Luke A. Adams

Synthesis and Characterization of High-Affinity 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene-Labeled Fluorescent Ligands for Human β-Adrenoceptors

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human β-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity β-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log KD of −9.53 and −8.46 as an antagonist of functional β2- and β1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human β2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent β2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) (J. Cardiovasc. Pharmacol.1983, 5, 430–437.)

Application of Fragment‐Based Screening to the Design of Inhibitors of Escherichia coli DsbA

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.

Protein conformation by EPR spectroscopy using gadolinium tags clicked to genetically encoded p-azido-L-phenylalanine

Quantitative cysteine-independent ligation of a Gd(3+) tag to genetically encoded p-azido-L-phenylalanine via Cu(I)-catalyzed click chemistry is shown to deliver an exceptionally powerful tool for Gd(3+)-Gd(3+) distance measurements by double electron-electron resonance (DEER) experiments, as the position of the Gd(3+) ion relative to the protein can be predicted with high accuracy.

A new synthesis of phosphoramidates: inhibitors of the key bacterial enzyme aspartate semi-aldehyde dehydrogenaseElectronic supplementary information (ESI) available: [??]D20 measurements for compounds 4, 5 and 11; analytical data for 11. See http://www.rsc.org/suppdata/cc/b2/b206199f/

A new, mild and high yielding synthesis of phosphoramidates is described: potassium salts of carboxylic acids are treated with ethylchloroformate and the resulting activated anhydride-carbonates are then treated with LiNH-P(O)(OEt)2 in situ--the methodology is especially suited to acid sensitive systems featuring BOC, tBu or acetal protecting groups.

Selective Distance Measurements Using Triple Spin Labeling with Gd3+, Mn2+, and a Nitroxide

Distance measurements by pulse electron paramagnetic resonance techniques, such as double electron-electron resonance (DEER, also called PELDOR), have become an established tool to explore structural properties of biomacromolecules and their assemblies. In such measurements a pair of spin labels provides a single distance constraint. Here we show that by employing three different types of spin labels that differ in their spectroscopic and spin dynamics properties it is possible to extract three independent distances from a single sample. We demonstrate this using the Antennapedia homeodomain orthogonally labeled with Gd3+ and Mn2+ tags in complex with its cognate DNA binding site labeled with a nitroxide.

Chemical Tagging with tert‐Butyl and Trimethylsilyl Groups for Measuring Intermolecular Nuclear Overhauser Effects in a Large Protein–Ligand Complex

Intermolecular 1 H-1 H nuclear Overhauser effects (NOE) present a powerful tool to assess contacts between proteins and binding partners, but are difficult to identify for complexes of high molecular weight. This report shows that intermolecular NOEs can readily be observed following chemical labeling with tert-butyl or trimethylsilyl (TMS) groups. Proteins can be furnished with tert-butyl or TMS groups site-specifically using genetically encoded unnatural amino acids or by chemical modification of single cysteine residues. No isotope labeling is required. The approach is demonstrated with the 95 kDa complex between tetrameric E. coli single-stranded DNA binding protein (SSB) and single-stranded DNA.

Trimethylsilyl tag for probing protein–ligand interactions by NMR

Protein–ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D 1H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B–NS3 protease and the human prolyl isomerase FK506 binding protein.

Site-Specific Incorporation of Selenocysteine by Genetic Encoding as a Photocaged Unnatural Amino Acid

Selenocysteine (Sec) is a naturally occurring amino acid that is also referred to as the 21st amino acid. Site-specific incorporation of Sec into proteins is attractive, because the reactivity of a selenol group exceeds that of a thiol group and thus allows site-specific protein modifications. It is incorporated into proteins by an unusual enzymatic mechanism which, in E. coli and other organisms, involves the recognition of a selenocysteine insertion sequence (SECIS) in the mRNA of the target protein. Reengineering of the natural machinery for Sec incorporation at arbitrary sites independent of SECIS elements, however, is challenging. Here we demonstrate an alternative route, whereby a photocaged selenocysteine (PSc) is incorporated as an unnatural amino acid in response to an amber stop codon, using a mutant Methanosarcina mazei pyrrolysyl-tRNA synthetase, Mm PCC2RS, and its cognate tRNACUA. Following decaging by UV irradiation, proteins synthesized with PSc are readily tagged, e.g., with NMR probes to study ligand binding by NMR spectroscopy. The approach provides a facile route for genetically encoded Sec incorporation. It allows the production of pure selenoproteins and the Sec residue enables site-specific covalent protein modification with reagents that would usually react first with naturally occurring cysteine residues. The much greater reactivity of Sec residues allows their selective alkylation in the presence of highly solvent-exposed cysteine residues.