E. Ben-zeev - Academia.edu (original) (raw)

Papers by E. Ben-zeev

Journal of Biological Chemistry, 2016

The Na,K-ATPase ␣ 2 subunit plays a key role in cardiac muscle contraction by regulating intracel... more The Na,K-ATPase ␣ 2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca 2؉ , whereas ␣ 1 has a more conventional role of maintaining ion homeostasis. The ␤ subunit differentially regulates maturation, trafficking, and activity of ␣-␤ heterodimers. It is not known whether the distinct role of ␣ 2 in the heart is related to selective assembly with a particular one of the three ␤ isoforms. We show here by immunofluorescence and co-immunoprecipitation that ␣ 2 is preferentially expressed with ␤ 2 in T-tubules of cardiac myocytes, forming ␣ 2 ␤ 2 heterodimers. We have expressed human ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 2 ␤ 2 , and ␣ 2 ␤ 3 in Pichia pastoris, purified the complexes, and compared their functional properties. ␣ 2 ␤ 2 and ␣ 2 ␤ 3 differ significantly from both ␣ 2 ␤ 1 and ␣ 1 ␤ 1 in having a higher K 0.5 K ؉ and lower K 0.5 Na ؉ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K ؉ and shift of the E 1 P-E 2 P conformational equilibrium toward E 1 P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of ␣ 2 ␤ 2 and ␣ 2 ␤ 3 over ␣ 1 ␤ 1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K ؉ affinity of ␣ 2 ␤ 2 could allow an acute response to raised ambient K ؉ concentrations in physiological conditions and explain the importance of ␣ 2 ␤ 2 for cardiac muscle contractility. The high sensitivity of ␣ 2 ␤ 2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of ␣ 2 ␤ 2selective digoxin derivatives for reducing cardiotoxicity.

Proteins: Structure, Function, and Genetics, 2003

We submitted predictions for all seven targets in the CAPRI experiment. For four targets, our sub... more We submitted predictions for all seven targets in the CAPRI experiment. For four targets, our submitted models included acceptable, medium accuracy predictions of the structures of the complexes, and for a fifth target we identified the location of the binding site of one of the molecules. We used a weighted-geometric docking algorithm in which contacts involving specified parts of the surfaces of either one or both molecules were up-weighted or down-weighted. The weights were based on available structural and biochemical data or on sequence analyses. The weighted-geometric docking proved very useful for five targets, improving the complementarity scores and the ranks of the nearly correct solutions, as well as their statistical significance. In addition, the weighted-geometric docking promoted formation of clusters of similar solutions, which include more accurate predictions.

Journal of Biomolecular Structure and Dynamics, 2003

Colicin E3 kills Escherichia coli cells by ribonucleolytic cleavage in the 16S rRNA. The cleavage... more Colicin E3 kills Escherichia coli cells by ribonucleolytic cleavage in the 16S rRNA. The cleavage occurs at the ribosomal decoding A-site between nucleotides A1493 and G1494. The breaking of this single phosphodiester bond results in a complete termination of protein biosynthesis leading to cell death. A model structure of the complex of the ribosomal subunit 30S and colicin E3 was constructed by means of a new weighted-geometric docking algorithm, in which interactions involving specified parts of the molecular surface can be up-weighted, allowing incorporation of experimental data in the docking search. Our model, together with available experimental data, predicts the role of the catalytic residues of colicin E3. In addition, it suggests that bound acidic immunity protein inhibits the enzymatic activity of colicin E3 by electrostatic repulsion of the negatively charged substrate.

Cancer Research, 2012

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pyruvate kinase (PK... more Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pyruvate kinase (PK) M2 which catalyzes the last step in glycolysis, is the alternative spliced isoform expressed in cancer cells, and a key player in exerting the Warburg effect. One of the mechanisms by which PKM2 modulate cancer cell metabolism is by switching between the low activity monomer and the high activity tetramer forms. This process is controlled by the varying concentration of an upstream glycolytic intermediate, FBP. These changes enable the cancer cell to manage its usage of glucose carbon backbones, whether for ATP production or for biomass generation, according to its changing demands. Further, it has been recently shown that the elevated levels of ROS in cancer cells contribute to the decreased activity of PKM2 to support NADPH production to increase cellular anti-oxidation capacity to sustain proliferation. Our goal is to disrupt the metabolic adaptation of cancer cells with small molecule PKM2 modulators. We hypothesized that an activator will redirect the consumption of nutrients, especially of glucose, away from biomass production and ultimately send the cells to die. Using our proprietary structure-based technology, we identified several series of novel allosteric PKM2 activators. Chemical optimization resulted in potent compounds with AC50 as low as 10nM, which were selective against the other PK isoforms. These compounds were proven to stabilize the active tetramer form of PKM2 in cancer cells. Bioenergetic experiments in several cell lines demonstrated that not only do these agents reduce lactate production; they also reduce the oxygen consumption rate. Analysis of cell cycle showed that treatment with PKM2 activators causes the cells to arrest at the G1 phase. In outcome-based assays, these compounds significantly reduced the proliferation rate of various cancer cell lines, and this effect was sensitive to media conditions, such as glucose levels. Taken together, our data supports the hypothesis that activation of PKM2 effectively deprives the cancer cell of building blocks and reduces the detoxification capacity that are required to support growth and proliferation. An in vivo colorectal cancer HT29 cell line mouse xenograft model with a modestly active compound (IC50=0.9uM) demonstrated tumor growth inhibition greater than 50% (100 mg/kg Q2D and 200 and 400 mg/kg IP QD). The compound was very safe in mouse, even at the highest exposure levels (200 and 400 mg/kg IP QD), indicating that these efficacious doses are significantly lower than the MTD. Additional xenograft models are ongoing. Taken together, there is strong support for the effect of potent PKM2 small molecule activators on the cellular metabolism of cancer cells, demonstrating statistically significant anti-cancer effect in an animal model of colorectal cancer. The favorable DMPK profile of these compounds further supports their development as anti-proliferative agents, both as a single agent and in combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3219. doi:1538-7445.AM2012-3219

Current Topics in Medicinal Chemistry, 2010

The application of structure-based in silico methods to drug discovery is still considered a majo... more The application of structure-based in silico methods to drug discovery is still considered a major challenge, especially when the x-ray structure of the target protein is unknown. Such is the case with human G protein-coupled receptors (GPCRs), one of the most important families of drug targets, where in the absence of x-ray structures, one has to rely on in silico 3D models. We report repeated success in using ab initio in silico GPCR models, generated by the PREDICT method, for blind in silico screening when applied to a set of five different GPCR drug targets. More than 100,000 compounds were typically screened in silico for each target, leading to a selection of <100 ''virtual hit'' compounds to be tested in the lab. In vitro binding assays of the selected compounds confirm high hit rates, of 12-21% (full dose-response curves, K i < 5 M). In most cases, the best hit was a novel compound (New Chemical Entity) in the 1-to 100-nM range, with very promising pharmacological properties, as measured by a variety of in vitro and in vivo assays. These assays validated the quality of the hits as lead compounds for drug discovery. The results demonstrate the usefulness and robustness of ab initio in silico 3D models and of in silico screening for GPCR drug discovery. modeling ͉ in silico screening ͉ structure-based G protein-coupled receptors (GPCRs) are membraneembedded proteins, responsible for communication between the cell and its environment (1). As a consequence, many major diseases, such as hypertension, cardiac dysfunction, depression, anxiety, obesity, inflammation, and pain, involve malfunction of these receptors (2), making them among the most important drug targets for pharmacological intervention (3-5). Thus, whereas GPCRs are only a small subset of the human genome, they are the targets for Ϸ50% of all recently launched drugs (6). As targets of paramount importance, it is expected that drug discovery for GPCRs would benefit from the introduction of computational methodologies (7), especially as these methods can be used in conjunction with such experimental methods as high-throughput screening (8, 9), NMR, and crystallography (10). Unfortunately, GPCRs, like other membrane-embedded proteins, have characteristics that make their 3D structure extremely difficult to determine experimentally. To date, the only GPCR for which a 3D structure was determined by x-ray crystallography is bovine rhodopsin (11), which is unique among GPCRs in that its ligand, retinal, is covalently bound and that it responds to light rather than to ligand binding. Hence, in the case of GPCRs, the limited availability of structural data has forced the computational design of ligands to heavily rely on ligand-based techniques. Indeed, for many GPCRs, the natural ligand can provide a good starting point, leading to useful pharmacophore models that can be used for identifying lead structures with novel scaffolds (6). These methods have been successfully applied for the discovery of peptide agonists to the somatostatin receptor (12) and for the discovery of nonpeptidic antagonists to the urotensin II receptor (13). Nonetheless, structure-based drug discovery remains highly desirable for GPCRs. It is known that all GPCRs structurally consist of seven transmembrane (TM) helices joined together by three extracellular and three intracellular loops. Of particular interest to

Journal of Clinical Oncology

Proteins: Structure, Function, and Bioinformatics, 2005

The diverse selection of targets in the CAPRI experiments provides grounds for determining the li... more The diverse selection of targets in the CAPRI experiments provides grounds for determining the limits of our rigid-body docking program MolFit, and for extending it. We find that the sensitivity of MolFit is high, enabling it to produce reasonably accurate docking solutions when the structures undergo moderate local conformation changes upon complex formation or when the docked molecules are modeled. Yet the ranks of these solutions are sometimes too low to meet the requirements of CAPRI assessment. This indicates that the selectivity of MolFit, which was optimized for docking of unbound X-ray structures, and which relies on the availability of external data from biochemical and bioinformatic sources, needs readjustment in order to meet the challenges presented by NMR or modeled structures. A different challenge is presented by large global conformation changes such as movements of domains. We show that such changes can be accommodated within the rigid-body approximation by employing rigid multibody multistage docking procedures. We also address the difficulty of ranking results from 2-body and multibody docking scans in cases in which there are no external data favoring one option over the other.

Journal of Immunological Methods, 1999

Phage display of antibody fragments has proved to be a powerful tool for the isolation and in vit... more Phage display of antibody fragments has proved to be a powerful tool for the isolation and in vitro evolution of these biologically important molecules. However, the general usefulness of this technology is still limited by some technical difficulties. One of the most debilitating obstacles to the widespread application of the technology is the accumulation of ''insert loss'' clones in the libraries; phagemid clones from which the DNA encoding part or all of the cloned antibody fragment had been deleted. Another difficulty arises when phage technology is applied for cloning hybridoma-derived antibody genes, where myeloma derived light chains, irrelevant to the hybridoma's antibody specificity may be fortuitously cloned. Here, we report the construction of a novel phage-display system designed to address these problems. In our system Ž . Ž . a single-chain Fv scFv is expressed as an in-frame fusion protein with a cellulose-binding domain CBD derived from the Clostridium thermocellum cellulosome. The CBD domain serves as an affinity tag allowing rapid phage capture and concentration from crude culture supernatants, and immunological detection of both displaying phage and soluble scFv produced thereof. We demonstrate the utility of our system in solving the technical difficulties described above, and in speeding up the process of scFv isolation from combinatorial antibody repertoires. q 1999 Elsevier Science B.V. All rights reserved.

Journal of Hepatology, 2004

To investigate the correlation between immune complex affinity to HCV and selection of a specific... more To investigate the correlation between immune complex affinity to HCV and selection of a specific monoclonal component. Material and Methods: HCV-RNA was quantified by bDNA in sera, extensively washed cryoprecipitates, supernatants from 8 patients with HCV-1b related MCII (IgM/k) and from 8 HCV control cases without MC. IgM-Vh sequences were PCR amplified and analysed by Genescan. Results: In 2 patients the cryoprecipitate contained more than 70% of the total circulating HCV-RNA, in 3 patients less than 10% and in the remaining 3 HCV-RNA was equally distributed in cryoprecipitate and supernatant. These percentages appear independent from viral load and cryocrit. PCR amplification of IgM-Vh revealed a prevalence of the Vh1 family in patients with MC while the Vh3 was the most represented family in controls. Genescan analysis revealed that only the 2 patients with 70% of HCV-RNA in cryoprecipitate had a monoclonal pick in Vh1. The E2 sequence was highly homogeneous in these 2 cases compared to the complex quasispecies in the others.

Journal of Computer-Aided Molecular Design, 2010

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizi... more Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, *tenfold higher than hit rates obtained in corresponding highthroughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of Electronic supplementary material The online version of this article (our knowledge this is the first report of structure-based discovery of CFTR modulators.

Biochimie, 2002

Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a... more Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone

Current Topics in Medicinal Chemistry, 2010

In silico drug discovery is a complex process requiring flexibility and ingenuity in method selec... more In silico drug discovery is a complex process requiring flexibility and ingenuity in method selection and a careful validation of work protocols. GPCR in silico drug discovery poses additional challenges due to the paucity of crystallographic data. This paper starts by reviewing selected GPCR in silico screening programs reported in the literature, including both structure-based and ligand-based approaches. Particular emphasis is given to library design, binding mode selection, process validation and compound selection for biological testing. Following literature review, we provide insights into in silico methodologies and process workflows used at EPIX to drive over 20 highly successful screening and lead optimization programs performed since 2001. Applications of the various methodologies discussed are demonstrated by examples from recent programs that have not yet been published.

Journal of Biological Chemistry, 2016

The Na,K-ATPase ␣ 2 subunit plays a key role in cardiac muscle contraction by regulating intracel... more The Na,K-ATPase ␣ 2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca 2؉ , whereas ␣ 1 has a more conventional role of maintaining ion homeostasis. The ␤ subunit differentially regulates maturation, trafficking, and activity of ␣-␤ heterodimers. It is not known whether the distinct role of ␣ 2 in the heart is related to selective assembly with a particular one of the three ␤ isoforms. We show here by immunofluorescence and co-immunoprecipitation that ␣ 2 is preferentially expressed with ␤ 2 in T-tubules of cardiac myocytes, forming ␣ 2 ␤ 2 heterodimers. We have expressed human ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 2 ␤ 2 , and ␣ 2 ␤ 3 in Pichia pastoris, purified the complexes, and compared their functional properties. ␣ 2 ␤ 2 and ␣ 2 ␤ 3 differ significantly from both ␣ 2 ␤ 1 and ␣ 1 ␤ 1 in having a higher K 0.5 K ؉ and lower K 0.5 Na ؉ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K ؉ and shift of the E 1 P-E 2 P conformational equilibrium toward E 1 P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of ␣ 2 ␤ 2 and ␣ 2 ␤ 3 over ␣ 1 ␤ 1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K ؉ affinity of ␣ 2 ␤ 2 could allow an acute response to raised ambient K ؉ concentrations in physiological conditions and explain the importance of ␣ 2 ␤ 2 for cardiac muscle contractility. The high sensitivity of ␣ 2 ␤ 2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of ␣ 2 ␤ 2selective digoxin derivatives for reducing cardiotoxicity.

Proteins: Structure, Function, and Genetics, 2003

We submitted predictions for all seven targets in the CAPRI experiment. For four targets, our sub... more We submitted predictions for all seven targets in the CAPRI experiment. For four targets, our submitted models included acceptable, medium accuracy predictions of the structures of the complexes, and for a fifth target we identified the location of the binding site of one of the molecules. We used a weighted-geometric docking algorithm in which contacts involving specified parts of the surfaces of either one or both molecules were up-weighted or down-weighted. The weights were based on available structural and biochemical data or on sequence analyses. The weighted-geometric docking proved very useful for five targets, improving the complementarity scores and the ranks of the nearly correct solutions, as well as their statistical significance. In addition, the weighted-geometric docking promoted formation of clusters of similar solutions, which include more accurate predictions.

Journal of Biomolecular Structure and Dynamics, 2003

Colicin E3 kills Escherichia coli cells by ribonucleolytic cleavage in the 16S rRNA. The cleavage... more Colicin E3 kills Escherichia coli cells by ribonucleolytic cleavage in the 16S rRNA. The cleavage occurs at the ribosomal decoding A-site between nucleotides A1493 and G1494. The breaking of this single phosphodiester bond results in a complete termination of protein biosynthesis leading to cell death. A model structure of the complex of the ribosomal subunit 30S and colicin E3 was constructed by means of a new weighted-geometric docking algorithm, in which interactions involving specified parts of the molecular surface can be up-weighted, allowing incorporation of experimental data in the docking search. Our model, together with available experimental data, predicts the role of the catalytic residues of colicin E3. In addition, it suggests that bound acidic immunity protein inhibits the enzymatic activity of colicin E3 by electrostatic repulsion of the negatively charged substrate.

Cancer Research, 2012

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pyruvate kinase (PK... more Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pyruvate kinase (PK) M2 which catalyzes the last step in glycolysis, is the alternative spliced isoform expressed in cancer cells, and a key player in exerting the Warburg effect. One of the mechanisms by which PKM2 modulate cancer cell metabolism is by switching between the low activity monomer and the high activity tetramer forms. This process is controlled by the varying concentration of an upstream glycolytic intermediate, FBP. These changes enable the cancer cell to manage its usage of glucose carbon backbones, whether for ATP production or for biomass generation, according to its changing demands. Further, it has been recently shown that the elevated levels of ROS in cancer cells contribute to the decreased activity of PKM2 to support NADPH production to increase cellular anti-oxidation capacity to sustain proliferation. Our goal is to disrupt the metabolic adaptation of cancer cells with small molecule PKM2 modulators. We hypothesized that an activator will redirect the consumption of nutrients, especially of glucose, away from biomass production and ultimately send the cells to die. Using our proprietary structure-based technology, we identified several series of novel allosteric PKM2 activators. Chemical optimization resulted in potent compounds with AC50 as low as 10nM, which were selective against the other PK isoforms. These compounds were proven to stabilize the active tetramer form of PKM2 in cancer cells. Bioenergetic experiments in several cell lines demonstrated that not only do these agents reduce lactate production; they also reduce the oxygen consumption rate. Analysis of cell cycle showed that treatment with PKM2 activators causes the cells to arrest at the G1 phase. In outcome-based assays, these compounds significantly reduced the proliferation rate of various cancer cell lines, and this effect was sensitive to media conditions, such as glucose levels. Taken together, our data supports the hypothesis that activation of PKM2 effectively deprives the cancer cell of building blocks and reduces the detoxification capacity that are required to support growth and proliferation. An in vivo colorectal cancer HT29 cell line mouse xenograft model with a modestly active compound (IC50=0.9uM) demonstrated tumor growth inhibition greater than 50% (100 mg/kg Q2D and 200 and 400 mg/kg IP QD). The compound was very safe in mouse, even at the highest exposure levels (200 and 400 mg/kg IP QD), indicating that these efficacious doses are significantly lower than the MTD. Additional xenograft models are ongoing. Taken together, there is strong support for the effect of potent PKM2 small molecule activators on the cellular metabolism of cancer cells, demonstrating statistically significant anti-cancer effect in an animal model of colorectal cancer. The favorable DMPK profile of these compounds further supports their development as anti-proliferative agents, both as a single agent and in combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3219. doi:1538-7445.AM2012-3219

Current Topics in Medicinal Chemistry, 2010

The application of structure-based in silico methods to drug discovery is still considered a majo... more The application of structure-based in silico methods to drug discovery is still considered a major challenge, especially when the x-ray structure of the target protein is unknown. Such is the case with human G protein-coupled receptors (GPCRs), one of the most important families of drug targets, where in the absence of x-ray structures, one has to rely on in silico 3D models. We report repeated success in using ab initio in silico GPCR models, generated by the PREDICT method, for blind in silico screening when applied to a set of five different GPCR drug targets. More than 100,000 compounds were typically screened in silico for each target, leading to a selection of <100 ''virtual hit'' compounds to be tested in the lab. In vitro binding assays of the selected compounds confirm high hit rates, of 12-21% (full dose-response curves, K i < 5 M). In most cases, the best hit was a novel compound (New Chemical Entity) in the 1-to 100-nM range, with very promising pharmacological properties, as measured by a variety of in vitro and in vivo assays. These assays validated the quality of the hits as lead compounds for drug discovery. The results demonstrate the usefulness and robustness of ab initio in silico 3D models and of in silico screening for GPCR drug discovery. modeling ͉ in silico screening ͉ structure-based G protein-coupled receptors (GPCRs) are membraneembedded proteins, responsible for communication between the cell and its environment (1). As a consequence, many major diseases, such as hypertension, cardiac dysfunction, depression, anxiety, obesity, inflammation, and pain, involve malfunction of these receptors (2), making them among the most important drug targets for pharmacological intervention (3-5). Thus, whereas GPCRs are only a small subset of the human genome, they are the targets for Ϸ50% of all recently launched drugs (6). As targets of paramount importance, it is expected that drug discovery for GPCRs would benefit from the introduction of computational methodologies (7), especially as these methods can be used in conjunction with such experimental methods as high-throughput screening (8, 9), NMR, and crystallography (10). Unfortunately, GPCRs, like other membrane-embedded proteins, have characteristics that make their 3D structure extremely difficult to determine experimentally. To date, the only GPCR for which a 3D structure was determined by x-ray crystallography is bovine rhodopsin (11), which is unique among GPCRs in that its ligand, retinal, is covalently bound and that it responds to light rather than to ligand binding. Hence, in the case of GPCRs, the limited availability of structural data has forced the computational design of ligands to heavily rely on ligand-based techniques. Indeed, for many GPCRs, the natural ligand can provide a good starting point, leading to useful pharmacophore models that can be used for identifying lead structures with novel scaffolds (6). These methods have been successfully applied for the discovery of peptide agonists to the somatostatin receptor (12) and for the discovery of nonpeptidic antagonists to the urotensin II receptor (13). Nonetheless, structure-based drug discovery remains highly desirable for GPCRs. It is known that all GPCRs structurally consist of seven transmembrane (TM) helices joined together by three extracellular and three intracellular loops. Of particular interest to

Journal of Clinical Oncology

Proteins: Structure, Function, and Bioinformatics, 2005

The diverse selection of targets in the CAPRI experiments provides grounds for determining the li... more The diverse selection of targets in the CAPRI experiments provides grounds for determining the limits of our rigid-body docking program MolFit, and for extending it. We find that the sensitivity of MolFit is high, enabling it to produce reasonably accurate docking solutions when the structures undergo moderate local conformation changes upon complex formation or when the docked molecules are modeled. Yet the ranks of these solutions are sometimes too low to meet the requirements of CAPRI assessment. This indicates that the selectivity of MolFit, which was optimized for docking of unbound X-ray structures, and which relies on the availability of external data from biochemical and bioinformatic sources, needs readjustment in order to meet the challenges presented by NMR or modeled structures. A different challenge is presented by large global conformation changes such as movements of domains. We show that such changes can be accommodated within the rigid-body approximation by employing rigid multibody multistage docking procedures. We also address the difficulty of ranking results from 2-body and multibody docking scans in cases in which there are no external data favoring one option over the other.

Journal of Immunological Methods, 1999

Phage display of antibody fragments has proved to be a powerful tool for the isolation and in vit... more Phage display of antibody fragments has proved to be a powerful tool for the isolation and in vitro evolution of these biologically important molecules. However, the general usefulness of this technology is still limited by some technical difficulties. One of the most debilitating obstacles to the widespread application of the technology is the accumulation of ''insert loss'' clones in the libraries; phagemid clones from which the DNA encoding part or all of the cloned antibody fragment had been deleted. Another difficulty arises when phage technology is applied for cloning hybridoma-derived antibody genes, where myeloma derived light chains, irrelevant to the hybridoma's antibody specificity may be fortuitously cloned. Here, we report the construction of a novel phage-display system designed to address these problems. In our system Ž . Ž . a single-chain Fv scFv is expressed as an in-frame fusion protein with a cellulose-binding domain CBD derived from the Clostridium thermocellum cellulosome. The CBD domain serves as an affinity tag allowing rapid phage capture and concentration from crude culture supernatants, and immunological detection of both displaying phage and soluble scFv produced thereof. We demonstrate the utility of our system in solving the technical difficulties described above, and in speeding up the process of scFv isolation from combinatorial antibody repertoires. q 1999 Elsevier Science B.V. All rights reserved.

Journal of Hepatology, 2004

To investigate the correlation between immune complex affinity to HCV and selection of a specific... more To investigate the correlation between immune complex affinity to HCV and selection of a specific monoclonal component. Material and Methods: HCV-RNA was quantified by bDNA in sera, extensively washed cryoprecipitates, supernatants from 8 patients with HCV-1b related MCII (IgM/k) and from 8 HCV control cases without MC. IgM-Vh sequences were PCR amplified and analysed by Genescan. Results: In 2 patients the cryoprecipitate contained more than 70% of the total circulating HCV-RNA, in 3 patients less than 10% and in the remaining 3 HCV-RNA was equally distributed in cryoprecipitate and supernatant. These percentages appear independent from viral load and cryocrit. PCR amplification of IgM-Vh revealed a prevalence of the Vh1 family in patients with MC while the Vh3 was the most represented family in controls. Genescan analysis revealed that only the 2 patients with 70% of HCV-RNA in cryoprecipitate had a monoclonal pick in Vh1. The E2 sequence was highly homogeneous in these 2 cases compared to the complex quasispecies in the others.

Journal of Computer-Aided Molecular Design, 2010

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizi... more Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, *tenfold higher than hit rates obtained in corresponding highthroughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of Electronic supplementary material The online version of this article (our knowledge this is the first report of structure-based discovery of CFTR modulators.

Biochimie, 2002

Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a... more Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone

Current Topics in Medicinal Chemistry, 2010

In silico drug discovery is a complex process requiring flexibility and ingenuity in method selec... more In silico drug discovery is a complex process requiring flexibility and ingenuity in method selection and a careful validation of work protocols. GPCR in silico drug discovery poses additional challenges due to the paucity of crystallographic data. This paper starts by reviewing selected GPCR in silico screening programs reported in the literature, including both structure-based and ligand-based approaches. Particular emphasis is given to library design, binding mode selection, process validation and compound selection for biological testing. Following literature review, we provide insights into in silico methodologies and process workflows used at EPIX to drive over 20 highly successful screening and lead optimization programs performed since 2001. Applications of the various methodologies discussed are demonstrated by examples from recent programs that have not yet been published.