Impact of Storage Conditions on the Stability of Volatile Sulfur Compounds in Sampling Bags.

Odorous emissions from agricultural and waste management operations can cause annoyance to local populations. Volatile sulfur compounds (VSCs) are dominant odorants that are often lost during collection using sample bags. The degree of VSC losses depends on factors such as storage time, bag materials, temperature, sample relative humidity (RH), light exposure, and the presence of volatile organic compounds (VOCs). To assess the impact of those factors on the stability of 10 VSCs (hydrogen sulfide, methanethiol, ethanethiol, dimethyl sulfide, tert-butanethiol, ethyl methyl sulfide, 1-butanethiol, dimethyl disulfide, diethyl disulfide, and dimethyl trisulfide), laboratory-based experiments were conducted according to a factorial experimental design. Linear mixed-effects models were constructed for loss predictions. The estimated recovery of HS in Tedlar bag was 8 to 10% higher than in Mylar and Nalophan between 6 and 30 h. At ≤20°C and without being exposed to light, at least 75% relative recovery of the 10 VSCs in Tedlar bags can be achieved after 18 h, whereas, a maximum of 12 h of storage should not be exceeded to ensure a minimum of 74% relative recovery of the VSCs in Mylar and Nalophan bags.

Fully activated MEK1 exhibits compromised affinity for binding of allosteric inhibitors U0126 and PD0325901.

Kinases catalyze the transfer of γ-phosphate from ATP to substrate protein residues triggering signaling pathways responsible for a plethora of cellular events. Isolation and production of homogeneous preparations of kinases in their fully active forms is important for accurate in vitro measurements of activity, stability, and ligand binding properties of these proteins. Previous studies have shown that MEK1 can be produced in its active phosphorylated form by coexpression with RAF1 in insect cells. In this study, using activated MEK1 produced by in vitro activation by RAF1 (pMEK1(in vitro)), we demonstrate that the simultaneous expression of RAF1 for production of activated MEK1 does not result in stoichiometric phosphorylation of MEK1. The pMEK1(in vitro) showed higher specific activity toward ERK2 protein substrate compared to the pMEK1 that was activated via coexpression with RAF1 (pMEK1(in situ)). The two pMEK1 preparations showed quantitative differences in the phosphorylation of T-loop residue serine 222 by Western blotting and mass spectrometry. Finally, pMEK1(in vitro) showed marked differences in the ligand binding properties compared to pMEK1(in situ). Contrary to previous findings, pMEK1(in vitro) bound allosteric inhibitors U0126 and PD0325901 with a significantly lower affinity than pMEK1(in situ) as well as its unphosphorylated counterpart (npMEK1) as demonstrated by thermal-shift, AS-MS, and calorimetric studies. The differences in inhibitor binding affinity provide direct evidence that unphosphorylated and RAF1-phosphorylated MEK1 form distinct inhibitor sites.

Affinity characterization-mass spectrometry methodology for quantitative analyses of small molecule protein binding in solution.

Affinity characterization by mass spectrometry (AC-MS) is a novel LC-MS methodology for quantitative determination of small molecule ligand binding to macromolecules. Its most distinguishing feature is the direct determination of all three concentration terms of the equilibrium binding equation, i.e., (M), (L), and (ML), which denote the macromolecule, ligand, and the corresponding complex, respectively. Although it is possible to obtain the dissociation constant from a single mixing experiment, saturation analyses are still valuable for assessing the overall binding phenomenon based on an established formalism. In addition to providing the prerequisite dissociation constant and binding stoichiometry, the technique also provides valuable information about the actual solubility of both macromolecule and ligand upon dilution and mixing in binding buffers. The dissociation constants and binding mode for interactions of DNA primase and thymidylate synthetase (TS) with high and low affinity small molecule ligands were obtained using the AC-MS method. The data were consistent with the expected affinity of TS for these ligands based on dissociation constants determined by alternative thermal-denaturation techniques: TdF or TdCD, and also consistent enzyme inhibition constants reported in the literature. The validity of AC-MS was likewise extended to a larger set of soluble protein-ligand systems. It was established as a valuable resource for counter screen and structure-activity relationship studies in drug discovery, especially when other classical techniques could only provide ambiguous results.

Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle protein motor domain.

Affinity selection-mass spectrometry (AS-MS) screening of kinesin spindle protein (KSP) followed by enzyme inhibition studies and temperature-dependent circular dichroism (TdCD) characterization was utilized to identify a series of benzimidazole compounds. This series also binds in the presence of Ispinesib, a known anticancer KSP inhibitor in phase I/II clinical trials for breast cancer. TdCD and AS-MS analyses support simultaneous binding implying existence of a novel non-Ispinesib binding pocket within KSP. Additional TdCD analyses demonstrate direct binding of these compounds to Ispinesib-resistant mutants (D130V, A133D, and A133D + D130V double mutant), further strengthening the hypothesis that the compounds bind to a distinct binding pocket. Also importantly, binding to this pocket causes uncompetitive inhibition of KSP ATPase activity. The uncompetitive inhibition with respect to ATP is also confirmed by the requirement of nucleotide for binding of the compounds. After preliminary affinity optimization, the benzimidazole series exhibited distinctive antimitotic activity as evidenced by blockade of bipolar spindle formation and appearance of monoasters. Cancer cell growth inhibition was also demonstrated either as a single agent or in combination with Ispinesib. The combination was additive as predicted by the binding studies using TdCD and AS-MS analyses. The available data support the existence of a KSP inhibitory site hitherto unknown in the literature. The data also suggest that targeting this novel site could be a productive strategy for eluding Ispinesib-resistant tumors. Finally, AS-MS and TdCD techniques are general in scope and may enable screening other targets in the presence of known drugs, clinical candidates, or tool compounds that bind to the protein of interest in an effort to identify potency-enhancing small molecules that increase efficacy and impede resistance in combination therapy.

Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli.

Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB(69-333) construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB(69-333). Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB(69-333) fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.

Purification and characterization of truncated human AMPK alpha 2 beta 2 gamma 3 heterotrimer from baculovirus-infected insect cells.

AMP-activated protein kinase (AMPK) is considered an important target for treatment of type II diabetes and the metabolic syndrome. The muscle-specific isoform of the regulatory gamma-subunit, gamma 3, within the context of AMPK alpha 2 beta 2 gamma 3 complex, is involved in glucose and fat metabolism in skeletal muscle. In an effort to identify gamma 3-specific activators of AMPK, we have produced truncated human recombinant AMPK alpha 2 beta 2 gamma 3 (hu alpha 2 beta 2 gamma 3(trunc)) for biochemical characterization. Infection of insect cells with three baculoviral stocks encoding the individual subunits resulted in soluble expression of a stable hu alpha 2 beta 2 gamma 3(trunc) heterotrimeric complex. Co-expression of the three subunits was essential for solubility since the individual protein components, when expressed separately, were almost completely insoluble. The hu alpha 2 beta 2 gamma 3(trunc) heterotrimer was purified to apparent homogeneity from baculovirus-infected insect cells in a 1:1:1 stoichiometric complex. The hu alpha 2 beta 2 gamma 3(trunc) heterotrimer had significant circular dichroism signal that was lost as a function of temperature, implying that the purified protein was folded. The hu alpha 2 beta 2 gamma 3(trunc) complex was capable of binding AMP and ATP, although the heterotrimer showed preference for AMP, in particular, as seen by thermal denaturation circular dichroism analyses. Further experiments showed that the truncated complex bound ZMP (AICAR-monophosphate) albeit with much lower affinity than AMP. To investigate whether there were isoform-specific differences in the nucleotide binding affinities, a well-characterized truncated mammalian alpha 1 beta 2 gamma 1 (m alpha 1 beta 2 gamma 1(trunc)) equivalent of hu alpha 2 beta 2 gamma 3(trunc) was also purified. The gamma 1 and gamma 3 isoforms showed comparable nucleotide binding affinities and solution behavior properties.

Crystal structures of MEK1 binary and ternary complexes with nucleotides and inhibitors.

MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. We have determined that the kinase domain spans residues 35-382 by proteolytic cleavage. The complete kinase domain has been crystallized and its X-ray crystal structure as a complex with magnesium and ATP-gammaS determined at 2.1 A. Unlike crystals of a truncated kinase domain previously published, the crystals of the intact domain can be grown either as a binary complex with a nucleotide or as a ternary complex with a nucleotide and one of a multitude of allosteric inhibitors. Further, the crystals allow for the determination of costructures with ATP competitive inhibitors. We describe the structures of nonphosphorylated MEK1 (npMEK1) binary complexes with ADP and K252a, an ATP-competitive inhibitor (see Table 1), at 1.9 and 2.7 A resolution, respectively. Ternary complexes have also been solved between npMEK1, a nucleotide, and an allosteric non-ATP competitive inhibitor: ATP-gammaS with compound 1 and ADP with either U0126 or the MEK1 clinical candidate PD325089 at 1.8, 2.0, and 2.5 A, respectively. Compound 1 is structurally similar to PD325901. These structures illustrate fundamental differences among various mechanisms of inhibition at the molecular level. Residues 44-51 have previously been shown to play a negative regulatory role in MEK1 activity. The crystal structure of the integral kinase domain provides a structural rationale for the role of these residues. They form helix A and repress enzymatic activity by stabilizing an inactive conformation in which helix C is displaced from its active state position. Finally, the structure provides for the first time a molecular rationale that explains how mutations in MEK may lead to the cardio-facio-cutaneous syndrome.

Thermodynamics of nucleotide and inhibitor binding to wild-type and ispinesib-resistant forms of human kinesin spindle protein.

Current antimitotic cancer chemotherapy based on vinca alkaloids and taxanes target tubulin, a protein required not only for mitotic spindle formation but also for the overall structural integrity of terminally differentiated cells. Among many innovations targeting specific mitotic events, inhibition of motor enzymes including KSP (or Eg5) has been validated as a highly productive approach. Many reported KSP inhibitors bind to an induced allosteric site near the site of ATP hydrolysis, and some have been tested in clinical trials with varying degrees of success. This allosteric site was defined in detail by X-ray crystallography of inhibitor complexes, yet complementary information on binding thermodynamics is still lacking. Using two model ATP-uncompetitive inhibitors, monastrol and ispinesib, we report here the results of thermal denaturation and isothermal titration calorimetric studies. These binding studies were conducted with the wild-type KSP motor domain as well as two ispinesib mutants (D130V and A133D) identified to confer resistance to ispinesib treatment. The thermodynamic parameters obtained were placed in the context of the available structural information and corresponding models of the two ispinesib-resistant mutants. The resulting overall information formed a strong basis for future structure-based design of inhibitors of KSP and related motor enzymes.

Correlations between growth and wool quality traits of genetically divergent Australian lambs in response to canola or flaxseed oil supplementation.

The correlations between growth and wool traits in response to canola and flaxseed oil supplementation were evaluated in Australian prime lambs. Sixty dual-purpose prime lambs including purebred Merino and crossbred lambs were allocated to one of five treatments of lucerne hay basal diet supplemented with isocaloric and isonitrogenous wheat-based pellets. Treatments were: no oil inclusion (Control); 2.5% canola oil; 5% canola oil; 2.5% flaxseed oil and 5% flaxseed oil, with lamb groups balanced by breed and gender. Each lamb was daily supplemented with 1kg of pellets and had free access to lucerne hay and water throughout the 7-week feeding trial, after a 3-week adaptation. Individual animal basal and supplementary pellet feed intakes were recorded daily, while body conformation traits, body condition scores and liveweights were measured on days 0, 21, 35 and 49. The lambs were dye-banded on the mid-side and shorn before commencing the feeding trial and mid-side wool samples were collected from the same dye-banded area of each lamb at the end of the experiment. Correlations between wool quality traits and lamb performance were non-significant (P>0.05). Oil supplementation had no detrimental effect on lamb growth and wool quality traits (P > 0.05). Gender significantly affected wither height gain and fibre diameter. There were significant interactions between oil supplementation and lamb breed on chest girth. The correlations between clean fleece yield (CFY) and other wool quality traits were moderate ranging from 0.29 to 0.55. Moderate to high correlations between fibre diameter (FD) and other wool quality traits were detected (0.46-0.99) with the strongest relationship between FD and wool spinning fineness (SF). The relationship between CFY and wool comfort factor (CF) were positive, while negative relationships between CFY and the others were observed. A combination of 5% oil supplementation and genetics is an effective and strategic management tool for enhancing feed efficiency and growth performance without negative effects on wool quality in dual-purpose lamb production. This is a good outcome for dual-purpose sheep farmers. It essentially means the absorbed nutrients in supplemented lambs yielded good growth performance without any detrimental impact on wool quality; a win-win case of nutrient partitioning into the synthesis of muscle and wool without compromising either traits.

Construction and characterization of a fully active PXR/SRC-1 tethered protein with increased stability.

The nuclear xenobiotic receptor PXR is a ligand-inducible transcription factor regulating drug-metabolizing enzymes and transporters and a master switch mediating potentially adverse drug-drug interactions. In addition to binding a coactivator protein such as SRC-1, the C-terminal ligand-binding domain (LBD) is solely responsible for ligand recognition and thus the ligand-dependent downstream effects. In an effort to facilitate structural studies of PXR to understand and abolish the interactions between PXR and its ligands, several recombinant PXR/SRC-1 constructs were designed and evaluated for expression, stability and activity. Expression strategies employing either dual expression or translationally coupled bicistronic expression were found to be unsuitable for producing stable PXR in a stochiometric complex with a peptide derived from SRC-1 (SRC-1p). A single polypeptide chain encompassing PXR and SRC-1p tethered with a peptidyl linker was designed to promote intramolecular complex formation. This tethered protein was overexpressed as a soluble protein and required no additional SRC-1p for further stabilization. X-ray crystal structures in the presence and absence of the known PXR agonist SR-12813 were determined to high resolution. In addition, a circular dichroism-based binding assay was developed to allow rapid evaluation of PXR ligand affinity, making this tethered protein a convenient and effective reagent for the rational attenuation of drug-induced PXR-mediated metabolism.

Supplementing Dairy Ewes Grazing Low Quality Pastures with Plant-Derived and Rumen-Protected Oils Containing Eicosapentaenoic Acid and Docosahexaenoic Acid Pellets Increases Body Condition Score and Milk, Fat, and Protein Yields.

The Australian dairy sheep industry is small and mostly based on a natural grass grazing system, which can limit productivity. The current study tested different plant oil-infused and rumen protected polyunsaturated fats and their interactions with sire breeds to improve lactation traits and body condition scores (BCS) of ewes grazing low quality pastures. It was hypothesised that supplementing lactating ewe's diets with plant-derived polyunsaturated oils would improve milk production and composition without compromising BCS. Sixty ewes (n = 10/treatment) in mid-lactation, balanced by sire breed, parity, milk yield, body condition score, and liveweight, were supplemented with: (1) control: wheat-based pellets without oil inclusion; wheat-based pellets including; (2) canola oil (CO); (3) rice bran oil (RBO); (4) flaxseed oil (FSO); (5); safflower oil (SFO); and (6) rumen protected marine oil containing eicosapentaenoic acid and docosahexaenoic acid (RPO). Except for the control group, all supplementary diets included the same level of 50 mL/kg DM of oil and all diets were isocaloric and isonitrogenous. Experimental animals were grazed in the same paddock with ad libitum access to pasture, hay, and water during the 10-week study. RPO was the most effective diet that enhanced milk, fat, and protein yields by approximately 30%, 13%, and 31%, respectively (p < 0.0001). A significant increase in milk production was also observed with CO, RBO, and SFO treatments (p < 0.0001). Breed significantly influenced animal performance with higher milk yields recorded for crossbred Awassi × East Friesian (AW × EF) (578 g/day) vs. purebred Awassi (452 g/day) (p < 0.0001). This study provides empirical evidence for the use of rumen-protected and plant-derived oil-infused pellets as supplements under low quality pasture grazing conditions to improve the production performance of purebred Awassi and crossbred AW × EF ewes.

Enhanced Omega-3 Polyunsaturated Fatty Acid Contents in Muscle and Edible Organs of Australian Prime Lambs Grazing Lucerne and Cocksfoot Pastures.

The enhancement of health-beneficial omega-3 long⁻chain (≥C20) polyunsaturated fatty acid (n-3 LC-PUFA) contents in the muscle, liver, heart, and kidney of Australian prime lambs through pasture grazing and supplementation with oil infused pellets was investigated. Forty-eight first-cross prime lambs were randomly assigned into a split-plot design with pasture type as the main plot effect and pellet supplementation as a sub-plot effect in a feeding trial that lasted for nine weeks. The n-3 LC-PUFA content in Longissimus dorsi muscle of all lambs was well above the 30 mg threshold for "omega-3 source" nutrition claim under the Australian Food Standards and Guidelines. Pasture type impacted the fatty acid contents in muscle, heart, and kidney of prime lambs. Lambs grazing cocksfoot grass only had high 18:3n-3 (ALA) and n-3 LC-PUFA contents (67.1 mg/100 g and 55.2 mg/100 g, respectively) in the Longissimus dorsi muscle, which was not significantly different (p > 0.8990) from the contents of lambs grazing only lucerne. Supplementation of pellets with or without oil infusion to grazing lambs generally decreased the ALA and n-3 LC-PUFA contents and increased the n-6/n-3 ratio in the Longissimus dorsi muscle. The fatty acid content in the internal organs of grazing lambs was also affected by pellet supplementation. The liver and kidney of grazing lambs were both "good sources" (60 mg/100 g) of omega-3. The cocksfoot grass showed considerable potential for producing healthy, premium quality meat with high contents of n-3 and n-3 LC-PUFA, which may consequently enhance the omega-3 intake of Australian lamb consumers.

Nutritional Supplements Fortified with Oils from Canola, Flaxseed, Safflower and Rice Bran Improve Feedlot Performance and Carcass Characteristics of Australian Prime Lambs.

This study investigated live animal performance and carcass characteristics of Australian prime lambs fed oil based polyunsaturated fatty acid (PUFA) enriched pellets in a feedlot system. The tested hypothesis was that supplementation of lambs with a variety of dietary oil based PUFA enriched pellets would enhance growth and carcass characteristics compared with the control lambs fed only with lucerne hay. Seventy-two, 6 months old White Suffolk x Corriedale first-cross prime lambs with an average liveweight (LWT) of 35.7 ± 0.9 kg were allocated to six treatment groups in a completely randomised experimental design. The treatments were: (1) control: lucerne hay only; or lucerne hay plus wheat-based pellets infused with 50 mL/kg dry matter (DM) of oils from (2) rice bran (RBO); (3) canola (CO); (4) rumen protected (RPO); (5) flaxseed (FO) and (6) safflower (SO) dietary sources. All lambs had ad libitum access to lucerne hay and clean fresh water. Supplemented lambs were fed 1 kg of pellet/head/day for 10 weeks. Feed intake, final LWT, average daily gain (ADG), body conformation and carcass characteristics of lambs in the supplemented groups were all greater than for the control group. SO lambs had the lowest ADG of 190.3 g/day. RBO and CO treatments had the lowest feed cost per unit gain of AU$3.0/kg. Supplemented lambs had similar over the hooks (OTH) incomes that were all higher than that of the control group. This empirical evidence-based data demonstrated that supplementation of lambs with RBO and CO had comparatively lower feed costs without compromising ADG, carcass characteristics and OTH income.

Stabilization of the autoproteolysis of TNF-alpha converting enzyme (TACE) results in a novel crystal form suitable for structure-based drug design studies.

The crystallization of TNF-alpha converting enzyme (TACE) has been useful in understanding the structure-activity relationships of new chemical entities. However, the propensity of TACE to undergo autoproteolysis has made enzyme handling difficult and impeded the identification of inhibitor soakable crystal forms. The autoproteolysis of TACE was found to be specific (Y352-V353) and occurred within a flexible loop that is in close proximity to the P-side of the active site. The rate of autoproteolysis was found to be proportional to the concentration of TACE, suggesting a bimolecular reaction mechanism. A limited specificity study of the S(1)' subsite was conducted using surrogate peptides and suggested substitutions that would stabilize the proteolysis of the loop at positions Y352-V353. Two mutant proteases (V353G and V353S) were generated and proved to be highly resistant to autoproteolysis. The kinetics of the more resistant mutant (V353G) and wild-type TACE were compared and demonstrated virtually identical IC(50) values for a panel of competitive inhibitors. However, the k(cat)/K(m) of the mutant for a larger substrate (P6 - P(6)') was approximately 5-fold lower than that for the wild-type enzyme. Comparison of the complexed wild-type and mutant structures indicated a subtle shift in a peripheral P-side loop (comprising the mutation site) that may be involved in substrate binding/turnover and might explain the mild kinetic difference. The characterization of this stabilized form of TACE has yielded an enzyme with similar native kinetic properties and identified a novel crystal form that is suitable for inhibitor soaking and structure determination.

Crystallization of glycosylated human BACE protease domain expressed in Trichoplusia ni.

Human beta-amyloid precursor protein cleaving enzyme (beta-secretase, or BACE) belongs to the aspartyl protease family, and is responsible for generating the N-terminus of beta-amyloid peptide (Abeta). BACE is a type I transmembrane glycoprotein with pre-, pro- and catalytic domains, a short transmembrane helix and a cytoplasmic region. In this study, a truncated form was engineered to produce the authentic catalytic domain of BACE in Trichoplusia ni (High 5) cells. The glycosylated BACE zymogen (proBACE) was secreted into the conditioned medium for facile purification by metal chelate and gel filtration chromatographies. The mature catalytic domain was obtained by a trans cleavage event under acidic conditions and crystallized in the absence of a bound inhibitor. A complete 3.4 A data set was collected on a single orthorhombic crystal with unit cell parameters a=74 A, b=130 A, c=134A. Successful molecular replacement shows two BACE molecules in the asymmetric unit.

Crystal structure of the catalytic domain of human ADAM33.

Adam33 is a putative asthma susceptibility gene encoding for a membrane-anchored metalloprotease belonging to the ADAM family. The ADAMs (a disintegrin and metalloprotease) are a family of glycoproteins implicated in cell-cell interactions, cell fusion, and cell signaling. We have determined the crystal structure of the Adam33 catalytic domain in complex with the inhibitor marimastat and the inhibitor-free form. The structures reveal the polypeptide fold and active site environment resembling that of other metalloproteases. The substrate-binding site contains unique features that allow the structure-based design of specific inhibitors of this enzyme.

Structural basis of CX-4945 binding to human protein kinase CK2.

Protein kinase CK2 (CK2), a constitutively active serine/threonine kinase, is involved in a variety of roles essential to the maintenance of cellular homeostasis. Elevated levels of CK2 expression results in the dysregulation of key signaling pathways that regulate transcription, and has been implicated in cancer. The adenosine-5'-triphosphate-competitive inhibitor CX-4945 has been reported to show broad spectrum anti-proliferative activity in multiple cancer cell lines. Although the enzymatic IC(50) of CX-4945 has been reported, the thermodynamics and structural basis of binding to CK2α remained elusive. Presented here are the crystal structures of human CK2α in complex with CX-4945 and adenylyl phosphoramidate at 2.7 and 1.3 Å, respectively. Biophysical analysis of CX-4945 binding is also described. This data provides the structural rationale for the design of more potent inhibitors against this emerging cancer target.

Evaluation of in vitro PXR-based assays and in silico modeling approaches for understanding the binding of a structurally diverse set of drugs to PXR.

The pregnane X-receptor (PXR) is a promiscuous nuclear receptor primarily responsible for the induction of genes from the cytochrome P450 3A family. In this study, we used a previously described PXR/SRC tethered protein to establish two in vitro assays for identifying PXR ligands: automated ligand identification system (ALIS) and temperature-dependent circular dichroism (TdCD). Kd values determined by ALIS and TdCD showed good correlations with the EC50 values determined by a PXR luciferase reporter-gene assay for 37 marketed drugs. The same set of compounds was modeled into the PXR ligand-binding domain that takes into consideration the structural variations of five published X-ray structures of PXR-ligand complexes. Major findings from our in silico analysis are as follows. First, the primary determinants for non-binders of PXR are molecular size and shape of the compounds. Low molecular weight (MW<300) compounds were in general found to be non-binders, and those molecules that do not match the shape of the PXR ligand-binding site may also act as a non-binder. Secondly, the favorable hydrophobic interactions, mostly through aromatic π-π interactions, and the presence of suitable hydrogen bond(s) between the compounds and PXR are attributes of strong binders. Thirdly, the structures of the PXR binding domain possess the flexibility that accommodates structurally diverse compounds, while some of the strong binders may also adapt flexible conformations for fitting into the binding site. The results from this study provide a molecular basis for future efforts in reducing/abolishing the PXR-dependent CYP3A4 induction liability.

Crystal structures of the pro-inflammatory cytokine interleukin-23 and its complex with a high-affinity neutralizing antibody.

Interleukin (IL)-23 is a pro-inflammatory cytokine playing a key role in the pathogenesis of several autoimmune and inflammatory diseases. We have determined the crystal structures of the heterodimeric p19-p40 IL-23 and its complex with the Fab (antigen-binding fragment) of a neutralizing antibody at 2.9 and 1.9 A, respectively. The IL-23 structure closely resembles that of IL-12. They share the common p40 subunit, and IL-23 p19 overlaps well with IL-12 p35. Along the hydrophilic heterodimeric interface, fewer charged residues are involved for IL-23 compared with IL-12. The binding site of the Fab is located exclusively on the p19 subunit, and comparison with published cytokine-receptor structures suggests that it overlaps with the IL-23 receptor binding site.

Structure-guided discovery of cyclin-dependent kinase inhibitors.

CDK2 inhibitors containing the related bicyclic heterocycles pyrazolopyrimidines and imidazopyrazines were discovered through high-throughput screening. Crystal structures of inhibitors with these bicyclic cores and two more related ones show that all but one have a common binding mode featuring two hydrogen bonds (H-bonds) to the backbone of the kinase hinge region. Even though ab initio computations indicated that the imidazopyrazine core would bind more tightly to the hinge, pyrazolopyrimidines gain an advantage in potency through participation of N4 in an H-bond network involving two catalytic residues and bridging water molecules. Further insight into inhibitor/CDK2 interactions was gained from analysis of additional crystal structures. Significant gains in potency were obtained by optimizing the fit of hydrophobic substituents to the gatekeeper region of the ATP binding site. The most potent inhibitors have good selectivity.