Isolation and synthesis of falcitidin, a novel myxobacterial-derived acyltetrapeptide with activity against the malaria target falcipain-2

Abstract

A 384-well microtitre plate fluorescence cleavage assay was developed to identify inhibitors of the cysteine protease falcipain-2, an important antimalarial drug target. Bioassay-guided isolation of a MeOH extract from a myxobacterium Chitinophaga sp. Y23 isolated from soil collected in Singapore, led to the identification of a new acyltetrapeptide, falcitidin (1), which displayed an IC50 value of 6 μM against falcipain-2. The planar structure of 1 was secured by NMR and MS/MS analysis. Attempts to isolate further material for biological testing were hampered by inconsistent production and by a low yield (<100 μg l(-1)). The absolute configuration of 1 was determined by Marfey's analysis and the structure was confirmed through total synthesis as isovaleric acid-D-His-L-Ile-L-Val-L-Pro-NH2. Falcitidin (1) is the first member of a new class of falcipain-2 inhibitors and, unlike other peptide-based inhibitors, does not contain reactive groups that irreversibly bind to active cysteine sites.

Identification, Characterization and Synthesis of Natural Parasitic Cysteine Protease Inhibitors – More Potent Falcitidin Analogs

“…16 Finally, advanced Marfey's analysis was conducted to determine the absolute configuration of the amino acids. 17 Pro configuration 10 and the incorporation of L-phenylalanine at position five by verifying the presence of L-phenylalaninol (Figure S5). All stereogenic centers were further confirmed by total synthesis and comparison of NMR data to natural isolated compounds (Table 1 and 2).…”

“…Exploration of the genomic and metabolomic potential of the Bacteroidetes genus Chitinophaga revealed a high potential to find chemical novelty. 14 In the framework of a previous metabolomics' study of Chitinophaga, an inhibitor of the antimalarial target falcipain-2, falcitidin (1), 10 was detected. 14 Originally wrongly described as myxobacterium-derived tetrapeptides produced by Chitinophaga sp.…”

“…Y23, falcitidin and its synthetic analogs are first members of a new class of cysteine protease inhibitors without a reactive group that covalent-reversibly or irreversibly binds to the active site cysteine residue. 10,11 Here, molecular networking was carried out to analyze the chemical profiles of all 25 previously cultured Chitinophaga strains (Figure 1a). This facilitated the discovery of more than 30 For analogs with a leucine/isoleucine at position five only analogs with a CHO-and CH2OH-group were detected, with neutral losses of 115.1031 Da (C6H13NO) and 117.1164 Da (C6H15NO), respectively (Figure 1c, Figure S1-4).…”

“…Instead of reactive groups that covalent-reversibly or irreversibly bind to active-site cysteines, these compounds share a Cterminal amidated proline. 10,11 High structural diversity in combination with diverse biological activities make natural products a valuable source in the search for new lead candidates for drug development. Technological improvements in resolution and accuracy of spectrometry methods followed by complex and large dataset analysis allowed the application of metabolomics techniques based on e.g.…”

Genomic and chemical decryption of the Bacteroidetes phylum for its potential to biosynthesize natural products

“…A complementary scan of LC-MS/MS data of the entire dataset was compared with in silico fragments of >40k NPs deposited in the commercial database AntiBase( 69 ). Congruently, no database recorded NP was identified within our dataset besides the falcitidin( 70 ), an acyltetrapeptide produced by a Chitinophaga strain. Although this finding shows the general applicability of this workflow, the underrepresentation of NPs isolated from the phylum Bacteriodetes also in public databases is still a severe limitation for comprehensive categorization of their omics-data today.…”