Using textons to rank crystallization droplets by the likely presence of crystals.

The visual inspection of crystallization experiments is an important yet time-consuming and subjective step in X-ray crystallography. Previously published studies have focused on automatically classifying crystallization droplets into distinct but ultimately arbitrary experiment outcomes; here, a method is described that instead ranks droplets by their likelihood of containing crystals or microcrystals, thereby prioritizing for visual inspection those images that are most likely to contain useful information. The use of textons is introduced to describe crystallization droplets objectively, allowing them to be scored with the posterior probability of a random forest classifier trained against droplets manually annotated for the presence or absence of crystals or microcrystals. Unlike multi-class classification, this two-class system lends itself naturally to unidirectional ranking, which is most useful for assisting sequential viewing because images can be arranged simply by using these scores: this places droplets with probable crystalline behaviour early in the viewing order. Using this approach, the top ten wells included at least one human-annotated crystal or microcrystal for 94% of the plates in a data set of 196 plates imaged with a Minstrel HT system. The algorithm is robustly transferable to at least one other imaging system: when the parameters trained from Minstrel HT images are applied to a data set imaged by the Rock Imager system, human-annotated crystals ranked in the top ten wells for 90% of the plates. Because rearranging images is fundamental to the approach, a custom viewer was written to seamlessly support such ranked viewing, along with another important output of the algorithm, namely the shape of the curve of scores, which is itself a useful overview of the behaviour of the plate; additional features with known usefulness were adopted from existing viewers. Evidence is presented that such ranked viewing of images allows faster but more accurate evaluation of drops, in particular for the identification of microcrystals.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part I.

Pyrrolo[2,3-f]isoquinoline based amino acids, tetracyclic lactams and cyclic ketone analogues are described as novel MK2 inhibitors with IC(50) as low as 5nM and good selectivity profiles against a number of related kinases including ERK, p38alpha and JNKs. TNFalpha release was suppressed from human peripheral blood mononuclear cells (hPBMCs), and a representative compound inhibited LPS induced TNFalpha release in mice illustrating the potential of this series to provide orally active MK2 inhibitors.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part II.

Spirocyclopropane- and spiroazetidine-substituted tetracycles 13D-E and 16A are described as orally active MK2 inhibitors. The spiroazetidine derivatives are potent MK2 inhibitors with IC(50)<3 nM and inhibit the release of TNFalpha (IC(50)<0.3 microM) from hPBMCs and hsp27 phosphorylation in anisomycin stimulated THP-1 cells. The spirocyclopropane analogues are less potent against MK2 (IC(50)=0.05-0.23 microM), less potent in cells (IC(50)<1.1 microM), but show good oral absorption. Compound 13E (100mg/kg po; bid) showed oral activity in rAIA and mCIA, with significant reduction of swelling and histological score.

Novel 3-aminopyrazole inhibitors of MK-2 discovered by scaffold hopping strategy.

New, selective 3-aminopyrazole based MK2-inhibitors were discovered by scaffold hopping strategy. The new derivatives proved to inhibit intracellular phosphorylation of hsp27 as well as LPS-induced TNFalpha release in cells. In addition, selected derivative 14e also inhibited LPS-induced TNFalpha release in vivo.

Structural basis for the exceptional in vivo efficacy of bisphosphonate drugs.

To understand the structural basis for bisphosphonate therapy of bone diseases, we solved the crystal structures of human farnesyl pyrophosphate synthase (FPPS) in its unliganded state, in complex with the nitrogen-containing bisphosphonate (N-BP) drugs zoledronate, pamidronate, alendronate, and ibandronate, and in the ternary complex with zoledronate and the substrate isopentenyl pyrophosphate (IPP). By revealing three structural snapshots of the enzyme catalytic cycle, each associated with a distinct conformational state, and details about the interactions with N-BPs, these structures provide a novel understanding of the mechanism of FPPS catalysis and inhibition. In particular, the accumulating substrate, IPP, was found to bind to and stabilize the FPPS-N-BP complexes rather than to compete with and displace the N-BP inhibitor. Stabilization of the FPPS-N-BP complex through IPP binding is supported by differential scanning calorimetry analyses of a set of representative N-BPs. Among other factors such as high binding affinity for bone mineral, this particular mode of FPPS inhibition contributes to the exceptional in vivo efficacy of N-BP drugs. Moreover, our data form the basis for structure-guided design of optimized N-BPs with improved pharmacological properties.

Structural basis of ubiquitin recognition by the deubiquitinating protease USP2.

Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs.

Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus.

The replication of flaviviruses requires the correct processing of their polyprotein by the viral NS3 protease (NS3pro). Essential for the activation of NS3pro is a 47-residue region of NS2B. Here we report the crystal structures of a dengue NS2B-NS3pro complex and a West Nile virus NS2B-NS3pro complex with a substrate-based inhibitor. These structures identify key residues for NS3pro substrate recognition and clarify the mechanism of NS3pro activation.

APRV - a program for automated data processing, refinement and visualization.

APRV (Automatic Processing, Refinement and Visualization) is a new program that enables high-throughput batch processing of crystallographic data. The program combines processing of raw diffraction images, initial structure refinement and visual inspection of resulting electron density into a seamless one-step procedure, during which all relevant parameters are refined automatically. It is controlled by a user-friendly graphical interface, facilitating operation by non-experts.

Structure and catalytic mechanism of L-rhamnulose-1-phosphate aldolase.

The structure of L-rhamnulose-1-phosphate aldolase has been established at 1.35 A resolution in a crystal form that was obtained by a surface mutation and has one subunit of the C(4)-symmetric tetramer in the asymmetric unit. It confirms an earlier 2.7 A resolution structure which was determined in a complicated crystal form with 20 subunits per asymmetric unit. The chain fold and the active center are similar to those of L-fuculose-1-phosphate aldolase and L-ribulose-5-phosphate 4-epimerase. The active center similarity is supported by a structural comparison of all three enzymes and by the binding mode of the inhibitor phosphoglycolohydroxamate at the site of the product dihydroxyacetone phosphate for the two aldolases. The sensitivity of the catalytic rate to several mutations and a comparison with the established mechanism of the related aldolase give rise to a putative catalytic mechanism. This mechanism involves the same binding mode of the second product L-lactaldehyde in both aldolases, except for a 180 degrees flip of the aldehyde group distinguishing between the two epimers rhamnulose and fuculose. The N-terminal domain exhibits a correlated anisotropic mobility that channels the isotropic Brownian motion into a directed movement of the catalytic base and the substrate phosphate on the N-domain toward the zinc ion and the lactaldehyde on the C-terminal domain. We suggest that this movement supports the catalysis mechanically.

The structure of L-rhamnulose-1-phosphate aldolase (class II) solved by low-resolution SIR phasing and 20-fold NCS averaging.

The enzyme L-rhamnulose-1-phosphate aldolase catalyzes the reversible cleavage of L-rhamnulose-1-phosphate to dihydroxyacetone phosphate and L-lactaldehyde. It is a homotetramer with an M(r) of 30 000 per subunit and crystallized in space group P3(2)21. The enzyme shows a low sequence identity of 18% with the structurally known L-fuculose-1-phosphate aldolase that splits a stereoisomer in a similar reaction. Structure analysis was initiated with a single heavy-atom derivative measured to 6 A resolution. The resulting poor electron density, a self-rotation function and the working hypothesis that both enzymes are C(4) symmetric with envelopes that resemble one another allowed the location of the 20 protomers of the asymmetric unit. The crystal-packing unit was a D(4)-symmetric propeller consisting of five D(4)-symmetric octamers around an internal crystallographic twofold axis. Presumably, the propellers associate laterally in layers, which in turn pile up along the 3(2) axis to form the crystal. The non-crystallographic symmetry was used to extend the phases to the 2.7 A resolution limit and to establish a refined atomic model of the enzyme. The structure showed that the two enzymes are indeed homologous and that they possess chemically similar active centres.