The role of water and protein flexibility in the structure-based virtual screening of allosteric GPCR modulators: an mGlu5 receptor case study

Abstract Stabilizing unique receptor conformations, allosteric modulators of G-protein coupled receptors (GPCRs) might open novel treatment options due to their new pharmacological action, their enhanced specificity and selectivity in both binding and signaling. Ligand binding occurs at intrahelical allosteric sites and involves significant induced fit effects that include conformational changes in the local protein environment and water networks. Based on the analysis of available crystal structures of metabotropic glutamate receptor 5 (mGlu5) we investigated these effects in the binding of mGlu5 receptor negative allosteric modulators. A large set of retrospective virtual screens revealed that the use of multiple protein structures and the inclusion of selected water molecules improves virtual screening performance compared to conventional docking strategies. The role of water molecules and protein flexibility in ligand binding can be taken into account efficiently by the proposed docking protocol that provided reasonable enrichment of true positives. This protocol is expected to be useful also for identifying intrahelical allosteric modulators for other GPCR targets.

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The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

Biochemical Journal ◽

10.1042/bj3330811 ◽

1998 ◽

Vol 333 (3) ◽

pp. 811-816 ◽

Cited By ~ 14

Author(s):

Antonio PÁRRAGA ◽

Isabel GARCÍA-SÁEZ ◽

Sinead B. WALSH ◽

Timothy J. MANTLE ◽

Miquel COLL

Keyword(s):

Conformational Changes ◽

Active Site ◽

Three Dimensional ◽

Dimensional Structure ◽

Water Molecules ◽

X Ray Diffraction ◽

Glutathione S Transferase ◽

X Ray ◽

The Right

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

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The β-tail domain (βTD) regulates physiologic ligand binding to integrin CD11b/CD18

Blood ◽

10.1182/blood-2005-11-056689 ◽

2006 ◽

Vol 109 (8) ◽

pp. 3513-3520 ◽

Cited By ~ 29

Author(s):

Vineet Gupta ◽

Annette Gylling ◽

José Luis Alonso ◽

Takashi Sugimori ◽

Petre Ianakiev ◽

...

Keyword(s):

Electron Microscopy ◽

Ligand Binding ◽

Conformational Changes ◽

Divalent Cations ◽

Contact Region ◽

Allosteric Modulator ◽

Tail Domain ◽

Wild Type ◽

High Affinity

Abstract Crystallographic and electron microscopy studies revealed genuflexed (bent) integrins in both unliganded (inactive) and physiologic ligandbound (active) states, suggesting that local conformational changes are sufficient for activation. Herein we have explored the role of local changes in the contact region between the membrane-proximal β-tail domain (βTD) and the ligand-binding βA domain of the bent conformation in regulating interaction of integrin CD11b/CD18 (αMβ2) with its physiologic ligand iC3b. We replaced the βTD CD loop residues D658GMD of the CD18 (β2) subunit with the equivalent D672SSG of the β3 subunit, with AGAA or with NGTD, expressed the respective heterodimeric receptors either transiently in epithelial HEK293T cells or stably in leukocytes (K562), and measured their ability to bind iC3b and to conformation-sensitive mAbs. In the presence of the physiologic divalent cations Ca2+ plus Mg2+ (at 1 mM each), the modified integrins showed increased (in HEK293) or constitutive (in K562) binding to iC3b compared with wild-type receptors. K562 expressing the βTD-modified integrins bound in Ca2+Mg2+ to the βA-directed high-affinity reporter mAb 24 but not to mAb KIM127, a reporter of the genu-straightened state. These data identify a role for the membrane proximal βTD as an allosteric modulator of integrin activation.

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GPCRs through the keyhole: the role of protein flexibility in ligand binding to β-adrenoceptors

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2016.1226197 ◽

2016 ◽

Vol 35 (12) ◽

pp. 2604-2619 ◽

Cited By ~ 3

Author(s):

Abigail L. Emtage ◽

Shailesh N. Mistry ◽

Peter M. Fischer ◽

Barrie Kellam ◽

Charles A. Laughton

Keyword(s):

Ligand Binding ◽

Protein Flexibility

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Thrombin-thrombomodulin interaction: energetics and potential role of water as an allosteric effector

Biochemical Journal ◽

10.1042/bj3100049 ◽

1995 ◽

Vol 310 (1) ◽

pp. 49-53 ◽

Cited By ~ 3

Author(s):

R De Cristofaro ◽

M Picozzi ◽

E De Candia ◽

B Rocca ◽

R Landolfi

Keyword(s):

Conformational Changes ◽

Potential Role ◽

Competitive Inhibition ◽

Entropy Change ◽

Bulk Water ◽

Water Molecules ◽

Rabbit Lung ◽

Allosteric Effector ◽

Free Energy Of Binding

The interaction of rabbit lung thrombomodulin (TM) and C-terminal hirudin 54-65 fragment (Hir54-65) with human alpha-thrombin were investigated by exploiting their competitive inhibition of thrombin-fibrinogen interaction. Measurements of Ki values for TM and Hir54-65 interactions with human alpha-thrombin performed over a temperature range spanning from 10 to 40 degrees C showed a constant enthalpy for both ligands. The enthalpic and entropic contributions to the free energy of binding, however, are different for TM and the hirudin peptide. The calculated values of delta H and delta S, in fact, were -47.3 +/- 2.51 kJ (-11.3 +/- 0.6 kcal)/mol and -42.7 +/- 7.9 J (-10.2 +/- 1.9 cal)/mol.K for the hirudin peptide, while being -22.9 +/- 2.09 kJ (-5.47 +/- 0.5 kcal)/mol and 102.50 +/- 6.69 J (24.5 +/- 1.6 cal)/mol.K respectively for TM binding. These findings indicate that the interaction between thrombin and Hir54-65 is largely driven by the enthalpic contribution, whereas the positive entropy change is the driving force for the formation of the thrombin-TM complex. In other experiments performed in the presence of various concentrations of either sorbitol or sucrose it could be demonstrated that the value of the equilibrium association constant for thrombin-TM interaction increases as a function of the osmotic pressure, while the thrombin-Hir54-65 interaction was not affected by the same conditions. Moreover, control experiments showed that no major conformational changes are produced on TM by osmotic pressures used in the present study. From these experiments it was calculated that roughly 35 water molecules are released into the bulk water upon TM binding. Such a phenomenon, which is likely to be responsible for the entropic change described above, indicates the relevance of hydration processes for the formation of the thrombin-TM adduct.

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Repositioning Dequalinium as Potent Muscarinic Allosteric Ligand by Combining Virtual Screening Campaigns and Experimental Binding Assays

International Journal of Molecular Sciences ◽

10.3390/ijms21175961 ◽

2020 ◽

Vol 21 (17) ◽

pp. 5961

Author(s):

Angelica Mazzolari ◽

Silvia Gervasoni ◽

Alessandro Pedretti ◽

Laura Fumagalli ◽

Rosanna Matucci ◽

...

Keyword(s):

Virtual Screening ◽

Allosteric Modulation ◽

Allosteric Modulators ◽

Binding Assays ◽

Docking Protocol ◽

Consensus Models ◽

Binding Cavity ◽

Orthosteric Ligands ◽

G Protein Coupled

Structure-based virtual screening is a truly productive repurposing approach provided that reliable target structures are available. Recent progresses in the structural resolution of the G-Protein Coupled Receptors (GPCRs) render these targets amenable for structure-based repurposing studies. Hence, the present study describes structure-based virtual screening campaigns with a view to repurposing known drugs as potential allosteric (and/or orthosteric) ligands for the hM2 muscarinic subtype which was indeed resolved in complex with an allosteric modulator thus allowing a precise identification of this binding cavity. First, a docking protocol was developed and optimized based on binding space concept and enrichment factor optimization algorithm (EFO) consensus approach by using a purposely collected database including known allosteric modulators. The so-developed consensus models were then utilized to virtually screen the DrugBank database. Based on the computational results, six promising molecules were selected and experimentally tested and four of them revealed interesting affinity data; in particular, dequalinium showed a very impressive allosteric modulation for hM2. Based on these results, a second campaign was focused on bis-cationic derivatives and allowed the identification of other two relevant hM2 ligands. Overall, the study enhances the understanding of the factors governing the hM2 allosteric modulation emphasizing the key role of ligand flexibility as well as of arrangement and delocalization of the positively charged moieties.

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Network analysis of dynamically important residues in protein structures mediating ligand-binding conformational changes

European Biophysics Journal ◽

10.1007/s00249-019-01384-1 ◽

2019 ◽

Vol 48 (6) ◽

pp. 559-568 ◽

Cited By ~ 2

Author(s):

Tadeo E. Saldaño ◽

Silvio C. E. Tosatto ◽

Gustavo Parisi ◽

Sebastian Fernandez-Alberti

Keyword(s):

Network Analysis ◽

Ligand Binding ◽

Conformational Changes ◽

Protein Structures

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Exploring the Role of Water Molecules in the Ligand Binding Domain of PDE4B and PDE4D: Virtual Screening Based Molecular Docking of Some Active Scaffolds

Current Computer - Aided Drug Design ◽

10.2174/1573409914666181105153543 ◽

2019 ◽

Vol 15 (4) ◽

pp. 334-366

Author(s):

Priya Singh ◽

Mitali Mishra ◽

Shivangi Agarwal ◽

Samaresh Sau ◽

Arun K. Iyer ◽

...

Keyword(s):

Molecular Docking ◽

Amino Acid ◽

Water Molecule ◽

Ligand Binding ◽

Binding Affinity ◽

Binding Domain ◽

Ligand Binding Domain ◽

Water Molecules ◽

Docking Experiment

Background: The phosphodiesterase (PDE) is a superfamily represented by four genes: PDE4A, B,C, and D which cause the hydrolysis of phosphodiester bond of cAMP to yield inactive AMP. c-AMP catalyzing enzyme is predominant in inflammatory and immunomodulatory cells. Therapy to treat Chronic Obstructive Pulmonary Disease (COPD) with the use of PDE4 inhibitors is highly envisaged. Objective: A molecular docking experiment with large dataset of diverse scaffolds has been performed on PDE4 inhibitors to analyze the role of amino acid responsible for binding and activation of the secondary transmitters. Apart from the general docking experiment, the main focus was to discover the role of water molecules present in the ligand-binding domain. Methods: All the compounds were docked in the PDE4B and PDE4D active cavity to produce the free binding energy scores and spatial disposition/orientation of chemical groups of inhibitors around the cavity. Under uniform condition, the experiments were carried out with and without water molecules in the LBD. The exhaustive study was carried out on the Autodock 4.2 software and explored the role of water molecules present in the binding domain. Results: In presence of water molecule, Roflumilast has more binding affinity (-8.48 Kcal/mol with PDE4B enzyme and -8.91 Kcal/mol with PDE4D enzyme) and forms two hydrogen bonds with Gln443 and Glu369 and amino acid with PDE4B and PDE4D enzymes respectively. While in absence of water molecule its binding affinity has decreased (-7.3 Kcal/mol with PDE4B enzyme and -5.17 Kcal/mol with PDE4D enzyme) as well as no H-bond interactions were observed. Similar observation was made with clinically tested molecules. Conclusion: In protein-ligand binding interactions, appropriate selection of water molecules facilitated the ligand binding, which eventually enhances the efficiency as well as the efficacy of ligand binding.

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