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Abstract: We report the functional and structural characterization of trehalose-6-phosphate phosphatase (TPP), from the Gram-negative bacterium B. pseudomallei that causes melioidosis, a severe infectious disease endemic in Southeast Asia and Northern Australia. TPP is a key enzyme in the trehalose biosynthesis pathway, which plays an important role in bacterial stress responses. Due to the absence of this biosynthetic pathway in mammals, TPP has drawn attention as a potential drug target, to combat antibiotic resistance. In this context, we present a detailed biochemical analysis of purified recombinant TPP, reporting its specific high catalytic activity toward the trehalose-6-phosphate substrate, and an absolute requirement for its Mg2+ cofactor. Furthermore, we present the crystal structure of TPP solved at 1.74 Å, revealing the canonical haloacid dehalogenase (HAD) superfamily fold and conserved substrate binding pocket, from which insights into the catalytic mechanism may be deduced. Our data represent a starting point for the rational design of antibacterial drugs.

Open Access

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Abstract: The Sondheimer dialkyne reagent has previously been employed in strain-promoted double-click cycloadditions with bis-azide peptides to generate stapled peptide inhibitors of protein–protein interactions. The substituted variants of the Sondheimer dialkyne can be used to generate functionalized stapled peptide inhibitors with improved biological properties; however, this remains a relatively underdeveloped field. Herein, we report the synthesis of new substituted variants of Sondheimer dialkyne and their application in the stapling of p53-based diazido peptides to generate potent stapled peptide-based inhibitors of the oncogenic p53-MDM2 interaction. The functionalized stapled peptide formed from a meta-fluoro-substituted Sondheimer dialkyne was found to be the most potent inhibitor. Furthermore, through experimental studies and density functional theory calculations, we investigated the impact of the substituent on the strain-promoted double-click reactivity of Sondheimer dialkyne.

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Abstract: The hormone melatonin, secreted from the pineal gland, mediates multiple physiological effects including modulation of Wnt/β-catenin signalling. The Wnt palmitoleate lipid modification is essential for its signalling activity, while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it. Notum enzyme inhibition can therefore upregulate Wnt signalling. While searching for Notum inhibitors by crystallographic fragment screening, a hit compound N-[2-(5-fluoro-1H-indol-3-yl)ethyl]acetamide that is structurally similar to melatonin, came to our attention. We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high resolution structures (≤ 1.5 Å) of their complexes. In each of the structures, two compound molecules bind with Notum: one at the enzyme's catalytic pocket, overlapping the space occupied by the acyl tail of the Wnt palmitoleate lipid; and the other at the edge of the pocket opposite the substrate entrance. Although the inhibitory activity of melatonin shown by in vitro enzyme assays is low (IC50 75 µM), the structural information reported here provides a basis for the design of potent and brain accessible drugs for neurodegenerative diseases such as Alzheimer's disease, in which up-regulation of Wnt signalling may be beneficial.

Open Access

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Abstract: Background: Malaria kills over 400,000 people each year and nearly half the world’s population live in at-risk areas. Progress against malaria has recently stalled, highlighting the need for developing novel therapeutics. The parasite haemoglobin degradation pathway, active in the blood stage of the disease where malaria symptoms and lethality manifest, is a well-established drug target. A key enzyme in this pathway is the papain-type protease falcipain-2. Methods: The crystallographic structure of falcipain-2 at 3.45 Å resolution was resolved in complex with an (E)-chalcone small-molecule inhibitor. The falcipain-2–(E)-chalcone complex was analysed with reference to previous falcipain complexes and their similarity to human cathepsin proteases. Results: The (E)-chalcone inhibitor binds falcipain-2 to the rear of the substrate-binding cleft. This is the first structure of a falcipain protease where the rear of the substrate cleft is bound by a small molecule. In this manner, the (E)-chalcone inhibitor mimics interactions observed in protein-based falcipain inhibitors, which can achieve high interaction specificity. Conclusions: This work informs the search for novel anti-malaria therapeutics that target falcipain-2 by showing the binding site and interactions of the medically privileged (E)-chalcone molecule. Furthermore, this study highlights the possibility of chemically combining the (E)-chalcone molecule with an existing active-site inhibitor of falcipain, which may yield a potent and selective compound for blocking haemoglobin degradation by the malaria parasite.

Open Access

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Abstract: The four member family of “Cyclin and Cystathionine β-synthase (CBS) domain divalent metal cation transport mediators”, CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.