BIO NMR Services

Verification of screening hits and definition of binding locations

BioNMR is the gold standard method for sorting out binding behaviors. A 15N-labeled version of the protein is used, so only the protein is observed in the NMR spectrum. Solution conditions can be changed in real time and additions can be made. The status of each residue in the protein is monitored. Upon addition of a potential binder, the response of the protein reveals if the added molecule binds to the protein or not, and if the binding is clean or causes the protein to unfold or aggregate. Based on which residues respond, the binding location can be determined (sometimes called “footprinting”).

After any screen, hit validation is essential, especially for difficult targets (e.g., protein-protein interactions) where false hits may bind more strongly than authentic hits.

For true binders, the binding footprint can immediately allow modeling of the docked ligand, which is essential for guiding a chemistry optimization strategy. Also, absence of a BioNMR response reliably indicates non-binding; no other technique can do this.

The amount of protein required for this type of experiment would be approximately 1-3 mg and the amount of compound needed would be about 0.2 mg. Labeling of the protein with 15N can be done easily if the protein is expressed in E. coli, but labeling can also be accomplished in yeast or mammalian cells, if necessary.

NMR screening of fragment libraries to obtain leads and Targeted Protein Degrader (TPD) ligands

Beyond examining molecules that are already identified as hits by other methods, BioNMR can be used as the primary detection method in a screen. Potential binders can be added as mixtures to increase throughput. When there is a positive response, the mixtures are broken out to find the actual hit. Because BioNMR is so sensitive, it can reliably detect weak binding and therefore is the method of choice for a fragment screen.

At first glance, BioNMR would appear to be a low-throughput method. However, it is exceptionally efficient since detection and validation are obtained simultaneously. The overall throughput from screen to authentic leads is therefore comparable to other approaches.

Because chemical shift patterns are unique to every protein, it is possible to perform a BioNMR screen with multiple proteins present. The response can clearly identify the protein to which the hit binds. This methodology can dramatically increase throughput for a screening library or can be used to assess selectivity. BioNMR is the only technique that can do this.

Elucidation of full detailed structures

BioNMR can provide 3D structures of proteins, protein-ligand complexes, oligonucleotides, peptides, macrocycles, and TPDs. While many proteins and oligonucleotides are amenable to X-ray crystallography and cryogenic electron microscopy, some are not. Further, because it is performed in solution, BioNMR can resolve concerns about the effects of crystal packing. BioNMR can also usually provide medium-resolution information faster than those other methods.

Typically, medium-sized macromolecules (e.g., peptides, macrocycles, TPDs) do not crystallize, but can be readily studied by BioNMR. BioNMR also allows the selection of any solvent condition. Detailed structures of these molecules can guide the design of analogs that are more potent, more stable, and/or more bioavailable.

Application of advanced NMR methods to small molecules

The sophisticated NMR techniques needed to deal with macromolecules can be applied to smaller molecules, to answer complex questions of structure or stereochemistry. NMR can provide a rigorous verification of primary structure.