Natural products and their semisynthetic derivatives are an important source of drugs for the pharmaceutical industry. Bacteria are prolific producers of natural products and encode a vast diversity of natural product biosynthetic gene clusters. However, much of this diversity is inaccessible to natural product discovery. Here, we use a combination of phylogenomic analysis of the microviridin biosynthetic pathway and chemo-enzymatic synthesis of bioinformatically predicted microviridins to yield new protease inhibitors. Phylogenomic analysis demonstrated that microviridin biosynthetic gene clusters occur across the bacterial domain and encode three distinct subtypes of precursor peptides. Our analysis shed light on the evolution of microviridin biosynthesis and enabled prioritization of their chemo-enzymatic production. Targeted one-pot synthesis of four microviridins encoded by the cyanobacterium Cyanothece sp. PCC 7822 identified a set of novel and potent serine protease inhibitors, the most active of which had an IC value of 21.5 nM. This study advances the genome mining techniques available for natural product discovery and obviates the need to culture bacteria.
Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases.
Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases.Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large group of compounds that combine great chemical complexity at low genetic costs. [1] The increasing knowledge about mechanisms and the scope of peptide-modifying enzymes has provided an avenue for the rational and random design of peptide libraries through synthetic biology methodologies. [2] Microviridins (1; see Scheme 1) are one of the most fascinating families of RiPPs with potent activities against various serine proteases. [3] Their unprecedented cage-like architecture results from the activity of two ATP-grasp ligases that introduce non-canonical lactone and lactam rings. [4] Full maturation of microviridins additionally requires the activity of a GNAT-type N-acetyltransferase. [4a] In vitro reconstitution studies have revealed a strict order of cyclization reactions and a stringent ring size requirement for microviridin with the large lactone ring being formed first, followed by the smaller lactone ring and the lactam ring. [4c] An increasing number of RiPPs was successfully reconstituted and diversified using chemo-enzymatic approaches that use chemically synthesized precursor peptides. [5] Expansion of the tool-kit for peptide modification has also enabled the design of "unnatural" peptide variants by modularizing different post-translational modification enzymes in vitro. [5] Microviridins feature a unique type of macrocyclization. [1,5] The full in vitro reconstitution of microviridins and thus their exploitation in chemo-enzymatic approaches was so far hampered by the long leader peptide that is essential for peptide maturation and the lack of an appropriate protease for leader peptide removal. Recent studies on lanthipeptide and cyanobactin biosynthetic enzymes have revealed that the functionality of the leader peptide does not necessarily require its direct linkage to the core peptide. [5a, 6] Leader peptides were shown to successfully activate the corresponding modifying enzymes when added in trans or were alternatively attached to the N-terminus of modifying enzymes. [5a...
Microviridins are a prominent family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) featuring characteristic lactone and lactam rings. Their unusual cage-like architecture renders them highly potent serine protease inhibitors of which individual variants specifically inhibit different types of proteases of pharmacological interest. While posttranslational modifications are key for the stability and bioactivity of RiPPs, additional attractive properties can be introduced by functional tags. To date -although highly desirable -no method has been reported to incorporate functional tags in microviridin scaffolds or the overarching class of graspetides. In this study, a chemoenzymatic in vitro platform is used to introduce functional tags in various microviridin variants yielding biotinylated, dansylated or propargylated congeners. This straightforward approach paves the way for customized protease inhibitors with built-in functionalities that can help to unravel the still elusive ecological roles and targets of this remarkable class of compounds and to foster applications based on protease inhibition.
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