Functional variants of human papillomavirus type 16 demonstrate host genome integration and transcriptional alterations corresponding to their unique cancer epidemiology.

BACKGROUND: Human papillomaviruses (HPVs) are a worldwide burden as they are a widespread group of tumour viruses in humans. Having a tropism for mucosal tissues, high-risk HPVs are detected in nearly all cervical cancers. HPV16 is the most common high-risk type but not all women infected with high-risk HPV develop a malignant tumour. Likely relevant, HPV genomes are polymorphic and some HPV16 single nucleotide polymorphisms (SNPs) are under evolutionary constraint instigating variable oncogenicity and immunogenicity in the infected host. RESULTS: To investigate the tumourigenicity of two common HPV16 variants, we used our recently developed, three-dimensional organotypic model reminiscent of the natural HPV infectious cycle and conducted various "omics" and bioinformatics approaches. Based on epidemiological studies we chose to examine the HPV16 Asian-American (AA) and HPV16 European Prototype (EP) variants. They differ by three non-synonymous SNPs in the transforming and virus-encoded E6 oncogene where AAE6 is classified as a high- and EPE6 as a low-risk variant. Remarkably, the high-risk AAE6 variant genome integrated into the host DNA, while the low-risk EPE6 variant genome remained episomal as evidenced by highly sensitive Capt-HPV sequencing. RNA-seq experiments showed that the truncated form of AAE6, integrated in chromosome 5q32, produced a local gene over-expression and a large variety of viral-human fusion transcripts, including long distance spliced transcripts. In addition, differential enrichment of host cell pathways was observed between both HPV16 E6 variant-containing epithelia. Finally, in the high-risk variant, we detected a molecular signature of host chromosomal instability, a common property of cancer cells. CONCLUSIONS: We show how naturally occurring SNPs in the HPV16 E6 oncogene cause significant changes in the outcome of HPV infections and subsequent viral and host transcriptome alterations prone to drive carcinogenesis. Host genome instability is closely linked to viral integration into the host genome of HPV-infected cells, which is a key phenomenon for malignant cellular transformation and the reason for uncontrolled E6 oncogene expression. In particular, the finding of variant-specific integration potential represents a new paradigm in HPV variant biology.

Molecular Dynamics of Fibrinogen Adsorption onto Graphene, but Not onto Poly(ethylene glycol) Surface, Increases Exposure of Recognition Sites That Trigger Immune Response.

Changes in the conformation of blood proteins due to their binding to nonbiological surfaces is the initial step in the chain of immunological reactions to foreign bodies. Despite the large number of experimental studies that have been performed on fibrinogen adsorption to nonbiological surfaces, a clear picture describing this complex process has eluded researchers to date. Developing a better understanding of the behavior of bioactive fibrinogen motifs upon their interaction with surfaces may facilitate the design of advanced materials with improved biocompatibility. This is especially important within the context of medical implants. Here we present results of explicit-solvent, all-atom MD simulations of the adsorption of the fibrinogen D-domain onto a graphene surface and a poly(ethylene glycol) (PEG) surface. Our results are consistent with experimental observations that interactions with PEG do not induce significant conformational changes on immune-reactive sites present in the D-domain of fibrinogen. In contrast, our results indicate that significant conformational changes induced by adsorption to graphene surfaces may occur under conditions that promote a high density of blood proteins on the surface. The structural rearrangements observed on graphene directly affect the secondary structure content of the D-domain, with consequent exposure of the recognition sites P1 (γ190-202) and P2 (γ377-395) and the subsite P2-C (γ383-395) involved in immune response. Analysis of the structural parameters of the MD conformers was shown to accurately assess the biocompatibility of the modeled surfaces.

Systematic discovery of molecular probes targeting multiple non-orthosteric and spatially distinct sites in the botulinum neurotoxin subtype A (BoNT/A).

The development of molecular probes targeting proteins has traditionally relied on labeling compounds already known to bind to the protein of interest. These known ligands bind to orthosteric or allosteric sites in their target protein as a way to control their activity. Binding pockets other than known orthosteric or allosteric sites may exist that are large enough to accommodate a ligand without significantly disrupting protein activity. Such sites may provide opportunities to discriminate between subtypes or other closely related proteins, since they are under less evolutionary pressure to be conserved. The Protein Scanning with Virtual Ligand Screening (PSVLS) approach was previously used to identify a novel inhibitor and a fluorescent probe against the catalytic site of the botulinum neurotoxin subtype A (BoNT/A). PSVLS screens compound databases against multiple sites within a target protein, and the results for all the sites probed against BoNT/A, not only the catalytic site, are available online. Here, we analyze the PSVLS data for multiple sites in order to identify molecular probes with affinity for binding pockets other than the catalytic site of BoNT/A. BoNT/A is a large protein with a light (LC) and a heavy (HC) chain that can be assayed separately. We used scintillation proximity assay (SPA) to test experimentally 5 probe candidates predicted computationally to have affinity for different non-orthosteric binding regions within the HC and LC, and one compound predicted not to have affinity for either domain. The binding profiles obtained experimentally confirmed the targeting of multiple and spatially distinct pockets within BoNT/A. Moreover, inhibition assay results indicate that some of these probes do not significantly interfere with the catalytic activity of BoNT/A.

Creating and virtually screening databases of fluorescently-labelled compounds for the discovery of target-specific molecular probes.

Our group has recently demonstrated that virtual screening is a useful technique for the identification of target-specific molecular probes. In this paper, we discuss some of our proof-of-concept results involving two biologically relevant target proteins, and report the development of a computational script to generate large databases of fluorescence-labelled compounds for computer-assisted molecular design. The virtual screening of a small library of 1,153 fluorescently-labelled compounds against two targets, and the experimental testing of selected hits reveal that this approach is efficient at identifying molecular probes, and that the screening of a labelled library is preferred over the screening of base compounds followed by conjugation of confirmed hits. The automated script for library generation explores the known reactivity of commercially available dyes, such as NHS-esters, to create large virtual databases of fluorescence-tagged small molecules that can be easily synthesized in a laboratory. A database of 14,862 compounds, each tagged with the ATTO680 fluorophore was generated with the automated script reported here. This library is available for downloading and it is suitable for virtual ligand screening aiming at the identification of target-specific fluorescent molecular probes.

Sweet taste receptor gene variation and aspartame taste in primates and other species.

Aspartame is a sweetener added to foods and beverages as a low-calorie sugar replacement. Unlike sugars, which are apparently perceived as sweet and desirable by a range of mammals, the ability to taste aspartame varies, with humans, apes, and Old World monkeys perceiving aspartame as sweet but not other primate species. To investigate whether the ability to perceive the sweetness of aspartame correlates with variations in the DNA sequence of the genes encoding sweet taste receptor proteins, T1R2 and T1R3, we sequenced these genes in 9 aspartame taster and nontaster primate species. We then compared these sequences with sequences of their orthologs in 4 other nontasters species. We identified 9 variant sites in the gene encoding T1R2 and 32 variant sites in the gene encoding T1R3 that distinguish aspartame tasters and nontasters. Molecular docking of aspartame to computer-generated models of the T1R2 + T1R3 receptor dimer suggests that species variation at a secondary, allosteric binding site in the T1R2 protein is the most likely origin of differences in perception of the sweetness of aspartame. These results identified a previously unknown site of aspartame interaction with the sweet receptor and suggest that the ability to taste aspartame might have developed during evolution to exploit a specialized food niche.

Genetic diversity in the IZUMO1-JUNO protein-receptor pair involved in human reproduction.

Fertilization in mammals begins with the union of egg and sperm, an event that starts a cascade of cellular processes. The molecular-level understanding of these processes can guide the development of new strategies for controlling and/or promoting fertilization, and inform researchers and medical professional on the best choice of interventions. The proteins encoded by the IZUMO1 and JUNO genes form a ligand-receptor protein pair involved in the recognition of sperm and egg. Due to their role in the fertilization process, these proteins are potential targets for the development of novel anti-contraceptive, as well as infertility treatments. Here we present a comprehensive analysis of these gene sequences, with the objective of identifying evolutionary patterns that may support their relevance as targets for preventing or improving fertility among humans. JUNO and IZUMO1 gene sequences were identified within the genomes of over 2,000 humans sequenced in the 1000 Genomes Project. The human sequences were subjected to analyses of nucleotide diversity, deviation from neutrality of genetic variation, population-based differentiation (FST), haplotype inference, and whole chromosome scanning for signals of positive or of balancing selection. Derived alleles were determined by comparison to archaic hominin and other primate genomes. The potential effect of common non-synonymous variants on protein-protein interaction was also assessed. IZUMO1 displays higher variability among human individuals than JUNO. Genetic differentiation between continental population pairs was within whole-genome estimates for all but the JUNO gene in the African population group with respect to the other 4 population groups (American, East Asian, South Asian, and European). Tajima's D values demonstrated deviation from neutrality for both genes in comparison to a group of genes identified in the literature as under balancing or positive selection. Tajima's D for IZUMO1 aligns with values calculated for genes presumed to be under balancing selection, whereas JUNO's value aligned with genes presumed to be under positive selection. These inferences on selection are both supported by SNP density, nucleotide diversity and haplotype analysis. A JUNO haplotype carrying 3 derived alleles out of 5, one of which is a missense mutation implicated in polyspermy, was found to be significant in a population of African ancestry. Polyspermy has a disadvantageous impact on fertility and its presence in approximately 30% of the population of African ancestry may be associated to a potentially beneficial role of this haplotype. This role has not been established and may be related to a non-reproductive role of JUNO. The high degree of conservation of the JUNO sequence combined with a dominant haplotype across multiple population groups supports JUNO as a potential target for the development of contraceptive treatments. In addition to providing a detailed account of human genetic diversity across these 2 important and related genes, this study also provides a framework for large population-based studies investigating protein-protein interactions at the genome level.

A portable bioinformatics course for upper-division undergraduate curriculum in sciences.

This article discusses the challenges that bioinformatics education is facing and describes a bioinformatics course that is successfully taught at the California State Polytechnic University, Pomona, to the fourth year undergraduate students in biological sciences, chemistry, and computer science. Information on lecture and computer practice topics, free for academic use software and web links required for the laboratory exercises and student surveys for two instances of the course, is presented. This course emphasizes hands-on experience and focuses on developing practical skills while providing a solid knowledge base for critically applying these skills. It is designed in blocks of 1-hour lecture followed by 2 hours of computer laboratory exercises, both covering the same general topic, for a total of 30 hours of lecture and 60 hours of computer practice. The heavy computational aspect of this course was designed to use a single multiprocessor computer server running Linux, accessible from laptops through Virtual Network Computing sessions. The laptops can be either provided by the institution or owned by the individual students. This configuration avoids the need to install and maintain bioinformatics software on each laptop. Only a single installation is required for most bioinformatics software on the Linux server. The content of this course and its software/hardware configuration are well suited for institutions with no dedicated computer laboratory. This author believes that the same model can be successfully implemented in other institutions, especially those who do not have a strong instructional computer technology support such as community colleges and small universities.

A molecular dynamics study of the correlations between solvent-accessible surface, molecular volume, and folding state.

We analyzed the correlations between molecular volume, solvent-accessible surface, and folding state (secondary structure content) for unfolded conformers of alpha (holo- and apomyoglobin) and beta (retinal-binding protein) proteins and a small water-soluble alanine-rich alpha-helical peptide. Conformers with different degrees of folding were obtained using molecular dynamics at constant temperature and pressure with implicit solvent (dielectric constant adjustment) for all four systems and with explicit solvent for the single helix peptide. Our results support the view that unfolded conformations are not necessary extended, that volume variation is not a good indication of folding state and that the simple model of water penetrating the interior of the protein does not explain the increase in volume upon unfolding.

Test of the Binding Threshold Hypothesis for olfactory receptors: explanation of the differential binding of ketones to the mouse and human orthologs of olfactory receptor 912-93.

We tested the Binding Threshold Hypothesis (BTH) for activation of olfactory receptors (ORs): To activate an OR, the odorant must bind to the OR with binding energy above some threshold value. The olfactory receptor (OR) 912-93 is known experimentally to be activated by ketones in mouse, but is inactive to ketones in human, despite an amino acid sequence identity of approximately 66%. To investigate the origins of this difference, we used the MembStruk first-principles method to predict the tertiary structure of the mouse OR 912-93 (mOR912-93), and the HierDock first-principles method to predict the binding site for ketones to this receptor. We found that the strong binding of ketones to mOR912-93 is dominated by a hydrogen bond of the ketone carbonyl group to Ser105. All ketones predicted to have a binding energy stronger than EBindThresh = 26 kcal/mol were observed experimentally to activate this OR, while the two ketones predicted to bind more weakly do not. In addition, we predict that 2-undecanone and 2-dodecanone both bind sufficiently strongly to activate mOR912-93. A similar binding site for ketones was predicted in hOR912-93, but the binding is much weaker because the human ortholog has a Gly at the position of Ser105. We predict that mutating this Gly to Ser in human should lead to activation of hOR912-93 by these ketones. Experimental substantiations of the above predictions would provide further tests of the validity of the BTH, our predicted 3D structures, and our predicted binding sites for these ORs.

N-(2,4)-dinitrophenyl-L-arginine Interacts with EphB4 and Functions as an EphB4 Kinase Modulator.

The erythropoietin-producing hepatocellular carcinoma receptor B4 is a receptor tyrosine kinase whose expression is preserved in various malignancies, including colon, gastric, and breast carcinoma. Hepatocellular carcinoma receptor B4 presence in tumor cells and involvement in cancer suppression makes it a potential therapeutic target for activating compounds. Moreover, modulators of its activity also have a strong potential to be used in diagnosis and therapy monitoring. We used virtual ligand screening to identify novel hepatocellular carcinoma receptor B4 kinase modulators for experimental testing. Three independent assay platforms confirmed that dinitrophenyl-L-arginine is likely to affect the kinase activity of hepatocellular carcinoma receptor B4. An enzyme-coupled spectrophotometric assay has been used to examine this possibility and may prove to be useful for assessing other novel kinase modulator candidates. Overall, our observations suggest that dinitrophenyl-L-arginine has an activating effect on hepatocellular carcinoma receptor B4 and, therefore, more efficient derivatives may have therapeutic effects in tumors where hepatocellular carcinoma receptor B4 exhibits antimalignant properties. The hepatocellular carcinoma receptor B4-activating effect is discussed with respect to previously described mechanisms, using predicted and experimental structures for docked ligands. As a novel kinase activity modulator, dinitrophenyl-L-arginine may provide new insights into molecular mechanisms by which kinases are activated or regulated, and may serve as a lead compound for the generation of novel hepatocellular carcinoma receptor B4-activating therapeutic compounds.

Principal component analysis of binding energies for single-point mutants of hT2R16 bound to an agonist correlate with experimental mutant cell response.

Directed evolution is a technique that enables the identification of mutants of a particular protein that carry a desired property by successive rounds of random mutagenesis, screening, and selection. This technique has many applications, including the development of G protein-coupled receptor-based biosensors and designer drugs for personalized medicine. Although effective, directed evolution is not without challenges and can greatly benefit from the development of computational techniques to predict the functional outcome of single-point amino acid substitutions. In this article, we describe a molecular dynamics-based approach to predict the effects of single amino acid substitutions on agonist binding (salicin) to a human bitter taste receptor (hT2R16). An experimentally determined functional map of single-point amino acid substitutions was used to validate the whole-protein molecular dynamics-based predictive functions. Molecular docking was used to construct a wild-type agonist-receptor complex, providing a starting structure for single-point substitution simulations. The effects of each single amino acid substitution in the functional response of the receptor to its agonist were estimated using three binding energy schemes with increasing inclusion of solvation effects. We show that molecular docking combined with molecular mechanics simulations of single-point mutants of the agonist-receptor complex accurately predicts the functional outcome of single amino acid substitutions in a human bitter taste receptor.

Interaction of E. coli outer-membrane protein A with sugars on the receptors of the brain microvascular endothelial cells.

Esherichia coli, the most common gram-negative bacteria, can penetrate the brain microvascular endothelial cells (BMECs) during the neonatal period to cause meningitis with significant morbidity and mortality. Experimental studies have shown that outer-membrane protein A (OmpA) of E. coli plays a key role in the initial steps of the invasion process by binding to specific sugar moieties present on the glycoproteins of BMEC. These experiments also show that polymers of chitobiose (GlcNAcbeta1-4GlcNAc) block the invasion, while epitopes substituted with the L-fucosyl group do not. We used HierDock computational technique that consists of a hierarchy of coarse grain docking method with molecular dynamics (MD) to predict the binding sites and energies of interactions of GlcNAcbeta1-4GlcNAc and other sugars with OmpA. The results suggest two important binding sites for the interaction of carbohydrate epitopes of BMEC glycoproteins to OmpA. We identify one site as the binding pocket for chitobiose (GlcNAcbeta1-4GlcNAc) in OmpA, while the second region (including loops 1 and 2) may be important for recognition of specific sugars. We find that the site involving loops 1 and 2 has relative binding energies that correlate well with experimental observations. This theoretical study elucidates the interaction sites of chitobiose with OmpA and the binding site predictions made in this article are testable either by mutation studies or invasion assays. These results can be further extended in suggesting possible peptide antagonists and drug design for therapeutic strategies.

HierVLS hierarchical docking protocol for virtual ligand screening of large-molecule databases.

To provide practical means for rapidly scanning the extensive experimental combinatorial chemistry libraries now available for high-throughput screening (HTS), it is essential to establish computational virtual ligand screening (VLS) techniques to rapidly identify out of a large library all active compounds against a particular protein target. Toward this goal we developed HierVLS, a fast hierarchical docking approach that starts with a coarse grain conformational search over a large number of configurations filtered with a fast but crude energy function, followed by a succession of finer grain levels, using successively more accurate but more expensive descriptions of the ligand-protein-solvent interactions to filter successively fewer cases. The final step of this procedure optimizes one configuration of the ligand in the protein site using our most accurate energy expression and description of the solvent, which would be impractical for all conformations and sites sampled in the coarse level. HierVLS is based on the HierDock approach, but rather than allowing an hour or more to determine the best binding site and energy for each ligands (as in HierDock), we have adapted our procedure so that it can lead to reliable results while using only 4 min (866 MHz Pentium III processor) per ligand. To validate the accuracy for HierVLS to predict the experimentally observed binding conformation, we considered 37 cocrystal structures comprising 11 target proteins. We find that HierVLS identifies the correct binding mode for all 37 cocrystals. In addition, the calculated binding energies correlate well with available experimental binding constants. To validate how well HierVLS can identify the correct ligand in an extensive library of decoys, we considered a library of over 10 000 molecules. HierVLS identifies 26 out of the 37 cases in the top 2% ranked by binding affinity among the 10 037 molecules. The failures result from either metal-containing sites on the protein or water-mediated ligand-protein interactions, which we anticipate can be solved within the constraints of practical VLS. We then applied HierVLS to screen a 55000-compound virtual library against the target protein-tyrosine phosphatase 1B (ptp1b). The top 250 compounds by binding affinity included all six ptp1b cocrystal ligands added to the library plus three other experimentally confirmed binders. The best (top 1) binder is an experimentally confirmed positive. We conclude that HierVLS is useful for selecting leads for a particular target out of large combinatorial databases.

Identifying potential selective fluorescent probes for cancer-associated protein carbonic anhydrase IX using a computational approach.

Carbonic anhydrase IX (CAIX) is a biomarker for tumor hypoxia. Fluorescent inhibitors of CAIX have been used to study hypoxic tumor cell lines. However, these inhibitor-based fluorescent probes may have a therapeutic effect that is not appropriate for monitoring treatment efficacy. In the search for novel fluorescent probes that are not based on known inhibitors, a database of 20,860 fluorescent compounds was virtually screened against CAIX using hierarchical virtual ligand screening (HierVLS). The screening database contained 14,862 compounds tagged with the ATTO680 fluorophore plus an additional 5998 intrinsically fluorescent compounds. Overall ranking of compounds to identify hit molecular probe candidates utilized a principal component analysis (PCA) approach. Four potential binding sites, including the catalytic site, were identified within the structure of the protein and targeted for virtual screening. Available sequence information for 23 carbonic anhydrase isoforms was used to prioritize the four sites based on the estimated "uniqueness" of each site in CAIX relative to the other isoforms. A database of 32 known inhibitors and 478 decoy compounds was used to validate the methodology. A receiver-operating characteristic (ROC) analysis using the first principal component (PC1) as predictive score for the validation database yielded an area under the curve (AUC) of 0.92. AUC is interpreted as the probability that a binder will have a better score than a non-binder. The use of first component analysis of binding energies for multiple sites is a novel approach for hit selection. The very high prediction power for this approach increases confidence in the outcome from the fluorescent library screening. Ten of the top scoring candidates for isoform-selective putative binding sites are suggested for future testing as fluorescent molecular probe candidates.

Paclitaxel is an inhibitor and its boron dipyrromethene derivative is a fluorescent recognition agent for botulinum neurotoxin subtype A.

We have successfully identified one new inhibitor and one new fluorescent recognition agent for the botulinum neurotoxin subtype A (BoNT/A) using the virtual screening protocol "protein scanning with virtual ligand screening" (PSVLS). Hit selection used an in-house developed holistic binding scoring method. Selected hits were tested experimentally for inhibitory activity using fluorescence resonance energy transfer (FRET) assays against the light chain (catalytic domain) of BoNT/A. Ligand binding was determined against the light and heavy chain BoNT/A complex through either radiolabeled ligand binding assays (nonfluorescent ligands) or fluorescence intensity assays (fluorescent ligands). These experimental assays have confirmed one compound (paclitaxel) to inhibit BoNT/A's proteolytic activity experimentally with an IC50 of 5.2 µM. A fluorescent derivative was also confirmed to bind to the toxin and therefore is a suitable candidate for the rational design of new detection agents and for the development of fluorescence-based multiprobe detection assays.

Genetics of sweet taste preferences.

Sweet taste is a powerful factor influencing food acceptance. There is considerable variation in sweet taste perception and preferences within and among species. Although learning and homeostatic mechanisms contribute to this variation in sweet taste, much of it is genetically determined. Recent studies have shown that variation in the T1R genes contributes to within- and between-species differences in sweet taste. In addition, our ongoing studies using the mouse model demonstrate that a significant portion of variation in sweetener preferences depends on genes that are not involved in peripheral taste processing. These genes are likely involved in central mechanisms of sweet taste processing, reward and/or motivation. Genetic variation in sweet taste not only influences food choice and intake, but is also associated with proclivity to drink alcohol. Both peripheral and central mechanisms of sweet taste underlie correlation between sweet-liking and alcohol consumption in animal models and humans. All these data illustrate complex genetics of sweet taste preferences and its impact on human nutrition and health. Identification of genes responsible for within- and between-species variation in sweet taste can provide tools to better control food acceptance in humans and other animals.

A cluster-aware graphical user interface for a virtual ligand screening tool.

In this paper, we describe the design and implementation of a Graphical User Interface (GUI) for Cassandra, a computer application for Virtual Ligand Screening (VLS). The GUI was designed using Trolltech QT4 and Perl, and serves the purpose of making the execution of Cassandra more user-friendly. Through this GUI, users can manage multiple concurrent, interactive data paths both on a single machine and on a High Performance Computing Cluster (HPCC). The GUI successfully decreased the complexity and steep learning curve of using Cassandra, and the average time to execute a VLS project. As result, we reduced the time necessary to train new users, increased the number of users, increased the overall efficiency of Cassandra, and optimized data handling and analysis.

Modeling the human PTC bitter-taste receptor interactions with bitter tastants.

We employed the first principles computational method MembStruk and homology modeling techniques to predict the 3D structures of the human phenylthiocarbamide (PTC) taste receptor. This protein is a seven-transmembrane-domain G protein-coupled receptor that exists in two main forms worldwide, designated taster and nontaster, which differ from each other at three amino-acid positions. 3D models were generated with and without structural similarity comparison to bovine rhodopsin. We used computational tools (HierDock and ScanBindSite) to generate models of the receptor bound to PTC ligand to estimate binding sites and binding energies. In these models, PTC binds at a site distant from the variant amino acids, and PTC binding energy was equivalent for both the taster and nontaster forms of the protein. These models suggest that the inability of humans to taste PTC is due to a failure of G protein activation rather than decreased binding affinity of the receptor for PTC. Amino-acid substitutions in the sixth and seventh transmembrane domains of the nontaster form of the protein may produce increased steric hindrance between these two alpha-helices and reduce the motion of the sixth helix required for G protein activation.

Predictions of CCR1 chemokine receptor structure and BX 471 antagonist binding followed by experimental validation.

A major challenge in the application of structure-based drug design methods to proteins belonging to the superfamily of G protein-coupled receptors (GPCRs) is the paucity of structural information (1). The 19 chemokine receptors, belonging to the Class A family of GPCRs, are important drug targets not only for autoimmune diseases like multiple sclerosis but also for the blockade of human immunodeficiency virus type 1 entry (2). Using the MembStruk computational method (3), we predicted the three-dimensional structure of the human CCR1 receptor. In addition, we predicted the binding site of the small molecule CCR1 antagonist BX 471, which is currently in Phase II clinical trials (4). Based on the predicted antagonist binding site we designed 17 point mutants of CCR1 to validate the predictions. Subsequent competitive ligand binding and chemotaxis experiments with these mutants gave an excellent correlation to these predictions. In particular, we find that Tyr-113 and Tyr-114 on transmembrane domain 3 and Ile-259 on transmembrane 6 contribute significantly to the binding of BX 471. Finally, we used the predicted and validated structure of CCR1 in a virtual screening validation of the Maybridge data base, seeded with selective CCR1 antagonists. The screen identified 63% of CCR1 antagonists in the top 5% of the hits. Our results indicate that rational drug design for GPCR targets is a feasible approach.