Synthesis and Biological Evaluation of Modified Peptide Derivatives Targeting the SARS-CoV-2 Nsp3 Macrodomain (Mac1) Replication Domain

Modified peptide derivatives (D1–D15) containing hydrophobic (phenylalanine and tryptophan) and positively charged (histidine) amino acid residues, were designed and synthesized. The cytotoxicity of the compounds was evaluated in healthy (CCD1079Sk) cell lines, revealing no significant cytotoxic effects and indicating favorable safety profiles. Molecular modeling studies were performed for all compounds and indicated that compounds D13, D14, and D15 form favorable binding interactions with the Nsp3 macrodomain 1 (Mac1) active site. The affinity of these compounds is expected to be moderate to high, although lower than that of ADP-ribose. Subsequent bioactivity assays, performed using a cell-based SARS-CoV-2 replicon system, revealed that compound D15 displays the most potent inhibition of viral replication (IC₅₀ = 22.2 μM (95% CI: 15.4–35.7 μM)), while compound D14 shows the second most potent inhibitory activity (IC₅₀ = 61.2 μM (95% CI: 39.2–143.9 μM)). Considering these results, it has been shown that compounds with a histidine side chain exhibit higher biological activity, suggesting a structure–activity relationship driven by positively charged residues.