Full Sequence Amino Acid Scanning of θ-Defensin RTD-1 Yields a Potent Anthrax Lethal Factor Protease Inhibitor

theta-Defensin RTD-1 is a noncompetitive inhibitor of anthrax lethal factor (LF) protease (IC50 = 390 +/- 20 nM, K-i = 365 +/- 20 nM) and a weak inhibitor of other mammalian metalloproteases such as TNF alpha converting enzyme (TACE) (K-i = 4.45 0.48 mu M). Using full sequence amino acid scanning in combination with a highly efficient one-pot cyclization-folding approach, we obtained an RTD-1-based peptide that was around 10 times more active than wild-type RTD-1 in inhibiting LF protease (IC50 = 43 +/- 3 nM, K-i = 18 +/- 1 nM). The most active peptide was completely symmetrical, rich in Arg and Trp residues, and able to adopt a native RTD-1-like structure. These results show the power of optimized chemical peptide synthesis approaches for the efficient production of libraries of disulfide-rich backbone-cyclized peptides to quickly perform structure activity relationship studies for optimizing protease inhibitors.