The Crystal Structure of the Hsp90-LA1011 Complex and the Mechanism by Which LA1011 May Improve the Prognosis of Alzheimer's Disease.

Functional changes in chaperone systems play a major role in the decline of cognition and contribute to neurological pathologies, such as Alzheimer's disease (AD). While such a decline may occur naturally with age or with stress or trauma, the mechanisms involved have remained elusive. The current models suggest that amyloid-ß (Aß) plaque formation leads to the hyperphosphorylation of tau by a Hsp90-dependent process that triggers tau neurofibrillary tangle formation and neurotoxicity. Several co-chaperones of Hsp90 can influence the phosphorylation of tau, including FKBP51, FKBP52 and PP5. In particular, elevated levels of FKBP51 occur with age and stress and are further elevated in AD. Recently, the dihydropyridine LA1011 was shown to reduce tau pathology and amyloid plaque formation in transgenic AD mice, probably through its interaction with Hsp90, although the precise mode of action is currently unknown. Here, we present a co-crystal structure of LA1011 in complex with a fragment of Hsp90. We show that LA1011 can disrupt the binding of FKBP51, which might help to rebalance the Hsp90-FKBP51 chaperone machinery and provide a favourable prognosis towards AD. However, without direct evidence, we cannot completely rule out effects on other Hsp90-co-chaprone complexes and the mechanisms they are involved in, including effects on Hsp90 client proteins. Nonetheless, it is highly significant that LA1011 showed promise in our previous AD mouse models, as AD is generally a disease affecting older patients, where slowing of disease progression could result in AD no longer being life limiting. The clinical value of LA1011 and its possible derivatives thereof remains to be seen.

Exotic Electronic Properties of 2D Nanosheets Isolated from Liquid Phase Exfoliated Phyllosilicate Minerals.

Spectrally inactive, electrically insulating, and chemically inert are adjectives broadly used to describe phyllosilicate minerals like mica and chlorite. Here, the above is disproved by demonstrating aqueous suspensions of liquid exfoliated nanosheets from five bulk mica types and chlorite schist. Nanosheet quality is confirmed via transmission electron and X-ray photoelectron spectroscopies, as well as electron diffraction. Through Raman spectroscopy, a previously unreported size- and layer-dependent spectral fingerprint is observed. When analyzing the high-yield suspensions (≈1 mg mL-1 ) through UV-vis spectroscopy, all phyllosilicates present bandgap (Eg ) narrowing from ≈7 eV in the bulk to ≈4 eV for monolayers. Unusually, the bandgap is inversely proportional to the areal size (A) of the nanosheets, measured via atomic force microscopy. Due to an unrecorded quantum confinement effect, nanosheet electronic properties scale toward semiconducting behavior (bandgap ≈3 eV) as nanosheet area increases. Furthermore, modeling X-ray diffraction spectra shows that the root cause of the initial bandgap narrowing is lattice relaxation. Finally, with their broad range of isomorphically substituted ions, phyllosilicate nanosheets show remarkable catalytic properties for hydrogen production.

Synthesis of a Thiazole Library via an Iridium-Catalyzed Sulfur Ylide Insertion Reaction.

A library of thiazoles and selenothiazoles were synthesized via Ir-catalyzed ylide insertion chemistry. This process is a functional group, particularly heterocycle-substituent tolerant. This was applied to the synthesis of fanetizole, an anti-inflammatory drug, and a thiazole-containing drug fragment that binds to the peptidyl-tRNA hydrolase (Pth) in Neisseria gonorrheae bacteria.

Uncovering an allosteric mode of action for a selective inhibitor of human Bloom syndrome protein.

BLM (Bloom syndrome protein) is a RECQ-family helicase involved in the dissolution of complex DNA structures and repair intermediates. Synthetic lethality analysis implicates BLM as a promising target in a range of cancers with defects in the DNA damage response; however, selective small molecule inhibitors of defined mechanism are currently lacking. Here, we identify and characterise a specific inhibitor of BLM's ATPase-coupled DNA helicase activity, by allosteric trapping of a DNA-bound translocation intermediate. Crystallographic structures of BLM-DNA-ADP-inhibitor complexes identify a hitherto unknown interdomain interface, whose opening and closing are integral to translocation of ssDNA, and which provides a highly selective pocket for drug discovery. Comparison with structures of other RECQ helicases provides a model for branch migration of Holliday junctions by BLM.

The structure-function relationship of oncogenic LMTK3.

Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.

Equilibrium and site selective analysis for DNA threading intercalation of a new phosphine copper(I) complex: Insights from X-ray analysis, spectroscopic and molecular modeling studies.

To clarify the interaction of phosphine copper(I) complex with DNA, our study reports the synthesis of a new phosphine copper(I) complex, along with a detailed analysis of the geometry characterization and its interaction with double-stranded DNA. The triclinic phase Cu(PPh3)2(L)(I) with a tetrahedral geometry was identified as the product of the reaction of copper(I) iodide with (E,E)-N,N'-1,2-Ethanediylbis[1-(3-pyridinyl)methanimine] ligand and triphenylphosphine by single-crystal X-ray analysis. Molecular interaction of the synthesized complex with the calf thymus deoxyribonucleic acid (ct-DNA) was investigated in the physiological buffer (pH 7.4) by multi-spectroscopic approaches associated with a competitive displacement towards Hoechst 33258 and methylene blue (MB) as groove and intercalator probes. The fluorescence and UV/Vis results detected the formation of a complex-DNA adduct in the ground-state with a binding affinity in order of 104 M-1, which is in keeping with both groove binders and intercalators. The thermodynamic parameters, ΔS0 = -200.31 ± 0.08 cal/mol·K and ΔH0 = -63.11 ± 0.24 kcal/mol, confirmed that the van der Waals interaction is the main driving force for the binding process. Moreover, the ionic strength and pH effect experiments demonstrated the electrostatic interactions between the complex and DNA is negligible. Analysis of the molecular docking simulation declared the flat (E,E)-N,N'-1,2-Ethanediylbis[1-(3-pyridinyl)methanimine] part of the complex was inserted between the sequential A…T/A…T base pairs, while the phosphine substituents were located in the groove, i.e. threading intercalation. Besides, the cytotoxicity of the complex against the MCF-7 human breast cancer cells was detected at IC50 = 10 µg/mL.

Conformational flexibility within the small domain of human serine racemase.

Serine racemase (SR) is a pyridoxal 5'-phosphate (PLP)-containing enzyme that converts L-serine to D-serine, an endogenous co-agonist for the N-methyl-D-aspartate receptor (NMDAR) subtype of glutamate ion channels. SR regulates D-serine levels by the reversible racemization of L-serine to D-serine, as well as the catabolism of serine by α,ß-elimination to produce pyruvate. The modulation of SR activity is therefore an attractive therapeutic approach to disorders associated with abnormal glutamatergic signalling since it allows an indirect modulation of NMDAR function. In the present study, a 1.89 Šresolution crystal structure of the human SR holoenzyme (including the PLP cofactor) with four subunits in the asymmetric unit is described. Comparison of this new structure with the crystal structure of human SR with malonate (PDB entry 3l6b) shows an interdomain cleft that is open in the holo structure but which disappears when the inhibitor malonate binds and is enclosed. This is owing to a shift of the small domain (residues 78-155) in human SR similar to that previously described for the rat enzyme. This domain movement is accompanied by changes within the twist of the central four-stranded ß-sheet of the small domain, including changes in the φ-ψ angles of all three residues in the C-terminal ß-strand (residues 149-151). In the malonate-bound structure, Ser84 (a catalytic residue) points its side chain at the malonate and is preceded by a six-residue ß-strand (residues 78-83), but in the holoenzyme the ß-strand is only four residues (78-81) and His82 has φ-ψ values in the α-helical region of the Ramachandran plot. These data therefore represent a crystallographic platform that enables the structure-guided design of small-molecule modulators for this important but to date undrugged target.

Co-crystallization of human inositol monophosphatase with the lithium mimetic L-690,330.

Lithium, which is still the gold standard in the treatment of bipolar disorder, has been proposed to inhibit inositol monophosphatase (IMPase) and is hypothesized to exert its therapeutic effects by attenuating phosphatidylinositol (PI) cell signalling. Drug-discovery efforts have focused on small-molecule lithium mimetics that would specifically inhibit IMPase without exhibiting the undesired side effects of lithium. L-690,330 is a potent bisphosphonate substrate-based inhibitor developed by Merck Sharp & Dohme. To aid future structure-based inhibitor design, determination of the exact binding mechanism of L-690,330 to IMPase was of interest. Here, the high-resolution X-ray structure of human IMPase in complex with L690,330 and manganese ions determined at 1.39 Šresolution is reported.

The X-ray structure of human calbindin-D28K: an improved model.

Calbindin-D28K is a widely expressed calcium-buffering cytoplasmic protein that is involved in many physiological processes. It has been shown to interact with other proteins, suggesting a role as a calcium sensor. Many of the targets of calbindin-D28K are of therapeutic interest: for example, inositol monophosphatase, the putative target of lithium therapy in bipolar disorder. Presented here is the first crystal structure of human calbindin-D28K. There are significant deviations in the tertiary structure when compared with the NMR structure of rat calbindin-D28K (PDB entry 2g9b), despite 98% sequence identity. Small-angle X-ray scattering (SAXS) indicates that the crystal structure better predicts the properties of calbindin-D28K in solution compared with the NMR structure. Here, the first direct visualization of the calcium-binding properties of calbindin-D28K is presented. Four of the six EF-hands that make up the secondary structure of the protein contain a calcium-binding site. Two distinct conformations of the N-terminal EF-hand calcium-binding site were identified using long-wavelength calcium single-wavelength anomalous dispersion (SAD). This flexible region has previously been recognized as a protein-protein interaction interface. SAXS data collected in both the presence and absence of calcium indicate that there are no large structural differences in the globular structure of calbindin-D28K between the calcium-loaded and unloaded proteins.

Synthesis of novel heteroleptic delocalised cationic pyrazole gold complexes as potent HepG2 cytotoxic agents.

A new series of cationic gold(i) pyrazole complexes were prepared in excellent yields as their perchlorate salts. Results of cell viability assays show that these novel complexes have good cytotoxic properties against the human HepG2 cancer cell line. These complexes showed promising anti-cancer activities and to our knowledge, pyrazoles have never been tested against this cell line. The regioselectivity of the complexation is also discussed in regards to the substitution pattern of the pyrazoles.

Author Correction: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex.

In the originally published version of this article, the affiliation details for Hugo Muñoz-Hernández, Carlos F. Rodríguez and Oscar Llorca incorrectly omitted 'Centro de Investigaciones Biológicas (CIB), Spanish National Research Council (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain'. This has now been corrected in both the PDF and HTML versions of the Article.

Trimerisation of carbon suboxide at a di-titanium centre to form a pyrone ring system.

The reaction of the syn-bimetallic bis(pentalene)dititanium complex Ti2(µ:η5,η5-Pn†)2 (Pn† = C8H4(1,4-SiiPr3)2) 1 with carbon suboxide (O[double bond, length as m-dash]C[double bond, length as m-dash]C[double bond, length as m-dash]C[double bond, length as m-dash]O, C3O2) results in trimerisation of the latter and formation of the structurally characterised complex [{Ti2(µ:η5,η5-Pn†)2}{µ-C9O6}]. The trimeric bridging C9O6 unit in the latter contains a 4-pyrone core, a key feature of both the hexamer and octamer of carbon suboxide which are formed in the body from trace amounts of C3O2 and are, for example, potent inhibitors of Na+/K+-ATP-ase. The mechanism of this reaction has been studied in detail by DFT computational studies, which also suggest that the reaction proceeds via the initial formation of a mono-adduct of 1 with C3O2. Indeed, the carefully controlled reaction of 1 with C3O2 affords [Ti2(µ:η5,η5-Pn†)2 (η2-C3O2)], as the first structurally authenticated complex of carbon suboxide.

RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex.

The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1-RUVBL2-RPAP3-PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, necessary for human R2TP assembly but absent from yeast. The mobile TPR domains of RPAP3 map to the opposite face of the ring, associating with PIH1D1, which mediates client protein recruitment. Thus, RPAP3 provides a flexible platform for bringing HSP90 into proximity with diverse client proteins.

The structure of FKBP38 in complex with the MEEVD tetratricopeptide binding-motif of Hsp90.

Tetratricopeptide (TPR) domains are known protein interaction domains. We show that the TPR domain of FKBP8 selectively binds Hsp90, and interactions upstream of the conserved MEEVD motif are critical for tight binding. In contrast FKBP8 failed to bind intact Hsp70. The PPIase domain was not essential for the interaction with Hsp90 and binding was completely encompassed by the TPR domain alone. The conformation adopted by Hsp90 peptides, containing the conserved MEEVD motif, in the crystal structure were similar to that seen for the TPR domains of CHIP, AIP and Tah1. The carboxylate clamp interactions with bound Hsp90 peptide were a critical component of the interaction and mutation of Lys 307, involved in the carboxylate clamp, completely disrupted the interaction with Hsp90. FKBP8 binding to Hsp90 did not substantially influence its ATPase activity.

Elaboration of tetra-orthogonally-substituted aromatic scaffolds towards novel EGFR-kinase inhibitors.

Nitration of three regioisomers of bromo-fluorobenzaldehyde proceeds regioselectively, notably with H2SO4/HNO3 at 0 °C. The thereby synthesized tetrasubstituted aromatics, endowed with orthogonal substituents, can be elaborated via Pd-catalysed coupling, reduction and reductive amination reactions. As a test-case, these compounds were converted into EGFR inhibitors related to Gefitinib, whose activity was rationalised by docking studies.

Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes.

Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6-(Hsp90)2-Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.

Mixed sandwich thorium complexes incorporating bis(tri-isopropylsilyl)cyclooctatetraenyl and pentamethylcyclopentadienyl ligands: synthesis, structure and reactivity.

The Th(iv) mixed-sandwich halide complexes Th(COT(TIPS2))Cp*X (where COT(TIPS2) = 1,4-{Si(i)Pr(3)}(2)C(8)H(6), X = Cl, I) have been synthesised, and structurally characterised. When Th(COT(TIPS2))Cp*I is reduced in situ in the presence of CO(2), a mixture of dimeric carboxylate and oxalate complexes {Th(COT(TIPS2))Cp*}(2)(µ-κ(1):κ(2)-CO(3)) and {Th(COT(TIPS2))Cp*}(2)(µ-κ(2):κ(2)-C(2)O(4)) are formed, possibly via a transient Th(iii) species. Th(COT(TIPS2))Cp*Cl is readily alkylated to yield the benzyl complex Th(COT(TIPS2))Cp*CH(2)Ph, which reacts with CO(2) to form a carboxylate and with H(2) to form a hydride; the latter inserts CO(2), giving the bridging formate complex {Th(COT(TIPS2))Cp*(µ-κ(1):κ(1)-O(2)CH)}(2).

Synthesis and electronic structure of the first cyaphide-alkynyl complexes.

The novel complexes trans-[Ru(dppe)2(C≡CR)(C≡P)] (R = CO2Me, C6H4OMe), the first to incorporate cyaphide as part of a conjugated system, are obtained in facile manner. The electronic structure of these compounds is probed by X-ray, DFT and UV/Vis studies.

Structural basis for phosphorylation-dependent recruitment of Tel2 to Hsp90 by Pih1.

Client protein recruitment to the Hsp90 system depends on cochaperones that bind the client and Hsp90 simultaneously and facilitate their interaction. Hsp90 involvement in the assembly of snoRNPs, RNA polymerases, PI3-kinase-like kinases, and chromatin remodeling complexes depends on the TTT (Tel2-Tti1-Tti2), and R2TP complexes-consisting of the AAA-ATPases Rvb1 and Rvb2, Tah1 (Spagh/RPAP3 in metazoa), and Pih1 (Pih1D1 in humans)-that together provide the connection to Hsp90. The biochemistry underlying R2TP function is still poorly understood. Pih1 in particular, at the heart of the complex, has not been described at a structural level, nor have the multiple protein-protein interactions it mediates been characterized. Here we present a structural and biochemical analysis of Hsp90-Tah1-Pih1, Hsp90-Spagh, and Pih1D1-Tel2 complexes that reveal a domain in Pih1D1 specific for binding CK2 phosphorylation sites, and together define the structural basis by which the R2TP complex connects the Hsp90 chaperone system to the TTT complex.