Structure-Based Dissection of the Natural Product Cyclopentapeptide Chitinase Inhibitor Argifin

Summary Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides, and antiasthmatics. Argifin, a natural product cyclopentapeptide, competitively inhibits family 18 chitinases in the nanomolar to micromolar range and shows extensive substrate mimicry. In an attempt to map the active fragments of this large natural product, the cyclopentapeptide was progressively dissected down to four linear peptides and dimethylguanylurea, synthesized using a combination of solution and solid phase peptide synthesis. The peptide fragments inhibit chitinase B1 from Aspergillus fumigatus (AfChiB1), the human chitotriosidase, and chitinase activity in lung homogenates from a murine model of chronic asthma, with potencies ranging from high nanomolar to high micromolar inhibition. X-ray crystallographic analysis of the chitinase-inhibitor complexes revealed that the conformations of the linear peptides were remarkably similar to that of the natural product. Strikingly, the dimethylguanylurea fragment, representing only a quarter of the natural product mass, was found to harbor all significant interactions with the protein and binds with unusually high efficiency. The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.

After coupling of Fmoc-Arg(Pmc)-OH, the resin was divided into two equal portions, which were then used for the preparation of (tetrapeptide) and (tripeptide) respectively. For (tetrapeptide), after coupling of Fmoc-D-Ala-OH, the resin-bound tetrapeptide was N(α)-deprotected and acetylated by treatment with acetic anhydride/ CH 2 Cl 2 /pyridine (v/v/v 0

Acylation:
A stirred solution of the crude peptide from above in DMF (2 mL) was treated with Nsuccinimidyl-N-methyl carbamate (24.9 mg, 0.145 mmol ) and DBU (14.5 µL, 0.096 mmol) at 40 °C for 36h. The reaction mixture was concentrated in vacuo and the crude product was purified by RP semi-preparative HPLC (gradient 2) to give The resin-bound tripeptide (see above) was N(α)-deprotected and acetylated as for (tetrapeptide). Following cleavage from the resin and isolation as before, the fully deprotected Arg-containing tripeptide intermediate was obtained as the trifluoroacetate salt as a white solid (10.9 mg, 0.017 mmol).

Acylation:
A stirred solution of the crude peptide from above in DMF (2 mL) was treated with

Ac-L-Arg(MC)-L-MePhe (Dipeptide)
This was performed as for (tetrapeptide), using 2-chlorotrityl polystyrene resin (0.20 g, 0.28 mmol). After cleavage from the resin and isolation as before, the fully deprotected Arg-containing dipeptide intermediate was obtained as the trifluoroacetate salt as a white solid (28.2 mg, 0.057 mmol).

Acylation:
A stirred solution of the crude peptide from above in DMF (2 mL g, 0.75 mmol). The reaction mixture was stirred for 1 h, then methylamine hydrochloride (51 mg, 0.75 mmol) and DIPEA (0.29 mL, 1.66 mmol) were added, and the mixture was allowed to warm to room temperature overnight. The solvent was evaporated and the residue was dissolved in EtOAc (15 mL) and washed with 5% citric acid (2 x 8 mL), 5% NaHCO 3 (2 x 8 mL), water (2 x 8 mL) and brine (8 mL).
The solvent was evaporated and the residue was dried thoroughly in vacuo, and then partitioned between CH 2 Cl 2 (2 mL) and water (2 mL). The organic extract was dried (MgSO 4 ) and the solvent was evaporated to give crude Ac-L-Arg-NHCH 3 as the TFA salt (15 mg), which was immediately neutralised with pre-washed Dowex hydroxide resin (200 mg) in MeOH (2 mL) for 1 h. The resin was filtered off and the solvent was evaporated to give Ac-Arg-NHCH 3 as the free base (7.5 mg, 0.033 mmol assumed). A solution of this material in dry DMF (0.35 mL) was cooled to 0 °C and was treated with a 0.31M solution of methylisocyanate in DMF (0.06 mL, 1.8 equiv).
The reaction mixture was allowed to warm to room temperature and was stirred for 18 h, then the solvent was evaporated and the crude product was analysed by mass spectrometry, which indicated partial conversion to (xxx) had occurred. The acylation reaction was repeated exactly as before, and the crude product now obtained was