Crystallographic Fragment Screening

Fragment-based approaches are now routinely applied for lead development in pharmaceutical drug research. Usually, a small but well selected library of low molecular weight compounds is pre-screened by biochemical or biophysical methods such as surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) or thermal shift assay; often followed for promising hit candidates by X-ray crystallography. We designed a small fragment library consisting of 364 compounds that is not strictly compliant to the otherwise often followed Astex rule of three for fragment library composition.[1] Thereafter, our library was validated on the pepsin-like aspartyl protease endothiapepsin, which serves as a model system for proteins that are involved in serious diseases such as malaria (plasmepsins), hypertension (renin) and Alzheimer's disease (ß-secretase) and therefore, is a valid target for further drug development. Due to the small size of fragments, they frequently exhibit only low affinity to the applied target protein and thus are often hard to detect in any screening approach, reflected in little overlap between different screening methods. After initial screening, we decided to validate the entire library by X-ray crystallography, which requires a steady supply of crystals, reproducible soaking conditions and a reliable setup at a synchrotron source, such as HZB BESSY II BL14.1 [2], preferably with some automation in initial data processing and refinement. A total hit rate greater than 10% was obtained, which will be compared to results from other screening methods. The resulting crystal structures will be discussed and provide an ideal basis for further lead development.

Dysprosium (Dy) modified BiFeO3 [Bi1−xDyxFeO3 (x = 0.05, 0.10, and 0.15)] nanoparticles have been synthesized by a low temperature co-precipitation method. Rietveld analysis of X-ray diffraction data reveals a transformation from rhombohedral structure to orthorhombic structure with increase in the Dy concentration. From the transmission electron microscopy analysis, it is observed that the particle sizes of Bi1−xDyxFeO3 (x = 0.05, 0.10, and 0.15) nanoparticles range between 50–70, 40–50, and 30–50 nm, respectively. Raman spectra of Bi1−xDyxFeO3 (x = 0.05) belonging to rhombohedral (R3c) structure show 4A1 and 7E fundamental Raman modes in the range of 100–650 cm−1 and two-phonon scattering modes such as 2A4 (longitudinal optical), 2E8 (transverse optical (TO)), and 2E9 (TO) in the range of 950–1270 cm−1. Suppression, broadening, and shifting of Raman modes have been observed with further increase in Dy concentration. The fundamental Raman modes of Bi1−xDyxFeO3 (x = 0.15) are assigned under orthorhombic (pnma) rather than rhombohedral (R3c) as the Rietveld analysis of X-ray diffraction data predicts a structural transformation for this particular composition. A complete suppression of two phonon modes has been noticed for this composition. An anomaly in the temperature dependent dielectric studies has been observed in all the samples at the vicinity of Neel temperature indicating a magnetic ordering, and an increase in magnetization with increase of Dy concentration in BiFeO3 is noticed from the room temperature magnetic studies. P–E hysteresis loop studies show a decrease of remnant polarization (Pr) with the increase in Dy concentration and disappearance of hysteresis loop occurs for Bi1−xDyxFeO3 (x = 0.15) depicting a transformation from non-centrosymmetric (R3c) to centrosymmetric (Pnma) system.

The role of the dispersion correction in the analysis of X-ray diffraction data of lithium fluoride has been considered by applying the recent theoretical and experimental values of the dispersion corrections.

Covalent fragment libraries in drug discovery-Design, synthesis, and screening methods.

High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density.

Structural studies of hydrated germanium X-type zeolite via Rietveld analysis of synchrotron powder X-ray diffraction data

According to Hann's model of molecular complexity an increased probability of detection binding to a target protein can be expected when small, low complex molecular fragments are screened with high sensitivity instead of full-sized ligands with lower sensitivity. Analysis of the HTS summary data of Novartis and comparison with NMR screening results obtained on generic fragment libraries indicate this expectation to be true with hitrates of 0.001% - 0.151% observed in the identification of ligands with an IC(50) threshold in the micromolar range in an HTS setup and hitrates above or equal to 3% observed in NMR screening of fragments with an affinity threshold in the millimolar range. It is however necessary to keep in mind that the sets of target studied were not identical for both method and the experience in NMR screening is too limited for a final conclusion. The term hitrate as used here reflects only the success rate in the observation of ligand binding event. It must not be confused with the overall success rate of fragment and high throughput screening in the lead finding process, which can be entirely different, since the steps required to follow-up a ligand binding event to a lead are different for both methods. A survey of fragment-based lead discovery case studies given in the literature shows that in approximately half of the cases the initial hit fragment was discovered by screening a generic library, whereas in the other cases some knowledge about an initial ligands or the protein binding site has been used, whereas systematic virtual screening of fragment databases has been only rarely reported. As comparatively high hitrates were obtained, further consideration to optimize the generic fragment screening library were directed to the chemical tractability of the fragment. As several functional groups preferred by chemists for modification and linking of the fragments are also preferentially involved in interactions between the fragments and the target protein, a set of screening fragments was derived from chemical building blocks by masking its linker group by a chemical transformation which can be later on used in the chemical follow-up of the fragment hit. For example primary amines can be masked as acetamides. If the screening fragment is active the related building block can then be used for synthesis of a follow-up library.

Using localized NMR spectroscopy on immobilized targets provides us with a method to simultaneously assess binding of small molecules to two different samples. This Target Immobilized NMR Screening (TINS) has a number of advantages, not least is the requirement for minimal quantities of non-isotopically labeled protein and the applicability to insoluble or unstable targets. The technique is sensitive to binding with K(D) values in the range of 100 nM to 20 mM, while careful selection of the reference protein reduces the number of false positive hits. This combination ensures a maximal number of valid hits from which to select starting points for hit elaboration projects. Hits can be prioritized using biological assays when appropriate, as well as an array of biophysical techniques. So far a variety of soluble proteins, including kinases, GTPases, viral targets and proteases, as well as a membrane protein, have been successfully screened against our fragment library. Here we illustrate our experiences with a number of examples which emphasize the usefulness of the method in selecting and prioritizing fragment hits for elaboration towards leads.

Crystallographic fragment screening (CFS) is an established method in academia and the pharmaceutical industry thanks to dedicated workflows established and optimized at several synchrotron sites.Apart from the hit identity, this technique also provides the structural 3D-information of the fragment hits on the protein surface and therefore fosters rational tool compound development and drug discovery.At Helmholtz-Zentrum Berlin (HZB), a dedicated CFS-workflow is available that enables stream-lined experiments and data analysis [1].The outcome of such a workflow depends to a large extend on the quality of the fragment library employed.Higher count and chemical diversity of the resulting fragment hits increases the chances for successful design of follow-up compounds.To this end, in collaborative effort with the drug design group at Marburg University, we designed libraries that are highly diverse in terms of their 3D-pharmacophores and representative for the chemical space of fragments.The resulting F2X-Universal Library of 1103 compounds and its representative sub-selection of 96 compounds -the F2X-Entry Screen -are now the libraries of choice for CFS user campaigns at our facility due to their high performance [2].Validation campaigns and several user campaigns were performed and showed hit rates of usually 15-25%, reaching 30% in exceptional cases.Another advantage of the libraries is their physical presentation as readyto-use 96-well plates with dried-in compounds which also enables CFS for sensitive crystals without DMSO tolerance.Apart from the exceptional library, a dedicated tool was developed at HZB to ease handling of large amounts of crystals -the EasyAccess Frame [3].By supplying the users with such and other tools, as well as the dried-in libraries we can provide CFS campaigns as "fragment screening to go", i.e., campaigns can be conducted inside our facility or in the user's home laboratory.Furthermore, the robot assisted, and remotely controllable beamlines BL 14.1 and BL 14.2 are a key part of the HZB workflow and deliver high-quality diffraction data.Subsequently, processing of the data up to the identification of even weakly bound fragments is highly automated and provided via FragMAXapp, a user friendly, web-based tool developed in collaboration with the FragMAX facility at MAX IV [4].Several CFS campaigns have been conducted successfully using the described workflow at HZB.As part of the EU-funded iNEXT Discovery project, application for access to the facility is straightforward and convenient for users from academia and industry.

Rietveld refinements and spectroscopic structural studies of a Na-free carbonate apatite made by hydrolysis of monetite

In this article, we have described various detailed features of the conformation of yeast tRNA(Phe) revealed by recent refinement analysis of x-ray diffraction data at 2.5 A resolution. The gross features of the molecule observed in the unrefined version have been largely confirmed and a number of new features found. The unique role of the ribose 2' hydroxyl groups in maintaining a series of nonhelical conformations in this RNA molecule has become apparent. Many of these features are a direct consequence of the geometry of the ribose phosphate backbone of RNA molecules, and these may also be found in structured regions of other RNA species as well. Special attention has been directed toward two conformational motifs revealed by this analysis. These include the striking similarity between the TpsiC and anticodon hairpin turns in the polynucleotide chain, which are stabilized by the participation of uridine in the U turn. In addition, there is frequent occurrence of an arch conformation in the polynucleotide chian which is stabilized by hydrogen bonds from 2' hydroxyl residues to phosphate groups across the base of the arch. The importance of the 2' hydroxyl interactions in defining tertiary structure is illustrated by the fact that, in the nonhelical regions, almost half of the ribose residues are involved in O2' hydrogen-bonding interactions which stabilize the conformation of the molecule.

Medium-range order (MRO) in amorphous (a-) SiO2 can be defined as the degree of orientational correlation between neighbouring SiO4/2 tetrahedra. Analysis of X-ray diffraction data on high surface area a-silicas shows that the MRO can be characterized quantitatively by means of (a) the first peak position in the interference function and (b) the ratio between O-O(II) and Si-O(II) coordination heights in the pair distribution function. These two quantities are physically related.

The hollow nanospheres were obtained by chemical vapor synthesis from diethylzinc as described by Markus Winterer and co-workers on page 434. The color coded TEM image displays hollow nanospheres of about 50 nm in diameter consisting of shells of polycrystalline ZnO with a thickness of 20 nm consistent with the column length obtained from Rietveld analysis of X-ray diffraction data. Experiments are consistent with a Kirkendall formation mechanism and metallic zinc nanoparticles as intermediate.

The analysis of x-ray diffraction data of liquid methanol at room temperature reveals a distinct possibility of the presence of hexamer molecular clusters due to intermolecular hydrogen bonding. On the assumption of hexamer domination in the structure, the center structure is derived, which in turn reproduces closely the available data on neutron diffraction and computer simulation for this system. The center–center correlations show special features of bonded structure.

The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi is a potential target for enzyme structure-based inhibitor design, based on previous studies which indicate that these parasites lack the metabolic enzymes required for de novo synthesis of purine nucleotides. By using a bacterial complement selection system, 59 purine analogs were assayed for their interaction with the HPRTs from T. cruzi and Homo sapiens. Eight compounds were identified from the bacterial assay to have an affinity for the trypanosomal enzyme. Inhibition constants for four of these compounds against purified recombinant trypanosomal and human HPRTs were determined and compared. The results confirm that the recombinant system can be used to identify compounds which have affinity for the trypanosomal HPRT. Furthermore, the results provide evidence for the importance of chemical modifications at positions 6 and 8 of the purine ring in the binding of these compounds to the HPRTs. An accurate three-dimensional structure of the trypanosomal enzyme will greatly enhance our understanding of the interactions between HPRTs and these compounds. Toward this end, crystallization conditions for the trypanosomal HPRT and preliminary analysis of X-ray diffraction data to a resolution of 2 A is reported. These results represent significant progress toward a structure-based approach to the design of inhibitors of the HPRT of trypanosomes with the long-range goal of developing new drugs for the treatment of Chagas' disease.