Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase

Abstract

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome.Methyltransferases (MTases) 3 play key roles in normal physiology and human diseases through methylating DNA, RNA, and proteins. Almost all MTases use S-adenosyl-L-methionine (SAM) as a methyl donor and generate S-adenosyl-Lhomocysteine (SAH) as a by-product. Pharmacological modulation of MTases by small molecules represents a novel approach to therapeutic intervention in cancer and other diseases (1). However, because the core domains of various MTases are conserved, designing inhibitors that specifically block the disease-related MTase without affecting other MTases, has been challenging. The ability to rationally design and generate selective inhibitors would have profound implications for development of new medicines for many methyltransferase-mediated diseases.Dengue virus (DENV), from genus Flavivirus in the family Flaviviridae, is the most prevalent mosquito-borne viral pathogen that infects humans. The four serotypes of DENV (DENV-1 to -4) pose a public health threat to 2.5 billion people worldwide, and cause 50 -100 million human infections each year. Neither vaccine nor antiviral therapy is currently available for DENV. The flavivirus MTase methylates the guanine N7 and ribose 2Ј-O positions of the viral RNA cap in a sequential manner (i.e. GpppA-RNA 3 m7GpppA-RNA 3 m7GpppAm-RNA) (2, 3). Recent studies have shown that flavivirus MTase is critical for viral replication and, therefore, represents a valid target for antiviral therapeutics (4 -6). We therefore examined the feasibility to design inhibitors that specifically modulate flavivirus MTase. EXPERIMENTAL PROCEDURESPreparation of DENV-3 MTases-The DNA fragment representing the MTase domain of DENV-3 was cloned into expression vector pGEX4T1 (Amersham Biosciences). Ala-substitution mutant MTases were prepared using a standard overlapping PCR procedure. Recombinant MTases, containing an N-terminal GST, were expressed in Escherichia coli. BL21 cells and purified through a GSTPrep TM FF 16/10 column (GE Healthcare). The GST tag was then cleaved by thrombin and removed from the MTases using the GST column. The MTases were further purified through gel filtration to ensure protein purity was Ͼ95%. The p...

An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity inE. Colicell lysate

“…As a proof of concept, we tested three known methyltransferase inhibitors and found that only (-)-epigallocatechin gallate inhibited hTGS1. Since specific RNA methyltransferases inhibitors, e.g., for Dengue virus methyltransferase 29 or even for PIMT 30 could be of therapeutic interest, a high-throughput screen is desirable to test large compound libraries.…”

“…Lim et al designed selective inhibitors of dengue virus methyltransferase responsible for viral cap methylation. 29 Even some HIV-1 RNAs have been found to be trimethylguanosine-capped and acquisition of this cap hypermethylations facilitates the optimal expression of these RNAs. 30 An assay to test potential small molecule inhibitors of RNA methyltransferases including trimethylguanosine synthases in high-throughput could thus speed up the discovery of novel inhibitors.…”

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

“…Enzyme-based screenings or structure-based drug design have identified DENV, WNV, and YFV MTase inhibitors targeting either the SAM/SAH (S-adenosyl-Lhomocysteine, the coproduct of the methylation) binding pocket (26)(27)(28), the cap binding pocket (29), or allosteric sites (30,31). As ZIKV is closely related, we initiated a comparative study of ZIKV MTase with other Flavivirus MTases with the aim of subsequently developing original inhibitors or repurposing inhibitors targeting DENV MTase for antiviral research on ZIKV.…”

Identification of a Novel Antiviral Inhibitor of the Flavivirus Guanylyltransferase Enzyme

“…The critical nature of the capping enzyme in viral replication and immune evasion and as its conservation across the flavivirus genus position the capping enzyme as an important target for antiviral development efforts. The methyltransferase activity has been the primary capping enzyme target for drug development (16,21), and ribavirin triphosphate has been observed to bind to GTP and displace it from the enzyme (1).…”