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and Keywords 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 2.0 A, 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.

We have developed a standardized and efficient workflow for high-throughput (HT) protein expression in E. coli and parallel purification which can be tailored to the downstream application of the target proteins. It includes a one-step purification for the purposes of functional assays and a two-step protocol for crystallographic studies, with the option of on-column tag removal.

Tissue transglutaminase 2 (TG2) is a multifunctional protein primarily known for its calcium-dependent enzymatic protein cross-linking activity via isopeptide bond formation between glutamine and lysine residues. TG2 overexpression and activity have been found to be associated with Huntington's disease (HD); specifically, TG2 is up-regulated in the brains of HD patients and in animal models of the disease. Interestingly, genetic deletion of TG2 in two different HD mouse models, R6/1 and R6/2, results in improved phenotypes including a reduction in neuronal death and prolonged survival. Starting with phenylacrylamide screening hit 7d, we describe the SAR of this series leading to potent and selective TG2 inhibitors. The suitability of the compounds as in vitro tools to elucidate the biology of TG2 was demonstrated through mode of inhibition studies, characterization of druglike properties, and inhibition profiles in a cell lysate assay.

Heat shock protein 90 (Hsp90) plays a key role in stress response and protection of the cell against the effects of mutation. Herein we report the identification of an Hsp90 inhibitor identified by fragment screening using a high-concentration biochemical assay, as well as its optimisation by in silico searching coupled with a structure-based drug design (SBDD) approach.

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays a major role in T-cell signaling downstream of the T-cell receptor (TCR), and considerable efforts have been directed toward discovery of ITK-selective inhibitors as potential treatments of inflammatory disorders such as asthma. Using a previously disclosed indazole series of inhibitors as a starting point, and using X-ray crystallography and solubility forecast index (SFI) as guides, we evolved a series of tetrahydroindazole inhibitors with improved potency, selectivity, and pharmaceutical properties. Highlights include identification of a selectivity pocket above the ligand plane, and identification of appropriate lipophilic substituents to occupy this space. This effort culminated in identification of a potent and selective ITK inhibitor (GNE-9822) with good ADME properties in preclinical species.

An X-ray crystal structure of Kelch-like ECH-associated protein(Keap1) co-crystallised with (1S,2R)-2-[(1S)-1-[(1,3-dioxo-2,3-di-hydro-1H-isoindol-2-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-carbonyl]cyclohexane-1-carboxylic acid (compound (S,R,S)-1a)was obtained. This X-ray crystal structure provides break-through experimental evidence for the true binding mode ofthe hit compound (S,R,S)-1a, as the ligand orientation wasfound to differ from that of the initial docking model, whichwas available at the start of the project. Crystallographic eluci-dation of this binding mode helped to focus and drive thedrug design process more effectively and efficiently.Neurodegenerative diseases are conditions with high unmetmedical needs for patients, and currently available treatmentsonly provide symptomatic relief. In recent years, nuclear factorerythroid 2-related factor 2 (Nrf2) has become an attractiveneuroprotective target, as the Nrf2 pathway represents a natu-ral cell defense mechanism.

The use of high concentration biochemical assays to identify weak binding fragment molecules can be an effective method to identify novel starting points for medicinal chemistry programmes. The combination of a high-quality fragment library with sensitive biochemical screening methods is a viable alternative to the more commonly used fragment screening methods such as nuclear magnetic resonance screening or high-throughput X-ray crystallography. Notably, there are a number of literature reports where fragment molecules have been identified by a high concentration biochemical assay. The use of high concentration screening of fragments using a portfolio of single-molecule fluorescence correlation spectroscopy detection techniques to ensure the highest reproducibility and sensitivity have been demonstrated, as well as the use of and X-ray crystallography to determine the binding mode of active fragments.

The unsaturated RuRu double-bonded complexes [Ru2(µ-CO)(µ-C2R2)(η-C5H5)2](R = Ph 1a or CF31b) react at room temperature with [Fe2(CO)9] to produce the new mixed-metal trinuclear complexes [Ru2Fe(CO)3(µ-CO)(µ3-CO)(µ3-C2R2)(η-C5H5)2](R = Ph 2 or CF33) in good yield. Similarly, complexes 1a and 1b react with [Ru(CO)4(C2H4)] at room temperature to afford the analogous triruthenium complexes [Ru3(CO)3(µ-CO)(µ3-CO)(µ3-C2R2)(η-C5H5)2](R = Ph 4 or CF35). The new complexes exist in two geometric forms which can be regarded as ‘rotamers’, differing only in the metal atom to which the alkyne ligand is π bound. The structure of complex 2 has been determined by X-ray diffraction and shows a triangular metal core with a triply bridging alkyne ligand bonding parallel to the (C5H5)Ru–Ru(C5H5) edge, π bound to the Fe(CO)3 unit, giving the molecule a plane of symmetry. The major isomers of 3 and 4 and the minor isomer of 5 also have this structure. An X-ray diffraction study on the major isomer of the latter, 5b, revealed that this adopts the other geometric form which has the alkyne π bound to a Ru(C5H5) unit, with no plane of symmetry. The rotation of the alkyne relative to the {M(C5H5)}2M(CO)3 triangle is accompanied by a shift of the µ-CO ligand to bridge a {M(C5H5)}M(CO)3 edge of the M3 triangle, i.e. as in the other isomer, the edge opposite the π-bound metal is bridged. A similar structure is observed for the minor isomers of 3 and 4. Fluxional interconversion was observed for the isomers of 4 at elevated temperatures. Reaction of 1a with [Co2(CO)8] at room temperature gives the tetranuclear cluster [Ru2Co2(CO)4(µ3-CO)2(µ4-C2Ph2)(η-C5H5)2]6 as one of the products. An X-ray diffraction study showed that this 60-electron complex has a closo-Co2Ru2C2 octahedral core with each CoRu2 face capped by a µ3-CO ligand. Complex 6 can also be regarded as a ‘butterfly’ type structure with the Co atoms at the wing-tips and the diphenylethyne ligand bridging all four metal atoms.

Reaction of molybdenum or tungsten atoms with hexafluorobenzene or 1,3,5-trifluorobenzene or with mixtures of these ligands and benzene or 1,3,5-trimethylbenzene gave the new compounds [M(C6F6)2], [M(C6H3F3-1,3,5)2], [M(C6F6)(C6H6)], [M(C6F6)(C6H3F3-1,3,5)] and [M(C6F6)(C6H3Me3-1,3,5)](M = Mo or W) in yields ranging from

The linking together of two fragment compounds that bind to distinct protein sub-sites can lead to a superadditivity of binding affinities, in which the binding free energy of the linked fragments exceeds the simple sum of the binding energies of individual fragments (linking coefficient E

The triruthenium µ3-alkyne complex [Ru3(CO)3(µ-CO)(µ3-CO){µ3-C2(CF3)2}(η-C5H5)2]1 reacted with Me3NO in MeCN to give [Ru3(MeCN)(CO)2(µ-CO)(µ3-CO){µ-C2(CF3)2}(η-C5H5)2]2. This with hexafluorobut-2-yne at or below room temperature gave [Ru3(CO)2(µ-CO){µ3-η3-C3(CF3)3}(µ3-CCF3)(η-C5H5)2]3, shown by X-ray diffraction to contain a ruthenium triangle with µ3-perfluoroethylidyne and µ3-η3-C3(CF3)3 ligands arising from cleavage of one of the hexafluorobut-2-yne molecules. In contrast, 2 reacted at room temperature with the alkynes diphenylethyne, methyl but-2-ynoate and but-2-yne to yield the species [Ru3(CO)2(µ-CO)2{µ3-C4(CF3)2R(R′)}(η-C5H5)2](R = R′= Ph; R = Me, R′= CO2Me; or R = R′= Me) respectively. X-Ray diffraction studies on the first two of these showed that the alkynes have linked to form a µ3-C4(CF3)2R(R′) ligand bound to a closed Ru3 triangle via a doubly bridging interaction with one pair of metal atoms and an η3-allyl mode with the other. Dimethyl acetylenedicarboxylate (dmad) reacted with 2 at room temperature differently again to give a structurally unidentified species [Ru3(CO)5{C2(CF3)2}{C2(CO2Me)2}(η-C5H5)2], which on heating afforded [Ru3(CO)3{µ3-C4(CF3)2(CO2Me)2}(η-C5H5)2]12, revealed by X-ray diffraction to contain a closo-pentagonal-bipyramidal Ru3C4 cluster in which a µ3-C4(CF3)2(CO2Me)2 ligand bridges an open ruthenium triangle. This complex was formed directly when 1 was heated with dmad in refluxing toluene. Analogous complexes [Ru3(CO)3{µ3-C4(CF3)2R(R′)}(η-C5H5)2](R = Me, R′= CO2Me; R = R′= Ph; or R = R′= Me) were produced on heating 1 with methyl but-2-ynoate, diphenylethyne and but-2-yne respectively. The last two reactions also gave isomers [Ru3(CO)3{µ3-C4(CF3)R2(CF3)}(η-C5H5)2](R = Ph or Me) in which formal insertion of the incoming alkyne into the co-ordinated CF3CCCF3 bond has occurred. In addition, the reaction of 1 with but-2-yne produced [Ru3(CO)2(µ-CO){µ3-η3-C3(CF3)Me2}(µ-CCF3)(η-C5H5)2] analogous to 3. Diruthenium species [Ru2(CO){µ-C4(CF3)2R(R′)}(η-C5H5)2] are formed in both toluene reflux reactions of 1 and room-temperature reactions of 2 as a result of triruthenium cluster degradation. Heating the room-temperature products [Ru3(CO)2(µ-CO)2{µ3-C4(CF3)2R2}(η-C5H5)2] confirms that these are intermediates in the formation of the complexes [Ru3(CO)2(µ-CO){µ3-η3-C3(CF3)R2}(µ3-CCF3)(η-C5H5)2] and the isomeric closo clusters [Ru3(CO)3{µ3-C4(CF3)2R2}(η-C5H5)2] and [Ru3(CO)3{µ3-C4(CF3)R2(CF3)}(η-C5H5)2]. Pathways for the processes leading to the various products are discussed.

Computational approaches have been increasingly employed for drug discovery in both academia and industry. The ultimate goal of rational drug design is the prediction of the biological activity of a compound, and such activity is driven, at the molecular level, by the specific intermolecular interactions between the small organic molecule, the biological target and the solvent. Similar molecular recognition processes are crucial for many biological functions and the strength of the recognition is characterized by its binding affinity. Thanks to important advances in theory and computing in the last decades, predictions of binding affinities using physics-based simulations are gaining popularity. In particular, binding free energy estimates based on alchemical pathways have been shown to be a rigorous approach for the affinity prediction problem and hold the promise to be able to guide lead optimisation. However, whilst relative calculations have made significant contributions in a drug-discovery context, absolute calculations have so far been mostly applied to model systems despite the advantages related to the ability to estimate affinities for largely diverse sets of molecules.We present the results of a study where we evaluated the performance of alchemical free energy estimates for a set of drug-like molecules that have been developed to target bromodomains, a family of epigenetic marks readers with established therapeutic potential. The study evaluates the performance of the protocol employed from a retrospective and perspective standpoint, with particular attention to the precision of the calculations and comparing the theoretical results with high-quality binding data. We show that, for this epigenetic target, good agreement between calculations and experiments is achievable even for very challenging compounds, suggesting that alchemical free energy calculations might be approaching the degree of reliability required in order to have an impact in drug discovery campaigns.