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Abstract: Seeking for new anticancer drugs with strong antiproliferative activity and simple molecular structure, we designed a novel series of compounds based on our previous reported pharmacophore model composed of five moieties. Antiproliferative assays on four tumoral cell lines and evaluation of Human Choline Kinase CKα1 enzymatic activity was performed for these compounds. Among tested molecules, those ones with biphenyl spacer showed betters enzymatic and antiproliferative activities (n-v). Docking and crystallization studies validate the hypothesis and confirm the results. The most active compound (t) induces a significant arrest of the cell cycle in G0/G1 phase that ultimately lead to apoptosis, following the mitochondrial pathway, as demonstrated for other choline kinase inhibitors. However additional assays reveal that the inhibition of choline uptake could also be involved in the antiproliferative outcome of this class of compounds.

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Abstract: TREM2 has been identified by genomic analysis as a potential and novel target for the treatment of Alzheimer’s disease. To enable structure-based screening of potential small molecule therapeutics, we sought to develop a robust crystallization platform for the TREM2 Ig-like domain. A systematic set of constructs containing the structural chaperone, maltose binding protein (MBP), fused to the Ig domain of TREM2, were evaluated in parallel expression and purification, followed by crystallization studies. Using protein crystallization and high-resolution diffraction as a readout, a MBP-TREM2 Ig fusion construct was identified that generates reproducible protein crystals diffracting at 2.0Å, which makes it suitable for soaking of potential ligands. Importantly, analysis of crystal packing interfaces indicates that most of the surface of the TREM2 Ig domain is available for small molecule binding. A proof of concept co-crystallization study with a small library of fragments validated potential utility of this system for the discovery of new TREM2 therapeutics.

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Abstract: O-GlcNAcylation is a post-translational modification of tau understood to lower the speed and yield of its aggregation, a pathological hallmark of Alzheimer’s disease (AD). O-GlcNAcase (OGA) is the only enzyme that removes O-linked N-acetyl-d-glucosamine (O-GlcNAc) from target proteins. Therefore, inhibition of OGA represents a potential approach for the treatment of AD by preserving the O-GlcNAcylated tau protein. Herein, we report the multifactorial optimization of high-throughput screening hit 8 to a potent, metabolically stable, and orally bioavailable diazaspirononane OGA inhibitor (+)-56. The human OGA X-ray crystal structure has been recently solved, but bacterial hydrolases are still widely used as structural homologues. For the first time, we reveal how a nonsaccharide series of inhibitors binds bacterial OGA and discuss the suitability of two different bacterial orthologues as surrogates for human OGA. These breakthroughs enabled structure–activity relationships to be understood and provided context and boundaries for the optimization of druglike properties.

Open Access

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Abstract: A protein’s amino acid sequence determines its structural, chemical and physical properties, yet how sequence variation influences protein function is still incompletely understood. Protein fitness landscapes powerfully describe the sequence-function relationship by dividing sequence space into functional hills and valleys. This representation is often invoked yet lacks experimental evidence; the immense vastness of possible sequence space makes comprehensive high-quality datasets difficult to obtain. Laboratory directed evolution has focused on optimal utilisation of substitution libraries, however examination of functional innovation in Nature shows that short insertions and deletions (InDels) also play a key role. Beyond rare targeted studies of specific InDels, high-throughput data on fitness landscape for mutations other than substitutions are lacking entirely. In my PhD, I worked towards experimentally describing the fitness landscapes of InDels and substitutions in three systems: GFP, phosphotriesterase (PTE) and the kinase MKK1 docking domain. Towards this goal, I established two experimental assays (GFP, PTE) for deep mutational scanning and a new software toolkit, InDelScanner, for interpreting resulting data that contain InDels. With GFP, I sorted the deletions and substitution libraries into three activity fractions using FACS, then deep sequenced them with Illumina MiSeq to obtain a pilot dataset. The comparison of deletion effects between different lengths of deletions (-3, -6 and -9 bp) indicates that deletions are partially tolerated in eGFP, with tolerance improved for short deletions and in the stabilised starting point GFP8. Further interpretation of data was complicated by limited resolution in the sequencing dataset stemming from poor FACS separation, so I optimised the conditions for better sorting resolution using the mKate2 fluorescent protein as an expression reporter. In the second iteration of the activity sorting I additionally included UMIs in the plasmid design to improve the utilisation of NGS capacity. In the case of PTE, I performed proof-of-concept experiments for microfluidic droplet sorting in an integrated device with an in-line incubation line and a fluorescent sorting design. In parallel, testing of solubility and activity of random InDel variants showed that functional InDels do not necessarily suffer from a stability handicap, making InDel mutagenesis a viable strategy for gene randomisation in directed evolution. One challenge of InDel library data analysis is that InDels are not compatible with existing, substitution-focused software. Using the GFP deletions dataset, I developed the InDelScanner scripts which accurately detect, aggregate and filter insertions, deletions and substitutions. Using the scripts for composition analysis of TRIAD libraries in PTE showed these libraries are well balanced and highly diverse. Finally, I used the InDelScanner scripts to interpret a deep mutational scanning dataset that recorded the sequence preferences in the MKK1 docking domain, acting to activate ERK2. This experiment showed that the fitness landscape in this kinase pair is shaped by the activating effect of hydrophobic residues in the docking groove, as well as widespread positive epistasis. Together, the projects in this thesis demonstrate that deep mutational scanning experiments are a powerful method for exploring the sequence-function relationship in proteins, which can extend into comparison of different types of mutations as well as probing their (epistatic) interactions.

Open Access

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Abstract: A novel transketolase has been reconstituted from two separate polypeptide chains encoded by a ‘split-gene’ identified in the genome of the hyperthermophilic bacterium, Carboxydothermus hydrogenoformans. The reconstituted active α2β2 tetrameric enzyme has been biochemically characterized and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to near 100% completion due to the release of the product carbon dioxide and can be used for the synthesis of a range of sugars of interest to the pharmaceutical industry. This novel reconstituted transketolase is thermally stable with no loss of activity after incubation for 1 h at 70°C and is stable after 1 h incubation with 50% of the organic solvents methanol, ethanol, isopropanol, DMSO, acetonitrile and acetone. The X-ray structure of the holo reconstituted α2β2 tetrameric transketolase has been determined to 1.4 Å resolution. In addition, the structure of an inactive tetrameric β4 protein has been determined to 1.9 Å resolution. The structure of the active reconstituted α2β2 enzyme has been compared to the structures of related enzymes; the E1 component of the pyruvate dehydrogenase complex and D-xylulose-5-phosphate synthase, in an attempt to rationalize differences in structure and substrate specificity between these enzymes. This is the first example of a reconstituted ‘split-gene’ transketolase to be biochemically and structurally characterized allowing its potential for industrial biocatalysis to be evaluated.