Biophysical and Structural Analyses of the Interaction between the SHANK1 PDZ Domain and an Internal SLiM.

The PDZ (Postsynaptic density protein-95[PSD-95]/Discs-large) domain, prevalent as a recognition module, has attracted significant attention given its ability to specifically recognize ligands with consensus motifs (also termed PDZ binding motifs [PBMs]). PBMs typically bear a C-terminal carboxylate as a recognition handle and have been extensively characterised, whilst internal ligands are less well known. Here we characterize a short linear motif (SLiM) - EESTSFQGP - as an internal PBM based on its strong binding affinity towards the SHANK1 PDZ domain (SHANK1656-762 hereafter referred to as SHANK1). Using the acetylated analogue Ac-EESTSFQGP-CONH2 as a competitor for the interaction of SHANK1 with FAM-Ahx-EESTSFQGP-CONH2 or a typical fluorophore-labelled C-terminal PBM - GKAP - FITC-Ahx-EAQTRL-COOH - the internal SLiM was demonstrated to show comparable low-micromolar IC50 by competition fluorescent anisotropy (FA). To gain further insight on the internal ligand interaction at the molecular level, we obtained the X-ray co-crystal structure of the Ac-EESTSFQGP-CONH2/SHANK1 complex and compared this to the Ac-EAQTRL-COOH/SHANK1 complex. The crystallographic studies reveal that the SHANK1 backbones for the two interactions overlap significantly. The main structural differences were shown to result from the flexible loops which reorganise to accommodate the two PBMs with distinct lengths and terminal groups. In addition, the two C-terminal residues Gly and Pro in Ac-EESTSFQGP-CONH2 were shown not to participate in interaction with the target protein, implying further truncation and structural modification using peptidomimetic approaches on this sequence may be feasible. Taken together, the SLiM Ac-EESTSFQGP-CONH2 holds potential as an internal ligand for targeting SHANK1.

Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d-Aminopeptidase.

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N-terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

Di-Iron(II) [2+2] Helicates of Bis-(Dipyrazolylpyridine) Ligands: The Influence of the Ligand Linker Group on Spin State Properties.

Four bis[2-{pyrazol-1-yl}-6-{pyrazol-3-yl}pyridine] ligands have been synthesized, with butane-1,4-diyl (L1 ), pyrid-2,6-diyl (L2 ), benzene-1,2-dimethylenyl (L3 ) and propane-1,3-diyl (L4 ) linkers between the tridentate metal-binding domains. L1 and L2 form [Fe2 (µ-L)2 ]X4 (X- =BF4 - or ClO4 - ) helicate complexes when treated with the appropriate iron(II) precursor. Solvate crystals of [Fe2 (µ-L1 )2 ][BF4 ]4 exhibit three different helicate conformations, which differ in the torsions of their butanediyl linker groups. The solvates exhibit gradual thermal spin-crossover, with examples of stepwise switching and partial spin-crossover to a low-temperature mixed-spin form. Salts of [Fe2 (µ-L2 )2 ]4+ are high-spin, which reflects their highly twisted iron coordination geometry. The composition and dynamics of assembly structures formed by iron(II) with L1 -L3 vary with the ligand linker group, by mass spectrometry and 1 H NMR spectroscopy. Gas-phase DFT calculations imply the butanediyl linker conformation in [Fe2 (µ-L1 )2 ]4+ influences its spin state properties, but show anomalies attributed to intramolecular electrostatic repulsion between the iron atoms.

A Deep-Blue-Emitting Heteroatom-Doped MR-TADF Nonacene for High-Performance Organic Light-Emitting Diodes.

We present a p- and n-doped nonacene compound, NOBNacene, that represents a rare example of a linearly extended ladder-type multiresonant thermally activated delayed fluorescence (MR-TADF) emitter. This compound shows efficient narrow deep blue emission, with a λPL of 410 nm, full width at half maximum, FWHM, of 38 nm, photoluminescence quantum yield, ΦPL of 71 %, and a delayed lifetime, τd of 1.18 ms in 1.5 wt % TSPO1 thin film. The organic light-emitting diode (OLED) using this compound as the emitter shows a comparable electroluminescence spectrum peaked at 409 nm (FWHM=37 nm) and a maximum external quantum efficiency (EQEmax ) of 8.5 % at Commission Internationale de l'Éclairage (CIE) coordinates of (0.173, 0.055). The EQEmax values were increased to 11.2 % at 3 wt % doping of the emitter within the emissive layer of the device. At this concentration, the electroluminescence spectrum broadened slightly, leading to CIE coordinates of (0.176, 0.068).

Algorithm-driven activity-directed expansion of a series of antibacterial quinazolinones.

Activity-directed synthesis (ADS) is a structure-blind, function driven approach that can drive the discovery of bioactive small molecules. In ADS, arrays of reactions are designed and executed, and the crude product mixtures are then directly screened to identify reactions that yield bioactive products. The design of subsequent reaction arrays is then informed by the hit reactions that are discovered. In this study, algorithms for reaction array design were developed in which the reactions to be executed were selected from a large set of virtual reactions; the reactions were selected on the basis of similarity to reactions known to yield bioactive products. The algorithms were harnessed to design arrays of photoredox-catalysed alkylation reactions whose crude products were then screened for inhibition of growth of S. aureus ATCC29213. It was demonstrated that the approach enabled expansion of a series of antibacterial quinazolinones. It is envisaged that such algorithms could ultimately enable fully autonomous activity-directed molecular discovery.

Ionic multiresonant thermally activated delayed fluorescence emitters for light emitting electrochemical cells.

We designed and synthesized two new ionic thermally activated delayed fluorescent (TADF) emitters that are charged analogues of a known multiresonant TADF (MR-TADF) compound, DiKTa. The emission of the charged derivatives is red-shifted compared to the parent compound. For instance, DiKTa-OBuIm emits in the green (λPL = 499 nm, 1 wt % in mCP) while DiKTa-DPA-OBuIm emits in the red (λPL = 577 nm, 1 wt % in mCP). In 1 wt % mCP films, both emitters showed good photoluminescence quantum yields of 71% and 61%, and delayed lifetimes of 316.6 µs and 241.7 µs, respectively, for DiKTa-OBuIm and DiKTa-DPA-OBuIm, leading to reverse intersystem crossing rates of 2.85 × 103 s-1 and 3.04 × 103 s-1. Light-emitting electrochemical cells were prepared using both DiKTa-OBuIm and DiKTa-DPA-OBuIm as active emitters showing green (λmax = 534 nm) and red (λmax = 656 nm) emission, respectively.

Pyrene Tags for the Detection of Carbohydrates by Label-Assisted Laser Desorption/Ionisation Mass Spectrometry*.

Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) is widely used for the analysis of biomolecules. Label-assisted laser desorption/ionisation mass spectrometry (LALDI-MS) is a matrix-free variant of MALDI-MS, in which only analytes covalently attached to a laser desorption/ionisation (LDI) enhancer are detected. LALDI-MS has shown promise in overcoming the limitations of MALDI-MS in terms of sample preparation and MS analysis. In this work, we have developed a series of pyrene-based LDI reagents (LALDI tags) that can be used for labelling and LALDI-MS analysis of reducing carbohydrates from complex (biological) samples without the need for additional chemical derivatisation or purification. We have systematically explored the suitability of four pyrene-based LDI enhancers and three aldehyde-reactive handles, optimised sample preparation, and demonstrated the use of LALDI tags for the detection of lactose. We have also exemplified the potential of LALDI tags for labelling carbohydrates in biological samples by direct detection of lactose in cow's milk. These results demonstrate that LALDI-MS is a promising technique for the analysis of reducing carbohydrates in biological samples, and pave the way for the development of LALDI-MS for glycomics and diagnostics.

Computational Mapping of Dirhodium(II) Catalysts.

The chemistry of dirhodium(II) catalysts is highly diverse, and can enable the synthesis of many different molecular classes. A tool to aid in catalyst selection, independent of mechanism and reactivity, would therefore be highly desirable. Here, we describe the development of a database for dirhodium(II) catalysts that is based on the principal component analysis of DFT-calculated parameters capturing their steric and electronic properties. This database maps the relevant catalyst space, and may facilitate exploration of the reactivity landscape for any process catalysed by dirhodium(II) complexes. We have shown that one of the principal components of these catalysts correlates with the outcome (e.g. yield, selectivity) of a transformation used in a molecular discovery project. Furthermore, we envisage that this approach will assist the selection of more effective catalyst screening sets, and, hence, the data-led optimisation of a wide range of rhodium-catalysed transformations.

Activity-Directed Synthesis of Inhibitors of the p53/hDM2 Protein-Protein Interaction.

Protein-protein interactions (PPIs) provide a rich source of potential targets for drug discovery and biomedical science research. However, the identification of structural-diverse starting points for discovery of PPI inhibitors remains a significant challenge. Activity-directed synthesis (ADS), a function-driven discovery approach, was harnessed in the discovery of the p53/hDM2 PPI. Over two rounds of ADS, 346 microscale reactions were performed, with prioritisation on the basis of the activity of the resulting product mixtures. Four distinct and novel series of PPI inhibitors were discovered that, through biophysical characterisation, were shown to have promising ligand efficiencies. It was thus shown that ADS can facilitate ligand discovery for a target that does not have a defined small-molecule binding site, and can provide distinctive starting points for the discovery of PPI inhibitors.

Supramolecular Iron Metallocubanes Exhibiting Site-Selective Thermal and Light-Induced Spin-Crossover.

Treatment of Fe[BF4]2·6H2O with 4,6-di(pyrazol-1-yl)-1H-pyrimid-2-one (HL1) or 4,6-di(4-methylpyrazol-1-yl)-1H-pyrimid-2-one (HL2) affords solvated crystals of [{FeIII(OH2)6}⊂FeII8(µ-L)12][BF4]7 (1, HL = HL1; 2, HL = HL2). The centrosymmetric complexes contain a cubic arrangement of iron(II) centers, with bis-bidentate [L]- ligands bridging the edges of the cube. The encapsulated [Fe(OH2)6]3+ moiety templates the assembly through 12 O-H···O hydrogen bonds to the [L]- hydroxylate groups. All four unique iron(II) ions in the cages are crystallographically high-spin at 250 K, but they undergo a gradual high → low spin-crossover on cooling, which is predominantly centered on one iron(II) site and its symmetry-related congener. This was confirmed by magnetic susceptibility data, light-induced excited spin state trapping (LIESST) effect measurements, and, for 1, Mössbauer spectroscopy and diffuse reflectance data. The clusters are stable in MeCN solution, and 1 remains high-spin above 240 K in that solvent. The cubane assembly was not obtained from reactions using other iron(II) salts or 4,6-di(pyrazol-1-yl)pyrimidine ligands, highlighting the importance of hydrogen bonding in templating the cubane assembly.

Construction of a Shape-Diverse Fragment Set: Design, Synthesis and Screen against Aurora-A Kinase.

Historically, chemists have explored chemical space in a highly uneven and unsystematic manner. As an example, the shape diversity of existing fragment sets does not generally reflect that of all theoretically possible fragments. To assess experimentally the added value of increased three dimensionality, a shape-diverse fragment set was designed and collated. The set was assembled by both using commercially available fragments and harnessing unified synthetic approaches to sp3 -rich molecular scaffolds. The resulting set of 80 fragments was highly three-dimensional, and its shape diversity was significantly enriched by twenty synthesised fragments. The fragment set was screened by high-throughput protein crystallography against Aurora-A kinase, revealing four hits that targeted the binding site of allosteric regulators. In the longer term, it is envisaged that the fragment set could be screened against a range of functionally diverse proteins, allowing the added value of more shape-diverse screening collections to be more fully assessed.

Covalent Label Transfer between Peroxisomal Importomer Components Reveals Export-driven Import Interactions.

Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal malate dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes because of high degrees of protease susceptibility or resistance, respectively. Here we present a means for analysis of in vitro import through a covalent biotin label transfer and employ this method to the import of PEX5C. Label transfer demonstrates that the PEX5C construct is monomeric under the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labeling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and, strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import.

Modular Synthesis of Diverse Natural Product-Like Macrocycles: Discovery of Hits with Antimycobacterial Activity.

A modular synthetic approach was developed in which variation of the triplets of building blocks used enabled systematic variation of the macrocyclic scaffolds prepared. The approach was demonstrated in the synthesis of 17 diverse natural product-like macrocyclic scaffolds of varied (12-20-membered) ring size. The biological relevance of the chemical space explored was demonstrated through the discovery of a series of macrocycles with significant antimycobacterial activity.

Homochiral Emissive Λ8 - and Δ8 -[Ir8 Pd4 ]16+ Supramolecular Cages.

Synthetic self-assembly is a powerful technique for the bottom-up construction of discrete and well-defined polyhedral nanostructures resembling the spherical shape of large biological systems. In recent years, numerous Archimedean-shaped coordination cages have been reported based on the assembly of bent monodentate organic ligands containing two or more distal pyridyl rings and square-planar PdII ions. The formation of photoactive PdII metallamacrocycles and cages, however, remain rare. Here we report the first examples of emissive and homochiral supramolecular cages of the form [Ir8 Pd4 ]16+ . These cages provide a suitably sized cavity to host large guest molecules. Importantly, encapsulation and energy transfer have been observed between the blue-emitting NBu4 [Ir(dFppy)2 (CN)2 ] guest and the red-emitting Δ8 -[Ir8 Pd4 ]16+ cage.

Optimizing protein stability in vivo.

Identifying mutations that stabilize proteins is challenging because most substitutions are destabilizing. In addition to being of immense practical utility, the ability to evolve protein stability in vivo may indicate how evolution has formed today's protein sequences. Here we describe a genetic selection that directly links the in vivo stability of proteins to antibiotic resistance. It allows the identification of stabilizing mutations within proteins. The large majority of mutants selected for improved antibiotic resistance are stabilized both thermodynamically and kinetically, indicating that similar principles govern stability in vivo and in vitro. The approach requires no prior structural or functional knowledge and allows selection for stability without a need to maintain function. Mutations that enhance thermodynamic stability of the protein Im7 map overwhelmingly to surface residues involved in binding to colicin E7, showing how the evolutionary pressures that drive Im7-E7 complex formation have compromised the stability of the isolated Im7 protein.

Probing Protein Surfaces: QSAR Analysis with Helix Mimetics.

α-Helix-mediated protein-protein interactions (PPIs) are important targets for small-molecule inhibition; however, generic approaches to inhibitor design are in their infancy and would benefit from QSAR analyses to rationalise the noncovalent basis of molecular recognition by designed ligands. Using a helix mimetic based on an oligoamide scaffold, we have exploited the power of a modular synthesis to access compounds that can readily be used to understand the noncovalent determinants of hDM2 recognition by this series of cell-active p53/hDM2 inhibitors.

Peroxisome protein import: a complex journey.

The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor-cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes.

Synthesis of highly functionalized oligobenzamide proteomimetic foldamers by late stage introduction of sensitive groups.

α-Helix proteomimetics represent an emerging class of ligands that can be used to inhibit an array of helix mediated protein-protein interactions. Within this class of inhibitor, aromatic oligobenzamide foldamers have been widely and successfully used. This manuscript describes alternative syntheses of these compounds that can be used to access mimetics that are challenging to synthesize using previously described methodologies, permitting access to compounds functionalized with multiple sensitive side chains and accelerated library assembly through late stage derivatisation.

Economical and scalable synthesis of 6-amino-2-cyanobenzothiazole.

2-Cyanobenzothiazoles (CBTs) are useful building blocks for: 1) luciferin derivatives for bioluminescent imaging; and 2) handles for bioorthogonal ligations. A particularly versatile CBT is 6-amino-2-cyanobenzothiazole (ACBT), which has an amine handle for straight-forward derivatisation. Here we present an economical and scalable synthesis of ACBT based on a cyanation catalysed by 1,4-diazabicyclo[2.2.2]octane (DABCO), and discuss its advantages for scale-up over previously reported routes.

Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana.

Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure-activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants.