CHARMM-GUI <i>Ligand Designer</i> for Template-Based Virtual Ligand Design in a Binding Site

Rational drug design involves a task of finding ligands that would bind to a specific target protein. This work presents CHARMM-GUI Ligand Designer that is an intuitive and interactive web-based tool to design virtual ligands that match the shape and chemical features of a given protein binding site. Ligand Designer provides ligand modification capabilities with 3D visualization that allow researchers to modify and redesign virtual ligands while viewing how the protein–ligand interactions are affected. Virtual ligands can also be parameterized for further molecular dynamics (MD) simulations and free energy calculations. Using 8 targets from 8 different protein classes in the directory of useful decoys, enhanced (DUD-E) data set, we show that Ligand Designer can produce similar ligands to the known active ligands in the crystal structures. Ligand Designer also produces stable protein–ligand complex structures when tested using short MD simulations. We expect that Ligand Designer can be a useful and user-friendly tool to design small molecules in any given potential ligand binding site on a protein of interest

Target-Specific Gene Silencing of Layer-by-Layer Assembled Gold–Cysteamine/siRNA/PEI/HA Nanocomplex

Target-specific intracellular delivery of small interfering RNA (siRNA) is regarded as one of the most important technologies for the development of siRNA therapeutics. In this work, a cysteamine modified gold nanoparticles (AuCM)/siRNA/polyethyleneimine (PEI)/hyaluronic acid (HA) complex was successfully developed using a layer-by-layer method for target-specific intracellular delivery of siRNA by HA receptor mediated endocytosis. Atomic force microscopic and zeta potential analyses confirmed the formation of a AuCM/siRNA/PEI/HA complex having a particle size of ca. 37.3 nm and a negative surface charge of ca. −12 mV. With a negligible cytotoxicity, AuCM/siRNA/PEI/HA complex showed an excellent target-specific gene silencing efficiency of ca. 70% in the presence of 50 vol % serum, which was statistically much higher than that of siRNA/Lipofectamine 2000 complex. In the competitive binding tests with free HA, dark-field bioimaging and inductively coupled plasma–atomic emission spectroscopy confirmed the target-specific intracellular delivery of AuCM/siRNA/PEI/HA complex to B16F1 cells with HA receptors. Moreover, the systemic delivery of AuCM/siRNA/PEI/HA complex using apolipoprotein B (ApoB) siRNA as a model drug resulted in a significantly reduced ApoB mRNA level in the liver tissue. Taken together, AuCM/siRNA/PEI/HA complex was thought to be developed as target-specific siRNA therapeutics for the systemic treatment of various liver diseases

Transmittance profile of ELP-lipid conjugates of (A) 1 mM, (B) 0.5 mM, (C) 0.2 mM, and (D) 0.1 mM in PBS at pH 7.4., and transition temperature as function of the concentration (mM) of various ELP-lipid conjugates (E).

<p>The turbidity of ELP-lipid conjugates were characterized by monitoring transmittance at 280 nm as a function of temperature. Solution of ELP-lipids conjugates were heated at a constant rate of 1°C/min. The transition temperature (Tt) was defined as the temperature at which the solution of ELP-lipid conjugate reached 50% of transmittance. Data is shown as mean ± S.D. (n = 3).</p

Structural Modeling of Cytokine-Receptor-JAK2 Signaling Complexes Using AlphaFold Multimer

Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains, and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all of the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here by using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts, with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices, causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to the TM α-helices of the active TPOR dimer was proposed. The models also help elucidate the molecular basis of oncogenic mutations that may involve a noncanonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available

DOX accumulation in the tumor at 1, 6, and 12 hr after i.v. injection combined with preheating.

<p>*, <i>p</i><0.005, #, <i>p</i><0.001, significant difference compared to e-TSL without preheating and free DOX with preheating.</p

List of all simulations.

<p>List of all simulations.</p

Structural Modeling of Cytokine-Receptor-JAK2 Signaling Complexes Using AlphaFold Multimer

Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains, and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all of the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here by using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts, with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices, causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to the TM α-helices of the active TPOR dimer was proposed. The models also help elucidate the molecular basis of oncogenic mutations that may involve a noncanonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available

Structural Modeling of Cytokine-Receptor-JAK2 Signaling Complexes Using AlphaFold Multimer

Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains, and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all of the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here by using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts, with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices, causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to the TM α-helices of the active TPOR dimer was proposed. The models also help elucidate the molecular basis of oncogenic mutations that may involve a noncanonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available

Antitumor efficacy of e-TSL and free DOX in the presence/absence of mild hyperthermia.

<p>e-TSL and free DOX were administered (5 mg DOX/kg) into tumor bearing BALB/c mice 5 min after preheating (30 min of water bath) and followed by mild hyperthermia (42°C, water bath) after 6 hrs. *, <i>p</i><0.05, significant difference compared to free DOX and PBS (control).</p

Snapshots at the (A) beginning (0 ns) and (B) end (2 µs) of the simulation of the bilayer system.

<p>The molar ratio of liposome formulation is DPPC:DSPE-PEG:cholesterol:SA-Vn 55∶2∶15∶0.41. Gray, red, dark-, and light-blue colors respectively represent the lipid-head phosphate, PEG, ELP head (peptide), and tail (carbon chain) groups. For clarity, lipid tail, water, and ion beads are omitted. The images were created with Visual Molecular Dynamics <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103116#pone.0103116-Humphrey1" target="_blank">[29]</a>.</p