Structure elucidation and structure confirmation
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The determination of the structure of unknown compounds extracted from marine organisms or other sources is one of the core activities of the NMR lab, through the research platform SmallStruct to which Mabcent-SFI has been the main client. Structure elucidation is done together with high resolution Mass Spectrometry (MS). MS is used to determine the exact mass from which the molecule formula and isotope pattern that best fits the mass is calculated. MS fragmentation of the molecule can give additional information about substructures split from the mother ion. NMR is then used to determine the exact molecule framework, using various experiments correlating the through-bond scalar couplings and the through-space dipolar couplings. The sensitivity limit to do a full characterization is approximately 1 mM of compound in 120 µl solvent, corresponding to 50 µg @ 400 Da and preferably at least 1 mg of a peptide < 5-10 kDa. Smaller amounts can be analyzed to some extent, which may be enough if closely related analogues are already known or if MS sequencing is sufficient. Read more…. |
Characterization of interactions between small molecules and proteins
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Knowledge of the interactions between small molecules and proteins or other macromolecules is extremely important in drug discovery. Characterization of interactions is therefore something that the NMR lab will be focusing on within the Drug Discovery and Design initiative together with partners in the pharmaceutical industry, like Lytix Biopharma: Particularly in the phase going from weakly binding fragment or early hit to a strong binding lead compound, NMR can provide useful binding data. Further, for macromolecules that are difficult to crystallize due to intrinsic properties, the binding mode of a molecule binding to a macromolecule can be characterized by a NMR, ITC and computational methods. This can be made to many different degrees, from plain confirmation of binding site or binding pose to high resolution structure determination of the bound molecule in complex with the target. Some of these methods do not require any isotope labeling. Read more… |
Fragment screening
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NMR is also a powerful technique for fragment/ligand screening. It’s strength lies in its ability to detect very weak binding (mM or stronger) and most methods do not require any isotop labelling or immobilization, but is performed directly on the native enzyme in aqueous solution. Cocktails of 10-30 compounds can be applied simultaneously and directly reveal which of the molecules in the cocktail that binds to the protein. Read more… |
Relative and absolute configuration/conformation
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The determination of the relative configuration of small molecules is a well established topic for NMR. A number of NMR parameters are sensitive to the relative positions of nuclei through space as well as the angles and dihedrals between the connecting bonds. Through accurate measurements of homonuclear and heteronuclear coupling constants, NOE buildup and residual dipolar couplings the relative configuration of a small molecule can in the majority of cases be determined. Recently, NMR has also been involved in the determination of absolute configuration, either directly by measuring RDCs in non-racemic gels (published success stories have been challenged, but robust protocols are coming, or so they say), or indirectly by validating structure ensembles for optical spectroscopy simulations. The pharmaceutical industry has also recently started to pay more attention to the three dimensional conformation distributions in solution of small flexible drugs candidates, something that NMR validated ensembles can describe. Read more… |
Macromolecule structure and dynamics
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One of the most traditional uses of NMR has been in structure determination of proteins and nucleic acids. Characterization of a target protein in solution can provide important knowledge for further drug discovery, especially for flexible proteins that are hard to crystallize or in the early stages of structural biology/drug discovery when crystallization conditions or strong enough inhibitors have not been discovered. For proteins, this typically requires the expression of 15N/13C isotope labelled protein in minimal media, for larger proteins also with partial och complete deuteration. Structure determination then involves assignment of the protein backbone and side chains using various 3D experiments, followed by the extraction of structural parameters like NOE contacts, backbone coupling constants and residual dipolar couplings. Once this work as been invested, a lot of interesting work can be done, including high resolution binding studies of ligands, metal binding, hydration, residual dynamics in various time scales, pH titrations of different protein protons etc etc. Read more… |
