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  • The isomeric phenyloxazole not only exhibited increased pote

    2020-11-20

    The isomeric 5-phenyloxazole not only exhibited increased potency against NEP, but also a decreased ACE inhibition thus providing encouraging selectivity. Changing the P1 group from an -propyl to a more polar methoxyethyl at the left hand melk inhibitor (as seen in compound pairs – and –) decreased ACE inhibition further such that showed around 1000-fold selectivity over ACE. Introduction of a second substituent onto the oxazole, such as with 4-methyl-5-phenyl-oxazole , gave a slight reduction in activity, whilst simple - chlorination of the phenyl group gave similar levels of activity to the parent . The two isomeric oxadiazoles and offered no pharmacology advantage over the oxazole systems. Compound is 25-fold more potent against NEP than Candoxatrilat , and significantly more selective over ACE than or . Interactions within the S2′ lipophilic pocket are well optimised for NEP. The i.v. pharmacokinetics of compound compare favourably with those of candoxatrilat. Compound has an i.v. clearance of 4.8ml/min/kg and a half-life of 2.1h in rat. Oral bioavailablity for was found to be low (1.7%) in a seperate rat study. In conclusion, this letter demonstrates that selective NEP inhibition can be achieved with heteroarylalanine derivatives . Natural amino acid stereochemistry for the novel heterocyclic substituents at P2′ in combination with a methoxyethyl S1 substituent enables phenyl oxazoles to exhibit potent and selective NEP inhibition. Compound has a useful pharmacokinetic half-life in rat. Acknowledgments
    Introduction The lysyl endopeptidase (Lys-C; EC 3.4.21.50) belongs to the peptidase S1 family. Lys-C is specific for the cleavage of peptidyl bonds on the C-terminal side of lysine residues. This kind of specific endopeptidase is produced from several bacterial species, including Achromobacter lyticus M497-1 [1], Pseudomonas aeruginosa[2] and Lysobacter sp. IB-9374 [3]. In the past, the Achromobacter protease 1 (AP1) from A. lyticus was part of detailed characterization studies [4], [5], [6], [7], [8]. This secretory peptidase (UniProt ID: P15636) is synthesized as a zymogen [653 amino acids (AA)], including signal peptide (pre-peptide; 20 AA), pro-peptide (185 AA), mature peptidase (268 AA), and an extension peptide (180 AA), from the N- to C-terminus. The recombinant production of AP1 in Escherichia coli has already been described [9]. This peptidase is a useful tool for the initial fragmentation of polypeptide chains in protein sequence analysis [10], [11], peptide bond synthesis [12], [13] and the processing of fusion proteins to biologically active peptides [14]. Lysobacter, for example, Lysobacter enzymogenes (type strain ATCC 29487), are Gram-negative, gliding bacteria [15]. In addition to Lys-C, further endopeptidases from L. enzymogenes are alpha-lytic endopeptidase (EC 3.4.21.12) and arginyl endopeptidase (Arg-C). A patent [16] describes the recombinant production of Lys-C from L. enzymogenes in E. coli. In this patent a synthetic lys-C gene, based on the proteolytically active form of Lys-C, which was optimized for the codon usage of E. coli was used. The synthetic lys-C gene was designed so that the amino acid sequence translated showed an artificial N-terminal pro-peptide (MGSK). The recombinant MGSK-Lys-C formed inclusion bodies, which were solubilized using guanidine hydrochloride and refolded using l-arginine as an unspecific folding additive. However, they did not use the N-terminal pro-peptide for the refolding of the recombinant Lys-C. It is reported for many enzymes that the pro-peptide had an important function in protein folding either in vivo or in vitro[17], [18], [19], [20]. The aim of the current project was to establish an E. coli based expression system to produce high amounts of recombinant Lys-C. Furthermore, the development of an optimized refolding process for Lys-C using the separately recombinant produced N-terminal pro-peptide of Lys-C in the refolding assay was investigated.