ATP-binding Region Research Articles

MAW point mutation impairs H. Seropedicae RecA ATP hydrolysis and DNA repair without inducing large conformational changes in its structure.

RecA is a multifunctional protein that plays a central role in DNA repair in bacteria. The structural Make ATP Work motif (MAW) is proposed to control the ATPase activity of RecA. In the present work, we report the biochemical activity and structural effects of the L53Q mutation at the MAW motif of the RecA protein from H. seropedicae (HsRecA L53Q). In vitro studies showed that HsRecA L53Q can bind ADP, ATP, and ssDNA, as does wild-type RecA. However, the ATPase and DNA-strand exchange activities were completely lost. In vivo studies showed that the expression of HsRecA L53Q in E. coli recA1 does not change its phenotype when cells were challenged with MMS and UV. Molecular dynamics simulations showed the L53Q point mutation did not cause large conformational changes in the HsRecA structure. However, there is a difference on dynamical cross-correlation movements of the residues involved in contacts within the ATP binding site and regions that hold the DNA binding sites. Additionally, a new hydrogen bond, formed between Q53 and T49, was hypothesized to allow an independent motion of the MAW motif from the hydrophobic core, what could explain the observed loss of activity of HsRecA L53Q.

Prediction Modeling of the MYH6 R443P Variant in Hypoplastic Left Heart Syndrome

Genetic variants in the alpha myosin heavy chain gene (MYH6; a‐MHC) are significantly enriched in Hypoplastic Left Heart Syndrome (HLHS), an anatomically and clinically severe form of Congenital Heart Disease (CHD). HLHS is characterized by atresia/stenosis of the aortic and mitral valves, and severe hypoplasia of the left ventricle and aorta. A novel variant resulting in an arginine to proline change (R443P) in the head domain of MYH6 was found to segregate with disease in a multigenerational family affected with HLHS and other forms of CHD.a‐MHC is a contractile protein that is important in the developing heart. After birth a‐MHC is located mainly in the atria, while the beta isoform (b‐MHC), which is encoded by the MYH7 gene, is mainly located in the ventricles Variants in both isoforms have been previously associated with hypertrophic and dilated cardiomyopathy, a leading cause of sudden cardiac death in children and adults, as well as CHD. . The two isoforms have ~92% amino acid homology, yet have distinct functional characteristics. At the cellular level, more is known about how variants in MYH7 cause disease because the majority of the b‐MHC protein has been crystallized, while crystal structures have not been solved for any portions of the human a‐MHC protein.The MUHS SMART team submitted two amino acid sequences from the MYH6 gene to I‐TASSER, a bioinformatics method for predicting three‐dimensional protein structure, in order to study the effect of the MYH6 R443P variant on protein structure and function. The first submission contained the wild‐type amino acid sequence, while the second contained the R443P variant. The predicted protein structures were 3‐D printed in order to visualize any structural differences between the wild‐type and variant. I‐TASSER results did not show a significant change in secondary structure but did predict altered ligand binding in the variant protein compared to wild‐type. The electrostatic interface changed from a positively charged, polar amino acid (arginine) to one with a neutral, non‐reactive side chain (proline).The R443P variant occurs within the helix O domain, which lies between the actin binding domain and the ATP binding region, and contains subdomains responsible for binding numerous other proteins, as well as, hydrolyzing ATP during contraction. Normal function of myosin during each contraction of the heart requires that interactions with this region occur at the appropriate place and time. We hypothesize that altered binding properties of myosin heavy chain could lead to altered contractility, affecting the maximum shortening velocity and force during muscle contraction in the developing heart. Future research will focus on the mechanism that this variant has on the functionality of these domains as well as drug targeting therapies as a means to correct or remediate the impact of variants such as R443P.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lack of hemizygosity for the insulin-like growth factor I receptor gene in a quantitative study of 33 Silver Russell syndrome probands and their families.

Previous studies have shown that individuals with a deletion of 15q26.1-->qter, which includes the insulin-like growth factor I receptor (IGFIR) gene, may exhibit phenotypic characteristics similar to those individuals with Silver-Russell Syndrome (SRS). Thirty-three SRS probands, with normal karyotypes, and their parents were investigated for the presence of both copies of IGFIR by gene dosage analysis of Southern blot hybridisation. All 33 SRS probands have both copies of IGFIR. Tetranucleotide repeat marker analysis for three locations on 15q also ruled out other deletions in these regions for those markers that were informative. Two important functional regions of IGFIR were also investigated for DNA mutations, using single-stranded conformational polymorphism analysis. No mutations were found in the cysteine-rich region involved in ligand binding (exon 3) or the ATP binding region (exon 16) which could contribute to the SRS phenotype. However, a silent mutation in the third position of one of the codons in the ATP region (3174G-->A, 1013 Glu-->Glu) was found.

Silencing of type II phosphatidylinositol 4-kinase β stabilizes prostate apoptosis response-4 and induces apoptosis in cancer cells.

Type II phosphatidylinositol 4-kinase β (PtdIns 4-kinase II β) is an enigma among the phosphatidylinositol 4-kinase family. The role of PtdIns 4-kinase II β in MCF-7 cells was addressed with the help of short hairpin RNA (shRNA). PtdIns 4-kinase II β shRNA transfection increased pan-caspase activity and induced apoptosis in cancerous MCF-7 cells. Non-cancerous MCF-10A cells were resistant to PtdIns 4-kinase II β shRNA-induced apoptosis. Caspase 8 and 9 inhibitors rescued MCF-7 cells from apoptosis. Shotgun proteomic studies with Flag-tagged PtdIns 4-kinase II β immunoprecipitates showed tumor suppressor prostate apoptosis response-4 (Par-4) as one of the interacting proteins in HEK293 cells. In reciprocal experiments, Par-4 antibodies co-precipitated PtdIns 4-kinase II β from MCF-7 cells. Deletion of membrane localization motif (ΔCCPCC) or a mutation in ATP-binding region (D304A) of PtdIns 4-kinase II β did not affect its interaction with Par-4. Pull-down assays with GST-PtdIns 4-kinase II β-truncated mutants showed that the region between 101 and 215 amino acid residues is essential for interaction with Par-4. At molecular level, PtdIns 4-kinase II β shRNA transfection increased Par-4 stability, its nuclear localization and inhibition of NF-κB binding to target DNA. Knocking down of Par-4 with siRNA (small interfering RNA) rescued MCF-7 cells from PtdIns 4-kinase II β shRNA-induced apoptosis. These results suggest that PtdIns 4-kinase II β may be a novel regulator of Par-4 through protein-protein interactions. These studies have potential implications in cancer therapy.

Dynamics-based community analysis and perturbation response scanning of allosteric interaction networks in the TRAP1 chaperone structures dissect molecular linkage between conformational asymmetry and sequential ATP hydrolysis

Allosteric interactions of the Hsp90 chaperones with cochaperones and diverse protein clients can often exhibit distinct asymmetric features that determine regulatory mechanisms and cellular functions in many signaling networks. The recent crystal structures of the mitochondrial Hsp90 isoform TRAP1 in complexes with ATP analogs have provided first evidence of significant asymmetry in the closed dimerized state that triggers independent activity of the chaperone protomers, whereby preferential hydrolysis of the buckled protomer is followed by conformational flipping between protomers and hydrolysis of the second protomer. Despite significant insights in structural characterizations of the TRAP1 chaperone, the atomistic details and mechanics of allosteric interactions that couple sequential ATP hydrolysis with asymmetric conformational switching in the TRAP1 protomers remain largely unknown. In this work, we explored atomistic and coarse-grained simulations of the TRAP1 dimer structures in combination with the ensemble-based network modeling and perturbation response scanning of residue interaction networks to probe salient features underlying allosteric signaling mechanism. This study has revealed that key effector sites that orchestrate allosteric interactions occupy the ATP binding region and N-terminal interface of the buckled protomer, whereas the main sensors of allosteric signals that drive functional conformational changes during ATPase cycle are consolidated near the client binding region of the straight protomer, channeling the energy of ATP hydrolysis for client remodeling. The community decomposition analysis of the interaction networks and reconstruction of allosteric communication pathways in the TRAP1 structures have quantified mechanism of allosteric regulation, revealing control points and interactions that coordinate asymmetric switching during ATP hydrolysis.

Identification of a unique loss-of-function mutation in IGF1R and a crosstalk between IGF1R and Wnt/β-catenin signaling pathways

IGF1R is a ubiquitous receptor tyrosine kinase that plays critical roles in cell proliferation, growth and survival. Clinical studies have demonstrated upregulation of IGF1R mediated signaling in a number of malignancies including colon, breast, and lung cancers. Overexpression of the IGF1R in these malignancies is associated with a poor prognosis and overall survival. IGF1R specific kinase inhibitors have failed in multiple clinical trials partly because of the complex nature of IGF1R signaling. Thus identifying new binding partners and allosteric sites on IGF1R are emerging areas of research. More recently, IGF1R has been shown to translocate into the nucleus and perform many functions. In this study, we generated a library of IGF1R deletion and point mutants to examine IGF1R subcellular localization and activation of downstream signaling pathways. We show that the nuclear localization of IGF1R is primarily defined by its cytoplasmic domain. We identified a cross-talk between IGF1R and Wnt/β-catenin signaling pathways and showed, for the first time, that IGF1R is associated with upregulation of TCF-mediated β-catenin transcriptional activity. Using loss-of-function mutants, deletion analysis and IGF1R specific inhibitor(s), we show that cytoplasmic and nuclear activities are two independent functions of IGF1R. Furthermore, we identified a unique loss-of-function mutation in IGF1R. This unique loss-of-function mutant retains only nuclear functions and sits in a pocket, outside ATP and substrate binding region, that is suited for designing allosteric inhibitors of IGF1R.

Alanine mutation of the catalytic sites of Pantothenate Synthetase causes distinct conformational changes in the ATP binding region

The enzyme Pantothenate synthetase (PS) represents a potential drug target in Mycobacterium tuberculosis. Its X-ray crystallographic structure has demonstrated the significance and importance of conserved active site residues including His44, His47, Asn69, Gln72, Lys160 and Gln164 in substrate binding and formation of pantoyl adenylate intermediate. In the current study, molecular mechanism of decreased affinity of the enzyme for ATP caused by alanine mutations was investigated using molecular dynamics (MD) simulations and free energy calculations. A total of seven systems including wild-type + ATP, H44A + ATP, H47A + ATP, N69A + ATP, Q72A + ATP, K160A + ATP and Q164A + ATP were subjected to 50 ns MD simulations. Docking score, MM-GBSA and interaction profile analysis showed weak interactions between ATP (substrate) and PS (enzyme) in H47A and H160A mutants as compared to wild-type, leading to reduced protein catalytic activity. However, principal component analysis (PCA) and free energy landscape (FEL) analysis revealed that ATP was strongly bound to the catalytic core of the wild-type, limiting its movement to form a stable complex as compared to mutants. The study will give insight about ATP binding to the PS at the atomic level and will facilitate in designing of non-reactive analogue of pantoyl adenylate which will act as a specific inhibitor for PS.

Discovery of Potent EGFR Inhibitors through the Incorporation of a 3D-Aromatic-Boron-Rich-Cluster into the 4-Anilinoquinazoline Scaffold: Potential Drugs for Glioma Treatment.

New 1,7-closo-carboranylanilinoquinazoline hybrids have been identified as EGFR inhibitors, one of them with higher affinity than the parent compound erlotinib. The comparative docking analysis with compounds bearing bioisoster-substructures, demonstrated the relevance of the 3D aromatic-boron-rich moiety for interacting into the EGFR ATP binding region. The capability to accumulate in glioma cells, the ability to cross the blood-brain barrier and the stability on simulated biological conditions, render these molecules as lead compounds for further structural modifications to obtain dual action drugs to treat glioblastoma.

Long-range coupling between ATP-binding and lever-arm regions in myosin via dielectric allostery.

A protein molecule is a dielectric substance, so the binding of a ligand is expected to induce dielectric response in the protein molecule, considering that ligands are charged or polar in general. We previously reported that binding of adenosine triphosphate (ATP) to molecular motor myosin actually induces such a dielectric response in myosin due to the net negative charge of ATP. By this dielectric response, referred to as "dielectric allostery," spatially separated two regions in myosin, the ATP-binding region and the actin-binding region, are allosterically coupled. In this study, from the statistically stringent analyses of the extensive molecular dynamics simulation data obtained in the ATP-free and the ATP-bound states, we show that there exists the dielectric allostery that transmits the signal of ATP binding toward the distant lever-arm region. The ATP-binding-induced electrostatic potential change observed on the surface of the main domain induced a movement of the converter subdomain from which the lever arm extends. The dielectric response was found to be caused by an underlying large-scale concerted rearrangement of the electrostatic bond network, in which highly conserved charged/polar residues are involved. Our study suggests the importance of the dielectric property for molecular machines in exerting their function.

Insight into the molecular mechanism of P-glycoprotein mediated drug toxicity induced by bioflavonoids: an integrated computational approach

In this work, molecular docking, pharmacophore modeling and molecular dynamics (MD) simulation were rendered for the mouse P-glycoprotein (P-gp) (code: 4Q9H) and bioflavonoids; amorphigenin, chrysin, epigallocatechin, formononetin and rotenone including a positive control; verapamil to identify protein–ligand interaction features including binding affinities, interaction characteristics, hot-spot amino acid residues and complex stabilities. These flavonoids occupied the same binding site with high binding affinities and shared the same key residues for their binding interactions and the binding region of the flavonoids was revealed that overlapped the ATP binding region with hydrophobic and hydrophilic interactions suggesting a competitive inhibition mechanism of the compounds. Root mean square deviations (RMSDs) analysis of MD trajectories of the protein–ligand complexes and NBD2 residues, and ligands pointed out these residues were stable throughout the duration of MD simulations. Thus, the applied preliminary structure-based molecular modeling approach of interactions between NBD2 and flavonoids may be gainful to realize the intimate inhibition mechanism of P-gp at NBD2 level and on the basis of the obtained data, it can be concluded that these bioflavonoids have the potential to cause herb–drug interactions or be used as lead molecules for the inhibition of P-gp (as anti-multidrug resistance agents) via the NBD2 blocking mechanism in future.

Penduliflaworosin, a Diterpenoid from Croton crassifolius, Exerts Anti-Angiogenic Effect via VEGF Receptor-2 Signaling Pathway.

Anti-angiogenesis targeting vascular endothelial growth factor receptor-2 (VEGFR-2) has been considered as an important strategy for cancer therapy. Penduliflaworosin is a diterpenoid isolated from the plant Croton crassifolius. Our previous study showed that this diterpenoid possesses strong anti-angiogenic activity by inhibiting vessel formation in zebrafish. This study was conducted to further investigate the anti-angiogenic activity and mechanism of penduliflaworosin. Results revealed that penduliflaworosin significantly inhibited VEGF-induced angiogenesis processes including proliferation, invasion, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). Moreover, it notably inhibited VEGF-induced sprout formation of aortic rings and blocked VEGF-induced vessel formation in mice. Western blotting studies showed that penduliflaworosin inhibited phosphorylation of the VEGF receptor-2 and its downstream signaling mediators in HUVECs, suggesting that the anti-angiogenic activity was due to an interference with the VEGF/VEGF receptor-2 pathway. In addition, molecular docking simulation indicated that penduliflaworosin could form hydrogen bonds within the ATP-binding region of the VEGF receptor-2 kinase unit. Finally, cytotoxicity assay showed that penduliflaworosin possessed little toxicity toward both cancer and normal cells. Taken together, our findings demonstrate that penduliflaworosin exerts its anti-angiogenic effect via the VEGF receptor-2 signaling pathway. The anti-angiogenic property and low cytotoxicity of penduliflaworosin suggest that it may be useful in cancer treatments.

Exploring the Molecular Basis for Binding of Inhibitors by Threonyl-tRNA Synthetase from Brucella abortus: A Virtual Screening Study.

Targeting threonyl-tRNA synthetase (ThrRS) of Brucella abortus is a promising approach to developing small-molecule drugs against bovine brucellosis. Using the BLASTp algorithm, we identified ThrRS from Escherichia coli (EThrRS, PDB ID 1QF6), which is 51% identical to ThrRS from Brucella abortus (BaThrRS) at the amino acid sequence level. EThrRS was used as the template to construct a BaThrRS homology model which was optimized using molecular dynamics simulations. To determine the residues important for substrate ATP binding, we identified the ATP-binding regions of BaThrRS, docked ATP to the protein, and identified the residues whose side chains surrounded bound ATP. We then used the binding site of ATP to virtually screen for BaThrRS inhibitors and got seven leads. We further characterized the BaThrRS-binding site of the compound with the highest predicted inhibitory activity. Our results should facilitate future experimental effects to find novel drugs for use against bovine brucellosis.

Insights into the structural dynamics of Liver kinase B1 (LKB1) by the binding of STe20 Related Adapterα (STRADα) and Mouse protein 25α (MO25α) co-activators

LKB1, the tumour suppressor, is found mutated in Peutz-Jeghers syndrome (PJS). The LKB1 is a serine-threonine kinase protein that is allosterically activated by the binding of STRADα and MO25α without phosphorylating the Thr212 present at activation loop. The present study aims to highlight the structural dynamics and complexation mechanism during the allosteric activation of LKB1 by these co-activators using molecular dynamics simulations. The all atom simulations performed on the complexes of LKB1 with ATP, STRADα, and MO25α for a period of 30 ns reveal that binding of STRADα and MO25α significantly stabilizes the highly flexible regions of LKB1 such as ATP binding region (β1-β2 loop), catalytic & activation loop segments and αG helix. Also, binding of STRADα and MO25α to LKB1 promotes coordinated motion between N- and C-lobes along with the catalytic & activation loops by forming H-bonds between LKB1 and co-activators, which further facilitate to establish the conserved attributes of active LKB1 such as (i) formation of salt bridge between Lys78 and Glu98, (ii) formation of stable hydrophobic R- and C-spines, and (iii) interaction between both catalytic and activation loops. Especially, the residues of LKB1 interacting with STRADα (Arg74, Glu342) and MO25α (Glu165, Pro203 and Phe204) are observed to play a significant role in stabilizing the (LKB1-ATP)-(STRADα-ATP)-MO25α complex. Overall, the present work highlighting the structural dynamics of LKB1 by the binding of allosteric co-activators is expected to provide a basic understanding on drug design specific to PJS syndrome.

Defects in Protein Folding Machinery Affect Cell Wall Integrity and Reduce Ethanol Tolerance in S. cerevisiae.

The chaperonin complex CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring complex) participates in the folding of many crucial proteins including actin and tubulin in eukaryotes. Mutations in genes encoding its subunits can affect protein folding and in turn, the physiology of the organism. Stress response in Saccharomyces cerevisiae is important in fermentation reactions and operates through overexpression and underexpression of genes, thus altering the protein profile. Defective protein folding machinery can disturb this process. In this study, the response of cct mutants to stress conditions in general and ethanol in specific was investigated. CCT1 mutants showed decreased resistance to different conditions tested including osmotic stress, metal ions, surfactants, reducing and oxidising agents. Cct1-3 mutant with the mutation in the conserved ATP-binding region showed irreversible defects than other mutants. These mutants were found to have inherent cell wall defects and showed decreased ethanol tolerance. This study reveals that cell wall defects and ethanol sensitivity are linked. Genetic and proteomic analyses showed that the yeast genes RPS6A (ribosomal protein), SCL1 (proteasomal subunit) and TDH3 (glyceraldehyde-3-phosphate dehydrogenase) on overexpression, improved the growth of cct1-3 mutant on ethanol. We propose the breakdown of common stress response pathways caused by mutations in CCT complex and the resulting scarcity of functional stress-responsive proteins, affecting the cell's defence against different stress agents in cct mutants. Defective cytoskeleton and perturbed cell wall integrity reduce the ethanol tolerance in the mutants which are rescued by the extragenic suppressors.

Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE.

Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the γ-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration.

Characterization of a PERK Kinase Inhibitor with Anti-Myeloma Activity

Abstract The malignant plasma cell of Multiple Myeloma (MM) is closely linked to dysregulated unfolded protein response in the ER due to increased production of immunoglobulin and the metabolic demands of malignant uncontrolled proliferation. Due to the immunoglobulin production, MM plasma cells are dependent on the unfolded protein response process (UPR), which controls protein production and ensures its proper translation and folding. A study by Michallet et al (2011) showed that knockdown of one of the three well-known arms of the UPR, PERK (protein kinase R (PKR)-like ER kinase) in MM cells resulted in autophagic cell death. This outcome indicated the important role of PERK activation for the metabolic shift of plasma cell to myeloma cell but it also showed that PERK knockdown could also impede the apoptotic effect. Therefore as PERK activity has been implicated in the survival of MM cells as well as with the apoptotic potential of the cells, targeting this pathway in MM may be an attractive therapeutic approach. In this study we used a small ATP competitive molecular inhibitor, GSK2606414, that acts by targeting PERK enzyme activity in its inactive DFG conformation at the ATP-binding region, while displaying ≥385 fold selectivity over c-kit, Aurora B, BRK and many other kinases. To examine the expression of PERK in MM, we initially screened 25 CD138+ MM patients and 6 human myeloma cell lines (HMCLs) for PERK mRNA expression. Our results showed that PERK mRNA is highly expressed in almost all patients (5-10 fold higher than the mean PERK expression of HMCLs) whereas differentially expressed in HMCLs. To test the effect of GSK2606414 on the proliferation of MM cells, 4 HMCLs were treated with different doses of GSK2606414 at two time points (24 and 48 hours). Treatment of cells with 3-30μM GSK2606414 resulted in a dose-dependent inhibition of cell proliferation in all HMCLs ranging for 20-95% reduction of proliferative activity, thus, indicating the dependency of these cells on PERK activity. We further studied the induction of apoptosis in GSK2606414 treated HMCLs samples using Annexin-PI staining. Treatment with GSK2606414 at 20μM in H929 and L363 HMCLs for 24 hours resulted 25 % and 15 % increase in apoptotic cells respectively compared to the untreated cells. However, the most important finding was a significant synergistic effect of GSK2606414 with bortezomib in these cells. Specifically when H929 and L363 cells were treated with 5nM bortezomib in combination with 20μM GSK2606414, the outcome resulted in a synergistic effect where apoptotic cells reached 99% and 77% respectively, whereas bortezomib only treated cells resulted in 87% and 42% Annexin-PI positively stained cells respectively. These finding show the ability of GSK2606414 to enhance the apoptotic effect of bortezomib in myeloma cells and act as an attractive therapeutic anti-myeloma strategy Under ER stress conditions, the activation of ATF6 and PERK/eIF2α leads to the induction of ATF4 translation and results in the upregulation of CHOP. To determine the gene target effects of GSK2606414, ATF4 and CHOP mRNA expression levels were determined in H929 cell line after 24 hour of treatment. Treatment with GSK2606414 alone did not alter the expression levels of CHOP but reduced more than 50% the expression levels of ATF4. When combined with bortezomib CHOP and ATF4 levels were reduced 20% and 60% respectively while treatment with bortezomib alone increased the levels of CHOP and ATF4 by 50-100%. To test the hypothesis that ATF4 and CHOP expression is also suppressed during sustained UPR response, two myeloma cell lines, H929 and L363, were pre-treated with GSK2606414 and then subjected to ER stress conditions by treatment with tunicamycin (TM). In the presence of exogeneous UPR inducers, such as TM GSK2656157 had a significant effect on the expression of the above genes at much lower IC50 range (≤100 nM) suggesting the in a more active UPR (sustained ER stress) the effects are more pronounced. Thus, the combination of GSK2606414 with bortezomib may inhibit one of the mechanisms of MM cells to escape ER stress induced by bortezomib. In conclusion, given the on-target pharmacological effects of PERK inhibitor on MM, development of PERK inhibitors may offer a therapeutic advantage by acting on another node of UPR signaling which directly or indirectly would affect MM pathogenesis and treatment. Disclosures Dimopoulos: Genesis: Honoraria; Novartis: Honoraria; Onyx: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Janssen-Cilag: Honoraria; Amgen: Honoraria.

Isolation and identification of hybrid recombinant E7 oncoprotein of type 16 human papilloma virus conjugated with heat shock protein 70

The recombinant E7 oncoprotein gene of type 16 human papilloma virus (conjugated with the heat shock protein 70 (E7(16)-HSP70)) was expressed in Saccharomyces cerevisiae yeast cells. A number of chromatographic isolations were conducted to obtain the target protein and its primary identification and to establish its originality. The use of metal chelate chromatography was based on selective binding of sorbent active groups with a poly-histidine fragment at C-terminus of the target protein polypeptide sequence, while the use of affine chromatography was based on the association with ATP-binding regions of E7 and HSP70. The degree of purity of the target protein after both procedures did not exceed 70%, indicating the presence of many low-molecular admixtures and products of the target molecule oligomerization. The use of chromatographic purification under denaturing conditions using strong reducing agents allowed isolation of the target protein, the monomer content in which exceeded 97%. The target protein originality was established by immunoblotting and electrophoresis; the correspondence of its primary structure to the declared sequence was demonstrated by mass-spectrophotometry.

3D-QSAR (CoMFA, CoMFA-RG, CoMSIA) and molecular docking study of thienopyrimidine and thienopyridine derivatives to explore structural requirements for aurora-B kinase inhibition

Aurora-B kinase plays a crucial role in cell cycle events and is identified as an important factor in regulation of spindle check point assembly. Thus, it can be proved as an important target in the field of oncology. 3D-QSAR model was generated using 54 molecules reported in literature containing thienopyrimidine and thienopyridine as scaffolds. All molecules were aligned using Distill function in Sybyl X1.2. This generated best model of CoMFA-RG (Region focusing) and CoMSIA were statistically significant with correlation coefficient r2ncv of 0.97, for both & Leave one out coefficient (LOO) q2 of 0.70 and 0.72, respectively. Best CoMSIA model was built up using various combination of descriptors and proved statistical significant among all models. Best CoMFA-RG and CoMSIA models were validated by 12 test set molecules giving satisfactory prediction (r2pred) values of 0.86 and 0.88, respectively. External test set validation was performed using 20 molecules and satisfactory prediction of their biological activity was found. Active compounds were docked on protein (PDB ID: 4C2V) by GOLD module and revealed important interactions with amino acids at ATP-binding region. These data explored insight requirements for Aurora-B inhibition which might be fruitful for understanding mechanisms with kinase ligand interactions.

RECOMBINANT FUSION E7 ONCOPROTEIN OF HUMAN PAPILLOMAVIRUS TYPE 16 CONJUGATED WITH HEAT SHOCK PROTEIN 70: ISOLATION AND IDENTIFICATION

A gene for a recombinant E7 oncoprotein of human papillomavirus type 16 conjugated with a heat shock protein 70 (E7(16)-HSP70) has been expressed in Saccharomyces cerevisiae. A number of chromatographic separations was performed in order to primarily identify the protein and establish its authenticity. Metal-chelate and affinity chromatography were aimed at selective binding of the active groups of sorbents with polyhistidine fragment at the C-terminus of the polypeptide sequence of the target protein and ATP-binding regions of E7 and HSP70, respectively. The purity of the target protein after two purification steps did not exceed 70% showing the presence of a number of low-molecular impurities and oligomerization products of the target molecule. Chromatographic purification under denaturing conditions using strong reducing agents made it possible to isolate the target protein with the content of the monomer exceeding 97%. Electrophoresis and immunoblotting methods permitted to establish the authenticity of the target protein, and mass spectrometry showed its set primary structure.

Rutin stimulates sarcoplasmic reticulum Ca(2+)-ATPase activity (SERCA1) and protects SERCA1 from peroxynitrite mediated injury.

In this study we analyzed the protective action of the flavonoid rutin on peroxynitrite (ONOO(-)) mediated impairment of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1 isoform), especially related to posttranslational and conformational changes. Rutin concentration dependently protected ONOO(-) induced SERCA1 activity decrease with effective concentration EC50 of 18±1.5µM. Upon treatment with ONOO(-), this flavonoid also prevented SERCA1 from thiol group oxidation and significantly reduced tyrosine nitration and protein carbonyl formation. In the absence of ONOO(-), rutin (250 and 350µM) stimulated SERCA1 activity at 2.1mM [ATP] and 10µM [Ca(2+)]free. According to changes in the kinetic parameters V max and K m with regard to [ATP], rutin (250µM) increased the rate of enzyme catalysis and decreased the affinity of SERCA1 to ATP. FITC fluorescence decreased in the presence of rutin (150 and 250µM), indicating conformational changes in the cytosolic ATP binding region of SERCA1. In silico study confirmed the binding of rutin in the cytosolic region of SERCA1, in the vicinity of the ATP binding site. Residue Glu183 localized within the conserved TGES loop was identified to play a key role in rutin-SERCA1 interaction (H-bond length of 1.7Å), elucidating the ability of rutin to affect the affinity of SERCA1 to ATP. The binding of rutin in the proximity of Lys515 is likely to cause a decrease in FITC fluorescence.