Methods to identify amino acid residues involved in macromolecular binding and uses therefor

Patent metadata

Field	Value
Publication number	US-10431325-B2
Application number	US-201314419229-A
Country	US
Kind code	B2
Filing date	Aug 2, 2013
Priority date	Aug 3, 2012
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

The present disclosure relates to methods and computational tools based, at least in part, on computer simulations that identify hot-spot amino acid residues and binding-region amino acid residues of a protein.

First claim

We claim:

1. An in vitro method of producing a protein variant which exhibits increased binding to a macromolecule, the method comprising
replacing or deleting one or more hot-spot amino acid residues in the protein by site-directed mutagenesis, wherein binding affinity of the modified protein to a macromolecule is increased, as compared to a corresponding protein lacking the replacement or deletion of one or more hot-spot amino acid residues,
(i) wherein the one or more hot-spot amino acid residues were identified using a computer-implemented method for predicting hot-spot amino acid residues of the protein, or portion thereof,
wherein the protein, or portion thereof, was represented in a structural model comprising a plurality of amino acid residues, and wherein
(i) a cluster of highly hydrophobic amino acid residues was selected from the plurality of amino acid residues of the model, wherein the cluster comprised two or more amino acid residues each having an effective-hydrophobicity greater than a chosen threshold, and wherein each amino acid residue in the cluster was within a first defined distance of at least one other amino acid residue in the cluster;
(ii) one or more solvent-exposed polar amino acid residues within a second defined distance of at least one amino acid residue in the cluster of highly hydrophobic amino acid residues was selected from the plurality of amino acid residues of the model;
(iii) amino acid residues that did not meet a criterion for evolutionary conservation were removed from the cluster of highly hydrophobic amino acid residues and from the one or more solvent-exposed polar amino acid residues, to produce a set of one or more predicted hot-spot amino acid residues; and
(iv) the one or more predicted hot-spot amino acid residues were stored;
wherein, if the hot-spot amino acid residue is replaced and is in the cluster of highly hydrophobic amino acid residues, it is replaced with an amino acid residue which is more hydrophobic such that the binding affinity is increases, or if the hot-spot amino acid residue is replaced and is from the one or more solvent-exposed polar amino acid residues, it is replaced with an amino acid residue which is more hydrophilic such that the binding affinity is increases.

2. The method of claim 1 , wherein additional hot-spot amino acid residues of the protein, or portion thereof, that are replaced or deleted to increase binding affinity of the protein variant to the macromolecule were predicted by repeating steps (i)-(iv) for at least two additional clusters of highly hydrophobic amino acid residues of the protein, or portion thereof.

3. The method of claim 1 , wherein additional hot-spot amino acid residues of the protein, or portion thereof, for all clusters of highly hydrophobic amino acid residues of the protein, or portion thereof, that can be replaced or deleted to increase binding affinity of the protein variant to the macromolecule when mutated were predicted by performing steps (i)-(iv) for each of the clusters, wherein each step was conducted in parallel for each cluster, or each series of steps (i)-(ii) was performed serially for each cluster.

4. The method of claim 1 , wherein the one or more predicted hot-spot amino acid residues that can be replaced or deleted to increase binding affinity of the protein variant to the macromolecule when mutated were presented as a contour map visual display.

5. The method of claim 4 , wherein the contour map was an electron density map.

6. The method of claim 4 , wherein the contour map was a map of a structural binding pocket.

7. The method of claim 1 , wherein the effective-hydrophobicity threshold was 0.10.

8. The method of claim 1 , wherein the first defined distance between at least two amino acid residues in the cluster was at most 15 Å.

9. The method of claim 1 , wherein the first defined distance between at least two amino acid residues in the cluster was the shortest distance between any two atoms of the at least two amino acid residues, wherein each of the two atoms were from separate amino acid residues.

10. The method of claim 1 , wherein the one or more solvent-exposed polar amino acid residues had a solvent accessible area (SAA) greater than 15 Å 2 .

11. The method of claim 1 , wherein the second defined distance between a solvent-exposed polar amino acid residue and at least one amino acid residue in the cluster of highly hydrophobic amino acid residues was at most 20 Å.

12. The method of claim 1 , wherein one or more of the solvent-exposed polar amino acid residues was selected to be within the cluster when the solvent-exposed charged amino acid residues were within a distance of at most 10 Å from a highly hydrophobic amino acid residue in the cluster.

13. The method of claim 1 , wherein the criterion for evolutionary conservation was a sequence-conservation threshold.

14. The method of claim 13 , wherein the sequence-conservation threshold was a ConSurf score that was 0.3.

15. The method of claim 1 , wherein the effective-hydrophobicity was calculated by:
(a) selecting an amino acid residue from the plurality of amino acid residues of the model;
(b) calculating, for all side-chain atoms of the amino acid residue, a ratio of the solvent accessible area (SAA) of the atoms to the SAA of atoms in an identical residue which is fully exposed; and
(c) multiplying each ratio by the hydrophobicity of the amino acid as determined by an amino acid hydrophobicity scale,

whereby the product of step (c) was the effective-hydrophobicity of the amino acid residue.

16. The method of claim 1 , wherein the macromolecule is selected from the group consisting of a protein binding partner, an antigen, an epitope, a peptide, a ligand, a receptor, a carbohydrate, a chemical, a small molecule, and an inhibitor.

17. The method of claim 1 , further comprising replacing at least one residue in the cluster of highly hydrophobic amino acid residues identified in step (ii) with at least one less hydrophobic amino acid residues, wherein the at least one residue in the cluster of highly hydrophobic amino acid residues is not a predicted hot-spot amino acid residue, and wherein replacing the at least one amino acid residues in the cluster of highly hydrophobic amino acid residues reduces protein aggregation without affecting macromolecule binding.

Assignees

• Novartis Ag
• Massachusetts Institute Of Tech Mit

Inventors

• Agrawal Neeraj J
• Helk Bernhard
• Trout Bernhardt L

Classifications

• G16B35/00

  ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides · CPC title

• G16C20/60

  In silico combinatorial chemistry · CPC title

• C07K1/107

  by chemical modification of precursor peptides · CPC title

• G16B5/00

  ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks · CPC title

• G16B15/00 Primary

  ICT specially adapted for analysing two-dimensional [2D] or three-dimensional [3D] molecular structures, e.g. structural or functional relations or structure alignment · CPC title