AbstractThe development of new HIV inhibitors with distinct resistance profiles is essential in order to combat the development of multi‐resistant viral strains. A drug discovery program based on the identification of compounds that are active against drug‐resistant viruses has produced PL‐100, a novel potent protease inhibitor (PI) that incorporates a lysine‐based scaffold. A selection for resistance against PL‐100 in cord blood mononuclear cells was performed, using the laboratory‐adapted IIIb strain of HIV‐1, and it was shown that resistance appears to develop slower against this compound than against amprenavir, which was studied as a control. Four mutations in protease (PR) were selected after 25 weeks: two flap mutations (K45R and M46I) and two novel active site mutations (T80I and P81S). Site‐directed mutagenesis revealed that all four mutations were required to develop low‐level resistance to PL‐100, which is indicative of the high genetic barrier of the compound. Importantly, these mutations did not cause cross‐resistance to currently marketed PIs. In contrast, the P81S mutation alone caused hypersensitivity to two other PIs, saquinavir (SQV) and nelfinavir (NFV). Analysis of p55Gag processing showed that a marked defect in protease activity caused by mutation P81S could only be compensated when K45R and M46I were present. These data correlated well with the replication capacity (RC) of the mutant viruses as measured by a standard viral growth assay, since only viruses containing all four mutations approached the RC of wild type virus. X‐ray crystallography provided insight on the structural basis of the resistance conferred by the identified mutations. J. Med. Virol. 80:2053–2063, 2008. © 2008 Wiley‐Liss, Inc.