Publications by authors named "Eleonora Gianti"

15 Publications

  • Page 1 of 1

TRPA1 modulation by piperidine carboxamides suggests an evolutionarily conserved binding site and gating mechanism.

Proc Natl Acad Sci U S A 2019 12 3;116(51):26008-26019. Epub 2019 Dec 3.

Biochemical and Cellular Pharmacology, Genentech, Inc., South San Francisco, CA 94080;

The transient receptor potential ankyrin 1 (TRPA1) channel functions as an irritant sensor and is a therapeutic target for treating pain, itch, and respiratory diseases. As a ligand-gated channel, TRPA1 can be activated by electrophilic compounds such as allyl isothiocyanate (AITC) through covalent modification or activated by noncovalent agonists through ligand binding. However, how covalent modification leads to channel opening and, importantly, how noncovalent binding activates TRPA1 are not well-understood. Here we report a class of piperidine carboxamides (PIPCs) as potent, noncovalent agonists of human TRPA1. Based on their species-specific effects on human and rat channels, we identified residues critical for channel activation; we then generated binding modes for TRPA1-PIPC interactions using structural modeling, molecular docking, and mutational analysis. We show that PIPCs bind to a hydrophobic site located at the interface of the pore helix 1 (PH1) and S5 and S6 transmembrane segments. Interestingly, this binding site overlaps with that of known allosteric modulators, such as A-967079 and propofol. Similar binding sites, involving π-helix rearrangements on S6, have been recently reported for other TRP channels, suggesting an evolutionarily conserved mechanism. Finally, we show that for PIPC analogs, predictions from computational modeling are consistent with experimental structure-activity studies, thereby suggesting strategies for rational drug design.
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http://dx.doi.org/10.1073/pnas.1913929116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926016PMC
December 2019

Suppression of Zika Virus Infection in the Brain by the Antiretroviral Drug Rilpivirine.

Mol Ther 2019 12 11;27(12):2067-2079. Epub 2019 Oct 11.

Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, 3500 N. Broad Street, Philadelphia, PA 19140, USA. Electronic address:

Zika virus (ZIKV) infection is associated with microcephaly in neonates and Guillain-Barré syndrome in adults. ZIKV produces a class of nonstructural (NS) regulatory proteins that play a critical role in viral transcription and replication, including NS5, which possesses RNA-dependent RNA polymerase (RdRp) activity. Here we demonstrate that rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of HIV-1 infection, inhibits the enzymatic activity of NS5 and suppresses ZIKV infection and replication in primary human astrocytes. Similarly, other members of the NNRTI family, including etravirine and efavirenz, showed inhibitory effects on viral infection of brain cells. Site-directed mutagenesis identified 14 amino acid residues within the NS5 RdRp domain (AA265-903), which are important for the RPV interaction and the inhibition of NS5 polymerase activity. Administration of RPV to ZIKV-infected interferon-alpha/beta receptor (IFN-A/R) knockout mice improved the clinical outcome and prevented ZIKV-induced mortality. Histopathological examination of the brains from infected animals revealed that RPV reduced ZIKV RNA levels in the hippocampus, frontal cortex, thalamus, and cerebellum. Repurposing of NNRTIs, such as RPV, for the inhibition of ZIKV replication offers a possible therapeutic strategy for the prevention and treatment of ZIKV-associated disease.
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http://dx.doi.org/10.1016/j.ymthe.2019.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904669PMC
December 2019

Computational Approaches to Studying Voltage-Gated Ion Channel Modulation by General Anesthetics.

Methods Enzymol 2018 19;602:25-59. Epub 2018 Mar 19.

Institute for Computational Molecular Science, College of Science and Technology, Temple University, Philadelphia, PA, United States. Electronic address:

Voltage-gated ion channels (VGICs) are responsible for the propagation of electrical signals in excitable cells. Small-molecule modulation of VGICs affects transmission of action potentials in neurons and thus can modulate the activity of the central nervous system. For this reason, VGICs are considered key players in the medically induced state of general anesthesia. Consistently, VGICs have been shown to respond to several general anesthetics. However, in spite of extensive electrophysiological characterizations, modulation of VGICs by anesthetics is still only partially understood. Among the challenging aspects are the presence of multiple binding sites and the observation of paradoxical effects, i.e., evidence, for the same channel, of inhibition and potentiation. In this context, molecular simulations emerged in the recent past as the tool of choice to complement electrophysiology studies with a microscopic picture of binding and allosteric regulation. In this chapter, we describe the most effective computational techniques to study VGIC modulation by general anesthetics. We start by reviewing the VGIC conduction cycle, the corresponding set of channel conformations, and the approaches used to model them. We then review the most successful strategies to identify binding sites and estimate binding affinities.
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http://dx.doi.org/10.1016/bs.mie.2018.01.002DOI Listing
April 2019

Sites Contributing to TRPA1 Activation by the Anesthetic Propofol Identified by Photoaffinity Labeling.

Biophys J 2017 Nov 19;113(10):2168-2172. Epub 2017 Sep 19.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts. Electronic address:

In addition to inducing anesthesia, propofol activates a key component of the pain pathway, the transient receptor potential ankyrin 1 ion channel (TRPA1). Recent mutagenesis studies suggested a potential activation site within the transmembrane domain, near the A-967079 cavity. However, mutagenesis cannot distinguish between protein-based and ligand-based mechanisms, nor can this site explain the complex modulation by propofol. Thus more direct approaches are required to reveal potentially druggable binding sites. Here we apply photoaffinity labeling using a propofol derivative, meta-azipropofol, for direct identification of binding sites in mouse TRPA1. We confirm that meta-azipropofol activates TRPA1 like the parent anesthetic, and identify two photolabeled residues (V954 and E969) in the S6 helix. In combination with docking to closed and open state models of TRPA1, photoaffinity labeling suggested that the A-967079 cavity is a positive modulatory site for propofol. Further, the photoaffinity labeling of E969 indicated pore block as a likely mechanism for propofol inhibition at high concentrations. The direct identification of drug-binding sites clarifies the molecular mechanisms of important TRPA1 agonists, and will facilitate drug design efforts to modulate TRPA1.
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http://dx.doi.org/10.1016/j.bpj.2017.08.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700380PMC
November 2017

On the role of water density fluctuations in the inhibition of a proton channel.

Proc Natl Acad Sci U S A 2016 12 12;113(52):E8359-E8368. Epub 2016 Dec 12.

Institute for Computational Molecular Science, Department of Chemistry, Temple University, Philadelphia, PA 19122;

Hv1 is a transmembrane four-helix bundle that transports protons in a voltage-controlled manner. Its crucial role in many pathological conditions, including cancer and ischemic brain damage, makes Hv1 a promising drug target. Starting from the recently solved crystal structure of Hv1, we used structural modeling and molecular dynamics simulations to characterize the channel's most relevant conformations along the activation cycle. We then performed computational docking of known Hv1 inhibitors, 2-guanidinobenzimidazole (2GBI) and analogs. Although salt-bridge patterns and electrostatic potential profiles are well-defined and distinctive features of activated versus nonactivated states, the water distribution along the channel lumen is dynamic and reflects a conformational heterogeneity inherent to each state. In fact, pore waters assemble into intermittent hydrogen-bonded clusters that are replaced by the inhibitor moieties upon ligand binding. The entropic gain resulting from releasing these conformationally restrained waters to the bulk solvent is likely a major contributor to the binding free energy. Accordingly, we mapped the water density fluctuations inside the pore of the channel and identified the regions of maximum fluctuation within putative binding sites. Two sites appear as outstanding: One is the already known binding pocket of 2GBI, which is accessible to ligands from the intracellular side; the other is a site located at the exit of the proton permeation pathway. Our analysis of the waters confined in the hydrophobic cavities of Hv1 suggests a general strategy for drug discovery that can be applied to any ion channel.
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http://dx.doi.org/10.1073/pnas.1609964114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206518PMC
December 2016

Does Proton Conduction in the Voltage-Gated H Channel hHv1 Involve Grotthuss-Like Hopping via Acidic Residues?

J Phys Chem B 2017 04 18;121(15):3340-3351. Epub 2016 Nov 18.

Laboratory of Computational Chemistry and Biochemistry, Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne (EPFL) , CH-1015 Lausanne, Switzerland.

Hv1s are ubiquitous highly selective voltage-gated proton channels involved in male fertility, immunology, and the invasiveness of certain forms of breast cancer. The mechanism of proton extrusion in Hv1 is not yet understood, while it constitutes the first step toward the design of high-affinity drugs aimed at this important pharmacological target. In this contribution, we explore the details of the mechanism via an integrative approach, using classical and QM/MM molecular dynamics simulations of a monomeric hHv1 model. We propose that protons localize in three binding sites along the channel lumen, formed by three pairs of conserved negatively charged residues lining the pore: D174/E153, D112/D185, and E119/D123. Local rearrangements, involving notably a dihedral transition of F150, a conserved phenylalanine lining the permeation pathway, appear to allow protons to hop from one acidic residue to the next through a bridging water molecule. These results constitute a first attempt at rationalizing hHv1 selectivity for H and the role played by D112 in this process. They pave the way for further quantitative characterization of H transport in hHv1.
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http://dx.doi.org/10.1021/acs.jpcb.6b08339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310143PMC
April 2017

Computational analysis of EBNA1 "druggability" suggests novel insights for Epstein-Barr virus inhibitor design.

J Comput Aided Mol Des 2016 Apr 5;30(4):285-303. Epub 2016 Apr 5.

Department of Chemistry and Biochemistry, University of the Sciences, 600 South, 43rd Street, Philadelphia, PA, 19104, USA.

The Epstein-Barr Nuclear Antigen 1 (EBNA1) is a critical protein encoded by the Epstein-Barr Virus (EBV). During latent infection, EBNA1 is essential for DNA replication and transcription initiation of viral and cellular genes and is necessary to immortalize primary B-lymphocytes. Nonetheless, the concept of EBNA1 as drug target is novel. Two EBNA1 crystal structures are publicly available and the first small-molecule EBNA1 inhibitors were recently discovered. However, no systematic studies have been reported on the structural details of EBNA1 "druggable" binding sites. We conducted computational identification and structural characterization of EBNA1 binding pockets, likely to accommodate ligand molecules (i.e. "druggable" binding sites). Then, we validated our predictions by docking against a set of compounds previously tested in vitro for EBNA1 inhibition (PubChem AID-2381). Finally, we supported assessments of pocket druggability by performing induced fit docking and molecular dynamics simulations paired with binding affinity predictions by Molecular Mechanics Generalized Born Surface Area calculations for a number of hits belonging to druggable binding sites. Our results establish EBNA1 as a target for drug discovery, and provide the computational evidence that active AID-2381 hits disrupt EBNA1:DNA binding upon interacting at individual sites. Lastly, structural properties of top scoring hits are proposed to support the rational design of the next generation of EBNA1 inhibitors.
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http://dx.doi.org/10.1007/s10822-016-9899-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5180362PMC
April 2016

An SH2 domain model of STAT5 in complex with phospho-peptides define "STAT5 Binding Signatures".

J Comput Aided Mol Des 2015 May 10;29(5):451-70. Epub 2015 Mar 10.

Department of Chemistry and Biochemistry, University of the Sciences, 600 S. 43rd Street, Philadelphia, PA, 19104, USA,

The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.
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http://dx.doi.org/10.1007/s10822-015-9835-6DOI Listing
May 2015

Flipping in the pore: discovery of dual inhibitors that bind in different orientations to the wild-type versus the amantadine-resistant S31N mutant of the influenza A virus M2 proton channel.

J Am Chem Soc 2014 Dec 18;136(52):17987-95. Epub 2014 Dec 18.

Department of Pharmaceutical Chemistry, University of California , Mission Bay Box 3122, San Francisco, California 94158, United States.

Influenza virus infections lead to numerous deaths and millions of hospitalizations each year. One challenge facing anti-influenza drug development is the heterogeneity of the circulating influenza viruses, which comprise several strains with variable susceptibility to antiviral drugs. For example, the wild-type (WT) influenza A viruses, such as the seasonal H1N1, tend to be sensitive to antiviral drugs, amantadine and rimantadine, while the S31N mutant viruses, such as the pandemic 2009 H1N1 (H1N1pdm09) and seasonal H3N2, are resistant to this class of drugs. Thus, drugs targeting both WT and the S31N mutant are highly desired. We report our design of a novel class of dual inhibitors along with their ion channel blockage and antiviral activities. The potency of the most active compound 11 in inhibiting WT and the S31N mutant influenza viruses is comparable with that of amantadine in inhibiting WT influenza virus. Solution NMR studies and molecular dynamics (MD) simulations of drug-M2 interactions supported our design hypothesis: namely, the dual inhibitor binds in the WT M2 channel with an aromatic group facing down toward the C-terminus, while the same drug binds in the S31N M2 channel with its aromatic group facing up toward the N-terminus. The flip-flop mode of drug binding correlates with the structure-activity relationship (SAR) and has paved the way for the next round of rational design of broad-spectrum antiviral drugs.
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http://dx.doi.org/10.1021/ja508461mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286326PMC
December 2014

Hydrogen-bonded water molecules in the M2 channel of the influenza A virus guide the binding preferences of ammonium-based inhibitors.

J Phys Chem B 2015 Jan 11;119(3):1173-83. Epub 2014 Nov 11.

Institute for Computational Molecular Science, Temple University , SERC Building, 1925 North 12th Street, Philadelphia, Pennsylvania 19122, United States.

The tetrameric M2 proton channel of influenza A virus is an integral membrane protein responsible for the acidification of the viral interior. Drugs such as amantadine target the transmembrane region of wild type M2 by acting as pore blockers. However, a number of mutations affecting this domain confer drug resistance, prompting the need for alternative inhibitors. The availability of high-resolution structures of drug-bound M2, paired with computational investigations, revealed that inhibitors can bind at different sites, and provided useful insights in understanding the principles governing proton conduction. Here, we investigated by computation the energetic and geometric factors determining the relative stability of pore blockers at individual sites of different M2 strains. We found that local free energy minima along the translocation pathway of positively charged chemical species correspond to experimentally determined binding sites of inhibitors. Then, by examining the structure of water clusters hydrating each site, as well as of those displaced by binding of hydrophobic scaffolds, we predicted the binding preferences of M2 ligands. This information can be used to guide the identification of novel classes of inhibitors.
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http://dx.doi.org/10.1021/jp506807yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4386636PMC
January 2015

Fragment-based Shape Signatures: a new tool for virtual screening and drug discovery.

J Comput Aided Mol Des 2013 Dec 24;27(12):1009-36. Epub 2013 Dec 24.

Department of Chemistry and Biochemistry, University of the Sciences, 600 S. 43rd Street, Philadelphia, PA, 19104, USA,

Since its introduction in 2003, the Shape Signatures method has been successfully applied in a number of drug design projects. Because it uses a ray-tracing approach to directly measure molecular shape and properties (as opposed to relying on chemical structure), it excels at scaffold hopping, and is extraordinarily easy to use. Despite its advantages, a significant drawback of the method has hampered its application to certain classes of problems; namely, when the chemical structures considered are large and contain heterogeneous ring-systems, the method produces descriptors that tend to merely measure the overall size of the molecule, and begin to lose selective power. To remedy this, the approach has been reformulated to automatically decompose compounds into fragments using ring systems as anchors, and to likewise partition the ray-trace in accordance with the fragment assignments. Subsequently, descriptors are generated that are fragment-based, and query and target molecules are compared by mapping query fragments onto target fragments in all ways consistent with the underlying chemical connectivity. This has proven to greatly extend the selective power of the method, while maintaining the ease of use and scaffold-hopping capabilities that characterized the original implementation. In this work, we provide a full conceptual description of the next generation Shape Signatures, and we underline the advantages of the method by discussing its practical applications to ligand-based virtual screening. The new approach can also be applied in receptor-based mode, where protein-binding sites (partitioned into subsites) can be matched against the new fragment-based Shape Signatures descriptors of library compounds.
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http://dx.doi.org/10.1007/s10822-013-9698-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880490PMC
December 2013

Modeling androgen receptor flexibility: a binding mode hypothesis of CYP17 inhibitors/antiandrogens for prostate cancer therapy.

J Chem Inf Model 2012 Oct 1;52(10):2670-83. Epub 2012 Oct 1.

Department of Chemistry & Biochemistry, University of the Sciences, 600 S. 43rd Street, Philadelphia, Pennsylvania 19104, USA.

Prostate Cancer (PCa), a leading cause of cancer death worldwide (www.cancer.gov), is a complex malignancy where a spectrum of targets leads to a diversity of PCa forms. A widely pursued therapeutic target is the Androgen Receptor (AR). As a Steroid Hormone Receptor, AR serves as activator of transcription upon binding to androgens and plays a central role in the development of PCa. AR is a structurally flexible protein, and conformational plasticity of residues in the binding-pocket is a key to its ability to accommodate ligands from various chemical classes. Besides direct modulation of AR activity by antagonists, inhibition of cytochrome CYP17 (17α-hydroxylase/17,20-lyase), essential in androgen biosynthesis, has widely been considered an effective strategy against PCa. Interestingly, Handratta et al. (2005) discovered new, potent inhibitors of CYP17 (C-17 steroid derivatives) with pure AR antagonistic properties. Although the antiandrogenic activity of their lead compound (VN/124-1) has been experimentally proven both in vitro and in vivo, no structural data are currently available to elucidate the molecular determinants responsible for these desirable dual inhibitory properties. We implemented a Structure-based Drug Design (SBDD) approach to generate a valuable hypothesis as to the binding modes of steroidal CYP17 inhibitors/antiandrogens against the AR. To deal with the plasticity of residues buried in the Ligand Binding Domain (LBD), we developed a flexible-receptor Docking protocol based on Induced-Fit (IFD) methodology (www.schrodinger.com/). Our results constitute an ideal starting point for the rational design of next-generation analogues of CYP17 inhibitors/antiandrogens as well as an attractive tool to suggest novel chemical classes of AR antagonists.
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http://dx.doi.org/10.1021/ci3002342DOI Listing
October 2012

Synthesis and biological evaluation of N-hydroxyphenylacrylamides and N-hydroxypyridin-2-ylacrylamides as novel histone deacetylase inhibitors.

J Med Chem 2010 Jan;53(2):822-39

Genextra Group, Congenia s.r.l., Via Adamello 16, 20139 Milan, Italy.

The histone deacetylases (HDACs) are able to regulate gene expression, and histone deacetylase inhibitors (HDACi) emerged as a new class of agents in the treatment of cancer as well as other human disorders such as neurodegenerative diseases. In the present investigation, we report on the synthesis and biological evaluation of compounds derived from the expansion of a HDAC inhibitor scaffold having N-hydroxy-3-phenyl-2-propenamide and N-hydroxy-3-(pyridin-2-yl)-2-propenamide as core structures and containing a phenyloxopropenyl moiety, either unsubstituted or substituted by a 4-methylpiperazin-1-yl or 4-methylpiperazin-1-ylmethyl group. The compounds were evaluated for their ability to inhibit nuclear HDACs, as well as for their in vitro antiproliferative activity. Moreover, their metabolic stability in microsomes and aqueous solubility were studied and selected compounds were further characterized by in vivo pharmacokinetic experiments. These compounds showed a remarkable stability in vivo, compared to hydroxamic acid HDAC inhibitors that have already entered clinical trials. The representative compound 30b showed in vivo antitumor activity in a human colon carcinoma xenograft model.
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http://dx.doi.org/10.1021/jm901502pDOI Listing
January 2010

Identification and selection of "privileged fragments" suitable for primary screening.

J Chem Inf Model 2008 Nov;48(11):2129-39

Computational Sciences Group, Department of Chemistry (Congenia s.r.l.), Genextra S.p.A., Milan MI 20100, Italy.

The use of small molecule libraries for fragment-based primary screening (FBS) is a well-known approach to identify protein binders in the low affinity range. However, the search, analysis, and selection of suitable screening fragments can be a lengthy process, because of the large number of compounds that must be analyzed for different levels of ring/substituents identification and submitted to selection/exclusion criteria based on their physicochemical properties. The purpose of the present work is to propose a strategy to identify substructures from databases of known drugs, which can be used as templates for the generation of libraries of "privileged fragments" that are able to provide high-quality hits. The entire process has been developed integrating Pipeline Pilot (Accelrys Inc., San Diego, CA; http://www.accelrys.com ) native components and user-defined molecular files containing ISIS-like substructure query features (Symyx, San Ramon, CA; http://www.symyx.com ). The method is effortless, easy to put in place, and fast enough to be iteratively applied to different sources of druglike compounds.
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http://dx.doi.org/10.1021/ci800219hDOI Listing
November 2008

PoInTree: a polar and interactive phylogenetic tree.

Genomics Proteomics Bioinformatics 2005 Feb;3(1):58-60

Nerviano Medical Sciences srl, 20014 Nerviano (MI), Italy.

PoInTree (Polar and Interactive Tree) is an application that allows to build, visualize and customize phylogenetic trees in a polar interactive and highly flexible view. It takes as input a FASTA file or multiple alignment formats. Phylogenetic tree calculation is based on a sequence distance method and utilizes the Neighbor Joining (NJ) algorithm. It also allows displaying precalculated trees of the major protein families based on Pfam classification. In PoInTree, nodes can be dynamically opened and closed and distances between genes are graphically represented. Tree root can be centered on a selected leaf. Text search mechanism, color-coding and labeling display are integrated. The visualizer can be connected to an Oracle database containing information on sequences and other biological data, helping to guide their interpretation within a given protein family across multiple species. The application is written in Borland Delphi and based on VCL Teechart Pro 6 graphical component (Steema software).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5172537PMC
http://dx.doi.org/10.1016/s1672-0229(05)03009-3DOI Listing
February 2005