Integrated Screening for Arginase Inhibitors

Arginase is an enzyme that catalyzes the formation of L-ornithine and urea from L-arginine. L-arginine is also a substrate for nitric oxide synthase (NOS), resulting in the formation of nitric oxide (NO) which is a key vasodilator. Not surprisingly, arginase inhibitors are being studied to treat various diseases, including hypertension, erectile dysfunction, atherosclerosis, wound healing and myocardial reperfusion injury. Recently, the use of virtual screening and docking to identify and characterize novel arginase inhibitors as potential therapeutics to treat leshmania infections has been reported in the literature. Hence, there is interest in the development of new and improved arginase inhibitors. Here, we describe the use of an iterative in silico and in vitro work-flow for identifying novel arginase inhibitors. The first in silico arm of the work-flow involves the use of library design, virtual screening, docking, and consensus scoring to identify predicted hit compounds. The in vitro arm involves rapid assaying of predicted hits in an optimized arginase assay. Confirmed hits are passed into the second in silico arm which involves ligand-based screening, docking, and consensus scoring. The crank is turned on the in silico – in vitro – in silico cycle until a promising candidate for hit-to-lead optimization has been identified. Preliminary results appear encouraging, providing hope that a novel arginase drug candidate will be identified and that our computational work-flow will prove useful on other targets

Computational Design of Novel Insulin Degrading Enzyme Inhibitors

Human insulin degrading enzyme (IDE) plays a role in the proteolytic cleavage of insulin, glucagon, and other short, hydrophobic peptides with roles in glucose and cellular metabolism. Because of IDE’s role in insulin clearance, IDE inhibitors may hold promise as therapies for potentiating insulin signaling in patients suffering from type 2 diabetes mellitus. IDE is a large (~100 kDa) chambered protease of the conserved M16A subfamily of zinc metalloproteases. The enzyme adopts a structure that is analogous to a clamshell formed by the joining of the N terminal and C terminal domains. The characteristic zinc binding and catalytic motif (HXXEH) is positioned within the enzyme’s N terminus, while C terminal residues also play important roles in substrate binding and catalysis. Here, we describe the use of a computational work-flow for identifying novel IDE inhibitors. The work flow integrates mutation-based active site structural analysis, virtual screening, docking and fragment-based design. Initial computational results appear promising and should lead to assay testing in the near future

Mammal responses to global changes in human activity vary by trophic group and landscape

Wildlife must adapt to human presence to survive in the Anthropocene, so it is critical to understand species responses to humans in different contexts. We used camera trapping as a lens to view mammal responses to changes in human activity during the COVID-19 pandemic. Across 163 species sampled in 102 projects around the world, changes in the amount and timing of animal activity varied widely. Under higher human activity, mammals were less active in undeveloped areas but unexpectedly more active in developed areas while exhibiting greater nocturnality. Carnivores were most sensitive, showing the strongest decreases in activity and greatest increases in nocturnality. Wildlife managers must consider how habituation and uneven sensitivity across species may cause fundamental differences in human–wildlife interactions along gradients of human influence.Peer reviewe

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

A Capillary Electrophoresis Method for Evaluation of Abeta Proteolysis in Vitro

According to the amyloid hypothesis, Aβ peptides are neurotoxic and underlie development and progression of Alzheimer’s disease (AD). Multiple Aβ clearance mechanisms, including destruction of the peptides by proteolytic enzymes, are hypothesized to regulate physiological Aβ peptide levels. The insulin-degrading enzyme (IDE) is considered one of the predominant enzymes having Aβ degrading activity. Despite its putative role in protecting against AD, relatively few methods exist for studying IDE activity in vitro. We report the application of capillary electrophoresis (CE) as a novel method for evaluating IDE-mediated Aβ 1–40 proteolysis. This method employs chemically unmodified substrates that are readily obtained from commercial sources. It involves minimal sample preparation, and requires no specialized equipment beyond a CE instrument equipped with a standard fused silica capillary. In the present analysis, we demonstrate that this CE-based method is amenable to kinetic analysis, and show that IDE-mediated Aβ proteolysis is significantly and disproportionately inhibited in the presence of insulin, an alternative IDE substrate

Bisubstrate Kinetics of Glutathione S-Transferase: A Colorimetric Experiment for the Introductory Biochemistry Laboratory

Most biochemical transformations involve more than one substrate. Bisubstrate enzymes catalyze multiple chemical reactions in living systems and include members of the transferase, oxidoreductase, and ligase enzyme classes. Working knowledge of bisubstrate enzyme kinetic models is thus of clear importance to the practicing biochemist. However, such models are infrequently explored in the undergraduate biochemistry laboratory. This deficiency suggests the need for well-defined, tractable, and economical methods for characterization of bisubstrate enzyme activity. The application of an established real-time colorimetric assay to investigate kinetic parameters of glutathione transfer by glutathione S-transferase is described. The reported method relies on commercially available materials and uses experimental conditions that are suited to application in the introductory biochemistry laboratory. Investigation of bisubstrate kinetic parameters was performed as part of a three-week laboratory experiment that emphasized protein extraction, purification, and characterization, leading to guided student development of bisubstrate enzyme kinetic models

Sir2 is Required for Clr4 to Initiate Centromeric Heterochromatin Assembly in Fission Yeast

Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4-mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi-deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly

Cell-Permeable, Small-Molecule Activators of the Insulin-Degrading Enzyme

The insulin-degrading enzyme (IDE) cleaves numerous small peptides, including biologically active hormones and disease-related peptides. The propensity of IDE to degrade neurotoxic Aβ peptides marks IDE as a potential therapeutic target for Alzheimer disease. Using a synthetic reporter based on the yeast a-factor mating pheromone precursor, which is cleaved by multiple IDE orthologs, we identified seven small molecules that stimulate rat IDE activity in vitro. Half-maximal activation of IDE by the compounds is observed in vitro in the range of 43 to 198 µM. All compounds decrease the K(m) of IDE. Four compounds activate IDE in the presence of the competing substrate insulin, which disproportionately inhibits IDE activity. Two compounds stimulate rat IDE activity in a cell-based assay, indicating that they are cell permeable. The compounds demonstrate specificity for rat IDE since they do not enhance the activities of IDE orthologs, including human IDE, and they appear specific for a-factor-based reporters since they do not enhance rat IDE-mediated cleavage of Aβ-based reporters. Our results suggest that IDE activators function in the context of specific enzyme-substrate pairs, indicating that the choice of substrate must be considered in addition to target validation in IDE activator screens

Anion Activation Site of Insulin-Degrading Enzyme

Insulin-degrading enzyme (IDE) (insulysin) is a zinc metallopeptidase that metabolizes several bioactive peptides, including insulin and the amyloid β peptide. IDE is an unusual metallopeptidase in that it is allosterically activated by both small peptides and anions, such as ATP. Here, we report that the ATP-binding site is located on a portion of the substrate binding chamber wall arising largely from domain 4 of the four-domain IDE. Two variants having residues in this site mutated, IDEK898A,K899A,S901A and IDER429S, both show greatly decreased activation by the polyphosphate anions ATP and PPPi. IDEK898A,K899A,S901A is also deficient in activation by small peptides, suggesting a possible mechanistic link between the two types of allosteric activation. Sodium chloride at high concentrations can also activate IDE. There are no observable differences in average conformation between the IDE-ATP complex and unliganded IDE, but regions of the active site and C-terminal domain do show increased crystallographic thermal factors in the complex, suggesting an effect on dynamics. Activation by ATP is shown to be independent of the ATP hydrolysis activity reported for the enzyme. We also report that IDEK898A,K899A,S901A has reduced intracellular function relative to unmodified IDE, consistent with a possible role for anion activation of IDE activity in vivo. Together, the data suggest a model in which the binding of anions activates by reducing the electrostatic attraction between the two halves of the enzyme, shifting the partitioning between open and closed conformations of IDE toward the open form