| 1. |
- Altincekic, Nadine, et al.
(författare)
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Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.
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| 2. |
- Altincekic, Nadide, et al.
(författare)
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Targeting the Main Protease (Mpro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies
- 2024
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 19:2, s. 563-574
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Tidskriftsartikel (refereegranskat)abstract
- The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.
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| 3. |
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| 4. |
- Dawed, Adem Y., et al.
(författare)
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Pharmacogenomics of GLP-1 receptor agonists : a genome- wide analysis of observational data and large randomised controlled trials
- 2023
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Ingår i: The Lancet Diabetes and Endocrinology. - : ELSEVIER SCIENCE INC. - 2213-8587 .- 2213-8595. ; 11:1, s. 33-41
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Tidskriftsartikel (refereegranskat)abstract
- Background: In the treatment of type 2 diabetes, GLP-1 receptor agonists lower blood glucose concentrations, body weight, and have cardiovascular benefits. The efficacy and side effects of GLP-1 receptor agonists vary between people. Human pharmacogenomic studies of this inter-individual variation can provide both biological insight into drug action and provide biomarkers to inform clinical decision making. We therefore aimed to identify genetic variants associated with glycaemic response to GLP-1 receptor agonist treatment. Methods:In this genome-wide analysis we included adults (aged & GE;18 years) with type 2 diabetes treated with GLP-1 receptor agonists with baseline HbA1c of 7% or more (53 mmol/mol) from four prospective observational cohorts (DIRECT, PRIBA, PROMASTER, and GoDARTS) and two randomised clinical trials (HARMONY phase 3 and AWARD). The primary endpoint was HbA1c reduction at 6 months after starting GLP-1 receptor agonists. We evaluated variants in GLP1R, then did a genome-wide association study and gene-based burden tests. Findings:4571 adults were included in our analysis, of these, 3339 (73%) were White European, 449 (10%) Hispanic, 312 (7%) American Indian or Alaskan Native, and 471 (10%) were other, and around 2140 (47%) of the participants were women. Variation in HbA1c reduction with GLP-1 receptor agonists treatment was associated with rs6923761G & RARR;A (Gly168Ser) in the GLP1R (0.08% [95% CI 0.04-0.12] or 0.9 mmol/mol lower reduction in HbA1c per serine, p=6.0 x 10-5) and low frequency variants in ARRB1 (optimal sequence kernel association test p=6.7 x 10-8), largely driven by rs140226575G & RARR;A (Thr370Met; 0.25% [SE 0.06] or 2.7 mmol/mol [SE 0.7] greater HbA1c reduction per methionine, p=5.2 x 10-6). A similar effect size for the ARRB1 Thr370Met was seen in Hispanic and American Indian or Alaska Native populations who have a higher frequency of this variant (6-11%) than in White European populations. Combining these two genes identified 4% of the population who had a 30% greater reduction in HbA1c than the 9% of the population with the worse response. Interpretation:This genome-wide pharmacogenomic study of GLP-1 receptor agonists provides novel biological and clinical insights. Clinically, when genotype is routinely available at the point of prescribing, individuals with ARRB1 variants might benefit from earlier initiation of GLP-1 receptor agonists.
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| 5. |
- Dawed, Adem Y., et al.
(författare)
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Variation in the plasma membrane monoamine transporter (PMAT) (encoded by SLC29A4) and organic cation transporter 1 (OCT1) (encoded by SLC22A1) and gastrointestinal intolerance to metformin in type 2 diabetes : An IMI direct study
- 2019
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Ingår i: Diabetes Care. - : American Diabetes Association. - 0149-5992 .- 1935-5548. ; 42:6, s. 1027-1033
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE Gastrointestinal adverse effects occur in 20–30% of patients with metformin-treated type 2 diabetes, leading to premature discontinuation in 5–10% of the cases. Gastrointestinal intolerance may reflect localized high concentrations of metformin in the gut. We hypothesized that reduced transport of metformin via the plasma membrane monoamine transporter (PMAT) and organic cation transporter 1 (OCT1) could increase the risk of severe gastrointestinal adverse effects. RESEARCH DESIGN AND METHODS The study included 286 severe metformin-intolerant and 1,128 metformin-tolerant individuals from the IMI DIRECT (Innovative Medicines Initiative: DIabetes REsearCh on patient straTification) consortium. We assessed the association of patient characteristics, concomitant medication, and the burden of mutations in the SLC29A4 and SLC22A1 genes on odds of intolerance. RESULTS Women (P < 0.001) and older people (P < 0.001) were more likely to develop metformin intolerance. Concomitant use of transporter-inhibiting drugs increased the odds of intolerance (odds ratio [OR] 1.72, P < 0.001). In an adjusted logistic regression model, the G allele at rs3889348 (SLC29A4) was associated with gastrointestinal intolerance (OR 1.34, P = 0.005). rs3889348 is the top cis-expression quantitative trait locus for SLC29A4 in gut tissue where carriers of the G allele had reduced expression. Homozygous carriers of the G allele treated with transporter-inhibiting drugs had more than three times higher odds of intolerance compared with carriers of no G allele and not treated with inhibiting drugs (OR 3.23, P < 0.001). Use of a genetic risk score derived from rs3889348 and SLC22A1 variants found that the odds of intolerance were more than twice as high in individuals who carry three or more risk alleles compared with those carrying none (OR 2.15, P = 0.01). CONCLUSIONS These results suggest that intestinal metformin transporters and concomitant medications play an important role in the gastrointestinal adverse effects of metformin.
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| 6. |
- Robertson, Angus J., et al.
(författare)
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NMR Observation of Sulfhydryl Signals in SARS-CoV-2 Main Protease Aids Structural Studies
- 2022
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 23:19
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Tidskriftsartikel (refereegranskat)abstract
- The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro/Nsp5), is a key antiviral drug target. NMR spectroscopy of this large system proved challenging and resonance assignments have remained incomplete. Here we present the near-complete (>97 %) backbone assignments of a C145A variant of Mpro (MproC145A) both with, and without, the N-terminal auto-cleavage substrate sequence, in its native homodimeric state. We also present SILLY (Selective Inversion of thioL and Ligand for NOESY), a simple yet effective pseudo-3D NMR experiment that utilizes NOEs to identify interactions between Cys-thiol or aliphatic protons, and their spatially proximate backbone amides in a perdeuterated protein background. High protection against hydrogen exchange is observed for 10 of the 11 thiol groups in MproC145A, even those that are partially accessible to solvent. A combination of SILLY methods and high-resolution triple-resonance NMR experiments reveals site-specific interactions between Mpro, its substrate peptides, and other ligands, which present opportunities for competitive binding studies in future drug design efforts.
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| 7. |
- Niemi, MEK, et al.
(författare)
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- 2021
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