Biomedical subjects

X Q Xie

Publications and source records attributed to X Q Xie.

14 recordsLinked to original sources

PubMed2001

PEBP2 and c-myb sites crucial for lambda5 core enhancer activity in pre-B cells.

The lambda5 gene is expressed exclusively in precursor (pre-) B cells where its gene product, as part of the pre-B cell receptor, is crucial for the proliferation of these cells. Several DNA regions regulate the activity and expression pattern of the lambda5 gene. Amongst these is an enhancer, B(lambda5), located 5' of the gene. Here we analyze the lambda5 enhancer core, b(lambda5), which in earlier experiments was demonstrated to retain 50% of the enhancer activity, and show that this activity is restricted to pre-B cells. We identify a DNA element within b(lambda5), PEBP2(lambda5), which is essential for enhancer activity: mutation within this site dramatically reduces core enhancer activity in pre-B cells. The PEBP2(lambda5) site binds bacterially produced polyoma enhancer binding proteins (PEBP) (Runx/AML/CBFA). Furthermore, PEBP2 proteins present in nuclear extracts from murine pre-B cells bind to the PEBP2(lambda5) element. PEBP2 proteins in mature B cells also bind to the PEBP2(lambda5 )element, implying that if PEBP2 proteins are responsible for the stage-specific expression, they have to be non-activating or inhibiting in mature B cells. We also demonstrate that a described partner of PEBP2, c-myb, binds to a sequence termed myb(lambda5) located just upstream of the PEBP2(lambda5) site in the core enhancer. The myb(lambda5) element is also crucial for enhancer activity, since mutating the myb site reduces core enhancer activity to the same extent as mutating the PEBP2 site. Earlier reports have shown that c-myb is expressed at high levels in pre-B cell lines whereas its expression is down-regulated in more mature B cell lines. Thus, c-myb may be involved in determining the stage-specific expression of the lambda5 gene.

Animals↗

PubMed2001

Stereochemical selectivity of methanandamides for the CB1 and CB2 cannabinoid receptors and their metabolic stability.

Several chiral, analogues of the endogenous cannabinoid receptor ligand, arachidonylethanolamide (anandamide), methylated at the 2,1' and 2' positions using asymmetric synthesis were evaluated in order to study (a) stereoselectivity of binding to CB1 and CB2 cannabinoid receptors; and (b) metabolic stability with regard to anandamide amidase. Enantiomerically pure 2-methyl arachidonic acids were synthesized through diastereoselective methylation of the respective chiral 2-oxazolidinone enolate derivatives and CB1 and CB2 receptor affinities of the resulting chiral anandamides were evaluated using a standard receptor binding assay. Introduction of a single 2-methyl group increased affinity for CB1, led to limited enantioselectivity and only modestly improved metabolic stability. However, a high degree of enantio- and diastereoselectivity was observed for the 2,1'-dimethyl analogues. (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (4) exhibited the highest CB1 receptor affinity in this series with a K(i) of 7.42 nM, an at least 10-fold improvement on anandamide (K(i)=78.2 nM). The introduction of two methyl groups at the 2-position of anandamide led to no change in affinity for CB1 but somewhat enhanced metabolic stability. Conversely, chiral headgroup methylation in the 2-gem-dimethyl series led to chiral analogues possessing a wide range of CB1 affinities. Of these the (S)-2,2,2'-trimethyl analogue (12) had the highest affinity for CB1 almost equal to that of anandamide. In agreement with our previous anandamide structure-activity relationship work, the analogues in this study showed high selectivity for the CB1 receptor over CB2. The results are evaluated in terms of stereochemical factors affecting the ligand's affinity for CB1 using receptor-essential volume mapping as an aid. Based on the results, a partial CB1 receptor site model is proposed, that bears two hydrophobic pockets capable of accommodating 1'- and 2-methyl groups

Arachidonic Acids↗

PubMed2001

Crystallization and preliminary studies of the DNA-binding runt domain of AML1.

The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.

Amino Acid Substitution↗

PubMed2000

Smad and AML proteins synergistically confer transforming growth factor beta1 responsiveness to human germ-line IgA genes.

Transcription of germ-line immunoglobulin heavy chain genes conditions them to participate in isotype switch recombination. Transforming growth factor-beta1 (TGF-beta1) stimulates promoter elements located upstream of the IgA1 and IgA2 switch regions, designated Ialpha1 and Ialpha2, and contributes to the development of IgA responses. We demonstrate that intracellular Smad proteins mediate activation of the Ialpha1 promoter by TGF-beta. TGF-beta type 1 receptor (ALK-5), activin type IB receptor (ALK-4), and the "orphan" ALK-7 trans-activate the Ialpha1 promoter, thus raising the possibility that other members of the TGF-beta superfamily can also modulate IgA synthesis. Smads physically interact with the AML family of transcription factors and cooperate with them to activate the Ialpha1 promoter. The Ialpha1 element provides a canapé of interspersed high and low affinity sites for Smad and AML factors, some of which are indispensable for TGF-beta responsiveness. While AML.Smad complexes are formed in the cytoplasm of DG75 and K562 cells constitutively, only after TGF-beta receptor activation, novel Smad3.Smad4.AML complexes are detected in nuclear extracts by EMSA with Ialpha1 promoter-derived probes. Considering the wide range of biological phenomena that AMLs and Smads regulate, the physical/functional interplay between them has implications that extend beyond the regulation of class switching to IgA.

Base Sequence↗

PubMed1999

High-resolution NMR and computer modeling studies of the cannabimimetic aminoalkylindole prototype WIN-55212-2.

Aminoalkylindoles (AAIs), although structurally dissimilar from the classical cannabinoids (CCs), are known to be capable of binding to cannabinoid receptors and of evoking cannabimimetic responses. However, their mode of binding remains unknown. In this communication, we have carried out further studies on the AAI prototype (R)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN-55212-2, 1) by the combined use of high-resolution 2D NMR and computer modeling. Our results suggest that the minimum energy conformations of the molecule 1 have distinct pharmacophoric features: (i) The naphthyl ring is oriented off the plane of the benzoxazine ring by approximately 59 degrees with the carbonyl C=O group pointing toward the C2-CH(3) group. (ii) At the C10-position the axial morpholinomethyl conformation is preferred over the equatorial in order to relieve a steric interaction with the C2-methyl group. The preferred conformer as defined by the three key pharmacophores, naphthyl, morpholino, and 3-keto groups, shows that the morpholinyl ring of the molecule 1 deviates from the plane of the benzoxazine ring by about 32 degrees and orients in the left molecular quadrant. This model supports the hypothesis that a certain deviation of the morpholino group from the plane of the indole ring in compound 1 is essential for cannabimimetic activity. We postulate that such an alignment by the respective pharmacophores allows them to interact optimally with the receptor. The results should help us to better understand the pharmacophoric requirements of the AAIs and serve as a basis for future SAR studies and drug design.

Benzoxazines↗

PubMed1999

Structural determinants for recognition and translocation by the anandamide transporter.

The biological actions of anandamide (arachidonylethanolamide), an endogenous cannabinoid lipid, are terminated by a two-step inactivation process consisting of carrier-mediated uptake and intracellular hydrolysis. Anandamide uptake in neurons and astrocytes is mediated by a high-affinity, Na+-independent transporter that is selectively inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). In the present study, we examined the structural determinants governing recognition and translocation of substrates by the anandamide transporter constitutively expressed in a human astrocytoma cell line. Competition experiments with a select group of analogs suggest that substrate recognition by the transporter is favored by a polar nonionizable head group of defined stereochemical configuration containing a hydroxyl moiety at its distal end. The secondary carboxamide group interacts favorably with the transporter, but may be replaced with either a tertiary amide or an ester, suggesting that it may serve as hydrogen acceptor. Thus, 2-arachidonylglycerol, a putative endogenous cannabinoid ester, also may serve as a substrate for the transporter. Substrate recognition requires the presence of at least one cis double bond situated at the middle of the fatty acid carbon chain, indicating a preference for ligands whose hydrophobic tail can adopt a bent U-shaped conformation. On the other hand, uptake experiments with radioactively labeled substrates show that no fewer than four cis nonconjugated double bonds are required for optimal translocation across the cell membrane, suggesting that substrates are transported in a folded hairpin conformation. These results outline the general structural requisites for anandamide transport and may assist in the development of selective inhibitors with potential clinical applications.

Arachidonic Acids↗

PubMed1999

AML and Ets proteins regulate the I alpha1 germ-line promoter.

The immunoglobulin heavy chain (IgH) class switch recombination of B lymphocytes preferentially targets unrearranged IgH genes that have already been rendered transcriptionally active. Transcription of the germ-line IgH genes is controlled by intervening (I) regions upstream of their switch regions. The I alpha1 promoter activates transcription of the human germ-line C alpha1 gene for IgA1 and mediates the transforming growth factor (TGF)-beta1 responsiveness of this locus. Here we show that the I alpha1 promoter contains several binding sites for the AML/PEBP2/CBF family of transcription factors and that AML and Ets proteins are major regulators of the basal and TGF-beta-inducible promoter activity. Our data constitute a starting point for studies to elucidate the molecular mechanism by which TGF-beta regulates IgA production.

Animals↗

PubMed1999

Solution properties of the free and DNA-bound Runt domain of AML1.

The Runt domain is responsible for specific DNA and protein-protein interactions in a family of transcription factors which includes human AML1. Structural data on the Runt domain has not yet become available, possibly due to solubility and stability problems with expressed protein fragments. Here we describe the optimization and characterization of a 140-residue fragment, containing the Runt domain of AML1, which is suitable for structural studies. The fragment of AML1 including amino acids 46-185 [AML1 Dm(46-185)] contains a double cysteine-->serine mutation which does not affect Runt domain structure or DNA-binding affinity. Purified AML1 Dm(46-185) is soluble and optimally stable in a buffer containing 200 mm MgSO4 and 20 mm sodium phosphate at pH 6.0. Nuclear magnetic resonance and circular dichroism spectroscopy indicate that the Runt domain contains beta-sheet, but little or no alpha-helical secondary structure elements. The 45 N-terminal residues of AML1 are unstructured and removal of the N-terminal enhances sequence-specific DNA binding. The NMR spectrum of AML1 Dm(46-185) displays a favorable chemical shift dispersion and resolved NOE connectivities are readily identified, suggesting that a structure determination of this Runt domain fragment is feasible. A titration of 15N-labelled AML1 Dm(46-185) with a 14-bp cognate DNA duplex results in changes in the 15N NMR heteronuclear single quantum coherence spectrum which indicate the formation of a specific complex and structural changes in the Runt domain upon DNA binding.

Base Sequence↗

PubMed1998

Conformational studies on a diastereoisomeric pair of tricyclic nonclassical cannabinoids by NMR spectroscopy and computer molecular modeling.

Among the nonclassical cannabinoids, CP-55,244 (4), which incorporates an axial 14 beta-hydroxymethyl group, is pharmacologically 30 times more potent than its prototype CP-47,497 (2) and 300 times more potent than delta 9-THC (1). It has a high degree of stereoselectivity (about 120:1) with respect to its diastereoisomer, CP-97,587 (5), which differs structurally by having the 14-hydroxymethyl group equatorial. Conformational studies of 4 and 5 were carried out using 2D NMR spectroscopy and molecular modeling in order to define and compare the similarities and differences between them. Specific structural features of interest are the conformation of the 1',1'-dimethylheptyl (DMH) side chain, the conformation of the cyclohexyl rings, the orientation of the phenolic ring (A ring) relative to the cyclohexyl ring (C ring), and the orientation of the hydroxymethyl group as well as the formation of intramolecular hydrogen bonding. Our results show that the conformations of the phenolic hydroxyl (Ph-OH) and DMH side chain for 4 are similar to those of 2. The proton of the phenolic hydroxyl is pointing away from the C ring while the DMH chain randomly adopts one of four dynamically averaged conformers in which it is almost perpendicular to the plane of the aromatic ring. The relative orientation of the A and C rings is such that the two rings interconvert between two low-energy conformations. Compound 5 prefers the conformer with the Ph-OH pointing toward the alpha-face of the cyclohexyl ring, while for 4, there is an increased preference for the conformer where the Ph-OH is directed toward the beta face. This may be due to intramolecular H-bonding between the Ph-OH and the axial 14 beta-hydroxymethyl group of 4 that stabilizes this conformation. Hydrogen bonding between the Ph-OH and the equatorial-14 alpha-hydroxymethyl of 5 was not detected. Thus, the orientation of the aliphatic hydroxyl group with respect to the D ring in 4 and 5 may play an important role with regard to the pharmacophoric requirements of the two analogs for the cannabinoid receptor and provide an explanation for the observed differences in their biological properties.

Cannabinoids↗

PubMed1997

Differential scanning calorimetry and small angle x-ray diffraction study of the interaction of (R)-PAF, (R)-ET-18-OME and (R)-Lyso-PAF with model membranes.

(R)-PAF, (R)-ET-18-OMe and (R)-Lyso-PAF are ether phospholipid analogs exhibiting different biological activities. All three molecules are highly lipophilic and interact with cell membranes. The manner in which their interactions occur is relevant to their biological activities and may provide information on their mechanism(s) of action at the molecular level. We have studied the interactions of (R)-PAF, (R)-ET-18-OMe and (R)-Lyso-PAF with model membranes using differential scanning calorimetry (DSC). A concentration-dependence study with DSC shows that all three analogs induce similar changes in the thermotropic behavior of model membranes. All produce a slight decrease in the main phase transition temperature of DPPC bilayers (deltaTc = 0.3 to 1.4 degrees C) but have a more substantial effect on the pretransition (deltaTc = 0.8 to 4.5 degrees C). All three disorder phosphatidylcholine (PC) membranes but impart order to PC/cholesterol membranes. We have used small angle x-ray diffraction experiments to supplement the DSC evidence on the topography of the ether lipid analogs in the bilayers. All three appear to insert themselves in the bilayer with their long chain parallel with the membrane bilayer chains.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1996

Unsaturated side chain beta-11-hydroxyhexahydrocannabinol analogs.

The cannabinoid side chain is a key pharmacophore in the interaction of cannabinoids with their receptors (CB1 and CB2). To study the stereochemical requirements of the side chain, we synthesized a series of cannabinoids in which rotation around the C1'-C2' bond is blocked. The key steps in the synthesis were the cuprate addition of a substituted resorcinol to (+)-apoverbenone, the TMSOTf-mediated formation of the dihydropyran ring, and the stereospecific introduction of the beta-11-hydroxymethyl group. All the analogs tested showed nanomolar affinity for the receptors, the cis-hept-1-ene side chain having the highest affinity for CB1 (Ki = 0.89 nM) and showing the widest separation between CB1 and CB2 affinities. The parent n-heptyl-beta-11-hydroxyhexahydrocannabinol was the least potent binding to CB1 (Ki = 8.9 nM) and had the lowest selectivity between CB1 and CB2.

Animals↗

PubMed1996

The conformational properties of the highly selective cannabinoid receptor ligand CP-55,940.

During a search for novel drugs possessing analgesic properties but devoid of the psychotropic effects of marijuana, a group of molecules designated as nonclassical cannabinoids was synthesized by Pfizer. Of these nonclassical cannabinoids CP-55,940 has received the most attention principally because it was used as the high affinity radioligand during the discovery and characterization of the G-protein-coupled cannabinoid receptor. In an effort to obtain information on the stereoelectronic requirements at the cannabinoid receptor active site, we have studied the conformational properties of CP-55,940 using a combination of solution NMR and computer modeling methods. Our data show that for the most energetically favored conformation, (i) the aromatic phenol ring is perpendicular to the cyclohexane ring, and the phenolic O-H bond is coplanar with the aromatic ring and points away from the cyclohexyl ring; ii) the dimethylheptyl chain adopts one of four preferred conformations in all of which the chain is almost perpendicular to the phenol ring; and iii) an intramolecular H-bond between the phenolic and hydroxypropyl groups allows all three hydroxyl groups of CP-55,940 to be oriented toward the upper face of the molecule. Such an orientation by the OH groups may be a characteristic requirement for cannabimimetic activity.

Cannabinoids↗

PubMed1995

Common cannabimimetic pharmacophoric requirements between aminoalkyl indoles and classical cannabinoids.

Aminoalkylindoles (AAIs) are structurally dissimilar from the classical cannabinoids (CCs), however, both AAIs and CCs appear to bind at the same site on the cannabinoid receptor. To obtain better insights on the structural correlation between AAIs and CCs, we have studied the conformational properties of the potent cannabimimetic AAI WIN 55212-2 and its inactive analogs using high resolution 2D NMR spectroscopy in combination with computer-assisted molecular modeling. The pharmacophoric similarities between the AAIs and the CCs were then investigated using superimposition techniques. The absolute stereochemistries of the biologically active enantiomer (-)HHC were used as superimposition points and considered as internal controls in order to test the molecular principles guiding this experiment. Our results show that the model is congruent with a superimposition in which the naphthoyl, morpholino and 3-keto groups in the AAI, respectively correspond to the side chain, cyclohexanol OH and phenolic OH of HHC. A good fit is obtained when the two biologically active antipodes are superimposed. Conversely, the fit is poor if the inactive AAI enantiomer is superimposed on the active HHC enantiomer. It can also be seen that in such an orientation a certain deviation of the C-ring from the plane of the phenol ring of the tricyclic HHC component and of the morpholinyl portion from the plane of the indole ring of WIN 55212-2 is essential for cannabimimetic activity. The inactive enantiomer WIN 55212-3 has its respective components aligned in the opposite quadrant. By comparing the stereoelectronic features of representative AAIs and CCs, we have developed a model which may help to uncover the pharmacophoric requirements of the AAIs and serve as a basis for future SAR and drug design.

Benzoxazines↗

PubMed1994

Conformational analysis of the prototype nonclassical cannabinoid CP-47,497, using 2D NMR and computer molecular modeling.

In an effort to determine the stereochemical requirements for pharmacological activity among the series of nonclassical cannabinoids synthesized at Pfizer, we have studied the conformational properties of the parent bicyclic analog CP-47,497. For this study, we have used a combination of solution NMR and theoretical computational approaches. The energetically favored conformation has the phenolic ring almost perpendicular to the cyclohexanol ring which exists in a chair conformation. The OH bond of the phenol is preferentially coplanar with the aromatic ring and points toward the C2 ring proton, while the dimethylheptyl side chain adopts a conformation almost perpendicular to the aromatic ring. The conformational features of this nonclassical cannabinoid analog closely resemble those of its classical counterparts. The only apparent difference is the small dihedral angle (psi 1 = 62 degrees) between the planes of the two rings of CP-47,497 compared to that of the tricyclic tetrahydro- or hexahydrocannabinol analogs (psi 1 = 137 degrees). However, CP-47,497 can be perfectly superimposed over the respective tricyclic analog by rotation around the Ph-cyclohexyl bond (C6-C7 bond) and assume a conformation which is energetically higher than the preferred one by 3.0 kcal/mol. It can be argued that such a conformation may be acquired by the nonclassical analog during its interaction with the active site.

Cannabinoids↗