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

Abstract

θ-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 TNFα converting enzyme (TACE) (K_i = 4.45 ± 0.48 μM). 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.

Figure 1

Primary and tertiary structure of rhesus θ-defensin 1 (RTD-1) (PDB ID code: 1HVZ). The backbone cyclized peptide (connecting bond shown in blue) is stabilized by the three disulfide bonds in a ladder formation (disulfide bonds shown in yellow). Scheme showing the amino acid scanning approach used in this work. Residues in RTD-1, except for the Cys residues, were replaced by a set of amino acids containing Ala, Asp, Glu, Leu, Val, Lys, Arg, Tyr or Trp. When the native the residue on the RTD-1 sequence was Arg, Leu, or Val, the corresponding amino in the set indicated above was replaced by His (Arg) or Ile (Val and Leu). Also, Leu was not included in the Phe2 sub-library therefore containing only 8 members. Accordingly, a total of 107 (=11 × 9 + 1 × 8) different f RTD-1 variants containing a single mutation were chemically prepared (Table S1).

Figure 2

Synthetic scheme used for the parallel production of the amino acid scanning library at position Gly10 of θ-defensin RTD-1 using a ‘tea-bag’ approach. A similar approach was used for the other positional sub-libraries, except for sub-libraries at positions R4 and L6, where the peptide bond between residues Phe2 and Cys3 was used as the cyclization site.

Figure 3

Chemical synthesis and characterization of several single mutant RTD-1 analogs. Analytical HPLC traces of the linear thioester precursor and the GSH-induced cyclization/folding crude after 24 h after desalting to remove buffer components. An asterisk indicates the desired folded peptide (Right panels). ES-MS characterization of the desalted folded/cyclized RTD-1 peptides. The expected average molecular weight is shown in parenthesis (Left panels).

Figure 4

Anti-LF activity for the RTD-1 single mutant analogs. A. Dose-dependent inhibition of LF by different single mutant RTD-1 analogs. Peptides RTD-1 and 1 were used a positive and negative controls respectively. B. Summary of the relative activities of all single mutants able to provide a folded product. The anti-LF of wild-type RTD-1 was used as a reference, i.e. 100% anti-LF activity. An asterisk denotes the corresponding mutant was not able to fold efficiently and therefore was not tested for biological activity. Experiments were performed in triplicate (N=3). Error bars indicate standard deviation.

Figure 5

Anti-LF activity of RTD-1 analogs containing multiple mutations, peptide 1 and peptides 8 through 13. A. K_i values and relative activity of the RTD-1 mutants when compared to wild-type RTD-1 is shown at the bottom). Experiments were performed in triplicate (N=3). Error bars indicate standard deviation. NA stands for non active. B. Dose-dependent inhibition of LF by different RTD-1 analogs.

Figure 6

Structural characterization of θ-defensin 13. A. Amide proton and nitrogen peaks in¹⁵N-HSQC of [U-15N]-peptide 13 exhibit extreme overlap due to highly symmetric nature of 13 primary structure. Chemical shift assignments of the backbone and side chain amides are indicated. B. Homology model of θ-defensin analog 13. Model was built using the structure of RTD-1 (PDB ID code: 1HVZ) as template. The resulting structure was subjected to molecular dynamics for 700 ns in a neutral water box containing 0.9% NaCl. One of 10 most stable structures is shown. The structure shows numerous cation-π interactions between the side-chains of different Arg and Trp residues. These interactions were highly dynamic during the simulation (Fig. S14) (Graphics were generated using the Yasara software package, www.yasara.org).