Unbound Form Research Articles

Risk Assessment of "Other Substances" – L-Glutamine and L-glutamic Acid

Risk Assessment of "Other Substances" – L-Glutamine and L-glutamic Acid

Structural Analysis of the SANT/Myb Domain of FLASH and YARP Proteins and Their Complex with the C-Terminal Fragment of NPAT by NMR Spectroscopy and Computer Simulations

FLICE-associated huge protein (FLASH), Yin Yang 1-Associated Protein-Related Protein (YARP) and Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) localize to discrete nuclear structures called histone locus bodies (HLBs) where they control various steps in histone gene expression. Near the C-terminus, FLASH and YARP contain a highly homologous domain that interacts with the C-terminal region of NPAT. Structural aspects of the FLASH–NPAT and YARP–NPAT complexes and their role in histone gene expression remain largely unknown. In this study, we used multidimensional NMR spectroscopy and in silico modeling to analyze the C-terminal domain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acids of NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a triple -helical bundle that resembles the DNA binding domain of Myb transcriptional factors and the SANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide contains a single -helix that makes multiple contacts with -helices I and III of the FLASH and YARP domains. Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPAT using a unique network of interactions, yielding two distinct complexes. In silico modeling suggests that both complexes are structurally compatible with DNA binding, raising the possibility that they may function in identifying specific sequences within histone gene clusters, hence initiating the assembly of HLBs and regulating histone gene expression during cell cycle progression.

Impacts of drug resistance mutations on the structural asymmetry of the HIV-2 protease

BackgroundDrug resistance is a severe problem in HIV treatment. HIV protease is a common target for the design of new drugs for treating HIV infection. Previous studies have shown that the crystallographic structures of the HIV-2 protease (PR2) in bound and unbound forms exhibit structural asymmetry that is important for ligand recognition and binding. Here, we investigated the effects of resistance mutations on the structural asymmetry of PR2. Due to the lack of structural data on PR2 mutants, the 3D structures of 30 PR2 mutants of interest have been modeled using an in silico protocol. Structural asymmetry analysis was carried out with an in-house structural-alphabet-based approach.ResultsThe systematic comparison of the asymmetry of the wild-type structure and a large number of mutants highlighted crucial residues for PR2 structure and function. In addition, our results revealed structural changes induced by PR2 flexibility or resistance mutations. The analysis of the highlighted structural changes showed that some mutations alter protein stability or inhibitor binding.ConclusionsThis work consists of a structural analysis of the impact of a large number of PR2 resistant mutants based on modeled structures. It suggests three possible resistance mechanisms of PR2, in which structural changes induced by resistance mutations lead to modifications in the dimerization interface, ligand recognition or inhibitor binding.

Potentials for a circular economy of mineral construction materials and demolition waste in urban areas: a case study from Vienna

Mineral construction and demolition wastes (CDW) are generated when buildings and infrastructures are renovated and when they reach their end of life. As one of the largest waste streams, they have a considerable potential for the reduction of waste generation, landfilling, and primary raw material consumption. To make use of this potential, sustainable development strategies of many cities include a circular management of CDW by measures in line with the waste hierarchy. The present study uses material flow analysis to determine how waste reduction, re-use and recycling of mineral CDW generated in a city can contribute to reduce the demand of raw material imports for construction minerals, using the case study of the city of Vienna. The results show that the annual consumption of construction minerals of 4.5 million tons can be reduced by 32% to 3 million tons by implementing the waste hierarchy to CDW. The most important measures are the use of recycling materials from mineral construction and demolition waste as recycling aggregate in concrete (575,000 t/yr), followed by the use of recycling material to substitute gravel in unbound form (463,000 t/yr), avoiding the demolition of historical buildings by extending their service life (230,000 t/yr), asphalt recycling (85,000 t/yr), and substitution of raw-mix in cement by recycling material from debris (84,000 t/yr). Re-use of full bricks (17,000 t/yr) is of lesser relevance. To implement this enhanced circularity scenario, however, efforts in installed technology, construction and demolition waste management as well as legal and entrepreneurial measures are required.

Protein dynamics and molecular motions study in relation to molecular interaction between proteins from sulfur oxidizing proteobacteria Allochromatium vinosum

The gamma-proteobacteria Allochromatium vinosum DSM 180T (A. vinosum) encodes the sulfur oxidizing dsr operon comprising of 15 genes. Dsr proteins are involved in oxidation of sulfur globules produced as an obligatory intermediate during the sulfur oxidation process. The dsrA and dsrB gene products are known to function as a α2β2 hetero-tetramer and the protein complex plays the catalytic role in sulfur oxidation process. DsrC has a highly conserved C-terminal domain that forms a flexible arm, where two strictly conserved cysteines were found to act as a substrate donating residue for DsrAB instead of being a subunit of this redox enzyme. Therefore, to elucidate the molecular mechanism of the sulfur oxidation process here an attempt was made to study the dynamics, stability and binding mechanisms of DsrAB and DsrC proteins through computational docking and molecular dynamics (MD) simulations. This structure function relationship investigation revealed that the C-terminal domain of DsrC interacts with DsrA of DsrAB protein complex for catalytic functions. Some basic amino acid residues of DsrC are found to form the catalytic pockets along with DsrAB protein complex where the sulfur anions bind to get oxidized. Structural dynamics and fluctuations as well as the secondary structural alterations study revealed the possible regions responsible for protein-protein interactions. Principal Component Analysis (PCA) of protein motions displayed that the collective motions of DsrAB-DsrC complex was higher and more anti-correlated than the unbound DsrAB form. The present molecular insight study would therefore help researchers to predict the plausible biochemical mechanism of sulfur oxidation process in sulfur metabolic pathways in near future. Communicated by Ramaswamy H. Sarma

Microbiome Catabolites as Novel Modulators of Cellular Glucose and Energy Metabolism

The gut microbiome supplies bioactive metabolites such as butyrate from dietary fiber to enhance the function of skeletal muscle mitochondria, and influences metabolism during exercise. Trained athletes have a distinctly different microbiome compared to sedentary individuals, since the composition and activity of the microbiota is in turn affected by muscle fitness.The gut microbiota regulate mitochondrial biogenesis through modulation of PGC‐1α, SIRT1 and AMPK, and the mitochondria regulate the gut microbiota by modulating mucosal immune responses and intestinal barrier function. Skeletal muscle is the main site of postprandial glucose disposal; the glucose transporter GLUT4 is activated by insulin by translocation to the plasma membrane from internal vesicles. In diabetic and pre‐diabetic insulin‐resistant tissues, this GLUT4 translocation is compromised. Therapeutic strategies against insulin resistance, including exercise and diet‐related interventions, aim to lower postprandial glycemia at least partly by restoring skeletal muscle glucose uptake and enhancing metabolism.Dietary phenolics can be considered as prebiotic‐type molecules, including precursors present in the unbound form as well as covalently‐attached to dietary fiber such as in cereal brans. After microbial action, many potentially bioactive phenolics are released and absorbed in the colon. These microbial products, once absorbed, interact with other tissues, including muscle and adipose cells, and stimulate the uptake of glucose by several mechanisms. For example, the microbial metabolite isovanillic acid sulfate (IVAS) stimulated a dose‐dependent increase in glucose transport in human LHCN‐M2 muscle cells through a GLUT4‐ and PI3K‐dependent mechanism. IVAS also upregulated GLUT1, GLUT4 and PI3K p85α protein, and increased phosphorylation of Akt.The stimulation of glucose uptake and metabolism provides a novel link between the diet, gut microbiota and skeletal muscle energy utilization.

Combined Magnetic Resonance Imaging and Photodynamic Therapy Using Polyfunctionalised Nanoparticles Bearing Robust Gadolinium Surface Units.

A robust dithiocarbamate tether allows novel gadolinium units based on DOTAGA (q=1) to be attached to the surface of gold nanoparticles (2.6-4.1 nm diameter) along with functional units offering biocompatibility, targeting and photodynamic therapy. A dramatic increase in relaxivity (r1 ) per Gd unit from 5.01 mm-1 s-1 in unbound form to 31.68 mm-1 s-1 (10 MHz, 37 °C) is observed when immobilised on the surface due to restricted rotation and enhanced rigidity of the Gd complex on the nanoparticle surface. The single-step synthetic route provides a straightforward and versatile way of preparing multifunctional gold nanoparticles, including examples with conjugated zinc-tetraphenylporphyrin photosensitizers. The lack of toxicity of these materials (MTT assays) is transformed on irradiation of HeLa cells for 30 minutes (PDT), leading to 75 % cell death. In addition to passive targeting, the inclusion of units capable of actively targeting overexpressed folate receptors illustrates the potential of these assemblies as targeted theranostic agents.

Identification of potential inhibitors of human methionine aminopeptidase (type II) for cancer therapy: Structure-based virtual screening, ADMET prediction and molecular dynamics studies

Methionine Aminopeptidases MetAPs are divalent-cofactor dependent enzymes that are responsible for the cleavage of the initiator Methionine from the nascent polypeptides. MetAPs are classified into two isoforms: namely, MetAP1 and MetAP2. Several studies have revealed that MetAP2 is upregulated in various cancers, and its inhibition has shown to suppress abnormal or excessive blood vessel formation and tumor growth in model organisms. Clinical studies show that the natural product fumagillin, and its analogs are potential inhibitors of MetAP2. However, due to their poor pharmacokinetic properties and neurotoxicities in clinical studies, their further developments have received a great setback. Here, we apply structure-based virtual screening and molecular dynamics methods to identify a new class of potential inhibitors for MetAP2. We screened Otava’s Chemical Library, which consists of about 3 200 000 tangible-chemical compounds, and meticulously selected the top 10 of these compounds based on their inhibitory potentials against MetAP2. The top hit compounds subjected to ADMET predictor using 3 independent ADMET prediction programs, were found to be drug-like. To examine the stability of ligand binding mode, and efficacy, the unbound form of MetAP2, its complexes with fumagillin, spiroepoxytriazole, and the best promising compounds compound-3369841 and compound-3368818 were submitted to 100 ns molecular dynamics simulation. Like fumagillin, spiroepoxytriazole, and both compound-3369841 and compound-3368818 showed stable binding mode over time during the simulations. Taken together, these uninherited-fumagillin compounds may serve as new class of inhibitors or provide scaffolds for further optimization towards the design of more potent MetAP2 inhibitors -development of such inhibitors would be essential strategy against various cancer types.

Specific Conformational Dynamics and Expansion Underpin a Multi-Step Mechanism for Specific Binding of p27 with Cdk2/Cyclin A

The protein p27, a prominent regulatory protein in eukaryotes and an intrinsically disordered protein (IDP), regulates cell division by causing cell cycle arrest when bound in ternary complex with cyclin-dependent kinase (Cdk2) and cyclins (e.g., Cdk2/Cyclin A). We present an integrative study of p27 and its binding to Cdk2/Cyclin A complex by performing single-molecule multiparameter fluorescence spectroscopy, stopped-flow experiments, and molecular dynamics simulations. Our results suggest that unbound p27 adopts a compact conformation and undergoes conformational dynamics across several orders of magnitude in time (nano-to milliseconds), reflecting a multi-step mechanism for binding Cdk2/Cyclin A. Mutagenesis studies reveal that the region D1 in p27 plays a significant role in mediating the association kinetics, undergoing conformational rearrangement upon initial binding. Additionally, FRET experiments indicate an expansion of p27 throughout binding. The detected local and long-range structural dynamics suggest that p27 exhibits a limited binding surface in the unbound form, and stochastic conformational changes in D1 facilitate initial binding to Cdk2/Cyclin A complex. Furthermore, the post-kinase inhibitory domain (post-KID) region of p27 exchanges between distinct conformational ensembles: an extended regime exhibiting worm-like chain behavior, and a compact ensemble, which may protect p27 against nonspecific interactions. In summary, the binding interaction involves three steps: (i) D1 initiates binding, (ii) p27 wraps around Cdk2/Cyclin A and D2 binds, and (iii) the fully-formed fuzzy ternary complex is formed concomitantly with an extension of the post-KID region. An understanding of how the IDP nature of p27 underpins its functional interactions with Cdk2/Cyclin A provides insight into the complex binding mechanisms of IDPs and their regulatory mechanisms.

The Role of HCN Channel Helices D and E in the Modulation of Camp Affinity

Hyperpolarization-activated cyclic-nucleotide regulated (HCN) channels are non-selective cation channels controlling key physiological functions such as cardiac pacemaking and repetitive neuronal firing. They are activated by voltage and modulated by binding of cAMP to their cytoplasmic C-terminal region named cyclic nucleotide binding domain (CNBD). Altered cAMP responses caused either by single point mutations in the protein or by non-physiological levels of the cyclic nucleotide in the cell lead to pathological conditions both in the heart and in the nervous system. The recently obtained cryo-EM structure of human HCN1 in the bound and unbound forms, confirms previous NMR studies on cAMP-induced conformational changes and reveals the presence of two additional helices at the C-terminus of the CNBD which fold in the presence of cAMP. We have previously shown, by ITC and patch clamp, that deletion of these helices (D and E) decreases the affinity for cAMP in the most studied HCN isoforms (HCN1, HCN2 and HCN4). Here we introduced single point mutations in HCN4 isoform, designed either to delete the single E helix or to selectively perturb the folding of the D helix, with the aim to dissect the role of the single D and E helices in this regulatory mechanism. Moreover, different truncations of the D helix were analyzed in order to clarify its role in the folding of the preceding C helix, known to be a key element in the transition of the CNBD from the unbound to the bound state. The patch clamp experiments were performed in whole cell configuration using a new all-in-one integrated amplifier, the ePatch.

Modified Physicochemical Properties of Acidic Model Drugs Immobilized on Fe3O4Magnetic Iron Oxide Nanoparticles

Nanoparticles are now extensively evaluated as drug carriers. The main objective of the present work was to evaluate the difference in physicochemical properties (solubility, permeability, lipophilicity) between acidic drugs immobilized on magnetic iron oxide nanoparticles and the same drugs in their unbound form. Synthesis of Fe3O4 magnetic iron oxide nanoparticles coated with (3-aminopropyl)triethoxysilane (APTES) was carried out with the chosen drugs attached. The obtained nanostructures were characterized using IR spectroscopy, atomic force microscopy (AFM), vibrating sample magnetometry (VSM) and dynamic light scattering (DLS) techniques, and physicochemical properties of the immobilized drugs were studied. The drug solubility was measured using the saturation shake-flask method. The permeability was measured using dialysis membranes, MWCO 50 kD with pores <10 nm. The lipophilicity was measured by partitioning in octanol. The drugs showed excellent solubility and permeability at low pH values (pH 2.0 and 5.0) and low solubility and permeability at higher pH values (pH 6.5 and 7.5) in comparison to the corresponding unbound drugs. The MNP[APTES]-immobilized drugs diffused to the organic phase very poorly, even in their non-ionized form (pH 2.0) and showed extremely low distribution coefficients. It was concluded that the immobilization of drugs on MNP[APTES] particles strongly modifies their physicochemical properties such as solubility, permeability and lipophilicity. This circumstance should be taken into account in designing new drug delivery systems, as the immobilization may strongly influence passive absorption. Extremely high release and dissolution rates of the tested drugs, followed by their efficient permeability through membrane pores at low pH, is the reason why the oral administration of therapeutic agents immobilized on MNPs should be considered as a method for more efficient drug delivery.

Large-size aggregates for road construction—a review of standard specifications and test methods

Construction aggregates are essential structural components in road structures, whether in unbound form or combined with cement or bitumen. Specifications and test methods for aggregates are standardised, but current standards are limited concerning the maximal aggregate size for which they apply. This paper deals with standard specifications and test methods for large-size road construction aggregates, reviewing a new Norwegian national standard for large-size aggregates. The standard test methods and their validity and applicability for use on large-size aggregates for road construction are evaluated. The differentiation between requirements regarding the rock material (single rocks) and the aggregate product is specifically discussed, as is the specific challenges related to the use of all-in aggregates. Current standards for construction aggregates do not cover all challenges related to description and quality assessment of construction aggregates. Quality assessment is particularly challenging for large-size aggregates, and this topic needs a better description by international standards. Field methods and digital image processing are introduced as solutions for some of the practical challenges related to sampling for large-size aggregates. The new Norwegian standard could be the first step towards a European standard for large-size aggregates.

New Green Determination of Cu, Fe, Mn, and Zn in Beetroot Juices along with Their Chemical Fractionation by Solid-Phase Extraction.

A new simple and rapid method for the determination of the total concentrations of Cu, Fe, Mn, and Zn in beetroot juices by flame atomic absorption spectrometry was developed and validated. The method included a very simple sample preparation, i.e., the two-fold dilution and acidification of the samples with HNO3 to 1 mol·L−1 and provided the precision within 2–3% and the trueness better than 6%. The method was applied for the rapid screening analysis of the different commercially available beetroot juices. The chemical fractionation of Cu, Fe, Mn, and Zn was also proposed by the two-column solid-phase extraction with the reversed-phase and cation exchange tubes. It was revealed that Cu, Fe, Mn, and Zn were primarily in beetroot juices in the form of the organically bound forms, contributing to the distinguished hydrophobic and residual fractions. The sums of the mean contributions of both fractions were up to 98% (Cu, Fe, Zn) and 100% (Mn), pointing out that no labile nor unbound forms of the studied metals were present in the matrix of beetroot juices.

MS-Based Approaches Enable the Structural Characterization of Transcription Factor/DNA Response Element Complex.

The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques.

Pre‐ and post‐docking sampling of conformational changes using ClustENM and HADDOCK for protein‐protein and protein‐DNA systems

Incorporating the dynamic nature of biomolecules in the modeling of their complexes is a challenge, especially when the extent and direction of the conformational changes taking place upon binding is unknown. Estimating whether the binding of a biomolecule to its partner(s) occurs in a conformational state accessible to its unbound form (“conformational selection”) and/or the binding process induces conformational changes (“induced‐fit”) is another challenge. We propose here a method combining conformational sampling using ClustENM—an elastic network‐based modeling procedure—with docking using HADDOCK, in a framework that incorporates conformational selection and induced‐fit effects upon binding. The extent of the applied deformation is estimated from its energetical costs, inspired from mechanical tensile testing on materials. We applied our pre‐ and post‐docking sampling of conformational changes to the flexible multidomain protein‐protein docking benchmark and a subset of the protein‐DNA docking benchmark. Our ClustENM‐HADDOCK approach produced acceptable to medium quality models in 7/11 and 5/6 cases for the protein‐protein and protein‐DNA complexes, respectively. The conformational selection (sampling prior to docking) has the highest impact on the quality of the docked models for the protein‐protein complexes. The induced‐fit stage of the pipeline (post‐sampling), however, improved the quality of the final models for the protein‐DNA complexes. Compared to previously described strategies to handle conformational changes, ClustENM‐HADDOCK performs better than two‐body docking in protein‐protein cases but worse than a flexible multidomain docking approach. However, it does show a better or similar performance compared to previous protein‐DNA docking approaches, which makes it a suitable alternative.

Monitoring DNA-Ligand Interactions in Living Human Cells Using NMR Spectroscopy.

Studies on DNA-ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA-ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA-ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro-validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off-target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of drug-like molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off-target binding.

Autoencoder-Based Detection of Dynamic Allostery Triggered by Ligand Binding Based on Molecular Dynamics.

Dynamic allostery on proteins, triggered by regulator binding or chemical modifications, transmits information from the binding site to distant regions, dramatically altering protein function. It is accompanied by subtle changes in side-chain conformations of the protein, indicating that the changes in dynamics, and not rigid or large conformational changes, are essential to understand regulation of protein function. Although a lot of experimental and theoretical studies have been dedicated to investigate this issue, the regulation mechanism of protein function is still being debated. Here, we propose an autoencoder-based method that can detect dynamic allostery. The method is based on the comparison of time fluctuations of protein structures, in the form of distance matrices, obtained from molecular dynamics simulations in ligand-bound and -unbound forms. Our method detected that the changes in dynamics by ligand binding in the PDZ2 domain led to the reorganization of correlative fluctuation motions among residue pairs, which revealed a different view of the correlated motions from the PCA and DCCM. In addition, other correlative motions were also found as a result of the dynamic perturbation from the ligand binding, which may lead to dynamic allostery. This autoencoder-based method would be usefully applied to the signal transduction and mutagenesis systems involved in protein functions and severe diseases.

Polyfunctionalised Nanoparticles Bearing Robust Gadolinium Surface Units for High Relaxivity Performance in MRI.

The first example of an octadentate gadolinium unit based on DO3A (hydration number q=1) with a dithiocarbamate tether has been designed and attached to the surface of gold nanoparticles (around 4.4 nm in diameter). In addition to the superior robustness of this attachment, the restricted rotation of the Gd complex on the nanoparticle surface leads to a dramatic increase in relaxivity (r1 ) from 4.0 mm-1 s-1 in unbound form to 34.3 mm-1 s-1 (at 10 MHz, 37 °C) and 22±2 mm-1 s-1 (at 63.87 MHz, 25 °C) when immobilised on the surface. The one-pot synthetic route provides a straightforward and versatile way of preparing a range of multifunctional gold nanoparticles. The incorporation of additional surface units for biocompatibility (PEG and thioglucose units) and targeting (folic acid) leads to little detrimental effect on the high relaxivity observed for these non-toxic multifunctional materials. In addition to the passive targeting attributed to gold nanoparticles, the inclusion of a unit capable of targeting the folate receptors overexpressed by cancer cells, such as HeLa cells, illustrates the potential of these assemblies.

Abstract 3883: High unbound plasma concentration of M-2, an active metabolite, is associated with shorter survival in patients with metastatic colorectal cancer who received regorafenib

Abstract Background and Aims: Regorafenib is an oral multikinase inhibitor which showed survival advantage in the later-line chemotherapy in patients with metastatic colorectal cancer (mCRC). This anticancer drug is sequentially metabolized to pharmacologically active metabolites M-2 and M-5 by the hepatic CYP3A. A randomized phase II trial has recently revealed that initial dose of 80 mg/day followed by dose escalations (40 mg/week) was superior to the standard dosing strategy (160 mg/day) with respect to overall survival (OS), resulting in the NCCN guideline recommendation for this dose escalation strategy, although scientific index for rational dose estimation is still limited. The aim of this prospective study was to clarify the association of pharmacokinetics (PK) with clinical outcome of regorafenib in patients with mCRC. Methods: Consecutive patients with mCRC administered regorafenib at Showa University Hospital were prospectively enrolled in this study. Patients received regorafenib 160 mg once daily for the first 3 weeks of each 4 weeks cycle. Blood samples for PK analysis were obtained on day1 (0-48 h) skipping the second dose on day2, and if possible day15 of treatment (0-24 h). Plasma concentrations of the regorafenib, M-2, and M-5 were analyzed by HPLC or LC-MS/MS. Unbound fraction of these compounds were measured by equilibrium dialysis method. Results: A total of 36 patients were enrolled between October 2013 and June 2017. The median progression free survival (PFS) was 1.9 months, and the median OS was 6.4 months. Area under the total (protein bound plus unbound) plasma concentration-time curve (AUC) on day1 was the highest in regorafenib (88.5 ± 53.8 µM·h), followed by M-2 and M-5 (37.0 ± 32.9 and 5.9 ± 6.5 µM·h, respectively), whereas AUC calculated based on unbound plasma concentration (uAUC) of M-2 was the highest (9.8 ± 12.0 nM·h), followed by M-5 and regorafenib (2.5 ± 4.3 and 1.8 ± 1.7 nM·h, respectively), reflecting ~10-fold higher unbound fraction in M-2 and M-5 than regorafenib. uAUC of M-2 measured on day15 was also higher than those of M-5 and regorafenib. Patients with the M-2 uAUC of 9.8 nM·h or higher had significantly shorter PFS than those with the uAUC of lower than 9.8 nM·h (30 vs. 74 days, p=0.0092). The highest uAUC value of M-2 may be compatible with its association with PFS, implying that unbound form of M-2 could be primarily important for clinical response. Conclusion: We thus found that uAUC of active M-2 was the highest as compared to those of regorafenib and M-5, which was associated with shorter PFS. Citation Format: Yutaro Kubota, Ken-ichi Fujita, Takehiro Takahashi, Yu Sunakawa, Hiroo Ishida, Kazuyuki Hamada, Wataru Ichikawa, Takuya Tsunoda, Yusuke Masuo, Yukio Kato, Yasutsuna Sasaki. High unbound plasma concentration of M-2, an active metabolite, is associated with shorter survival in patients with metastatic colorectal cancer who received regorafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3883.

Design, synthesis, in vitro anticancer, antioxidant and antibacterial activity; DNA/BSA binding, photoleavage and docking studies of Cu(II) ternary metal complexes

Three mononuclear, mixed ligand ternary Cu(II) complexes of 3-((Z)-1-(2-hydroxyphenylimino)ethyl)-4-hydroxy-6-methyl-2H-pyran-2-one (HEHMP) viz; [Cu-(Phen) (HEHMP)] (1a), [Cu-(Bpy)(HEHMP)] (1 b) and [Cu-Bpy(NCS)(HEHMP)] (1c) were synthesized and characterized by data obtained from various spectral techniques. The binding affinities of these complexes with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) protein were explored by absorption and fluorescence quenching titrations. The results indicated strong affinity of the title compounds to bind with both CT-DNA and BSA. The antioxidant properties of the synthesized compounds evaluated by free-radical scavenging method using spectrophotometric technique indicated their affirmative potential activity. Gel electrophoresis experiments revealed the efficacy of metal complexes in resulting the cleavage of pBR322 supercoiled DNA. In vitro cytotoxicity studies of these complexes evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against HeLa and MCF-7 cancer cell lines indicated relatively high effectiveness of the complex 1c. Confocal microscopy signified the potential of the complexes to induce apoptosis in HeLa cell lines. In addition, the antibacterial activity of the compounds carried out by disc diffusion method revealed significantly enhanced antibacterial activity in Cu (II) ternary complexes compared to the activity of ligands in unbound form signifying the implicit role of metal ion in inducing lipophilic character.