Identification, Characterization, and Synthesis of Natural Parasitic Cysteine Protease Inhibitors: Pentacitidins Are More Potent Falcitidin Analogues

Abstract

Protease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogues of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C-terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A–D. The isolation, characterization, and absolute structure elucidation of two falcitidin-related pentapeptide aldehyde analogues by extensive MS/MS spectrometry and NMR spectroscopy in combination with advanced Marfey’s analysis was in agreement with the in silico analysis of the corresponding biosynthetic gene cluster. Total synthesis of chosen pentapeptide analogues followed by in vitro testing against a panel of proteases revealed selective parasitic cysteine protease inhibition and, additionally, low-micromolar inhibition of α-chymotrypsin. The pentapeptides investigated here showed superior inhibitory activity compared to falcitidin.

Bioactive natural products from Bacteroidetes

“…13). 76 It is congruent with the pentapeptide structure, strengthening the degradation hypothesis. The BGC contains a terminal reductase domain probably involved in the formation of the C-terminal aldehyde and alcohol analogs via reductive release.…”

“…NMR spectroscopy revealed that both pentacitidins were puried as diastereomers for the Phe residue. 76 Interestingly, a pentapeptide and its corresponding falcitidin tetrapeptide analog with a C-terminal amidated proline were detected by UHPLC-MS, following the NMR analysis of pure pentacitidins, suggesting that falcitidin-like tetrapeptides are the degradation products of the mature natural productsthe pentacitidins. 76 Further studies are needed to test this hypothesis and elucidate the degradation mechanism.…”

“…74,75 Metabolic networking analysis revealed the presence of more than 30 natural analogs of falcitidin, including further falcitidin-like tetrapeptides. 76 They are produced by various Chitinophaga strains and two of these analogs were isolated from Chitinophaga eiseniae DSM 22224. Pentacitidins A (41) and B ( 42) are falcitidin-like pentapeptides with Phe as an additional C-terminal amino acid with an aldehyde group (phenylalaninal, H-Phe-al; Fig.…”

“…75 Pentacitidins were produced by solid-phase peptide synthesis. 76 Aldehydes are electrophilic warheads that form covalent-reversible hemithioacetal adducts with catalytic Cys residues and covalentreversible hemiacetal adducts with Ser residues. Testing pentacitidins against human cysteine proteases (cathepsins B and L) and related proteases from parasites (falcipain-2/3 and rhodesain) revealed selective inhibition of the parasitic enzymes with sub-micromolar IC 50 values.…”

“…Docking studies showed favorable distances between the catalytic Cys residue and the pentacitidin aldehyde in its L-conguration. 76 A pentacitidin BGC was tentatively identied, consisting of a single NRPS gene (18.5 kb) with ve A-domains and a C-starter unit (Fig. 13).…”