METHOD: Literature search was performed within the PubMed, ScienceDirect.com and Google Scholar.

RESULTS: The presence of proline at the C-terminal tripeptide of ACE inhibitor can competitively inhibit the ACE activity. The effects of other amino acids are less studied leading to difficulties in predicting potent peptide sequences. The broad specificity of the enzyme may be due to the dual active sites observed on the somatic ACE. The inhibitors may not necessarily competitively inhibit the enzyme which explains why some reported inhibitors do not have the common ACE inhibitor characteristics. Finally, the in vivo assay has to be carried out before the peptides as the antihypertensive agents can be claimed. The peptides must be absorbed into circulation without being degraded, which will affect their bioavailability and potency. Thus, peptides with strong in vitro IC50 values do not necessarily have the same effect in vivo and vice versa.

OBJECTIVE: To augment the affinity of AnkGAG1D4 scaffold towards its CA target, through computational predictions and experimental designs.

METHOD: Three dimensional structure of the binary complex formed by AnkGAG1D4 docked to the CA was used as a model for van der Waals (vdW) binding energy calculation. The results generated a simple guideline to select the amino acids for modifications. Following the predictions, modified AnkGAG1D4 proteins were produced and further evaluated for their CA-binding activity, using ELISA-modified method and bio-layer interferometry (BLI).

RESULTS: Tyrosine at position 56 (Y56) in AnkGAG1D4 was experimentally identified as the most critical residue for CA binding. Rational substitutions of this residue diminished the binding affinity. However, vdW calculation preconized to substitute serine for tyrosine at position 45. Remarkably, the affinity for the viral CA was significantly enhanced in AnkGAG1D4-S45Y mutant, with no alteration of the target specificity.