A rice GT61 glycosyltransferase possesses dual activities mediating 2-O-xylosyl and 2-O-arabinosyl substitutions of xylan.

MAIN CONCLUSION: A member of the rice GT61 clade B is capable of transferring both 2-O-xylosyl and 2-O-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-O-xylosyl residue onto xylan. Grass xylan is substituted predominantly with 3-O-arabinofuranose (Araf) as well as with some minor side chains, such as 2-O-Araf and 2-O-(methyl)glucuronic acid [(Me)GlcA]. 3-O-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-O-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Araf residues from UDP-Xyl and UDP-Araf, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Araf residues were transferred onto O-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-O-xylosyl/2-O-arabinosyl transferase and OsXYXT2 is a xylan 2-O-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.

Interaction between Permeation Enhancers and Lipid Bilayers.

Permeation enhancers (PEs) are a class of molecules that interact with the epithelial membrane and transiently increase its transcellular permeability. Although there have been few clinical trials of PE coformulated drugs, the mechanism of action of PEs remains elusive. In this paper, the interaction between two archetypes of PEs [salcaprozate sodium (SNAC) and sodium caprate (C10)] and membranes is investigated with extensive all-atom molecular dynamics simulations. The simulations show that (1) the association between the neutral PEs and membranes is favored in free energy, (2) the propensity of neutral PE aggregation is larger in aqueous solution than in lipid bilayers, (3) the equilibrium distribution of neutral PEs in membranes is fast, e.g., accessible with unbiased MD simulations, and (4) the micelle of neutral PEs formed in aqueous solution does not rupture the membranes (e.g., not forming pores or breaking up the membrane) under simulation conditions. All results combined, this study indicates that PEs insert into the membranes in an equilibrium or near equilibrium process. This study lays the foundation for future investigations of how PEs impact the free energy of permeation for small molecules.

Lilly Absorption Modeling Platform: A Tool for Early Absorption Assessment.

Oral drug absorption modeling has developed at a rapid pace in the 40 years or so since the first ideas for mathematical approaches to oral absorption were introduced. The success of compartmental approaches accelerated the uptake of absorption modeling, and over the last 20 years, work on absorption modeling has shifted almost exclusively to the compartmental framework. This report describes a new noncompartmental absorption modeling framework, the Lilly Absorption Modeling Platform (LAMP). LAMP connects a well-mixed stomach to a continuous tube model of the small intestine with plug flow. Within the continuous tube framework, the model includes intestinal mixing and a novel highly tunable precipitation model that can describe a combination of rapid nucleation and slow growth. The framework is designed to balance speed, consistency, and ease of use with a minimum of model complexity to capture the essential features of gastrointestinal (GI) physiology and critical elements of the oral absorption process. The model was validated based on predictions of the fraction absorbed and the maximum absorbable dose for a set of Eli Lilly and Company clinical compounds.

Assessing the Intestinal Permeability of Small Molecule Drugs via Diffusion Motion on a Multidimensional Free Energy Surface.

A protocol that accurately assesses the intestinal permeability of small molecule compounds plays an essential role in decreasing the cost and time in inventing a new drug. This manuscript presents a novel computational method to study the passive permeation of small molecule drugs based on the inhomogeneous solubility-diffusion model. The multidimensional free energy surface of the drug transiting through a lipid bilayer is computed with transition-tempered metadynamics that accurately captures the mechanisms of passive permeation. The permeability is computed by following the diffusion motion of the drug molecules along the minimal free energy path found on the multidimensional free energy surface. This computational method is assessed by studying the permeability of five small molecule drugs (ketoprofen, naproxen, metoprolol, propranolol, and salicylic acid). The results demonstrate a remarkable agreement between the computed permeabilities and those measured with the intestinal assay. The in silico method reported in this manuscript also reproduces the permeability measured from the intestinal assay (in vivo) better than the cell-based assays (e.g., PAMPA and Caco-2) do. In addition, the multidimensional free energy surface reveals the interplay between the structure of the small molecule and its permeability, shedding light on strategies of drug optimization.

Relative Bioavailability Risk Assessment: A Systematic Approach to Assessing In Vivo Risk Associated With CM&C-Related Changes.

Relative bioavailability (RBA) studies are often carried out to bridge changes made between drug products used for clinical studies. In this work, we describe the development of a risk assessment (RA) tool that comprehensively and objectively assesses the risk of noncomparable in vivo performance associated with Chemistry, Manufacturing, and Controls (CM&C)-related changes. The RA tool is based on a risk grid that provides a quantitative context to facilitate discussions to determine the need for an in vivo RBA study. Relevant regulatory guidances and the required in vitro and in silico absorption modeling data, on which the RA is based, are discussed. In addition, an analysis of previously executed RBA studies at Eli Lilly and Company over a period of several years is presented. The risk grid incorporates individual risk factors for a given study and provides a recommendation on the risk associated with bypassing an RBA study. The outcome of an RA results in 1 of 3 possible risk zones; lower tier risk, intermediate tier risk, and upper tier risk. In cases where the outcome from the RA falls into the intermediate tier risk zone, further in depth data analysis is required.

Molecular transport through membranes: Accurate permeability coefficients from multidimensional potentials of mean force and local diffusion constants.

Estimating the permeability coefficient of small molecules through lipid bilayer membranes plays an important role in the development of effective drug candidates. In silico simulations can produce acceptable relative permeability coefficients for a series of small molecules; however, the absolute permeability coefficients from simulations are usually off by orders of magnitude. In addition to differences between the lipid bilayers used in vitro and in silico, the poor convergence of permeation free energy profiles and over-simplified diffusion models have contributed to these discrepancies. In this paper, we present a multidimensional inhomogeneous solubility-diffusion model to study the permeability of a small molecule drug (trimethoprim) passing through a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer. Our approach improves the permeation model in three ways: First, the free energy profile (potential of mean force, PMF) is two-dimensional in two key coordinates rather than simply one-dimensional along the direction normal to the bilayer. Second, the 2-D PMF calculation has improved convergence due to application of the recently developed transition-tempered metadynamics with randomly initialized replicas, while third, the local diffusivity coefficient was calculated along the direction of the minimum free energy path on the two-dimensional PMF. The permeability is then calculated as a line integral along the minimum free energy path of the PMF. With this approach, we report a considerably more accurate permeability coefficient (only 2-5 times larger than the experimental value). We also compare our approach with the common practice of computing permeability coefficients based only on the translation of the center of mass of the drug molecule. Our paper concludes with a discussion of approaches for minimizing the computational cost for the purpose of more rapidly screening a large number of drug candidate molecules.

Transition-Tempered Metadynamics Is a Promising Tool for Studying the Permeation of Drug-like Molecules through Membranes.

Metadynamics is an important enhanced sampling technique in molecular dynamics simulation to efficiently explore potential energy surfaces. The recently developed transition-tempered metadynamics (TTMetaD) has been proven to converge asymptotically without sacrificing exploration of the collective variable space in the early stages of simulations, unlike other convergent metadynamics (MetaD) methods. We have applied TTMetaD to study the permeation of drug-like molecules through a lipid bilayer to further investigate the usefulness of this method as applied to problems of relevance to medicinal chemistry. First, ethanol permeation through a lipid bilayer was studied to compare TTMetaD with nontempered metadynamics and well-tempered metadynamics. The bias energies computed from various metadynamics simulations were compared to the potential of mean force calculated from umbrella sampling. Though all of the MetaD simulations agree with one another asymptotically, TTMetaD is able to predict the most accurate and reliable estimate of the potential of mean force for permeation in the early stages of the simulations and is robust to the choice of required additional parameters. We also show that using multiple randomly initialized replicas allows convergence analysis and also provides an efficient means to converge the simulations in shorter wall times and, more unexpectedly, in shorter CPU times; splitting the CPU time between multiple replicas appears to lead to less overall error. After validating the method, we studied the permeation of a more complicated drug-like molecule, trimethoprim. Three sets of TTMetaD simulations with different choices of collective variables were carried out, and all converged within feasible simulation time. The minimum free energy paths showed that TTMetaD was able to predict almost identical permeation mechanisms in each case despite significantly different definitions of collective variables.

SAD phasing: History, current impact and future opportunities.

Single wavelength anomalous diffraction (SAD) can trace its beginnings to the early 1950s. Researchers at the time recognized that SAD offers some unique features that might be advantageous for crystallographic phasing, despite the fact that at that time recording accurate SAD data was problematic. In this review we will follow the trail from those early days, highlighting key advances in the field and interpreting them in terms on how they stimulated continued phasing development that produced the theoretical foundation for the routine macromolecular structure determination by SAD today. The technological advances over the past three decades in both hardware and software, which played a significant role in making SAD phasing a 'first choice method', will also be described.

Native SAD is maturing.

Native SAD phasing uses the anomalous scattering signal of light atoms in the crystalline, native samples of macromolecules collected from single-wavelength X-ray diffraction experiments. These atoms include sodium, magnesium, phosphorus, sulfur, chlorine, potassium and calcium. Native SAD phasing is challenging and is critically dependent on the collection of accurate data. Over the past five years, advances in diffraction hardware, crystallographic software, data-collection methods and strategies, and the use of data statistics have been witnessed which allow 'highly accurate data' to be routinely collected. Today, native SAD sits on the verge of becoming a 'first-choice' method for both de novo and molecular-replacement structure determination. This article will focus on advances that have caught the attention of the community over the past five years. It will also highlight both de novo native SAD structures and recent structures that were key to methods development.

The structure of augmenter of liver regeneration crystallized in the presence of 50 mM CdCl2 reveals a novel Cd2Cl4O6 cluster that aids in crystal packing.

The crystal structure of the protein augmenter of liver regeneration containing a 14-residue hexahistidine purification tag (hsALR) has been determined to 2.4 Å resolution by Cd-SAD using a highly redundant data set collected on a rotating-anode home X-ray source and processed in 1998. The hsALR crystal structure is a tetramer composed of two homodimers bridged by a novel Cd(2)Cl(4)O(6) cluster via binding to the side-chain carboxylate groups of two solvent-exposed aspartic acid residues. A comparison with the native sALR tetramer shows that the cluster dramatically changes the hsALR dimer-dimer interface, which can now better accommodate the extra 14 N-terminal residues associated with the purification tag. The refined 2.4 Šresolution structure is in good agreement with both the X-ray data (R(cryst) of 0.165, R(free) of 0.211) and the expected stereochemistry (r.m.s. deviations from ideality for bond lengths and bond angles of 0.007 Å and 1.15°, respectively).

Effective absorption modeling in relative bioavailability study risk assessment.

Absorption modeling is an excellent strategic fit to perform a risk assessment for relative bioavailability (RBA) studies as it provides direct input into the question that is at the core of the RBA decision, namely, how does the absorption of the test drug product compare to the reference and is it likely to be different enough to justify an RBA study. The main limitation to absorption modeling in risk assessment is the inherent uncertainty associated with modeling. The extent to which the absorption modeling is integrated into the risk assessment should depend on the level of confidence in the modeling. It is difficult, however, to quantify the level of confidence on a case by case basis. The effective application of absorption modeling for RBA risk assessment therefore requires a general understanding of when modeling is expected to be reliable and also how to build reliability directly into the modeling. This paper describes a framework for effective modeling in RBA risk assessment that is based on four fundamental building blocks: (1) relate severity of drug product change and API properties to reliability of modeling, (2) use critical model variables to express the critical differences in the drug products, (3) generate a fraction-absorbed response surface expressed in terms of the critical model variables to evaluate the relative performance of the drug products, and (4) tie the first three building blocks together by following good model building practices that assure the highest quality model is built. The building blocks are demonstrated by a simple but common example of a change in solid state from free base to HCl salt.

Developability assessment of clinical drug products with maximum absorbable doses.

Maximum absorbable dose refers to the maximum amount of an orally administered drug that can be absorbed in the gastrointestinal tract. Maximum absorbable dose, or D(abs), has proved to be an important parameter for quantifying the absorption potential of drug candidates. The purpose of this work is to validate the use of D(abs) in a developability assessment context, and to establish appropriate protocol and interpretation criteria for this application. Three methods for calculating D(abs) were compared by assessing how well the methods predicted the absorption limit for a set of real clinical candidates. D(abs) was calculated for these clinical candidates by means of a simple equation and two computer simulation programs, GastroPlus and an program developed at Eli Lilly and Company. Results from single dose escalation studies in Phase I clinical trials were analyzed to identify the maximum absorbable doses for these compounds. Compared to the clinical results, the equation and both simulation programs provide conservative estimates of D(abs), but in general D(abs) from the computer simulations are more accurate, which may find obvious advantage for the simulations in developability assessment. Computer simulations also revealed the complex behavior associated with absorption saturation and suggested in most cases that the D(abs) limit is not likely to be achieved in a typical clinical dose range. On the basis of the validation findings, an approach is proposed for assessing absorption potential, and best practices are discussed for the use of D(abs) estimates to inform clinical formulation development strategies.

Structure based mechanism of the Ca(2+)-induced release of coelenterazine from the Renilla binding protein.

The crystal structure of the Ca(2+)-loaded coelenterazine-binding protein from Renilla muelleri in its apo-state has been determined at resolution 1.8 A. Although calcium binding hardly affects the compact scaffold and overall fold of the structure before calcium addition, there are easily discerned shifts in the residues that were interacting with the coelenterazine and a repositioning of helices, to expose a cavity to the external solvent. Altogether these changes offer a straightforward explanation for how following the addition of Ca(2+), the coelenterazine could escape and become available for bioluminescence on Renilla luciferase. A docking computation supports the possibility of a luciferase-binding protein complex.

Structural and transcriptional analyses of a purine nucleotide-binding protein from Pyrococcus furiosus: a component of a novel, membrane-bound multiprotein complex unique to this hyperthermophilic archaeon.

The open-reading frame PF0895 in the genome of the hyperthermophilic archaeon, Pyrococcus furiosus, encodes a 206-residue protein (M(R )23,152). The structure of the recombinant protein was solved by single isomorphous replacement with anomalous scattering (SIRAS) using a mercury derivative. It has been refined to 1.70 A with a crystallographic R and R(free )values of 19.7% and 22.3%, respectively. The PF0895 structure is similar to those of the ATP binding cassettes observed in the ABC transporter family. However, bioinformatics and molecular analyses indicate that PF0895 is not part of the expected five-gene operon that encodes a typical prokaryotic solute-binding ABC transporter. Rather, transcriptional profiling data show that PF0895 is part of a novel four-gene operon (PF0895-PF0896-PF0897-PF0897.1) where only PF0895 has homologs in other organisms. Interestingly, from genome analysis, P. furiosus itself contains a second version of this complex, encoded by PF1090-PF1093. From the structural studies we can only conclude that one of the subunits of this novel membrane complex, PF0895, and its homolog PF1090, likely bind a purine nucleotide. PF0895 is therefore predicted to be part of a membrane-bound multiprotein complex unrelated to ABC transporters that is so far unique to P. furiosus. It appears to play a role in the stress response, as its expression is down regulated when the organism is subjected to cold-shock, where cells are transferred from 95 degrees C, near the optimal growth temperature, to 72 degrees C, near the minimal growth temperature. The related PF1090-containing operon is unaffected by cold-shock and is independently regulated.

The first agmatine/cadaverine aminopropyl transferase: biochemical and structural characterization of an enzyme involved in polyamine biosynthesis in the hyperthermophilic archaeon Pyrococcus furiosus.

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k(cat)/K(m) value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108 degrees C that increases to 112 degrees C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-A resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica.

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.

Reconstruction of ancient genome and gene order from complete microbial genome sequences.

Microbial genome sequences provide us with the fossil records for inferring their origination and evolution. Assuming that current microbial genomes are the evolutionary results of ancient genomes or fragments and the neighboring genes in ancient genomes are more likely neighbors in current genomes, in this paper we proposed a paleontological algorithm and assembled the orthologous gene groups from 66 complete and current microbial genome sequences into a pseudo-ancient genome, which consists of continuous fragments of various sizes. We performed bootstrap resampling and correlation analyses and the results showed that the assembled ancient genome and fragments are statistically significant and the genes of the same fragment are inherently related and likely derived from common ancestors. This method provides a new computational tool for studying microbial genome structure and evolution.

Structural basis of CoA recognition by the Pyrococcus single-domain CoA-binding proteins.

The single-domain coenzyme A (CoA)-binding protein is conserved in bacteria, archaea, and a few eukaryal taxa. It consists of a Rossmann-fold domain, belonging to the FAD/NAD(P)-binding ;superfamily. The crystal structure of the Thermus thermophilus single-domain CoA-binding protein, TTHA1899, has been determined and it has been demonstrated, by isothermal titration calorimetry, that the protein interacts with CoA [Wada T. et al. Acta Crystallogr D Biol Crystallogr 59 (2003) 1213]. In the present study, we determined the crystal structures of an orthologous protein from the archaeon Pyrococcus horikoshii (PH1109), alone and complexed with CoA, at 1.65 A and 1.70 A resolutions, respectively, and that of P. furiosus protein (PF0725) in the CoA-bound form at 1.70 A. The CoA-bound structures are very similar to each other, revealing that the Pyrococcus proteins bind CoA in a 1:1 stoichiometry. Five loop-containing regions form the CoA-binding groove, to which the CoA molecule is docked. A comparison of the structures and the sequences of the Pyrococcus proteins with those of the T. theromphilus orthologue TTHA1899 indicated that archaeal and bacterial single-domain CoA-binding proteins share the same CoA-binding mode. Nevertheless, many of the peripheral residues involved in the hydrogen-bonding/electrostatic interactions with CoA are not strictly conserved in the family. The CoA interaction of the single-domain CoA-binding proteins is significantly different and much more extensive than that of the multi-subunit/multi-domain CoA-binding protein succinyl-CoA synthetase.

A non-natural dinucleotide containing an isomeric L-related deoxynucleoside: dinucleotide inhibitors of anti-HIV integrase activity.

The first X-ray crystal structure of a non-natural dinucleotide, 5'-O-phosphoryl-1'-deoxy-2'-isoadenylyl-(3' --> 5')-cytidine 6.5-hydrate (pIsodApC), C19H26N8O13P2 x 6.5H2O, belonging to a family of dinucleotides that contain an isomeric nucleoside component, is described. A complex system of hydrogen bonds between water molecules and various sites on the dinucleotide was found. All H atoms were located from electron-density difference maps, which allowed identification of protonation sites. Compounds of this family have been found to bind at the active site of HIV integrase and to be inhibitors of this key viral enzyme. These dinucleotides are completely resistant to cleavage by exonucleases; an abnormal dihedral angle twist in an internucleotide phosphate bond revealed in the X-ray crystal structure may be contributing to this unusual stability towards nucleases.