone 3 tail (H3K9 and H3K36). We present a molecular dynamics simulations of mono-, di- and trimethylated histone tails in complex with JMJD2A catalytic domain to gain insight into how JMJD2A discriminates between the methylation states of H3K9. The methyl groups are located at specific distances and orientations with respect to Fe(II) in methylammonium binding pocket. For the trimethyllysine the mechanism which provides the effectual orientation of methyl groups is the symmetry, whereas for the dimethyllysine case the determining factors are the interactions between methyllysine head and its environment and subsequently the restriction on angular motion. The occurrence frequency of methyl groups in a certain proximity of Fe(II) comes out as the explanation of the enzyme activity difference on di- and tri-methylated peptides. Energy analysis suggests that recognition is mostly driven by van der Waals and followed by Coulombic interactions in the enzyme-substrate interface. The number (mono, di or tri) and orientations of methyl groups and water molecules significantly affect the extent of van der Waals interaction strengths. Hydrogen bonding analysis suggests that the interaction between JMJD2A and its substrates mainly comes from main chain-side chain interactions. Binding free energy analysis points out Arg8 as an important residue forming an intra-substrate hydrogen bond with tri and dimethylated Lys9 of the H3 chain. Our study provides new insights into how JMJD2A discriminates between its substrates from both a structural and dynamical point of view.