NMR-solve method for rapid identification of bi-ligand drug candidates

A method for identifying an atom of a common ligand mimic that is proximal to an interface region of an enzyme;

• wherein the enzyme binds a common ligand (CL) or a common ligand mimic (CL mimic) at a common ligand site (CL site) and binds a specificity ligand (SL) at an adjacent specificity ligand site (SL site);
• wherein an interface region is defined as the atoms of the enzyme between the CL site and SL site, and atoms of an SL if bound to the enzyme;
• wherein the enzyme catalyzes a reaction involving the SL and a reactive atom of the CL;
• and
• wherein a CL reactive region is defined as the reactive atom of the CL and immediately adjacent atoms of the CL;
• comprising the steps of
• (a) identifying an atom of the interface region, comprising the steps of
• (1) binding a CL to the CL site of said enzyme, and then performing a multidimensional NMR experiment;
• (2) binding a chemically modified CL to the CL site of the enzyme, wherein the modification is to an atom of the CL reactive region, and then repeating the same NMR experiment;
• (3) comparing the spectra from steps (a)(1) and (a)(2) to identify an NMR cross-peak corresponding to a nucleus that is affected by the chemical modification, thereby identifying an atom of the interface region;
• then
• (b) identifying an atom in the CL mimic that is proximal to the interface region, comprising the steps of
• (1) binding a CL mimic to the CL site of said enzyme;
• (2) irradiating the nucleus of the interface atom identified in step (a);
• and
• (3) identifying in a multidimensional NMR experiment, an NMR cross-peak corresponding to a nucleus of the CL mimic that is perturbed by the irradiation of the interface nucleus, thereby identifying an atom of the CL mimic that is proximal to the interface region.

A method for identifying an atom of a common ligand mimic that is proximal to an interface region;

• wherein the enzyme binds a common ligand (CL) or a common ligand mimic (CL mimic) at a common ligand site (CL site) and binds a specificity ligand (SL) at an adjacent specificity ligand site (SL site);
• wherein an interface region is defined as the atoms of the enzyme between the CL site and SL site, and atoms of an SL if bound to the enzyme;
• wherein the enzyme catalyzes a reaction mechanism involving the SL and a reactive atom of the CL;
• wherein a CL reactive region is defined as the reactive atom of the CL and immediately adjacent atoms of the CL;
• and wherein an atom of the interface region has been identified;
• comprising the steps of
• (1) binding a CL mimic to the CL site;
• (2) perturbing the identified atom of the interface region;
• and
• (3) identifying an NMR cross-peak corresponding to an atom of the CL mimic that is perturbed by the perturbation of the interface atom, thereby identifying an atom of the CL mimic that is proximal to the interface region.

A method for identifying an atom of a common ligand mimic that is proximal to an interface region of an enzyme;

• wherein the enzyme binds a common ligand (CL) or a common ligand mimic (CL mimic) at a common ligand site (CL site) and binds a specificity ligand (SL) at an adjacent specificity ligand site (SL site);
• wherein an interface region is defined as the atoms of the enzyme between the CL site and SL site, and atoms of an SL if bound to the enzyme;
• wherein the enzyme catalyzes a reaction involving the SL and a reactive atom of the CL;
• wherein a CL reactive region is defined as the reactive atom of the CL and immediately adjacent atoms of the CL;
• and wherein an atom of the interface region has been identified;
• comprising the steps of
• (1) binding a CL mimic to the CL site of said enzyme and then performing a multidimensional NMR experiment;
• (2) binding a CL mimic to the CL site of a chemically modified enzyme, wherein the modification is isotopic labeling at the atom of the interface region that has been identified, and then repeating the same NMR experiment;
• (3) comparing the spectra from steps (1) and (2) to identify an NMR cross-peak corresponding to a nucleus that is affected by the chemical modification, thereby identifying an atom of the CL mimic that is proximal to the interface region.