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Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor.

ABSTRACT: Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.

SUBMITTER: Igonet S

PROVIDER: S-EPMC5970182 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor.

Igonet Sébastien S Raingeval Claire C Cecon Erika E Pučić-Baković Maja M Lauc Gordan G Cala Olivier O Baranowski Maciej M Perez Javier J Jockers Ralf R Krimm Isabelle I Jawhari Anass A

Scientific reports 20180525 1

Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A ...[more]

PMID: 29802269

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Project description:ObjectiveThe American Indian/Alaska Native (AI/AN) population in the U.S. is thriving in spite of settler colonialist efforts of erasure. AI/AN people, however, continue to experience persistent health disparities including a disproportionate burden of substance use and sexually transmitted diseases/infections (STDs/STIs), as well as a disproportionate lack of public health STD screening services and STD prevention interventions grounded in AI/AN social contexts, experiences, and epistemologies. The present study explored how stressors and protective factors based on the Indigenist Stress Coping framework predict STD screening outcomes among Native adults.MethodsWe analyzed baseline self-report data from 254 Native adults ages 18-55 years with recent binge substance use who were enrolled in an evaluation of "EMPWR," a two-session STD risk reduction program in a rural, reservation-based community in the U.S. Southwest. Logistic regression models with robust variance were used to estimate odds ratios of lifetime STD testing for the theoretical stressors and cultural buffers.ResultsA little over half the sample were males (52.5%, n = 136), with a mean age of 33.6 years (SD = 8.8). The majority (76.7%, n = 195) reported having ever been screened for STD in their life. Discrimination score were significantly associated with lifetime STD testing: The higher discrimination was associated with lower odds of STD testing in the fully adjusted model (aOR = 0.40, 95%CI: 0.18, 0.92). The effects of AI/AN-specific cultural buffer such as participation in traditional practices on STD testing outcomes was in the expected positive direction, even though the association was not statistically significant. Household size was significantly associated with STD screening: The higher the number of people lived together in the house, the higher the odds of STD testing in the fully adjusted model (aOR = 1.19, 95%CI: 1.04, 1.38).ConclusionOur findings suggest that STD prevention programs should take into consideration AI/AN-specific historical traumatic stressors such as lifetime discrimination encounters and how these interact to drive or discourage sexual health services at local clinics. In addition, larger household size may be a protective factor functioning as a form of social support, and the extended family's role should be taken into consideration. Future research should consider improvement in measurements of AI/AN enculturation constructs.

Dataset Information

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor.

Publications

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor.

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