ABSTRACT
Tetracyclic triterpenes and steroids are pharmacologically important molecules, and acetylation could improve their bioactivities. In this study, a highly regio- and stereo-specific acetyltransferase, AmAT19, was discovered from Astragalus membranaceus. AmAT19 could selectively catalyze the 6α-OH acetylation of four tetracyclic triterpenes and steroids. The strict selectivity is associated with different orientations of the 6α/ß-OH as indicated by molecular docking. Acetylated products 1a, 3a and 4a remarkably increased the inhibitory activity against the 3-chymotrypsin-like protease (3CLpro) of SARS-CoV-2, compared to 1, 3, and 4. AmAT19 could be a promising catalyst for specific 6α-OH acetylation to expand the molecular diversity of triterpenes and steroids.
Subject(s)
Acetyltransferases/metabolism , Astragalus Plant/enzymology , Steroids/metabolism , Triterpenes/metabolism , Acetylation , CatalysisABSTRACT
Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.