scholarly journals Molecular Determinants of Ligand Residence in Galectin

2021 ◽  
Author(s):  
Jaya Krishna Koneru ◽  
Suman Sinha ◽  
Jagannath Mondal
Keyword(s):  
Binding Site ◽  
Specific Binding ◽  
Binding Pocket ◽  
Cellular Adhesion ◽  
Dynamics Simulation ◽  
Natural Ligand ◽  
Molecular Determinants ◽  
Galectin 3 ◽  
Synthetic Ligand ◽  
Mechanistic Basis

The recognition of carbohydrates by lectins play key roles in diverse cellular processes such as cellular adhesion, proliferation and apoptosis which makes it a promising therapeutic target against cancers. One of the most functionally active lectins, galectin-3 is distinctively known for its specific binding affinity towards β-galactoside. Despite the prevalence of high-resolution crystallographic structures, the mechanistic basis and the molecular determinants of the sugar recognition process by galectin-3 are currently elusive. Here we address this question by capturing the complete dynamical binding process of human galectin-3 with its native ligand N-acetyllactosamine (LacNAc) and one of its synthetic derivatives by unbiased Molecular Dynamics simulation. In our simulations, both the natural ligand LacNAc and its synthetic derivative, initially solvated in water, diffuse around the protein and eventually recognise the designated binding site at the S-side of galectin-3, in crystallographic precision and identifies key metastable intermediate ligand-states around the galectin on their course to eventual binding. The simulations highlight that the origin of the experimentally observed multi-fold efficacy of synthetically designed ligand-derivative over its native natural ligand LacNAc lies in the derivative's relatively longer residence time in the bound pocket. A kinetic analysis demonstrates that the LacNAc-derivative would be more resilient compared to the parent ligand against unbinding from the protein binding site. In particular, the analysis identifies that interactions of the binding pocket residues Trp181, Arg144 and Arg162 with the tetrafuorophenyl ring of the derivative as the key determinant for the synthetic ligand to latch into the pocket.

scholarly journals Mutagenesis Analysis of the rGTP-Specific Binding Site of Hepatitis C Virus RNA-Dependent RNA Polymerase

2005 ◽  
Vol 79 (18) ◽  
pp. 11607-11617 ◽  
Author(s):  
Zhaohui Cai ◽  
MinKyung Yi ◽  
Chen Zhang ◽  
Guangxiang Luo
Keyword(s):  
Binding Site ◽  
Specific Binding ◽  
Rna Replication ◽  
Binding Pocket ◽  
Hcv Rna ◽  
Amino Acid Substitutions ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Cell Colony

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is the virus-encoded RNA-dependent RNA polymerase (RdRp) essential for HCV RNA replication. An earlier crystallographic study identified a rGTP-specific binding site lying at the surface between the thumb domain and the fingertip about 30 Å away from the active site of the HCV RdRp (S. Bressanelli, L. Tomei, F. A. Rey, and R. De Francesco, J. Virol 76:3482-3492, 2002). To determine its physiological importance, we performed a systematic mutagenesis analysis of the rGTP-specific binding pocket by amino acid substitutions. Effects of mutations of the rGTP-specific binding site on enzymatic activity were determined by an in vitro RdRp assay, while effects of mutations on HCV RNA replication were examined by cell colony formation, as well as by transient replication of subgenomic HCV RNAs. Results derived from these studies demonstrate that amino acid substitutions of the rGTP-specific binding pocket did not significantly affect the in vitro RdRp activity of purified recombinant NS5B proteins, as measured by their abilities to synthesize RNA on an RNA template containing the 3′ untranslated region of HCV negative-strand RNA. However, most mutations of the rGTP-specific binding site either impaired or completely ablated the ability of subgenomic HCV RNAs to induce cell colony formation. Likewise, these mutations caused either reduction in or lethality to transient replication of the human immunodeficiency virus Tat-expressing HCV replicon RNAs in the cell. Collectively, these findings demonstrate that the rGTP-specific binding site of the HCV NS5B is not required for in vitro RdRp activity but is important for HCV RNA replication in vivo.

scholarly journals Molecular determinants underlying DS2 activity at δ-containing GABAA receptors

2021 ◽  
Author(s):  
Christina B. Falk-Petersen ◽  
Frederik Rostrup ◽  
Rebekka Löffler ◽  
Stine Buchleithner ◽  
Kasper Harpsøe ◽  
...  
Keyword(s):  
Binding Site ◽  
Drug Targets ◽  
Transmembrane Domain ◽  
Direct Interaction ◽  
Binding Pocket ◽  
Docking Studies ◽  
Tonic Inhibition ◽  
Site Directed Mutagenesis ◽  
Receptor Subtypes ◽  
Molecular Determinants

AbstractDelta selective compound 2 (DS2) is one of the most widely used tools to study selective actions mediated by δ subunit-containing GABAA receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive.Using a combination of computational modeling, site-directed mutagenesis and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α4β1δ receptors: an α4(+)δ(-) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αβγ2 receptors, and two sites in the transmembrane domain (TMD); one in the α4(+)β1(-) and one in the α4(-)β1(+) interface, with the α4(-)β1(+) site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α4(+)β1(-) interface, either α4(S303L) of the α4(+)-side or β1(I289Q) of the β1(-)-side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patchclamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points towards a functional selectivity of DS2 and its analogs via a surprisingly well-conserved binding pocket in the TMD.Significance statementδ-Containing GABAA receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition. Yet, no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the know compound DS2, the ground is laid for design of ligands that selectively target δ-containing GABAA receptor subtypes, for better understanding of tonic inhibition, and, ultimately, for rational development of novel drugs.

Alteration of ssRNA Torsion and Water Influx Into ssRNA Pocket in K309A and S247A mutations

2019 ◽  
Vol 15 ◽  
Author(s):  
I. Olaposi Omotuyi
Keyword(s):  
Hydrogen Bond ◽  
Salt Bridge ◽  
Binding Pocket ◽  
Dynamics Simulation ◽  
Lassa Virus ◽  
Partial Loss ◽  
Hydrogen Bond Interaction ◽  
Phosphodiester Bond ◽  
Water Influx ◽  
Mechanistic Basis

Background: Lassa virus (LV) infection is a endemic disease from West Africa posing threat to the entire world. A thorough understanding of the mechanistic workings of the genome products of LV may be key to developing drugs candidates for the treatment of LV infection. Methods: Molecular dynamics simulation has been used to provide insight into the mechanistic basis for total loss of ssRNA interaction in nucleoprotein (NP) K309A, partial loss in S247A, and no loss in S237A by following the hydrogen bond interaction, water influx into the ssRNA pocket and glycosidic torsion angle (?) of the ssRNA. Results: The results revealed that K309A mutation is associated with complete loss of salt-bridge interaction between lysine ?-amino and U4-O2P phosphodiester linkage but not in S237A where S247-OG atom played a redundant role. S247A is also associated with partial loss of hydrogen bond between OG atom of S247 and C5-O2P phosphodiester bond as T178-OG1 group seem to have a seemingly redundant interaction with C5-O2P. While S247A only is also associated with alteration of ? rotation in U6/C7, both K309A and S247 but not S237A is associated with increased water influx into the ssRNA binding pocket. Conclusion: K309A mutation may result in non-viable Lassa viron as loss of ssRNA interaction may negatively affect genome biochemistry, semi-viable Lassa viron in S247A mutation may be due to loss of 3D arrangement of ssRNA due to splayed out nucleotides.

Unveiling the Structural Insights into the Selective Inhibition of Protein Kinase D1

2019 ◽  
Vol 25 (10) ◽  
pp. 1059-1074 ◽  
Author(s):  
Raju Dash ◽  
Md. Arifuzzaman ◽  
Sarmistha Mitra ◽  
Md. Abdul Hannan ◽  
Nurul Absar ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
Protein Kinase ◽  
Binding Site ◽  
Free Energy Calculations ◽  
Dynamics Simulation ◽  
Selective Inhibitors ◽  
Conserved Residues ◽  
Protein Kinase D1 ◽  
Binding Mechanisms

Background: Although protein kinase D1 (PKD1) has been proved to be an efficient target for anticancer drug development, lack of structural details and substrate binding mechanisms are the main obstacles for the development of selective inhibitors with therapeutic benefits. Objective: The present study described the in silico dynamics behaviors of PKD1 in binding with selective and non-selective inhibitors and revealed the critical binding site residues for the selective kinase inhibition. Methods: Here, the three dimensional model of PKD1 was initially constructed by homology modeling along with binding site characterization to explore the non-conserved residues. Subsequently, two known inhibitors were docked to the catalytic site and the detailed ligand binding mechanisms and post binding dyanmics were investigated by molecular dynamics simulation and binding free energy calculations. Results: According to the binding site analysis, PKD1 serves several non-conserved residues in the G-loop, hinge and catalytic subunits. Among them, the residues including Leu662, His663, and Asp665 from hinge region made polar interactions with selective PKD1 inhibitor in docking simulation, which were further validated by the molecular dynamics simulation. Both inhibitors strongly influenced the structural dynamics of PKD1 and their computed binding free energies were in accordance with experimental bioactivity data. Conclusion: The identified non-conserved residues likely to play critical role on molecular reorganization and inhibitor selectivity. Taken together, this study explained the molecular basis of PKD1 specific inhibition, which may help to design new selective inhibitors for better therapies to overcome cancer and PKD1 dysregulated disorders.

scholarly journals The effect of protein mutations on drug binding suggests ensuing personalised drug selection

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shunzhou Wan ◽  
Deepak Kumar ◽  
Valentin Ilyin ◽  
Ussama Al Homsi ◽  
Gulab Sher ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Ligand Binding ◽  
Binding Free Energy ◽  
Personalised Medicine ◽  
Clinical Decision ◽  
Drug Binding ◽  
Dynamics Simulation ◽  
Breast Cancer Patients ◽  
Natural Ligand ◽  
Affinity Interaction

AbstractThe advent of personalised medicine promises a deeper understanding of mechanisms and therefore therapies. However, the connection between genomic sequences and clinical treatments is often unclear. We studied 50 breast cancer patients belonging to a population-cohort in the state of Qatar. From Sanger sequencing, we identified several new deleterious mutations in the estrogen receptor 1 gene (ESR1). The effect of these mutations on drug treatment in the protein target encoded by ESR1, namely the estrogen receptor, was achieved via rapid and accurate protein–ligand binding affinity interaction studies which were performed for the selected drugs and the natural ligand estrogen. Four nonsynonymous mutations in the ligand-binding domain were subjected to molecular dynamics simulation using absolute and relative binding free energy methods, leading to the ranking of the efficacy of six selected drugs for patients with the mutations. Our study shows that a personalised clinical decision system can be created by integrating an individual patient’s genomic data at the molecular level within a computational pipeline which ranks the efficacy of binding of particular drugs to variant proteins.

scholarly journals Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K

2021 ◽  
Author(s):  
Afsar Ali Mian ◽  
Isabella Haberbosch ◽  
Hazem Khamaisie ◽  
Abed Agbarya ◽  
Larissa Pietsch ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Inhibitors ◽  
Therapeutic Potential ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Binding Pocket ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Lung Cancer Patients

AbstractResistance remains the major clinical challenge for the therapy of Philadelphia chromosome–positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common “gatekeeper” mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph− cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia–like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

scholarly journals Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

2021 ◽  
Vol 22 (14) ◽  
pp. 7362
Author(s):  
Amina Ben Abla ◽  
Guilhem Boeuf ◽  
Ahmed Elmarjou ◽  
Cyrine Dridi ◽  
Florence Poirier ◽  
...  
Keyword(s):  
Specific Binding ◽  
Cellular Adhesion ◽  
High Yield ◽  
Adhesion Assay ◽  
Cell Binding ◽  
Affinity Tag ◽  
Binding Domains ◽  
Rational Development ◽  
Fibronectin Type Iii ◽  
Arginine Glycine Aspartic Acid

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.

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