Lists of Chemical Warfare Agents and Precursors from International Nonproliferation Frameworks: Structural Annotation and Chemical Fingerprint Analysis.

To support efforts to stem the proliferation of chemical weapons (CWs), we have curated and structurally annotated CW-control lists from three key international nonproliferation frameworks: the Chemical Weapons Convention (CWC), the Australia Group (AG), and the Wassenaar Arrangement. The curated lists are available as web tables at the Costanzi Research website (https://costanziresearch.com/cw-control-lists/). The annotations include manually curated 2D structural images, which provide a means to appreciate at a glance the similarities and differences between different entries, as well as downloadable 2D structures, in two different formats and three different structural identifiers, namely, simplified molecular-input line-entry system, standard InChI, and standard InChIKey, which are intended to provide a platform for cheminformatics analyses. The tables also include links to National Center for Biotechnology Information's PubChem and National Institute of Standards and Technology's Chemistry WebBook cards, hence providing prompt access to a wealth of physicochemical, analytical chemistry, and toxicological information. To showcase the importance of structural annotations, we discuss a discrepancy in a CW-control list covering the defoliant Agent Orange, which we identified through our curation process, and propose a solution to address it. Moreover, we present the results of chemical fingerprinting analyses, through which we clustered the entries of the three CW-control lists under study into structurally related groups and studied the overlaps between the three lists. As an application of this study, we examine the recent updates of CWC Schedule 1 and the AG precursors list, highlighting the relationships between the two amendments and proposing the possible addition of further chemicals. Our research is intended to facilitate the communication between scientific advisors and policymakers as well as the work of chemists and cheminformaticians involved in the CW nonproliferation field. Ultimately, we seek to provide tools to bolster the control of CWs and support the global efforts to rid the world of this category of weapons.

Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library.

The ß2-adrenergic receptor (ß2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known ß-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human ß2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the ß2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the ß2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the ß2AR. In cell-signaling studies, 15 inhibits cAMP production through the ß2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits ß-arrestin recruitment to the activated ß2AR. This study presents an allosteric small-molecule ligand for the ß2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

Influence of the Structural Accuracy of Homology Models on Their Applicability to Docking-Based Virtual Screening: The ß2 Adrenergic Receptor as a Case Study.

How accurate do structures of the ß2 adrenergic receptor (ß2AR) need to be to effectively serve as platforms for docking-based virtual screening campaigns? To answer this research question, here, we targeted through controlled virtual screening experiments 23 homology models of the ß2AR endowed with different levels of structural accuracy. Subsequently, we studied the correlation between virtual screening performance and structural accuracy of the targeted models. Moreover, we studied the correlation between virtual screening performance and template/target receptor sequence identity. Our study demonstrates that docking-based virtual screening campaigns targeting homology models of the ß2AR, in the majority of the cases, yielded results that exceeded random expectations in terms of area under the receiver operating characteristic curve (ROC AUC). Moreover, with the most effective scoring method, over one-third and one-quarter of the models yielded results that exceeded random expectation also in terms of enrichment factors (EF1, EF5, and EF10) and BEDROC (α = 160.9), respectively. Not surprisingly, we found a detectable linear correlation between virtual screening performance and structural accuracy of the ligand-binding cavity. We also found a detectable linear correlation between virtual screening performance and structural accuracy of the second extracellular loop (EL2). Finally, our data indicate that, although there is no detectable linear correlation between virtual screening performance and template/ß2AR sequence identity, models built on the basis of templates that show high sequence identity with the ß2AR, especially within the ligand-biding cavity, performed consistently well. Conversely, models with lower sequence identity displayed performance levels that ranged from very good to random, with no apparent correlation with the sequence identity itself.

Chiral analogues of (+)-cyclazosin as potent α1B-adrenoceptor selective antagonist.

(+)-Cyclazosin [(+)-1] is one of most selective antagonists of the α1B-adrenoceptor subtype (selectivity ratios, α1B/α1A = 13, α1B/α1D = 38-39). To improve the selectivity, we synthesized and pharmacologically studied the blocking activity against α1-adrenoceptors of several homochiral analogues of (+)-cyclazosin featuring different substituents on the carbonyl or amine groups, namely (-)-2, (+)-3, (-)-4-(-)-8, (+)-9. Moreover, we studied the activity of some their opposite enantiomers, namely (-)-1, (-)-3, (+)-6, and (-)-9, to evaluate the influence of stereochemistry on selectivity. The benzyloxycarbonyl and methyl (4aS,8aR) analogues (+)-3 and (-)-6 improved in a significant way the α1B selectivity of the progenitor compound: 4 and 14 time vs. the α1D subtype and 35 and 77 times vs. the α1A subtype, respectively. The study confirmed the importance of the hydrophobic cis-octahydroquinoxaline moiety of these molecules for the establishment of interactions with the α1-adrenoceptors as well that of their (4aS,8aR) stereochemistry to grant selectivity for the α1B subtype. Hypotheses on the mode of interaction of these compounds were advanced on the basis of molecular modeling studies performed on compound (+)-3.

Gigantism and acromegaly due to Xq26 microduplications and GPR101 mutation.

BACKGROUND: Increased secretion of growth hormone leads to gigantism in children and acromegaly in adults; the genetic causes of gigantism and acromegaly are poorly understood. METHODS: We performed clinical and genetic studies of samples obtained from 43 patients with gigantism and then sequenced an implicated gene in samples from 248 patients with acromegaly. RESULTS: We observed microduplication on chromosome Xq26.3 in samples from 13 patients with gigantism; of these samples, 4 were obtained from members of two unrelated kindreds, and 9 were from patients with sporadic cases. All the patients had disease onset during early childhood. Of the patients with gigantism who did not carry an Xq26.3 microduplication, none presented before the age of 5 years. Genomic characterization of the Xq26.3 region suggests that the microduplications are generated during chromosome replication and that they contain four protein-coding genes. Only one of these genes, GPR101, which encodes a G-protein-coupled receptor, was overexpressed in patients' pituitary lesions. We identified a recurrent GPR101 mutation (p.E308D) in 11 of 248 patients with acromegaly, with the mutation found mostly in tumors. When the mutation was transfected into rat GH3 cells, it led to increased release of growth hormone and proliferation of growth hormone-producing cells. CONCLUSIONS: We describe a pediatric disorder (which we have termed X-linked acrogigantism [X-LAG]) that is caused by an Xq26.3 genomic duplication and is characterized by early-onset gigantism resulting from an excess of growth hormone. Duplication of GPR101 probably causes X-LAG. We also found a recurrent mutation in GPR101 in some adults with acromegaly. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others.).

Novel structural and functional insights into M3 muscarinic receptor dimer/oligomer formation.

Class A G protein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M3 muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M., Heusel, M., Liu, T., Costanzi, S., and Wess, J. (2011) J. Biol. Chem. 286, 28584-28598). To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within two distinct cytoplasmic M3R regions, the C-terminal portion of the second intracellular loop (i2) and helix H8 (H8). The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of two structurally distinct M3R dimer interfaces, one involving i2/i2 contacts (TM4-TM5-i2 interface) and the other one characterized by H8-H8 interactions (TM1-TM2-H8 interface). Specific H8-H8 disulfide cross-links led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation or mobility of H8 are critical for efficient receptor-G protein coupling. Our findings provide novel structural and functional insights into the mechanisms involved in M3R dimerization (oligomerization). Because the M3R shows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest.

The GPCR crystallography boom: providing an invaluable source of structural information and expanding the scope of homology modeling.

G protein-coupled receptors (GPCRs) are integral membrane proteins of high pharmaceutical interest. Until relatively recently, their structures have been particularly elusive, and rhodopsin has been for many years the only member of the superfamily with experimentally elucidated structures. However, a number of recent technical and scientific advancements made the determination of GPCR structures more feasible, thus leading to the solution of the structures of several receptors. Besides providing direct structural information, these experimental GPCR structures also provide templates for the construction of GPCR models. In depth studies have been performed to probe the accuracy of these models, in particular with respect to the interactions with their ligands, and to assess their applicability the rational discovery of GPCR modulators. Given the current state of the art and the pace of the field, the future of GPCR structural studies is likely to be characterized by a landscape populated by an increasingly higher number of experimental and theoretical structures.

GPR101: Modeling a constitutively active receptor linked to X-linked acrogigantism.

GPR101 is a G protein-coupled receptor (GPCR) implicated in a rare form of genetic gigantism known as X-linked acrogigantism, or X-LAG. In particular, X-LAG patients harbor microduplications in the long arm of the X-chromosome that invariably include the GPR101 gene. Duplications of the GPR101 gene lead to the formation of a new chromatin domain that causes over-expression of the receptor in the pituitary tumors of the patients. Notably, GPR101 is a constitutively active receptor, which stimulates cells to produce the second messenger cyclic AMP (cAMP) in the absence of ligands. Moreover, GPR101 was recently reported to constitutively activate not only the cAMP pathway via Gs, but also other G protein subunits (Gq/11 and G12/13). Hence, chemicals that block the constitutive activity of GPR101, known as inverse agonists, have the potential to be useful for the development of pharmacological tools for the treatment of X-LAG. In this study, we provide structural insights into the putative structure of GPR101 based on in-house built homology models, as well as third party models based on the machine learning methods AlphaFold and AlphaFold-Multistate. Moreover, we report a molecular dynamics study, meant to further probe the constitutive activity of GPR101. Finally, we provide a structural comparison with the closest GPCRs, which suggests that GPR101 does not share their natural ligands. While this manuscript was under review, cryo-electron microscopy structures of GPR101 were reported. These structures are expected to enable computer-aided ligand discovery efforts targeting GPR101.

Structural aspects of M3 muscarinic acetylcholine receptor dimer formation and activation.

To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M(3) muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. [(35)S]GTPγS binding/Gα(q/11) immunoprecipitation assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (∼50% reduction in receptor-G-protein coupling, as compared to control M3R). These data support the novel concept that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Given the high degree of structural homology among family A GPCRs, these findings should be of broad significance.

A2B adenosine receptor blockade inhibits growth of prostate cancer cells.

The role of the A2B adenosine receptor (AR) in prostate cell death and growth was studied. The A2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A1, A2A, A2B, and A3) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A2B AR using PC-3 cells as a model. The A2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A2B AR agonist NECA and the selective A2B AR agonist BAY60-6583, but not the A2A AR agonist CGS21680, concentration-dependently induced adenosine 3',5'-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A2B AR. The selective A2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A2B AR antagonists as potential novel therapeutics.

New insights for drug design from the X-ray crystallographic structures of G-protein-coupled receptors.

Methodological advances in X-ray crystallography have made possible the recent solution of X-ray structures of pharmaceutically important G protein-coupled receptors (GPCRs), including receptors for biogenic amines, peptides, a nucleoside, and a sphingolipid. These high-resolution structures have greatly increased our understanding of ligand recognition and receptor activation. Conformational changes associated with activation common to several receptors entail outward movements of the intracellular side of transmembrane helix 6 (TM6) and movements of TM5 toward TM6. Movements associated with specific agonists or receptors have also been described [e.g., extracellular loop (EL) 3 in the A(2A) adenosine receptor]. The binding sites of different receptors partly overlap but differ significantly in ligand orientation, depth, and breadth of contact areas in TM regions and the involvement of the ELs. A current challenge is how to use this structural information for the rational design of novel potent and selective ligands. For example, new chemotypes were discovered as antagonists of various GPCRs by subjecting chemical libraries to in silico docking in the X-ray structures. The vast majority of GPCR structures and their ligand complexes are still unsolved, and no structures are known outside of family A GPCRs. Molecular modeling, informed by supporting information from site-directed mutagenesis and structure-activity relationships, has been validated as a useful tool to extend structural insights to related GPCRs and to analyze docking of other ligands in already crystallized GPCRs.

Structural basis of M3 muscarinic receptor dimer/oligomer formation.

Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.

In silico screening for agonists and blockers of the ß(2) adrenergic receptor: implications of inactive and activated state structures.

Ten crystal structures of the ß(2) adrenergic receptor have been published, reflecting different signaling states. Here, through controlled-docking experiments, we examined the implications of using inactive or activated structures on the in silico screening for agonists and blockers of the receptor. Specifically, we targeted the crystal structures solved in complex with carazolol (2RH1), the neutral antagonist alprenalol, the irreversible agonist FAUC50 (3PDS), and the full agonist BI-167017 (3P0G). Our results indicate that activated structures favor agonists over blockers, whereas inactive structures favor blockers over agonists. This tendency is more marked for activated than for inactive structures. Additionally, agonists tend to receive more favorable docking scores when docked at activated rather than inactive structures, while blockers do the opposite. Hence, the difference between the docking scores attained with an activated and an inactive structure is an excellent means for the classification of ligands into agonists and blockers as we determined through receiver operating characteristic curves and linear discriminant analysis. With respect to virtual screening, all structures prioritized well agonists and blockers over nonbinders. However, inactive structures worked better for blockers and activated structures worked better for agonists, respectively. Notably, the combination of individual docking experiments through receptor ensemble docking resulted in an excellent performance in the retrieval of both agonists and blockers. Finally, we demonstrated that the induced-fit docking of agonists is a viable way of modifying an inactive crystal structure and bias it toward the in silico recognition of agonists rather than blockers.

Structural basis of G protein-coupled receptor-G protein interactions.

The interaction of G protein-coupled receptors (GPCRs) with heterotrimeric G proteins represents one of the most fundamental biological processes. However, the molecular architecture of the GPCR-G protein complex remains poorly defined. In the present study, we applied a comprehensive GPCR-G protein alpha subunit (Galpha) chemical cross-linking strategy to map a receptor-Galpha interface, both before and after agonist-induced receptor activation. Using the M(3) muscarinic acetylcholine receptor (M3R)-Galpha(q) system as a model system, we examined the ability of approximately 250 combinations of cysteine-substituted M3R and Galpha(q) proteins to undergo cross-link formation. We identified many specific M3R-Galpha(q) contact sites, in both the inactive and active receptor conformations, allowing us to draw conclusions regarding the basic architecture of the M3R-Galpha(q) interface and the nature of the conformational changes following receptor activation. As heterotrimeric G proteins as well as most GPCRs share a high degree of structural homology, our findings should be of broad general relevance.

Virtual screening leads to the discovery of novel non-nucleotide P2Y1 receptor antagonists.

The P2Y(1) receptor (P2Y(1)R) is a G protein-coupled receptor naturally activated by extracellular ADP. Its stimulation is an essential requirement of ADP-induced platelet aggregation, thus making antagonists highly sought compounds for the development of antithrombotic agents. Here, through a virtual screening campaign based on a pharmacophoric representation of the common characteristics of known P2Y(1)R ligands and the putative shape and size of the receptor binding pocket, we have identified novel antagonist hits of µM affinity derived from a N,N'-bis-arylurea chemotype. Unlike the vast majority of known P2Y(1)R antagonists, these drug-like compounds do not have a nucleotidic scaffold or highly negatively charged phosphate groups. Hence, our compounds may provide a direction for the development of receptor probes with altered physicochemical properties.

Pharmacochemistry of the platelet purinergic receptors.

Platelets contain at least five purinergic G protein-coupled receptors, e.g., the pro-aggregatory P2Y(1) and P2Y(12) receptors, a P2Y(14) receptor (GPR105) of unknown function, and anti-aggregatory A(2A) and A(2B) adenosine receptor (ARs), in addition to the ligand-gated P2X1 ion channel. Probing the structure-activity relationships (SARs) of the P2X and P2Y receptors for extracellular nucleotides has resulted in numerous new agonist and antagonist ligands. Selective agents derived from known ligands and novel chemotypes can be used to help define the subtypes pharmacologically. Some of these agents have entered into clinical trials in spite of the challenges of drug development for these classes of receptors. The functional architecture of P2 receptors was extensively explored using mutagenesis and molecular modeling, which are useful tools in drug discovery. In general, novel drug delivery methods, prodrug approaches, allosteric modulation, and biased agonism would be desirable to overcome side effects that tend to occur even with receptor subtype-selective ligands. Detailed SAR analyses have been constructed for nucleotide and non-nucleotide ligands at the P2Y(1), P2Y(12), and P2Y(14) receptors. The thienopyridine antithrombotic drugs Clopidogrel and Prasugrel require enzymatic pre-activation in vivo and react irreversibly with the P2Y(12) receptor. There is much pharmaceutical development activity aimed at identifying reversible P2Y(12) receptor antagonists. The screening of chemically diverse compound libraries has identified novel chemotypes that act as competitive, non-nucleotide antagonists of the P2Y(1) receptor or the P2Y(12) receptor, and antithrombotic properties of the structurally optimized analogues were demonstrated. In silico screening at the A(2A) AR has identified antagonist molecules having novel chemotypes. Fluorescent and other reporter groups incorporated into ligands can enable new technology for receptor assays and imaging. The A(2A) agonist CGS21680 and the P2Y(1) receptor antagonist MRS2500 were derivatized for covalent attachment to polyamidoamine dendrimeric carriers of MW 20,000, and the resulting multivalent conjugates inhibited ADP-promoted platelet aggregation. In conclusion, a wide range of new pharmacological tools is available to control platelet function by interacting with cell surface purine receptors.

Ligand and structure-based models for the prediction of ligand-receptor affinities and virtual screenings: Development and application to the beta(2)-adrenergic receptor.

In this study, we evaluated the applicability of ligand-based and structure-based models to quantitative affinity predictions and virtual screenings for ligands of the beta(2)-adrenergic receptor, a G protein-coupled receptor (GPCR). We also devised and evaluated a number of consensus models obtained through partial least square regressions, to combine the strengths of the individual components. In all cases, the bioactive conformation of each ligand was derived from molecular docking at the crystal structure of the receptor. We identified the most effective models applicable to the different scenarios, in the presence or in the absence of a training set. For ranking the affinity of closely related analogs when a training set is available, a ligand-based consensus model (LI-CM) seems to be the best choice, while the structure-based MM-GBSA score seems the best alternative in the absence of a training set. For virtual screening purposes, the structure-based MM-GBSA score was found to be the method of choice. Consensus models consistently had performances superior or close to those of the best individual components, and were endowed with a significantly increased robustness. Given multiple models with no a priori knowledge of their predictive capabilities, constructing a consensus model ensures results very close to those that the best model alone would have yielded.

Functionalized congeners of P2Y1 receptor antagonists: 2-alkynyl (N)-methanocarba 2'-deoxyadenosine 3',5'-bisphosphate analogues and conjugation to a polyamidoamine (PAMAM) dendrimer carrier.

The P2Y(1) receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y(1) receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y(1) receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of an intermediate amide group revealed high affinity of carboxylic congener 8 (K(i) 23 nM) and extended amine congener 15 (K(i) 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended epsilon-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y(1) receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y(1) receptor modeling and ligand docking. Attempted P2Y(1) antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM dendrimer to produce a multivalent conjugate exhibiting a desired biological effect, i.e., antithrombotic action.

Low level exposure to cadmium increases the risk of chronic kidney disease: analysis of the NHANES 1999-2006.

BACKGROUND: Environmental factors have been associated with the outbreak of chronic kidney disease (CKD). We evaluated the association of Cadmium (Cd) exposure with the risk of CKD in U.S. adults who participated in the 1999-2006 National Health and Nutrition Examination Surveys (NHANES). METHODS: 5426 subjects > or = 20 years were stratified for values of urinary and blood Cd and a multivariate logistic regression was performed to test the association between blood and urinary Cd, CKD and albuminuria (ALB) after adjustment for age, gender, race/ethnicity, body mass index and smoking habits. RESULTS: Subjects with urinary Cd > 1 mcg/g and subjects with blood Cd > 1 mcg/L showed a higher association with ALB (OR 1.63, 95% CI 1.23, 2.16; P = 0.001). Subjects with blood Cd > 1 mcg/L showed a higher association with both CKD (OR 1.48, 95% CI 1.01, 2.17; P = 0.046) and ALB (OR 1.41, 95% CI 1.10, 1.82; P = 0.007). An interaction effect on ALB was found for high levels of urinary and blood Cd (P = 0.014). CONCLUSIONS: Moderately high levels of urinary and blood Cd are associated with a higher proportion of CKD and ALB in the United States population.