| 1. |
- Gutlerrez-de-Teran, Hugo, et al.
(författare)
-
The Role of a Sodium Ion Binding Site in the Allosteric Modulation of the A(2A) Adenosine G Protein-Coupled Receptor
- 2013
-
Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 21:12, s. 2175-2185
-
Tidskriftsartikel (refereegranskat)abstract
- The function of G protein-coupled receptors (GPCRs) can be modulated by a number of endogenous allosteric molecules. In this study, we used molecular dynamics, radioligand binding, and thermostability experiments to elucidate the role of the recently discovered sodium ion binding site in the allosteric modulation of the human A(2A) adenosine receptor, conserved among class A GPCRs. While the binding of antagonists and sodium ions to the receptor was noncompetitive in nature, the binding of agonists and sodium ions appears to require mutually exclusive conformational states of the receptor. Anniloride analogs can also bind to the sodium binding pocket, showing distinct patterns of agonist and antagonist modulation. These findings suggest that physiological concentrations of sodium ions affect functionally relevant conformational states of GPCRs and can help to design novel synthetic allosteric modulators or bitopic ligands exploiting the sodium ion binding pocket.
|
|
| 2. |
- Liu, Wei, et al.
(författare)
-
Serial Femtosecond Crystallography of G Protein-Coupled Receptors
- 2013
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 342:6165, s. 1521-1524
-
Tidskriftsartikel (refereegranskat)abstract
- X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment.
|
|
| 3. |
- Luttens, Andreas, 1993-
(författare)
-
Discovery of Chemical Probes through Structure-based Virtual Screening of Vast Compound Databases
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bioactive molecules have traditionally been discovered through labor-intensive screening methods in which individual compounds are tested against specific protein targets or cells to identify those that produce the desired biological effect. However, these approaches have significant limitations. Firstly, the number of molecules that can be tested in a standard laboratory is restricted, and the acquisition and curation of these compounds come at a high cost. Secondly, these methods are time-consuming because each compound must be tested individually, and they are confined to small libraries with very limited chemical space coverage. In contrast, structure-based virtual screening can rapidly predict a molecule's interaction with a target protein, allowing for the evaluation of enormous libraries of chemical substances. Furthermore, this approach is not restricted to physically available molecules and can be extended to virtual compounds. Commercial chemical space has recently grown exponentially and currently contains several billion molecules that can be readily synthesized and delivered for experimental testing within weeks. Despite the enormous potential of these databases for drug discovery, they also pose new challenges, and development of effective strategies is required to explore ultralarge libraries. The goal of this thesis was to develop and apply novel strategies focused on exploring the potential of ultralarge chemical libraries using structure-based virtual screening. Publication I summarizes best practices on large-scale virtual screening and benchmarking protocols for molecular docking calculations. Publication II describes a docking screen of several hundred million lead-like molecules against the SARS-CoV-2 main protease, leading to promising starting points for development of coronavirus inhibitors. The binding modes predicted by docking were confirmed experimentally by X-ray crystallography. After several rounds of optimization, nanomolar broad-spectrum inhibitors with antiviral effects against coronaviruses in cell models were discovered. Manuscript III demonstrates how machine learning can be used to accelerate virtual screening campaigns. Classification models were trained on docking scores to identify promising molecules in ultralarge libraries relevant to the protein target of interest. The classification algorithms were able to reduce a multi-billion-scale library to a subset of high-confidence candidates with improved docking scores. Manuscript IV focuses on large-scale fragment docking to identify compounds binding to 8-oxoguanine glycosylase 1 and how to efficiently optimize them to potent inhibitors. The docking scoring function was able to correctly predict binding modes of the experimental hits and optimization led to submicromolar inhibitors with anti-inflammatory and anti-cancer effects in cell models. Publication V presents how docking of tailored virtual libraries of nature-inspired macrocycles led to potent disruptors of the KEAP1-Nrf2 complex. The results of this thesis highlight that large-scale virtual screening is a resourceful tool to discover ligands of a wide variety of drug targets.
|
|
| 4. |
- Massink, Arnault, et al.
(författare)
-
Sodium Ion Binding Pocket Mutations and Adenosine A(2A) Receptor Function
- 2015
-
Ingår i: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0026-895X .- 1521-0111. ; 87:2, s. 305-313
-
Tidskriftsartikel (refereegranskat)abstract
- Recently we identified a sodium ion binding pocket in a high-resolution structure of the human adenosine A(2A) receptor. In the present study we explored this binding site through site-directed mutagenesis and molecular dynamics simulations. Amino acids in the pocket were mutated to alanine, and their influence on agonist and antagonist affinity, allosterism by sodium ions and amilorides, and receptor functionality was explored. Mutation of the polar residues in the Na+ pocket were shown to either abrogate (D52A(2.50) and N284A(7.49)) or reduce (S91A(3.39), W246A(6.48), and N280A(7.45)) the negative allosteric effect of sodium ions on agonist binding. Mutations D52A(2.50) and N284A(7.49) completely abolished receptor signaling, whereas mutations S91A(3.39) and N280A(7.45) elevated basal activity and mutations S91A(3.39), W246A(6.48), and N280A(7.45) decreased agonist-stimulated receptor signaling. In molecular dynamics simulations D52A(2.50) directly affected the mobility of sodium ions, which readily migrated to another pocket formed by Glu13(1.39) and His278(7.43). The D52A(2.50) mutation also decreased the potency of amiloride with respect to ligand displacement but did not change orthosteric ligand affinity. In contrast, W246A(6.48) increased some of the allosteric effects of sodium ions and amiloride, whereas orthosteric ligand binding was decreased. These new findings suggest that the sodium ion in the allosteric binding pocket not only impacts ligand affinity but also plays a vital role in receptor signaling. Because the sodium ion binding pocket is highly conserved in other class A G protein-coupled receptors, our findings may have a general relevance for these receptors and may guide the design of novel synthetic allosteric modulators or bitopic ligands.
|
|
| 5. |
- Weierstall, Uwe, et al.
(författare)
-
Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography
- 2014
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5, s. 3309-
-
Tidskriftsartikel (refereegranskat)abstract
- Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-mu m-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.
|
|
