Locating binding poses in protein-ligand systems using reconnaissance metadynamics

A molecular dynamics-based protocol is proposed for finding and scoring protein-ligand binding poses. This protocol uses the recently developed reconnaissance metadynamics method, which employs a self-learning algorithm to construct a bias that pushes the system away from the kinetic traps where it would otherwise remain. The exploration of phase space with this algorithm is shown to be roughly six to eight times faster than unbiased molecular dynamics and is only limited by the time taken to diffuse about the surface of the protein. We apply this method to the well-studied trypsin-benzamidine system and show that we are able to refind all the poses obtained from a reference EADock blind docking calculation. These poses can be scored based on the length of time the system remains trapped in the pose. Alternatively, one can perform dimensionality reduction on the output trajectory and obtain a map of phase space that can be used in more expensive free-energy calculations.

Binding of Calcium and Carbonate to Polyacrylates

Polyacrylate molecules can be used to slow the growth of calcium carbonate. However, little is known about the mechanism by which the molecules impede the growth rate. A recent computational study (Bulo et al. Macromolecules 2007, 40, 3437) used metadynamics to investigate the binding of calcium to polyacrylate chains and has thrown some light on the coiling and precipitation of these polymers. We extend these simulations to examine the binding of calcium and carbonate to polyacrylate chains. We show that calcium complexed with both carbonate and polyacrylate is a very stable species. The free energies of calcium-carbonate-polyacrylate complexes, with different polymer configurations, are calculated, and differences in the free energy of the binding of carbonate are shown to be due to differences in the amount of steric hindrance about the calcium, which prevents the approach of the carbonate ion.

Metamaterials Nanosensor for Label-free Analysis of Conformation and Molecular Interaction of Biomolecules

Analysis of molecular interaction and conformational dynamics of biomolecules is of paramount importance in understanding of their vital functions in complex biological systems, disease detection, and new drug development. Plasmonic biosensors based upon surface plasmon resonance and localized surface plasmon resonance have become the predominant workhorse for detecting accumulated biomass caused by molecular binding events. However, unlike surface-enhanced Raman spectroscopy (SERS), the plasmonic biosensors indeed are not suitable tools to interrogate vibrational signatures of conformational transitions required for biomolecules to interact. Here, we show that plasmonic metamaterials can offer two transducing channels for parallel acquisition of optical transmission and sensitive SERS spectra at the biointerface, simultaneously probing the conformational states and binding affinity of biomolecules, e.g. G-quadruplexes, in different environments (Fig. 1). We further demonstrate the use of the metamaterials for fingerprinting and detection of arginine-glycine-glycine domain of nucleolin, a cancer biomarker which specifically binds to a G-quadruplex, with the picomolar sensitivity. The dual-mode nanosensor will significantly contribute to unraveling the complexes of the conformational dynamics of biomolecules as well as to improving specificity of biodetection assays.

Label-free Analysis of Conformational Dynamics and Molecular Interaction of Biomolecules by Vis-NIR Metasensor

Analysis of binding recognition and conformation of biomolecules is of paramount important in understanding of their vital functions in complex biological systems. By enabling sub-wavelength light localization and strong local field enhancement, plasmonic biosensors have become dominant tools used for such analysis owing to their label-free and real-time attributes1,2. However, the plasmonic biosensors are not well-suited to provide information regarding conformation or chemical fingerprint of biomolecules. Here, we show that plasmonic metamaterials, consisting of periodic arrays of artificial split-ring resonators (SRRs)3, can enable capabilities of both sensing and fingerprinting of biomolecules. We demonstrate that by engineering geometry of individual SRRs, localized surface plasmon resonance (LSPR) frequency of the metamaterials could be tuned to visible-near infrared regimes (Vis-NIR) such that they possess high local field enhancement for surface-enhanced Raman scattering spectroscopy (SERS). This will provide the basis for the development of a dual mode label-free conformational-resolving and quantitative detection platform. We present here the ability of each sensing mode to independently detect binding adsorption and to identify different conformational states of Guanine (G)-rich DNA monolayers in different environment milieu. Also shown is the use of the nanosensor for fingerprinting and detection of Arginine-Glycine-Glycine (RGG) peptide binding to the G-quadruplex aptamer. The dual-mode nanosensor will significantly contribute to unraveling the complexes of the conformational dynamics of biomolecules as well as to improving specificity of biodetection assays that the conventional, population-averaged plasmonic biosensors cannot achieve.

Metamaterials-Based Label-Free Nanosensor for Conformation and Affinity Biosensing

Analysis of molecular interaction and conformational dynamics of biomolecules is of paramount importance in understanding of their vital functions in complex biological systems, disease detection, and new drug development. Plasmonic biosensors based upon surface plasmon resonance and localized surface plasmon resonance have become the predominant workhorse for detecting accumulated biomass caused by molecular binding events. However, unlike surface-enhanced Raman spectroscopy (SERS), the plasmonic biosensors indeed are not suitable tools to interrogate vibrational signatures of conformational transitions required for biomolecules to interact. Here, we show that highly tunable plasmonic metamaterials can offer two transducing channels for parallel acquisition of optical transmission and sensitive SERS spectra at the biointerface, simultaneously probing the conformational states and binding affinity of biomolecules, e.g. G-quadruplexes, in different environments. We further demonstrate the use of the metamaterials for fingerprinting and detection of arginine-glycine-glycine domain of nucleolin, a cancer biomarker which specifically binds to a G-quadruplex, with the picomolar sensitivity.

Polymerisable squaramide receptors for anion binding and sensing

A novel series of polymerisable squaramides has been synthesised in high yields using simple chemical reactions, and evaluated in the binding of anionic species. These vinyl monomers can be used as functional building blocks in co-polymerisations with a plethora of co-monomers or cross-linkers, grace to their compatibility with free-radical polymerisation reactions. Aromatic substituted squaramides were found to be the strongest receptors, while binding of certain anions was accompanied by a strong colour change, attributed to the de-protonation of the squaramide. The best performing squaramide monomers were incorporated in molecularly imprinted polymers (MIPs) targeting a model anion and their capacities and selectivity were evaluated by rebinding experiments. Polymers prepared using the new squaramide monomers were compared to urea based co-polymers, and were found to contain up to 80% of the theoretical maximum number of binding sites, an exceptionally high value compared to previous reports. Strong polymer colour changes were observed upon rebinding of certain anions, equivalent to those witnessed in solution, paving the way for application of such materials in anion sensing devices.



Graphical abstract: Polymerisable squaramide receptors for anion binding and sensing

Sex hormone binding globulin and insulin resistance

Sex hormone binding globulin (SHBG) is a glycoprotein composed of two 373-amino-acid subunits. The SHBG gene and a promotor region have been identified. The SHBG receptor has yet to be cloned but is known to act through a G-protein-linked second-messenger system following plasma membrane binding. The principal function of SHBG has traditionally been considered to be that of a transport protein for sex steroids, regulating circulating concentrations of free (unbound) hormones and their transport to target tissues. Recent research suggests that SHBG has functions in addition to the binding and transport of sex steroids. Observational studies have associated a low SHBG concentration with an increased incidence of type 2 diabetes mellitus (DM) independent of sex hormone levels in men and women. Genetic studies using Mendelian randomization analysis linking three single nucleotide polymorphisms of the SHBG gene to risk of developing type 2 DM suggest SHBG may have a role in the pathogenesis of type 2 DM. The correlation between SHBG and insulin resistance that is evident in a number of cross-sectional studies is in keeping with the suggestion that the association between SHBG and incidence of type 2 DM is explained by insulin resistance. Several potential mechanisms may account for this association, including the identification of dietary factors that influence SHBG gene transcription. Further research to characterize the SHBG-receptor and the SHBG second messenger system is required. An interventional study examining the effects on insulin resistance of altering SHBG concentrations may help in determining whether this association is causal.

Abnormal alterations in the Ca2+/CaV1.2/calmodulin/caMKII signaling pathway in a tremor rat model and in cultured hippocampal neurons exposed to Mg2+-free solution

Voltage-dependent calcium channels (VDCCs) are key elements in epileptogenesis. There are several binding-sites linked to calmodulin (CaM) and several potential CaM-dependent protein kinase II (CaMKII)-mediated phosphorylation sites in CaV1.2. The tremor rat model (TRM) exhibits absence‑like seizures from 8 weeks of age. The present study was performed to detect changes in the Ca2+/CaV1.2/CaM/CaMKII pathway in TRMs and in cultured hippocampal neurons exposed to Mg2+‑free solution. The expression levels of CaV1.2, CaM and phosphorylated CaMKII (p‑CaMKII; Thr‑286) in these two models were examined using immunofluorescence and western blotting. Compared with Wistar rats, the expression levels of CaV1.2 and CaM were increased, and the expression of p‑CaMKII was decreased in the TRM hippocampus. However, the expression of the targeted proteins was reversed in the TRM temporal cortex. A significant increase in the expression of CaM and decrease in the expression of CaV1.2 were observed in the TRM cerebellum. In the cultured neuron model, p‑CaMKII and CaV1.2 were markedly decreased. In addition, neurons exhibiting co‑localized expression of CaV1.2 and CaM immunoreactivities were detected. Furthermore, intracellular calcium concentrations were increased in these two models. For the first time, o the best of our knowledge, the data of the present study suggested that abnormal alterations in the Ca2+/CaV1.2/CaM/CaMKII pathway may be involved in epileptogenesis and in the phenotypes of TRMs and cultured hippocampal neurons exposed to Mg2+‑free solution.

A let-7 microRNA-binding site polymorphism in 3'-untranslated region of KRAS gene predicts response in wild-type KRAS patients with metastatic colorectal cancer treated with cetuximab monotherapy

PURPOSE: recent studies have found that KRAS mutations predict resistance to monoclonal antibodies targeting the epidermal growth factor receptor in metastatic colorectal cancer (mCRC). A polymorphism in a let-7 microRNA complementary site (lcs6) in the KRAS 3' untranslated region (UTR) is associated with an increased cancer risk in non-small-cell lung cancer and reduced overall survival (OS) in oral cancers. We tested the hypothesis whether this polymorphism may be associated with clinical outcome in KRAS wild-type (KRASwt) mCRC patients treated with cetuximab monotherapy.

PATIENTS AND METHODS: the presence of KRAS let-7 lcs6 polymorphism was evaluated in 130 mCRC patients who were enrolled in a phase II study of cetuximab monotherapy (IMCL-0144). Genomic DNA was extracted from dissected formalin-fixed paraffin-embedded tumor tissue, KRAS mutation status and polymorphism were assessed using direct sequencing and PCR restriction fragment length polymorphism technique.

RESULTS: KRAS let-7 lcs6 polymorphism was found to be related to object response rate (ORR) in mCRC patients whose tumors had KRASwt. The 12 KRASwt patients harboring at least a variant G allele (TG or GG) had a 42% ORR compared with a 9% ORR in 55 KRASwt patients with let-7 lcs6 TT genotype (P = 0.02, Fisher's exact test). KRASwt patients with TG/GG genotypes had trend of longer median progression-free survival (3.9 versus 1.3 months) and OS (10.7 versus 6.4 months) compared to those with TT genotypes.

CONCLUSIONS: these results are the first to indicate that the KRAS 3'UTR polymorphism may predict for cetuximab responsiveness in KRASwt mCRC patients, which warrants validation in other clinical trials.

Developing Food Products For Consumers on a Gluten Free Diet

There is a significant increase in people that choose to follow an avoidance diet, especially excluding gluten. Unlike previously, there is now a demand for ‘no compromise’ gluten-free cereal products that have the same properties as their wheat contain counterparts. This is very challenging for the bakers and the cereal technologists due to the functional role of the gluten network in some of these products. Numerous combinations of raw materials form natural sources have been studied and critically evaluated in this review. Most of the gluten-free products are made of native and modified starches blended with different hydrocolloids due to their structure-building and water binding properties. These ingredients are added to a gluten-free flour, such as rice and corn. The legislation framework, formulations for manufacturing of highl nutritional value bread, pasta and cakes/biscuits as well as quality assurance aspects for the gluten-free manufacturers are discussed in this review.

Novel free fatty acid receptor 1 (GPR40) agonists based on 1,3,4-thiadiazole-2-carboxamide scaffold

Free fatty acid receptor 1 (FFA1), previously known as GPR40 is a G protein-coupled receptor and a new target for treatment of type 2 diabetes. Two series of FFA1 agonists utilizing a 1,3,4-thiadiazole-2-caboxamide scaffold were synthetized. Both series offered significant improvement of the potency compared to the previously described 1,3,4-thiadiazole-based FFA1 agonists and high selectivity for FFA1. Molecular docking predicts new aromatic interactions with the receptor that improve agonist potency. The most potent compounds from both series were profiled for in vitro ADME properties (plasma and metabolic stability, LogD, plasma protein binding, hERG binding and CYP inhibition). One series suffered very rapid degradation in plasma and in presence of mouse liver microsomes. However, the other series delivered a lead compound that displayed a reasonable ADME profile together with the improved FFA1 potency.

Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

Schistosomiasis is a chronically debilitating helminth infection with a significant socio-economic and public health impact. Accurate diagnostics play a pivotal role in achieving current schistosomiasis control and elimination goals. However, many of the current diagnostic procedures, which rely on detection of schistosome eggs, have major limitations including lack of accuracy and the inability to detect pre-patent infections. DNA-based detection methods provide a viable alternative to the current tests commonly used for schistosomiasis diagnosis. Here we describe the optimisation of a novel droplet digital PCR (ddPCR) duplex assay for the diagnosis of Schistosoma japonicum infection which provides improved detection sensitivity and specificity. The assay involves the amplification of two specific and abundant target gene sequences in S. japonicum; a retrotransposon (SjR2) and a portion of a mitochondrial gene (nad1). The assay detected target sequences in different sources of schistosome DNA isolated from adult worms, schistosomules and eggs, and exhibits a high level of specificity, thereby representing an ideal tool for the detection of low levels of parasite DNA in different clinical samples including parasite cell free DNA in the host circulation and other bodily fluids. Moreover, being quantitative, the assay can be used to determine parasite infection intensity and, could provide an important tool for the detection of low intensity infections in low prevalence schistosomiasis-endemic areas.

Free fatty acid receptor 1 (GPR40) agonists containing spirocyclic periphery inspired by LY2881835

The free fatty acid receptor 1 (FFA1), a G protein-coupled receptor (GPCR) naturally activated by long-chain fatty acids is a novel target for the treatment of metabolic diseases. The basic amine spirocyclic periphery of Eli Lilly's drug candidate LY2881835 for treatment of type 2 diabetes mellitus (which reached phase I clinical trials) inspired a series of novel FFA1 agonists. These were designed to incorporate the 3-[4-(benzyloxy)phenyl]propanoic acid pharmacophore core decorated with a range of spirocyclic motifs. The latter were prepared via the Prins cyclization and subsequent modification of the 4-hydroxytetrahydropyran moiety in the Prins product. Here, we synthesize 19 compounds and test for FFA1 activity. Within this pilot set, a nanomolar potency (EC50=55nM) was reached. Four lead compounds (EC50 range 55-410nM) were characterized for aqueous solubility, metabolic stability, plasma protein binding and Caco-2 permeability. While some instability in the presence of mouse liver microsomes was noted, mouse pharmacokinetic profile of the compound having the best overall ADME properties was evaluated to reveal acceptable bioavailability (F=10.3%) and plasma levels achieved on oral administration.

Application of GPCR Structures for Modelling of Free Fatty Acid Receptors

Five G protein-coupled receptors (GPCRs) have been identified to be activated by free fatty acids (FFA). Among them, FFA1 (GPR40) and FFA4 (GPR120) bind long-chain fatty acids, FFA2 (GPR43) and FFA3 (GPR41) bind short-chain fatty acids and GPR84 binds medium-chain fatty acids. Free fatty acid receptors have now emerged as potential targets for the treatment of diabetes, obesity and immune diseases. The recent progress in crystallography of GPCRs has now enabled the elucidation of the structure of FFA1 and provided reliable templates for homology modelling of other FFA receptors. Analysis of the crystal structure and improved homology models, along with mutagenesis data and structure activity, highlighted an unusual arginine charge pairing interaction in FFA1-3 for receptor modulation, distinct structural features for ligand binding to FFA1 and FFA4 and an arginine of the second extracellular loop as a possible anchoring point for FFA at GPR84. Structural data will be helpful for searching novel small molecule modulators at the FFA receptors.