Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

Iatmanen-Harbi, Soria; Senicourt, Lucile; Papadopoulos, Vassilios; Lequin, Olivier; Lacapère, J

doi:10.3390/ijms20061444

Cited by 12 publications

(21 citation statements)

References 47 publications

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“…The drug and cholesterol binding domains were found to be in distinct domains of the protein results confirmed by NMR [87,88,90]. Moreover, these findings were further confirmed in structural studies by NMR and crystallography studies that reported the atomic structure of TSPO [91][92][93][94][95][96]. These studies also proposed that the functional TSPO is a dimer, that ligand binding to TSPO can promote cholesterol movement, and that cholesterol is an allosteric regulator of TSPO [91,93,94,97].…”

Section: Molecular Structure and Cellular Functionsmentioning

confidence: 52%

Cellular sources of TSPO expression in healthy and diseased brain

Nutma

Ceyzériat

Amor

et al. 2021

Eur J Nucl Med Mol Imaging

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115

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The 18 kDa translocator protein (TSPO) is a highly conserved protein located in the outer mitochondrial membrane. TSPO binding, as measured with positron emission tomography (PET), is considered an in vivo marker of neuroinflammation. Indeed, TSPO expression is altered in neurodegenerative, neuroinflammatory, and neuropsychiatric diseases. In PET studies, the TSPO signal is often viewed as a marker of microglial cell activity. However, there is little evidence in support of a microglia-specific TSPO expression. This review describes the cellular sources and functions of TSPO in animal models of disease and human studies, in health, and in central nervous system diseases. A discussion of methods of analysis and of quantification of TSPO is also presented. Overall, it appears that the alterations of TSPO binding, their cellular underpinnings, and the functional significance of such alterations depend on many factors, notably the pathology or the animal model under study, the disease stage, and the involved brain regions. Thus, further studies are needed to fully determine how changes in TSPO binding occur at the cellular level with the ultimate goal of revealing potential therapeutic pathways.

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Section: Molecular Structure and Cellular Functionsmentioning

confidence: 52%

Cellular sources of TSPO expression in healthy and diseased brain

Nutma

Ceyzériat

Amor

et al. 2021

Eur J Nucl Med Mol Imaging

Self Cite

110

115

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“…The spontaneous change that we observe in the orientation of the F100 side chain may indicate that it is involved in placing the ligand inside the binding pocket, but not crucial for its binding, and that this role is left to F99 and Y34, as suggested by [58]. Deeper studies will of course need to be done to explore in detail the exact role of these three residues.…”

Section: Pk11195 Interactions With the Htspo Modelmentioning

confidence: 82%

“…(iii) The W95XPXF99 motif is fully conserved among human, mouse, and Rs TSPO, while the Bc TSPO sequence differs significantly in this region ( Figure 1 , blue rectangle). This motif is conserved also in other prokaryotic and eukaryotic TSPO sequences [ 40 , 57 ], and it was suggested to play a role in oligomerization processes [ 49 ], as well as in ligand binding [ 34 , 58 ]. In the Mo TSPO experimental structure, W95 points into the binding cavity and F99 is oriented toward the membrane, whereas in the Rs TSPO structure, both residues point into the binding pocket.…”

Section: Results and Discussionmentioning

confidence: 99%

The Interplay of Cholesterol and Ligand Binding in hTSPO from Classical Molecular Dynamics Simulations

et al. 2021

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The translocator protein (TSPO) is a 18kDa transmembrane protein, ubiquitously present in human mitochondria. It is overexpressed in tumor cells and at the sites of neuroinflammation, thus representing an important biomarker, as well as a promising drug target. In mammalian TSPO, there are cholesterol–binding motifs, as well as a binding cavity able to accommodate different chemical compounds. Given the lack of structural information for the human protein, we built a model of human (h) TSPO in the apo state and in complex with PK11195, a molecule routinely used in positron emission tomography (PET) for imaging of neuroinflammatory sites. To better understand the interactions of PK11195 and cholesterol with this pharmacologically relevant protein, we ran molecular dynamics simulations of the apo and holo proteins embedded in a model membrane. We found that: (i) PK11195 stabilizes hTSPO structural fold; (ii) PK11195 might enter in the binding site through transmembrane helices I and II of hTSPO; (iii) PK11195 reduces the frequency of cholesterol binding to the lower, N–terminal part of hTSPO in the inner membrane leaflet, while this impact is less pronounced for the upper, C–terminal part in the outer membrane leaflet, where the ligand binding site is located; (iv) very interestingly, cholesterol most frequently binds simultaneously to the so-called CRAC and CARC regions in TM V in the free form (residues L150–X–Y152–X(3)–R156 and R135–X(2)–Y138–X(2)–L141, respectively). However, when the protein is in complex with PK11195, cholesterol binds equally frequently to the CRAC–resembling motif that we observed in TM I (residues L17–X(2)–F20–X(3)–R24) and to CRAC in TM V. We expect that the CRAC–like motif in TM I will be of interest in future experimental investigations. Thus, our MD simulations provide insight into the structural features of hTSPO and the previously unknown interplay between PK11195 and cholesterol interactions with this pharmacologically relevant protein.

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“…Drug-protein interactions can be observed as spectral changes in the far-UV region rather than in the near-UV region because they are frequently accompanied by changes in protein secondary structures. [18][19][20][21][22] However, not only proteins and drug substances but also solvents generally show light absorption in the far-UV region. Such light absorption imposes limits on the concentrations of target proteins and drug substances for CD spectroscopy, which can make it difficult to detect changes in the CD spectra originating from drug-protein interactions.…”

Section: Circular Dichroism and Absorption Spectroscopymentioning

confidence: 99%

“…CD spectroscopy is used for observing structural changes of proteins and nucleic acids upon ligand binding. [18][19][20][21][22] DSC is used for observing the binding of ligands to proteins as an increase in melting temperature of the proteins, [22][23][24][25][26] while ITC is used for directly measuring the heat that is associated with ligand binding. 22,27 In addition, the solubilization capacities of DMSO, ACN, and dioxane for poorly soluble compounds were examined to evaluate their potential for practical use in drug-protein interaction analyses.…”

Section: Introductionmentioning

confidence: 99%

Technical Capabilities and Limitations of Optical Spectroscopy and Calorimetry Using Water-Miscible Solvents: The Case of Dimethyl Sulfoxide, Acetonitrile, and 1,4-Dioxane

Hirano

Nagatoishi

Wada

et al. 2020

Journal of Pharmaceutical Sciences

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In drug development, water-miscible solvents are commonly used to dissolve drug substances. Typical routine procedures in drug development include dilution of the stock drug solution into an aqueous solution containing target macromolecules for drug binding assays. However, water-miscible solvents impose some technical limitations on the assays on account of their light absorption and heat capacity. Here, we examined the effects of the dilution of 3 water-miscible solvents, that is, dimethyl sulfoxide, acetonitrile, and 1,4-dioxane, on the baseline stability and signal/noise ratio in circular dichroism spectroscopy, isothermal titration calorimetry, and differential scanning calorimetry. Dimethyl sulfoxide and 1,4-dioxane affect the signal/noise ratio of circular dichroism spectra at typically used concentrations due to their light absorbance. The water-miscible solvents generate interfering signals in the isothermal titration calorimetry due to their mixing heat. They show negative or positive slope in the differential scanning calorimetry. Such interfering effects of the solvents are reduced by appropriate dilution according to the analytical techniques. Because the water-miscible solvents have solubilization capacity for alkyl chain moieties and aromatic moieties of chemicals, drug substances containing these moieties can be dissolved into the solvents and then subjected to the analyses to examine their interactions with target proteins after appropriate dilution of the drug solutions.

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Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

Cited by 12 publications

References 47 publications

Cellular sources of TSPO expression in healthy and diseased brain

Cellular sources of TSPO expression in healthy and diseased brain

The Interplay of Cholesterol and Ligand Binding in hTSPO from Classical Molecular Dynamics Simulations

Technical Capabilities and Limitations of Optical Spectroscopy and Calorimetry Using Water-Miscible Solvents: The Case of Dimethyl Sulfoxide, Acetonitrile, and 1,4-Dioxane

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