Before carrying out the drug overlay process, it is important to ensure that the molecules are in their respective active conformations. It is possible to carry out overlays, where one or both the molecules undergo conformational changes for their best fitting, if active conformation of the molecules is unknown.
It is relatively more expensive to conduct these because of extremely large computer times. The two properties that should be known about the two drugs before overlaying are as follows:
1. Root mean square distance (RMSD) between all their matched atom pairs.
2. Relative orientation of the molecules, where the RMSD value is minimum.
For example, in the overlay of cocaine and procaine, the matched atom pairs are identified as the respective two nitrogen atoms and aromatic rings in their structure. The distance between the respective nitrogen atoms and corresponding aromatic carbons in each molecule is calculated.
The RMSD for pairs of all the atoms is then calculated. One of the structures is moved in stages with respect to the other and the step is repeated until a minimum RMSD is obtained. This will ultimately, lead to the best fit.