Development of Allosteric NIK Ligands from Fragment-Based NMR Screening.

NF-κB inducing kinase (NIK) is vital for the induction of many immune responses, and as such, NIK dysregulation has been implicated in various inflammatory diseases and cancers. NIK has been pursued as a potential therapeutic target, and small-molecule inhibitors that bind the orthosteric site on NIK have been reported. However, despite the established chemical matter, NIK inhibitors have not yet reached the clinic. With the goal of developing allosteric NIK ligands using a fragment-based NMR screening approach, we report the identification and development of a series of allosteric, fragment-sized NIK ligands that bind with micromolar potency and good ligand efficiency.

Characterization and engineering of a two-enzyme system for plastics depolymerization.

Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.

Evaluation of the SO2 and NOx offset ratio method to account for secondary PM2.5 formation.

UNLABELLED: The U.S. Environmental Protection Agency (EPA), state and local agencies have focused their efforts in assessing secondary fine particulate matter (aerodynamic diameter < or = 2.5 microm; PM2.5) formation in prevention of significant deterioration (PSD) air dispersion modeling. The National Association of Clean Air Agencies (NACAA) developed a method to account for secondary PM2.5 formation by using sulfur dioxide (SO2) and nitrogen oxides (NOx) offset ratios. These ratios are used to estimate the secondary formation of sulfate and nitrate PM2.5. These ratios were first introduced by the EPA for nonattainment areas in the Implementation of the New Source Review (NSR) Program for Particulate Matter Less than 2.5 Micrometers (PM2.5), 73 FR 28321, to offset emission increases of direct PM2.5 emissions with reductions of PM2.5 precursors and vice versa. Some regulatory agencies such as the Minnesota Pollution Control Agency (MPCA) have developed area-specific offset ratios for SO2 and NOx based on Comprehensive Air Quality Model with Extensions (CAMx) evaluations for air dispersion modeling analyses. The current study evaluates the effect on American Meteorological Society/Environmental Protection Agency Regulatory Model (AERMOD) predicted concentrations from the use of EPA and MPCA developed ratios. The study assesses the effect of these ratios on an electric generating utility (EGU), taconite mine, food processing plant, and a pulp and paper mill. The inputs used for these four scenarios are based on common stack parameters and emissions based on available data. The effect of background concentrations also evaluates these scenarios by presenting results based on uniform annual PM2.5 background values. This evaluation study helps assess the viability of the offset ratio method developed by NACAA in estimating primary and secondary PM2.5 concentrations. An alternative Tier 2 approach to combine modeled and monitored concentrations is also presented. IMPLICATIONS: On January 4, 2012, the EPA committed to engage in rulemaking to evaluate updates to the Guideline on Air Quality Models (Appendix W of 40 CFR 51) and, as appropriate, incorporate new analytical techniques or models for secondary PM2.5. As a result, the National Association of Clean Air Agencies (NACAA) developed a screening method involving offset ratios to account for secondary PM2.5 formation. The use of this method is promising to evaluate total (direct and indirect) PM2.5 impacts for permitting purposes. Therefore, the evaluation of this method is important to determine its viability for widespread use.