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Cyclin-dependent kinases (CDKs) are historic therapeutic targets implicated in tumorigenic events due to their critical involvement in the cell cycle phase. However, selectivity has proven to be a bottleneck, causing repeated failures. Previously, we reported CR6-interacting factor 1 (CRIF1), acting as a cell cycle negative regulator through interaction with CDK2. In the current report, we identified the CRIF1-CDK2 interaction interface by in silico studies and shortlisted interface inhibitors through virtual screening on CRIF1 using 40 678 drug-like compounds. These compounds were tested by cell proliferation assay, and four of these molecules were found to selectively inhibit the proliferation of osteosarcoma (OS) cell lines, but do not affect normal bone mesenchymal stem cells (BMSC). A binding study reveals significant affinities of the inhibitors on CRIF1. More importantly, treatment of the OS cells with a combination of ionizing radiation (IR) and the best-performing inhibitors remarkably increased IR inhibition potential from 19.9% to 59.6%. This occurred by selectively promoting G2/M arrest and apoptosis related to CDK2 overactivation in OS cells but not in BMSC and was supported by significant CDK2 phosphorylation modifications. Knocking down of CRIF1 by siRNA treatment showed similar effects to the interface inhibitors. Together we substantiate the identification of novel lead molecules, which may provide a new treatment to overcome selectivity issues and enhance the radiosensitivity of tumor cells, opening a conceptually novel strategy of CDK-targeting for different cancer types.

Zika virus (ZIKV) is an emerging mosquito-borne virus recently linked to intrauterine growth restriction including abnormal fetal brain development. The recent outbreak of ZIKV reached pandemic level resulting in an alarming public health emergency. At present, there is limited understanding of the infectious mechanism and no approved therapy. Nonstructural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase (MTase) and RNA dependent RNA polymerase domain. Here we used molecular modeling to obtain the structure of ZIKV MTase and molecular docking to identify the additional hydrophobic region uniquely conserved in flavivirus MTase that can be used as a druggable site. Subsequently, a virtual screening with a library of 28 341 compounds identified 10 best hits showing decisive contacts with the MTase. In vitro efficacy analysis of these compounds against ZIKV, by plaque reduction assay, has confirmed four of the top scored ligands (Life Chemicals ID: F3043-0013, F0922-0796, F1609-0442, and F1750-0048) having EC50 (50% effective concentration) values of 4.8 ± 2.3, 12.5 ± 7.4, 17.5 ± 8.4, and 17.6 ± 3.1 μM respectively, identifying lead compounds for anti-ZIKV drug development.

Annexin B1 (AnxB1) is a calcium-dependent phospholipid binding protein from Taenia solium cysticercus and has been reported to possess anticoagulant activity, to inhibit phospholipase A2, and to regulate membrane transport. Native AnxB1 and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. The results of dynamic light scattering analysis showed that Hepes buffer combined with low concentration salts (NaCl or CaCl2) was beneficial for preventing aggregation and for AnxB1 stabilization in the storage. After the additive screen, crystals have been yielded in the presence of guanidine hydrochloride (Gn-HCl). We determined that a low concentration of Gn-HCl significantly delayed clotting time and increased anticoagulant activity. Analysis of the crystal showed that in the presence of Gn-HCl, AnxB1 crystallizes in orthorhombic space group, which is modified from the cubic space group for crystals grown in the absence of Gn-HCl. A high quality data set (at 1.9 A) has been co...

A new parameter “relative crystallizability” for protein crystallization has been proposed, and its relationship with protein solubility and crystallization success has been studied (Zhu et al. J. Struct. Biol. 2006, 154, 297). Here we further construct the phase diagrams of a larger number of proteins, study the phase modification as a function of temperature, and establish the relationship between the nucleation zone area (SN) and crystallization success. The phase diagrams of 10 proteins were constructed and their SN were compared, demonstrating that temperature modifies the protein nucleation zone. Such modification can significantly enlarge the SN and increase protein crystallizability. For example, the SN of ribonuclease S and trypsin increases by 2.4- and 1.6-fold when the temperature moves to 277 K from 295 K, while at the same time the crystallization hits increase from 20.8% to 42.9% and 12.5% to 25%, respectively. SN of chymotrypsinogen A and concanavalin A increases by 1.6- and 1.7-fold (277 t...

We describe a straightforward crystallogenesis protocol leading to the preparation of protein crystals suitable for structure determination that involves protein expression and purification, refolding of the overexpressed protein, search of optimal crystallization conditions and diffraction data collection on native and selenomethionine substituted crystals. The protocol is exemplified with epididymal-specific lipocalin (rLcn6), a newly discovered monomeric protein of 19 kDa that may play an important role in sperm maturation. This protein was cloned from Norway rat (Rattus norvegicus) genome and expressed as insoluble inclusion bodies in Escherichia coli. After refolding of the purified protein, microcrystals were obtained after sparse matrix screening. Optimization of conditions after stepwise incremental initial conditions led to single crystals belonging to space group P212121 and data set at 1.90 A. After attempts with several models, initial phasing was not found by molecular replacement. A similar ...

Crystal growth of steroid enzymes in complex with hydrophobic ligands is hindered by the low solubility of both partners. However, their interaction can promote highly soluble complexes, leading to well diffracting crystals and high-quality structures. The moderate amphiphilic property of polyethylene glycol increases steroid solubility and facilitates complex formation, but altered orders of soaking can lead to a variety of complex formations and different structures. Understanding the latter mechanism is important for inhibitor design. Thus, different enzyme–ligand complex structures have provided detailed pictures of steroid alternative binding and multispecificity of the enzymes, as, for example, in the case of human 17β-hydroxysteroid dehydrogenases (17β-HSD) type 1 due to C19 steroid pseudosymmetry or in human 17β-HSD type 5 due to the spatial binding site. These complex structures also provide insights into the dynamics of the enzyme binding and catalytic process. Hybrid inhibitors constituted by t...

Crystallographic studies of ligand-protein complexes reveal most preferable ligand binding modes, but do not show less populated modes that may contribute to measurable biochemical and biophysical characteristics of the complexes. In some cases, a ligand may bind a protein in essentially different modes. An example is 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), a steroidogenic enzyme that catalyzes reduction of estrone to estradiol in gonadal and peripheral tissues. The enzyme exhibits a high specificity for estrogens which bind with their C17 atom in the proximity of the NADP(H) cofactor. 17Beta-HSD1 can also bind androgens, but in a reverse binding mode, in which the steroid C3 atom is the closest carbon atom to the cofactor. Here we map the interaction energy of estradiol and dihydrotestosterone binding to 17beta-HSD1. Positions and orientations of the steroids in the ligand-binding tunnel were sampled systematically, and at each combination of these generalized coordinates, the energy was Monte Carlo minimized. The computed maps show energy minima corresponding to the X-ray structures and predict alternative binding modes, in particular, an upside-down orientation in which steroidal face alpha is exposed to protein residues that normally interact with face beta. The methodology can be used for mapping ligand-receptor interactions in various systems, for example, in ion channels and G-protein-coupled receptors that bind elongated ligands in confined space between transmembrane helices.