the effect of mutations on the drug binding affinity (Fig. 2). The total free energy
change in a thermodynamic cycle in any alchemical transformation is equal to zero.
DG 1 À DG 4 À DG 2 À DG 3
ð
Þ¼0
ð16aÞ
DG 1 À DG 4 ¼ DG 2 À DG 3
ð16bÞ
3 Application of Computational Methods to Understand
Drug-Resistant Mutations
3.1 Computational Mutation Scanning
Computational mutation scanning [92] is a useful method to explore the sensitivity
to changes in the composition of the amino acid in a protein-binding site (Fig. 3). In
computational mutation scanning, the wild-type amino acid residue is mutated to
another amino acid in the binding pocket or elsewhere. However, the most widely
practised method is to mutate any amino acid residue to an alanine, since it is the
simplest amino acid with a side chain (not glycine because it is devoid of a side
chain). Hence, this method is equivalent to the experimental “alanine-scanning
mutagenesis”, which is a powerful tool to investigate and confirm the important
interactions in the protein–protein interface and protein–ligand interactions. In
computational alanine scanning, all atoms from the C b carbon atom of the amino
acid under study are replaced by three hydrogen atoms to convert it to an alanine.
After the mutation, the change in the binding energy is estimated either using
docking with an appropriate scoring function or by MM-PBSA or MM-GBSA to
compute DDG (Eq. 17c). By scanning with alanine at various positions in the
binding cavity, important residues can be identified, as mutating an important
amino acid will drastically decrease the binding energy.
DG
Wild
bind ¼ DG
Wild
complex À DG
Wild
receptor À DG ligand
ð17aÞ
DG
Mut
bind ¼ DG
Mut
complex À DG
Mut
receptor À DG ligand
ð17bÞ
DDG ¼ DG
Mut
bind À DG
Wild
bind ¼ DG
Mut
complex À DG
Wild
complex
h
i
À DG
Mut
receptor À DG
Wild
receptor
h
i
ð17cÞ
In the context of predicting drug-resistant mutations, one must perform alanine
scanning in the binding site on two complexes, i.e. with the substrate bound complex
and the inhibitor-bound complex. The change in the binding energy after mutation is
computed for both the systems, viz., for inhibitor and the substrate. A decrease in the
binding affinity for the inhibitor with negligible or no change in the binding affinity
for the substrate indicates a hotspot amenable to resistant mutation, these spots are
14
E. A. F. Martis and E. C. Coutinho
change in a thermodynamic cycle in any alchemical transformation is equal to zero.
DG 1 À DG 4 À DG 2 À DG 3
ð
Þ¼0
ð16aÞ
DG 1 À DG 4 ¼ DG 2 À DG 3
ð16bÞ
3 Application of Computational Methods to Understand
Drug-Resistant Mutations
3.1 Computational Mutation Scanning
Computational mutation scanning [92] is a useful method to explore the sensitivity
to changes in the composition of the amino acid in a protein-binding site (Fig. 3). In
computational mutation scanning, the wild-type amino acid residue is mutated to
another amino acid in the binding pocket or elsewhere. However, the most widely
practised method is to mutate any amino acid residue to an alanine, since it is the
simplest amino acid with a side chain (not glycine because it is devoid of a side
chain). Hence, this method is equivalent to the experimental “alanine-scanning
mutagenesis”, which is a powerful tool to investigate and confirm the important
interactions in the protein–protein interface and protein–ligand interactions. In
computational alanine scanning, all atoms from the C b carbon atom of the amino
acid under study are replaced by three hydrogen atoms to convert it to an alanine.
After the mutation, the change in the binding energy is estimated either using
docking with an appropriate scoring function or by MM-PBSA or MM-GBSA to
compute DDG (Eq. 17c). By scanning with alanine at various positions in the
binding cavity, important residues can be identified, as mutating an important
amino acid will drastically decrease the binding energy.
DG
Wild
bind ¼ DG
Wild
complex À DG
Wild
receptor À DG ligand
ð17aÞ
DG
Mut
bind ¼ DG
Mut
complex À DG
Mut
receptor À DG ligand
ð17bÞ
DDG ¼ DG
Mut
bind À DG
Wild
bind ¼ DG
Mut
complex À DG
Wild
complex
h
i
À DG
Mut
receptor À DG
Wild
receptor
h
i
ð17cÞ
In the context of predicting drug-resistant mutations, one must perform alanine
scanning in the binding site on two complexes, i.e. with the substrate bound complex
and the inhibitor-bound complex. The change in the binding energy after mutation is
computed for both the systems, viz., for inhibitor and the substrate. A decrease in the
binding affinity for the inhibitor with negligible or no change in the binding affinity
for the substrate indicates a hotspot amenable to resistant mutation, these spots are
14
E. A. F. Martis and E. C. Coutinho