Distinct binding of cetirizine enantiomers to human serum albumin and the human histamine receptor H1.

Cetirizine, a major metabolite of hydroxyzine, became a marketed second-generation H1 antihistamine that is orally active and has a rapid onset of action, long duration of effects and a very good safety record at recommended doses. The approved drug is a racemic mixture of (S)-cetirizine and (R)-cetirizine, the latter being the levorotary enantiomer that also exists in the market as a third-generation, non-sedating and highly selective antihistamine. Both enantiomers bind tightly to the human histamine H1 receptor (hH1R) and behave as inverse agonists but the affinity and residence time of (R)-cetirizine are greater than those of (S)-cetirizine. In blood plasma, cetirizine exists in the zwitterionic form and more than 90% of the circulating drug is bound to human serum albumin (HSA), which acts as an inactive reservoir. Independent X-ray crystallographic work has solved the structure of the hH1R:doxepin complex and has identified two drug-binding sites for cetirizine on equine serum albumin (ESA). Given this background, we decided to model a membrane-embedded hH1R in complex with either (R)- or (S)-cetirizine and also the complexes of both ESA and HSA with these two enantiomeric drugs to analyze possible differences in binding modes between enantiomers and also among targets. The ensuing molecular dynamics simulations in explicit solvent and additional computational chemistry calculations provided structural and energetic information about all of these complexes that is normally beyond current experimental possibilities. Overall, we found very good agreement between our binding energy estimates and extant biochemical and pharmacological evidence. A much higher degree of solvent exposure in the cetirizine-binding site(s) of HSA and ESA relative to the more occluded orthosteric binding site in hH1R is translated into larger positional fluctuations and considerably lower affinities for these two nonspecific targets. Whereas it is demonstrated that the two known pockets in ESA provide enough stability for cetirizine binding, only one such site does so in HSA due to a number of amino acid replacements. At the histamine-binding site in hH1R, the distinct interactions established between the phenyl and chlorophenyl moieties of the two enantiomers with the amino acids lining up the pocket and between their free carboxylates and Lys179 in the second extracellular loop account for the improved pharmacological profile of (R)-cetirizine.

Engineering Erg10 Thiolase from Saccharomyces cerevisiae as a Synthetic Toolkit for the Production of Branched-Chain Alcohols.

Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium's 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.

A novel p38 MAPK docking-groove-targeted compound is a potent inhibitor of inflammatory hyperalgesia.

The MAPK (mitogen-activated protein kinase) p38 is an important mediator of inflammation and of inflammatory and neuropathic pain. We have described recently that docking-groove-dependent interactions are important for p38 MAPK-mediated signal transduction. Thus virtual screening was performed to identify putative docking-groove-targeted p38 MAPK inhibitors. Several compounds of the benzo-oxadiazol family were identified with low micromolar inhibitory activity both in a p38 MAPK activity assay, and in THP-1 human monocytes acting as inhibitors of LPS (lipopolysaccharide)-induced TNFα (tumour necrosis factor α) secretion. Positions 2 and 5 in the phenyl ring are essential for the described inhibitory activity with a chloride in position 5 and a methyl group in position 2 yielding the best results, giving an IC50 value of 1.8 µM (FGA-19 compound). Notably, FGA-19 exerted a potent and long-lasting analgesic effect in vivo when tested in a mouse model of inflammatory hyperalgesia. A single intrathecal injection of FGA-19 completely resolved hyperalgesia, being 10-fold as potent and displaying longer lasting effects than the established p38 MAPK inhibitor SB239063. FGA-19 also reversed persistent pain in a model of post-inflammatory hyperalgesia in LysM (lysozyme M)-GRK2 (G-protein-coupled-receptor kinase)(+/-) mice. These potent in vivo effects suggested p38 MAPK docking-site-targeted inhibitors as a potential novel strategy for the treatment of inflammatory pain.

Novel HLA-B27-restricted epitopes from Chlamydia trachomatis generated upon endogenous processing of bacterial proteins suggest a role of molecular mimicry in reactive arthritis.

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na(+)-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27(+) cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330-338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211-223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211-223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.

ALFA: automatic ligand flexibility assignment.

ALFA is a fast computational tool for the conformational analysis of small molecules that uses a custom-made iterative algorithm to provide a set of representative conformers in an attempt to reproduce the diversity of states in which small molecules can exist, either isolated in solution or bound to a target. The results shown in this work prove that ALFA is fast enough to be integrated into massive high-throughput virtual screening protocols with the aim of incorporating ligand flexibility and also that ALFA reproduces crystallographic X-ray structures of bound ligands with great accuracy. Furthermore, the application includes a graphical user interface that allows its use through the popular molecular graphics program PyMOL to make it accessible to nonexpert users. ALFA is distributed free of charge upon request from the authors.

A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

Interactions of bacterial cell division protein FtsZ with C8-substituted guanine nucleotide inhibitors. A combined NMR, biochemical and molecular modeling perspective.

FtsZ is the key protein of bacterial cell-division and target for new antibiotics. Selective inhibition of FtsZ polymerization without impairing the assembly of the eukaryotic homologue tubulin was demonstrated with C8-substituted guanine nucleotides. By combining NMR techniques with biochemical and molecular modeling procedures, we have investigated the molecular recognition of C8-substituted-nucleotides by FtsZ from Methanococcus jannaschii (Mj-FtsZ) and Bacillus subtilis (Bs-FtsZ). STD epitope mapping and trNOESY bioactive conformation analysis of each nucleotide were employed to deduce differences in their recognition mode by each FtsZ species. GMP binds in the same anti conformation as GTP, whereas 8-pyrrolidino-GMP binds in the syn conformation. However, the anti conformation of 8-morpholino-GMP is selected by Bs-FtsZ, while Mj-FtsZ binds both anti- and syn-geometries. The inhibitory potencies of the C8-modified-nucleotides on the assembly of Bs-FtsZ, but not of Mj-FtsZ, correlate with their binding affinities. Thus, MorphGTP behaves as a nonhydrolyzable analog whose binding induces formation of Mj-FtsZ curved filaments, resembling polymers formed by the inactive forms of this protein. NMR data, combined with molecular modeling protocols, permit explanation of the mechanism of FtsZ assembly impairment by C8-substituted GTP analogs. The presence of the C8-substituent induces electrostatic remodeling and small structural displacements at the association interface between FtsZ monomers to form filaments, leading to complete assembly inhibition or to formation of abnormal FtsZ polymers. The inhibition of bacterial Bs-FtsZ assembly may be simply explained by steric clashes of the C8-GTP-analogs with the incoming FtsZ monomer. This information may facilitate the design of antibacterial FtsZ inhibitors replacing GTP.

A structure-based design of new C2- and C13-substituted taxanes: tubulin binding affinities and extended quantitative structure-activity relationships using comparative binding energy (COMBINE) analysis.

Ten novel taxanes bearing modifications at the C2 and C13 positions of the baccatin core have been synthesized and their binding affinities for mammalian tubulin have been experimentally measured. The design strategy was guided by (i) calculation of interaction energy maps with carbon, nitrogen and oxygen probes within the taxane-binding site of ß-tubulin, and (ii) the prospective use of a structure-based QSAR (COMBINE) model derived from an earlier series comprising 47 congeneric taxanes. The tubulin-binding affinity displayed by one of the new compounds (CTX63) proved to be higher than that of docetaxel, and an updated COMBINE model provided a good correlation between the experimental binding free energies and a set of weighted residue-based ligand-receptor interaction energies for 54 out of the 57 compounds studied. The remaining three outliers from the original training series have in common a large unfavourable entropic contribution to the binding free energy that we attribute to taxane preorganization in aqueous solution in a conformation different from that compatible with tubulin binding. Support for this proposal was obtained from solution NMR experiments and molecular dynamics simulations in explicit water. Our results shed additional light on the determinants of tubulin-binding affinity for this important class of antitumour agents and pave the way for further rational structural modifications.

An aspartate residue in the external vestibule of GLYT2 (glycine transporter 2) controls cation access and transport coupling.

Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+-Cl--glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes.

Rationale for the opposite stereochemistry of the major monoadducts and interstrand crosslinks formed by mitomycin C and its decarbamoylated analogue at CpG steps in DNA and the effect of cytosine modification on reactivity.

Mitomycin C (MMC) is a potent antitumour agent that forms a covalent bond with the 2-amino group of selected guanines in the minor groove of double-stranded DNA following intracellular reduction of its quinone ring and opening of its aziridine moiety. At some 5'-CG-3' (CpG) steps the resulting monofunctional adduct can evolve towards a more deleterious bifunctional lesion, which is known as an interstrand crosslink (ICL). MMC reactivity is enhanced when the cytosine bases are methylated (5 MC) and decreased when they are replaced with 5-F-cytosine (5FC) whereas the stereochemical preference of alkylation changes upon decarbamoylation. We have studied three duplex oligonucleotides of general formula d(CGATAAXGCTAACG) in which X stands for C, 5MC or 5FC. Using a combination of molecular dynamics simulations in aqueous solution, quantum mechanics and continuum electrostatics, we have been able to (i) obtain a large series of snapshots that facilitate an understanding in atomic detail of the distinct stereochemistry of monoadduct and ICL formation by MMC and its decarbamoylated analogue, (ii) provide an explanation for the altered reactivity of MMC towards DNA molecules containing 5MC or 5FC, and (iii) show the distinct accommodation in the DNA minor groove of the different covalent modifications, particularly the most cytotoxic C1α and C1ß ICLs.

CRDOCK: an ultrafast multipurpose protein-ligand docking tool.

An ultrafast docking and virtual screening program, CRDOCK, is presented that contains (1) a search engine that can use a variety of sampling methods and an initial energy evaluation function, (2) several energy minimization algorithms for fine tuning the binding poses, and (3) different scoring functions. This modularity ensures the easy configuration of custom-made protocols that can be optimized depending on the problem in hand. CRDOCK employs a precomputed library of ligand conformations that are initially generated from one-dimensional SMILES strings. Testing CRDOCK on two widely used benchmarks, the ASTEX diverse set and the Directory of Useful Decoys, yielded a success rate of ~75% in pose prediction and an average AUC of 0.66. A typical ligand can be docked, on average, in just ~13 s. Extension to a representative group of pharmacologically relevant G protein-coupled receptors that have been recently cocrystallized with some selective ligands allowed us to demonstrate the utility of this tool and also highlight some current limitations. CRDOCK is now included within VSDMIP, our integrated platform for drug discovery.

A reverse combination of structure-based and ligand-based strategies for virtual screening.

A new approach is presented that combines structure- and ligand-based virtual screening in a reverse way. Opposite to the majority of the methods, a docking protocol is first employed to prioritize small ligands ("fragments") that are subsequently used as queries to search for similar larger ligands in a database. For a given chemical library, a three-step strategy is followed consisting of (1) contraction into a representative, non-redundant, set of fragments, (2) selection of the three best-scoring fragments docking into a given macromolecular target site, and (3) expansion of the fragments' structures back into ligands by using them as queries to search the library by means of fingerprint descriptions and similarity criteria. We tested the performance of this approach on a collection of fragments and ligands found in the ZINC database and the directory of useful decoys, and compared the results with those obtained using a standard docking protocol. The new method provided better overall results and was several times faster. We also studied the chemical diversity that both methods cover using an in-house compound library and concluded that the novel approach performs similarly but at a much smaller computational cost.

AtlasCBS: a web server to map and explore chemico-biological space.

New approaches are needed that can help decrease the unsustainable failure in small-molecule drug discovery. Ligand Efficiency Indices (LEI) are making a great impact on early-stage compound selection and prioritization. Given a target-ligand database with chemical structures and associated biological affinities/activities for a target, the AtlasCBS server generates two-dimensional, dynamical representations of its contents in terms of LEI. These variables allow an effective decoupling of the chemical (angular) and biological (radial) components. BindingDB, PDBBind and ChEMBL databases are currently implemented. Proprietary datasets can also be uploaded and compared. The utility of this atlas-like representation in the future of drug design is highlighted with some examples. The web server can be accessed at http://ub.cbm.uam.es/atlascbs and https://www.ebi.ac.uk/chembl/atlascbs.

Molecular basis of the differential interaction with lithium of glycine transporters GLYT1 and GLYT2.

Glycine synaptic levels are controlled by glycine transporters (GLYTs) catalyzing Na(+)/Cl(-)/glycine cotransport. GLYT1 displays a 2:1 :1 stoichiometry and is the main regulator of extracellular glycine concentrations. The neuronal GLYT2, with higher sodium coupling (3:1 :1), supplies glycine to the pre-synaptic terminal to refill synaptic vesicles. In this work, using structural homology modelling and molecular dynamics simulations of GLYTs, we predict the conservation of the two sodium sites present in the template (leucine transporter from Aquifex aeolicus), and confirm its use by mutagenesis and functional analysis. GLYTs Na1 and Na2 sites show differential cation selectivity, as inferred from the action of lithium, a non-transport-supporting ion, on Na(+)-site mutants. GLYTs lithium responses were unchanged in Na1-site mutants, but abolished or inverted in mutants of Na2 site, which binds lithium in the presence of low sodium concentrations and therefore, controls lithium responses. Here, we report, for the first time, that lithium exerts opposite actions on GLYTs isoforms. Glycine transport by GLYT1 is inhibited by lithium whereas GLYT2 transport is stimulated, and this effect is more evident at increased glycine concentrations. In contrast to GLYT1, high and low affinity lithium-binding processes were detected in GLYT2.

Synthesis, biological assessment and molecular modeling of 14-aryl-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]quinolin-13-amines.

The synthesis and pharmacological evaluation of racemic 14-aryl-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]quinolin-13-amines (19-28), prepared by Friedländer reaction of 3-amino-1-aryl-1H-benzo[f]chromene-2-carbonitriles (10-18) with suitable cycloalkanones is described. These molecules are potent, in the nanomolar range [IC(50) (EeAChE)=7-101 nM], and selective inhibitors of acetylcholinesterase (AChE). The most potent inhibitor, 4-(13-amino-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]quinolin-14-yl)phenol (20) [IC(50) (EeAChE)=7±2 nM] is four-fold more active than tacrine. Kinetic studies on compound 20 showed that this is a mixed-type inhibitor of EeAChE with a K(i) of 5.00 nM. However, racemic 20 was unable to displace propidium iodide, suggesting that the inhibitor does not strongly bind to the peripheral anionic site (PAS) of AChE. Docking, molecular dynamics stimulations, and MM-GBSA calculations agree well with this behavior.

VSDMIP 1.5: an automated structure- and ligand-based virtual screening platform with a PyMOL graphical user interface.

A graphical user interface (GUI) for our previously published virtual screening (VS) and data management platform VSDMIP (Gil-Redondo et al. J Comput Aided Mol Design, 23:171-184, 2009) that has been developed as a plugin for the popular molecular visualization program PyMOL is presented. In addition, a ligand-based VS module (LBVS) has been implemented that complements the already existing structure-based VS (SBVS) module and can be used in those cases where the receptor's 3D structure is not known or for pre-filtering purposes. This updated version of VSDMIP is placed in the context of similar available software and its LBVS and SBVS capabilities are tested here on a reduced set of the Directory of Useful Decoys database. Comparison of results from both approaches confirms the trend found in previous studies that LBVS outperforms SBVS. We also show that by combining LBVS and SBVS, and using a cluster of ~100 modern processors, it is possible to perform complete VS studies of several million molecules in less than a month. As the main processes in VSDMIP are 100% scalable, more powerful processors and larger clusters would notably decrease this time span. The plugin is distributed under an academic license upon request from the authors.

The amino terminal domain from Mrt4 protein can functionally replace the RNA binding domain of the ribosomal P0 protein.

In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein.

Binding of non-canonical peptidoglycan controls Vibrio cholerae broad spectrum racemase activity.

Broad-spectrum amino acid racemases (Bsrs) enable bacteria to generate non-canonical D-amino acids (NCDAAs), whose roles and impact on microbial physiology, including modulation of cell wall structure and dissolution of biofilms, are just beginning to be appreciated. Here we used a diverse array of structural, biochemical and molecular simulation studies to define and characterize how BsrV is post-translationally regulated. We discovered that contrary to Vibrio cholerae alanine racemase AlrV highly compacted active site, BsrV's is broader and can be occupied by cell wall stem peptides. We found that peptidoglycan peptides modified with NCDAAs are better stabilized by BsrV's catalytic cavity and show better inhibitory capacity than canonical muropeptides. Notably, BsrV binding and inhibition can be recapitulated by undigested peptidoglycan sacculi as it exists in the cell. Docking simulations of BsrV binding the peptidoglycan polymer generate a model where the peptide stems are perfectly accommodated and stabilized within each of the dimers active sites. Taking these biochemical and structural data together, we propose that inhibition of BsrV by peptidoglycan peptides underlies a negative regulatory mechanism to avoid excessive NCDAA production. Our results collectively open the door to use "à la carte" synthetic peptides as a tool to modulate DAAs production of Bsr enzymes.

Insights into nucleotide recognition by cell division protein FtsZ from a mant-GTP competition assay and molecular dynamics.

Essential cell division protein FtsZ forms the bacterial cytokinetic ring and is a target for new antibiotics. FtsZ monomers bind GTP and assemble into filaments. Hydrolysis to GDP at the association interface between monomers leads to filament disassembly. We have developed a homogeneous competition assay, employing the fluorescence anisotropy change of mant-GTP upon binding to nucleotide-free FtsZ, which detects compounds binding to the nucleotide site in FtsZ monomers and measures their affinities within the millimolar to 10 nM range. We have employed this method to determine the apparent contributions of the guanine, ribose, and the α-, ß-, and γ-phosphates to the free energy change of nucleotide binding. Similar relative contributions have also been estimated through molecular dynamics and binding free energy calculations, employing the crystal structures of FtsZ-nucleotide complexes. We find an energetically dominant contribution of the ß-phosphate, comparable to the whole guanosine moiety. GTP and GDP bind with similar observed affinity to FtsZ monomers. Loss of the regulatory γ-phosphate results in a predicted accommodation of GDP which has not been observed in the crystal structures. The binding affinities of a series of C8-substituted GTP analogues, known to inhibit FtsZ but not eukaryotic tubulin assembly, correlate with their inhibitory capacity on FtsZ polymerization. Our methods permit testing of FtsZ inhibitors targeting its nucleotide site, as well as compounds from virtual screening of large synthetic libraries. Our results give insight into the FtsZ-nucleotide interactions, which could be useful in the rational design of new inhibitors, especially GTP phosphate mimetics.

gCOMBINE: A graphical user interface to perform structure-based comparative binding energy (COMBINE) analysis on a set of ligand-receptor complexes.

We present gCOMBINE, a Java-written graphical user interface (GUI) for performing comparative binding energy (COMBINE) analysis (Ortiz et al. J Med Chem 1995; 38:2681-2691) on a set of ligand-receptor complexeswith the aim of deriving highly informative quantitative structure-activity relationships. The essence of the method is to decompose the ligand-receptor interaction energies into a series of terms, explore the origins of the variance within the set using Principal Component Analysis, and then assign weights to selected ligandresidue interactions using partial least squares analysis to correlate with the experimental activities or binding affinities. The GUI allows plenty of interactivity and provides multiple plots representing the energy descriptors entering the analysis, scores, loadings, experimental versus predicted regression lines, and the evolution of parameterssuch as r(2) (correlation coefficient), q(2) (cross-validated r(2)), and prediction errors as the number of extracted latent variables increases. Other representative features include the implementation of a sigmoidal dielectric function for electrostatic energy calculations, alternative cross-validation procedures (leave-N-out and random groups), drawing of confidence ellipses, and the possibility to carry out several additional tasks such as optional truncation of positive interaction energy values and generation of ready-to-use PDB files containing information related to the importance for activity of individual protein residues. This information can be displayed and color-coded using a standard molecular graphics program such as PyMOL. It is expected that this user-friendly tool will expand the applicability of the COMBINE analysis method and encourage more groups to use it in their drug design research programs.