Discovery of 2-amino-3-amido-5-aryl-pyridines as highly potent, orally bioavailable, and efficacious PERK kinase inhibitors.

The protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is one of the three endoplasmic reticulum (ER) transmembrane sensors of the unfolded protein response (UPR) that regulates protein synthesis, alleviates cellular ER stress and has been implicated in tumorigenesis and prolonged cancer cell survival. In this study, we report a series of 2-amino-3-amido-5-aryl-pyridines that we have identified as potent, selective, and orally bioavailable PERK inhibitors. Amongst the series studied herein, compound (28) a (R)-2-Amino-5-(4-(2-(3,5-difluorophenyl)-2-hydroxyacetamido)-2-ethylphenyl)-N-isopropylnicotinamide has demonstrated potent biochemical and cellular activity, robust pharmacokinetics and 70% oral bioavailability in mice. Given these data, this compound (28) was studied in the 786-O renal cell carcinoma xenograft model. We observed dose-dependent, statistically significant tumor growth inhibition, supporting the use of this tool compound in additional mechanistic studies.

The detection and subsequent volume optimization of biological nanocrystals.

Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography. Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging. The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions. It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions. A crystal volume optimization method is described and associated with a phase diagram for crystallization.

A new view on crystal harvesting.

X-ray crystallography typically requires the mounting of crystals, which can make the sample difficult to manipulate when it is small and the microscope objective is close to the crystallization plate. By simply moving the objective to the bottom of a clear crystallization plate (inverting the normal view), crystals were able to be manipulated and harvested from wells having a 0.9 mm diameter and 5.0 mm depth. The mounting system enabled the structural solution of the 187 amino acid N-terminal domain of Saccharomyces cerevisiae glutaminyl-tRNA synthetase from crystals that appeared during high-throughput screening but proved recalcitrant to scale-up and optimization. While not a general mounting solution, the simple expedient of removing the objective lens from the area where manipulation and harvesting occur greatly facilitates the manual, or even automated, process.

The structure of yeast glutaminyl-tRNA synthetase and modeling of its interaction with tRNA.

Eukaryotic glutaminyl-tRNA synthetase (GlnRS) contains an appended N-terminal domain (NTD) whose precise function is unknown. Although GlnRS structures from two prokaryotic species are known, no eukaryotic GlnRS structure has been reported. Here we present the first crystallographic structure of yeast GlnRS, finding that the structure of the C-terminal domain is highly similar to Escherichia coli GlnRS but that 214 residues, including the NTD, are crystallographically disordered. We present a model of the full-length enzyme in solution, using the structures of the C-terminal domain, and the isolated NTD, with small-angle X-ray scattering data of the full-length molecule. We proceed to model the enzyme bound to tRNA, using the crystallographic structures of GatCAB and GlnRS-tRNA complex from bacteria. We contrast the tRNA-bound model with the tRNA-free solution state and perform molecular dynamics on the full-length GlnRS-tRNA complex, which suggests that tRNA binding involves the motion of a conserved hinge in the NTD.

Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase.

In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNA(Gln) is primarily synthesized by first forming Glu-tRNA(Gln), followed by conversion to Gln-tRNA(Gln) by a tRNA-dependent amidotransferase. We report a functional and structural analysis of the NTD of Saccharomyces cerevisiae GlnRS, Gln4. Yeast mutants lacking the NTD exhibit growth defects, and Gln4 lacking the NTD has reduced complementarity for tRNA(Gln) and glutamine. The 187-amino acid Gln4 NTD, crystallized and solved at 2.3 Å resolution, consists of two subdomains, each exhibiting an extraordinary structural resemblance to adjacent tRNA specificity-determining domains in the GatB subunit of the GatCAB amidotransferase, which forms Gln-tRNA(Gln). These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNA(Gln), consistent with a hinge-closing mechanism proposed for GatB recognition of tRNA. Our results suggest a possible origin and function of the NTD that would link the phylogenetically diverse mechanisms of Gln-tRNA(Gln) synthesis.

What's in a drop? Correlating observations and outcomes to guide macromolecular crystallization experiments.

Observations of crystallization experiments are classified as specific outcomes and integrated through a phase diagram to visualize solubility and thereby direct subsequent experiments. Specific examples are taken from our high-throughput crystallization laboratory which provided a broad scope of data from 20 million crystallization experiments on 12,500 different biological macromolecules. The methods and rationale are broadly and generally applicable in any crystallization laboratory. Through a combination of incomplete factorial sampling of crystallization cocktails, standard outcome classifications, visualization of outcomes as they relate chemically and application of a simple phase diagram approach we demonstrate how to logically design subsequent crystallization experiments.

Small angle X-ray scattering as a complementary tool for high-throughput structural studies.

Structural crystallography and nuclear magnetic resonance (NMR) spectroscopy are the predominant techniques for understanding the biological world on a molecular level. Crystallography is constrained by the ability to form a crystal that diffracts well and NMR is constrained to smaller proteins. Although powerful techniques, they leave many soluble, purified structurally uncharacterized protein samples. Small angle X-ray scattering (SAXS) is a solution technique that provides data on the size and multiple conformations of a sample, and can be used to reconstruct a low-resolution molecular envelope of a macromolecule. In this study, SAXS has been used in a high-throughput manner on a subset of 28 proteins, where structural information is available from crystallographic and/or NMR techniques. These crystallographic and NMR structures were used to validate the accuracy of molecular envelopes reconstructed from SAXS data on a statistical level, to compare and highlight complementary structural information that SAXS provides, and to leverage biological information derived by crystallographers and spectroscopists from their structures. All the ab initio molecular envelopes calculated from the SAXS data agree well with the available structural information. SAXS is a powerful albeit low-resolution technique that can provide additional structural information in a high-throughput and complementary manner to improve the functional interpretation of high-resolution structures.

Crystal cookery - using high-throughput technologies and the grocery store as a teaching tool.

Crystallography is a multidisciplinary field that links divergent areas of mathematics, science and engineering to provide knowledge of life on an atomic scale. Crystal growth, a key component of the field, is an ideal vehicle for education. Crystallization has been used with a 'grocery store chemistry' approach and linked to high-throughput remote-access screening technologies. This approach provides an educational opportunity that can effectively teach the scientific method, readily accommodate different levels of educational experience, and reach any student with access to a grocery store, a post office and the internet. This paper describes the formation of the program through the students who helped develop and prototype the procedures. A summary is presented of the analysis and preliminary results and a description given of how the program could be linked with other aspects of crystallography. This approach has the potential to bridge the gap between students in remote locations and with limited funding, and access to scientific resources, providing students with an international-level research experience.

The application and use of chemical space mapping to interpret crystallization screening results.

Macromolecular crystallization screening is an empirical process. It often begins by setting up experiments with a number of chemically diverse cocktails designed to sample chemical space known to promote crystallization. Where a potential crystal is seen a refined screen is set up, optimizing around that condition. By using an incomplete factorial sampling of chemical space to formulate the cocktails and presenting the results graphically, it is possible to readily identify trends relevant to crystallization, coarsely sample the phase diagram and help guide the optimization process. In this paper, chemical space mapping is applied to both single macromolecules and to a diverse set of macromolecules in order to illustrate how visual information is more readily understood and assimilated than the same information presented textually.

Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing.

Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways. sam1(-) sam2(-) mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography. The structure of yeast tryptophanyl-tRNA synthetase was solved to 1.8 A by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1(-) sam2(-) strain grown in selenomethionine. Six of eight selenium residues were located in the structure.

Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature.

An efficient optimization method for the crystallization of biological macromolecules has been developed and tested. This builds on a successful high-throughput technique for the determination of initial crystallization conditions. The optimization method takes an initial condition identified through screening and then varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner. The amount of sample and number of steps is minimized and no biochemical reformulation is required. In the current application a robotic liquid handling system enables high-throughput use, but the technique can easily be adapted in a nonautomated setting. This method has been applied successfully for the rapid optimization of crystallization conditions in nine representative cases.