Preparation and flocculation properties of biodegradable konjac glucomannan-grafted poly(trimethyl allyl ammonium chloride)

Polymer Bulletin - Preparation of konjac glucomannan-grafted poly(trimethyl allyl ammonium chloride) (KGM-g-PTMAAC) was carried out using KGM as polysaccharide matrix and TMAAC as cationic...     

Advancements in the Development of non-BET Bromodomain Chemical Probes

The bromodomain and extra terminal (BET) family of bromodomain-containing proteins (BCPs) have been the subject of extensive research over the past decade, resulting in a plethora of high-quality chemical probes for their tandem bromodomains. In turn, these chemical probes have helped reveal the profound biological role of the BET bromodomains and their role in disease, ultimately leading to a number of molecules in active clinical development. However, the BET subfamily represents just 8/61 of the known human bromodomains, and attention has now expanded to the biological role of the remaining 53 non-BET bromodomains. Rapid growth of this research area has been accompanied by a greater understanding of the requirements for an effective bromodomain chemical probe and has led to a number of new non-BET bromodomain chemical probes being developed. Advances since December 2015 are discussed, highlighting the strengths/caveats of each molecule, and the value they add toward validating the non-BET bromodomains as tractable therapeutic targets.     

Characterization of a Tryptophan 6-Halogenase from Streptomyces albus and Its Regioselectivity Determinants

Tryptophan halogenases are found in diverse organisms and catalyze regiospecific halogenation. They play an important role in the biosynthesis of halogenated indole alkaloids, which are biologically active and of therapeutic importance. Here, a tryptophan 6-halogenase (SatH) from Streptomyces albus was characterized by using a whole-cell reaction system in Escherichia coli. SatH showed substrate specificity for chloride and bromide ions, leading to regiospecific halogenation at the C6-position of l -tryptophan. In addition, SatH exhibited higher performance in bromination than that of previously reported tryptophan halogenases in the whole-cell reaction system. Through structure-based protein mutagenesis, it has been revealed that two consecutive residues, A78/V79 in SatH and G77/I78 in PyrH, are key determinants in the regioselectivity difference between tryptophan 6- and 5-halogenases. Substituting the AV with GI residues switched the regioselectivity of SatH by moving the orientation of tryptophan. These data contribute to an understanding of the key residues that determine the regioselectivity of tryptophan halogenases.     

ACE2, the Receptor that Enables Infection by SARS-CoV-2: Biochemistry,Structure, Allostery and Evaluation of the Potential Development of ACE2 Modulators

Angiotensin converting enzyme 2 (ACE2) is the human receptor that interacts with the spike protein of coronaviruses, including the one that produced the 2020 coronavirus pandemic (COVID-19). Thus, ACE2 is a potential target for drugs that disrupt the interaction of human cells with SARS-CoV-2 to abolish infection. There is also interest in drugs that inhibit or activate ACE2, that is, for cardiovascular disorders or colitis. Compounds binding at alternative sites could allosterically affect the interaction with the spike protein. Herein, we review biochemical, chemical biology, and structural information on ACE2, including the recent cryoEM structures of full-length ACE2. We conclude that ACE2 is very dynamic and that allosteric drugs could be developed to target ACE2. At the time of the 2020 pandemic, we suggest that available ACE2 inhibitors or activators in advanced development should be tested for their ability to allosterically displace the interaction between ACE2 and the spike protein.     

Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. Gd^III−Gd^III distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-Gd^III were indistinguishable from Gd^III−Gd^III distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.     

Chemical-Shift Perturbations Reflect Bile Acid Binding to Norovirus Coat Protein: Recognition Comes in Different Flavors

Bile acids have been reported as important cofactors promoting human and murine norovirus (NoV) infections in cell culture. The underlying mechanisms are not resolved. Through the use of chemical shift perturbation (CSP) NMR experiments, we identified a low-affinity bile acid binding site of a human GII.4 NoV strain. Long-timescale MD simulations reveal the formation of a ligand-accessible binding pocket of flexible shape, allowing the formation of stable viral coat protein–bile acid complexes in agreement with experimental CSP data. CSP NMR experiments also show that this mode of bile acid binding has a minor influence on the binding of histo-blood group antigens and vice versa. STD NMR experiments probing the binding of bile acids to virus-like particles of seven different strains suggest that low-affinity bile acid binding is a common feature of human NoV and should therefore be important for understanding the role of bile acids as cofactors in NoV infection.     

Deliberately Losing Control of C−H Activation Processes in the Design of Small-Molecule-Fragment Arrays Targeting Peroxisomal Metabolism

Combined photochemical arylation, “nuisance effect” (S_NAr) reaction sequences have been employed in the design of small arrays for immediate deployment in medium-throughput X-ray protein–ligand structure determination. Reactions were deliberately allowed to run “out of control” in terms of selectivity; for example the ortho-arylation of 2-phenylpyridine gave five products resulting from mono- and bisarylations combined with S_NAr processes. As a result, a number of crystallographic hits against NUDT7, a key peroxisomal CoA ester hydrolase, have been identified.     

Magnetic Resonance Signal Amplification by Reversible Exchange of Selective PyFALGEA Oligopeptide Ligands Towards Epidermal Growth Factor Receptors

The biorelevant PyFALGEA oligopeptide ligand, which is selective towards the epidermal growth factor receptor (EGFR), has been successfully employed as a substrate in magnetic resonance signal amplification by reversible exchange (SABRE) experiments. It is demonstrated that PyFALGEA and the iridium catalyst IMes form a PyFALGEA:IMes molecular complex. The interaction between PyFALGEA:IMes and H₂ results in a ternary SABRE complex. Selective 1D EXSY experiments reveal that this complex is labile, which is an essential condition for successful hyperpolarization by SABRE. Polarization transfer from parahydrogen to PyFALGEA is observed leading to significant enhancement of the ¹H NMR signals of PyFALGEA. Different iridium catalysts and peptides are inspected to discuss the influence of their molecular structures on the efficiency of hyperpolarization. It is observed that PyFALGEA oligopeptide hyperpolarization is more efficient when an iridium catalyst with a sterically less demanding NHC ligand system such as IMesBn is employed. Experiments with shorter analogues of PyFALGEA, that is, PyLGEA and PyEA, show that the bulky phenylalanine from the PyFALGEA oligopeptide causes steric hindrance in the SABRE complex, which hampers hyperpolarization with IMes. Finally, a single-scan ¹H NMR SABRE experiment of PyFALGEA with IMesBn revealed a unique pattern of NMR lines in the hydride region, which can be treated as a fingerprint of this important oligopeptide.     

Household endotoxin reduction in the Louisa Environmental Intervention Project for rural childhood asthma

Endotoxin exacerbates asthma. We designed the Louisa Environmental Intervention Project (LEIP) and assessed its effectiveness in reducing household endotoxin and improving asthma symptoms in rural Iowa children. Asthmatic school children (N = 104 from 89 homes) of Louisa and Keokuk counties in Iowa (aged 5-14 years) were recruited and block-randomized to receive extensive (education + professional cleaning) or educational interventions. Environmental sampling collection and respiratory survey administration were done at baseline and during three follow-up visits. Mixed-model analyses were used to assess the effect of the intervention on endotoxin levels and asthma symptoms in the main analysis and of endotoxin reduction on asthma symptoms in exploratory analysis. In the extensive intervention group, dust endotoxin load was significantly reduced in post-intervention visits. The extensive compared with the educational intervention was associated with significantly decreased dust endotoxin load in farm homes and less frequent nighttime asthma symptoms. In exploratory analysis, dust endotoxin load reduction from baseline was associated with lower total asthma symptoms score (Odds ratio: 0.52, 95% confidence interval: 0.29-0.92). In conclusion, the LEIP intervention reduced household dust endotoxin and improved asthma symptoms. However, endotoxin reductions were not sustained post-intervention by residents.