•Reducing salt and sugar resulted in an increase in extrudate expansion.•Salt and sugar has synergistic effect on processing energy and mechanical strength.•More porous structure was achieved with ...lower levels of salt and sugar.•Effects of salt and sugar on processing and product parameters are not independent.
The effect of salt (0–2%) and sugar (0–10%) on the extrusion of wheat–corn blend was studied using a co-rotating twin screw extruder under the same processing settings. Reducing salt and sugar levels increased torque, die pressure and specific mechanical energy. The increased die pressure resulted in higher expansion of the extrudate with more porous structure. This was particularly evident when both salt and sugar were eliminated in the formulation. Reducing salt and sugar did not appear to influence the shear viscosity of the melt except when eliminated from the formulation. Similarly the maximum force to fracture the extrudates was also not significantly affected, except when salt and sugar were removed completely. Salt and sugar has synergistic effect on processing energy, extrudate expansion and mechanical strength. This provided a potential to decrease the sugar and salt in extruded products but still maintain textural quality.
The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory ...effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.
Prolonged drug-target occupancy has become increasingly important in lead optimization, and biophysical assays that measure residence time are in high demand. Here we report a practical label-free ...assay methodology that provides kinetic and affinity measurements suitable for most target classes without long preincubations and over comparatively short sample contact times. The method, referred to as a “chaser” assay, has been applied to three sets of unrelated kinase/inhibitor panels in order to measure the residence times, where correlation with observed efficacy was suspected. A lower throughput chaser assay measured a residence time of 3.6 days ±3.4% (95% CI) and provided single digit pM sensitivity. A higher throughput chaser methodology enabled a maximum capacity of 108 compounds in duplicate/day with an upper residence time limit of 9 h given an assay dissociation time of 34 min.
Incorporation of functional ingredients into foods Day, Li; Seymour, Robert B.; Pitts, Keith F. ...
Trends in food science & technology,
09/2009, Letnik:
20, Številka:
9
Journal Article, Conference Proceeding
Recenzirano
The market for functional ingredients and foods has experienced growth in recent years due to the increased consumer awareness and promotion of healthy eating and lifestyle. Challenges remain to ...ensure that functional ingredients survive and remain ‘active’ and ‘bioavailable’ after food processing and storage. Food can be used as a vehicle for the delivery of bioactives and micronutrients at suitable levels that provide health benefits for increased wellbeing. Demonstration of successful and effective incorporation of bioactives into foods is important for the commercialization of new bioactives and functional food ingredients. In this paper, dermatan sulphate and black carrot concentrate were used as model bioactive substances to demonstrate the approaches that can be taken for the development of functional ingredients and foods.
Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator ...of T cell receptor signaling, which dampens T cell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented T cell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context.
•The active and inactive HPK1 kinase in apo and ligand bound forms are characterized•Structures show rare domain-swapped dimer involving helical activation segment•Dimerization observed in solution suggesting potential biologically relevance•Ligand complex structure provides insights for structure-based drug design
Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is one of the intracellular regulators that dampens T cell receptor signaling. Wu et al. studied the molecular structure of HPK1 kinase with bound ligand, which provides insights for structure-based drug design.
Citrus fibre was used as a substitute for sucrose in the formulation of wheat-corn-based extrudates produced using twin screw extruder. Replacing 5 % sugar with an equal quantity of fibre resulted in ...an increase in die pressure, torque and specific mechanical energy (SME), and consequently the expansion ratio increased. A further increase in fibre content to 10 % (e.g. reducing sugar to 0 %) had little effect on the extrusion processing parameters. The microstructure of extrudates examined by X-ray computed tomography shows that the porosity of the extrudates increased with increasing fibre content, which was attributed to an increased number of large pores. As the citrus fibre content increased, the maximum force required to fracture the extrudate increased; however, the cell wall thickness decreased. This suggests that the fibre component may have contributed to the strength of the cell wall. This study demonstrated a potential utilisation of fibre from by-product of juice extraction as a source of dietary fibre in low sugar-extruded products.
A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the ...compound specifically interacted with human LDHA. Structural variation of the screening hit resulted in improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of an improved compound bound to human LDHA was also obtained and it explained many of the observed structure–activity relationships.
Abstract Canonical Wnt signaling has been demonstrated to increase bone formation, and Wnt pathway components are being pursued as potential drug targets for osteoporosis and other metabolic bone ...diseases. Deletion of the Wnt antagonist secreted frizzled-related protein (sFRP)-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit sFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 440,000 drug-like compounds was screened for inhibitors of human sFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T-cell factor (TCF)-luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone sulfonamide, bound to sFRP-1 with a KD of 0.35 μM in a tryptophan fluorescence quenching assay. This compound also selectively inhibited sFRP-1 with an EC50 of 3.9 μM in the cell-based functional assay. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads (WAY-316606) bound to sFRP-1 with a KD of 0.08 μM and inhibited it with an EC50 of 0.65 μM. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 μM. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation.
A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface ...plasmon resonance experiments performed with a screening hit (LDHA IC50=1.7μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.18μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure–activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F=45%).
The bacterial plasmid-encoded organomercurial lyase, MerB (EC 4.99.1.2), catalyzes the protonolysis of organomercury compounds yielding Hg(II) and the corresponding protonated hydrocarbon. A small, ...soluble protein with no known homologues, MerB is widely distributed among eubacteria in three phylogenetically distinct subfamilies whose most prominent motif includes three conserved cysteine residues. We found that the 212-residue MerB encoded by plasmid R831b is a cytosolic enzyme, consistent with its high thiol requirement in vitro. MerB is inhibited by the nonphysiological dithiol DTT but uses the physiological thiols, glutathione and cysteine, equally well. Highly conserved Cys96 and Cys159 are essential for activity, whereas weakly conserved Cys160 is not. Proteins mutant in highly conserved Cys117 are insoluble. All MerB cysteines are DTNB-reactive in native and denatured states except Cys117, which fails to react with DTNB in the native form, suggesting it is buried. Mass spectrometric analysis of trypsin fragments of reduced proteins treated with N-ethylmaleimide or iodoacetamide revealed that all cysteines form covalent adducts and remain covalently modifiable even when exposed to 1:1 PHMB prior to treatment with NEM or IAM. Stable PHMB adducts were also observed on all cysteines in mutant proteins, suggesting rapid exchange of PHMB among the remaining protein thiols. However, PHMB exposure of reduced wild-type MerB yielded only Hg adducts on the Cys159/Cys160 peptide, suggesting a trapped reaction intermediate. Using HPLC to follow release of benzoic acid from PHMB, we confirmed that fully reduced wild-type MerB and mutant C160S can carry out a single protonolysis without exogenous thiols. On the basis of the foregoing we refine the previously proposed SE2 mechanism for protonolysis by MerB.
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