Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein-protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l-peptides; their ability to cyclize d-amino-acid-containing peptides has rarely been documented. Herein we show that macrocycles consisting of d-amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp-specific ligase. Furthermore, by using a peptide-library approach, we show that butelase 1 tolerates most of the d-amino acid residues at the P1'' and P2'' positions.

Peptides featuring an N-terminal cysteine residue and the unnatural amino acid 3-(2-cyano-4-pyridyl)alanine (Cpa) cyclize spontaneously in aqueous solution at neutral pH. Cpa is readily available and easily introduced into peptides using standard solid-phase peptide synthesis. The reaction is orthogonal to all proteinogenic amino acids, including cysteine residues that are not at the N-terminus. A substrate peptide of the Zika virus NS2B-NS3 protease cyclized in this way produced an inhibitor of high affinity and proteolytic stability. 1a CGKRKSCpa-NH2 1b 98 8.3 ± 0.2 2a CCKRKSCpa-NH2 2b 90 2.5 ± 0.1 3a CFKRKSCpa-NH2 3b 97 3.4 ± 0.2 4a CEKRKSCpa-NH2 4b 93 3.3 ± 0.1 5a CGKRKSCpaLI-NH2 5b 96 4.4 ± 0.1 6a CKRKSCpa-NH2 6b 92 8.5 ± 0.1 7a CKRKWCpa-NH2 7b 97 7.3 ± 0.1 8a CGKRKCpa-NH2 8b 97 8.4 ± 0.1 9a CRKSCpa-NH2 9b 91 2.7 ± 0.1 10a CKRKCpa-NH2 10b 94 2.8 ± 0.1 a Peptides synthesized by Fmoc solid-phase peptide synthesis using Rink amide resin. b Cpa, L-3-(2-cyano-4-pyridyl)alanine. c Cyclization was monitored by LC-MS in 10 mM Tris-HCl pH 7.5, 1 mM TCEP buffer at a peptide concentration of 75 µM. d Yields were calculated from LC-MS data. e Half-times were calculated from LC-MS data assuming first-order reaction kinetics.

A series of cyclic active‐site‐directed inhibitors of the NS2B‐NS3 proteases from Zika (ZIKV), West Nile (WNV), and dengue‐4 (DENV4) viruses has been designed. The most potent compounds contain a reversely incorporated d ‐lysine residue in the P1 position. Its side chain is connected to the P2 backbone, its α‐amino group is converted into a guanidine to interact with the conserved Asp129 side chain in the S1 pocket, and its C terminus is connected to the P3 residue via different linker segments. The most potent compounds inhibit the ZIKV protease with K i values <5 nM. Crystal structures of seven ZIKV protease inhibitor complexes were determined to support the inhibitor design. All the cyclic compounds possess high selectivity against trypsin‐like serine proteases and furin‐like proprotein convertases. Both WNV and DENV4 proteases are inhibited less efficiently. Nonetheless, similar structure‐activity relationships were observed for these enzymes, thus suggesting their potential application as pan‐flaviviral protease inhibitors.

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein–protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l‐peptides; their ability to cyclize d‐amino‐acid‐containing peptides has rarely been documented. Herein we show that macrocycles consisting of d‐amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp‐specific ligase. Furthermore, by using a peptide‐library approach, we show that butelase 1 tolerates most of the d‐amino acid residues at the P1′′ and P2′′ positions.

Flaviviruses are vector-borne RNA viruses, many of which are clinically relevant human viral pathogens, such as dengue, Zika, Japanese encephalitis, West Nile and yellow fever viruses. Millions of people are infected with these viruses around the world each year. Vaccines are only available for some members of this large virus family, and there are no effective antiviral drugs to treat flavivirus infections. The unmet need for vaccines and therapies against these flaviviral infections drives research towards a better understanding of the epidemiology, biology and immunology of flaviviruses. In this review, we discuss the basic biology of the flavivirus replication process and focus on the molecular aspects of viral genome replication. Within the virus-induced intracellular membranous compartments, flaviviral RNA genome replication takes place, starting from viral poly protein expression and processing to the assembly of the virus RNA replication complex, followed by the delivery of the progeny viral RNA to the viral particle assembly sites. We attempt to update the latest understanding of the key molecular events during this process and highlight knowledge gaps for future studies.