An affinity-based probe for methyltransferase enzymes based on sinefungin

Abstract

Epigenetics control numerous cellular processes such as gene transcription, signal transduction, and protein stabilization. An understanding of epigenetic mechanisms can lead to the development of therapeutic agents for various diseases. Herein, we report the design and synthesis of a sinefungin affinity-probe (BpyneSF) that targets methyltranferase enzymes and proteins involved in recognition of methylation. This probe contains a bioorthogonal alkyne residue for conjugation using the copper-catalyzed azide–alkyne cycloaddition and a photoactivatable crosslinker group for covalent attachment of the probe to its proteomic targets. We investigate the efficiency and selectivity of the probe to inhibit and label methyltransferase enzymes, and we demonstrate, through in-gel fluorescence, on-bead digestion, and tandem mass spectrometry, that BpyneSF can label methyltransferase SETD2 and reader proteins in vitro. These results establish the utility of BpyneSF as a tool for affinity-based protein profiling in complex biological environments.

Dibromopinocembrin and Dibromopinostrobin Are Potential Anti-Dengue Leads with Mild Animal Toxicity

“…The crystal structures of all four interested DENV proteins were downloaded from the protein databank (PDB), including E protein (1OKE), NS2B/3 protease (2FOM), NS5 MTase (5EHI), and Pol domain (3VWS). The three-dimensional (3D) structures of all flavanones and reported inhibitors were constructed using GaussView 6 program and they were optimized at the B3LYP/6-31G* basis set [ 27 ] using Gaussian 16 software [ 40 ]. Each flavanone was docked with 100 independent runs into the four targets using AutoDock VinaXB [ 23 ] and compared with the known inhibitor in order to predict the binding pattern and affinity.…”

“…Each flavanone was docked with 100 independent runs into the four targets using AutoDock VinaXB [ 23 ] and compared with the known inhibitor in order to predict the binding pattern and affinity. The docking grid dimension; 20 × 20 × 20 = 8000 Å 3 were set as the previously reported binding sites for each protein target [ 27 ]. The x, y, and z coordinates of each grid box are listed in Table S1 (in Supplementary Materials ).…”

“… Binding affinity (kcal/mol) for the studied pinocembrins ( TH022 and TH011 ) and pinostrobins ( TH019, TH002, TH012, and TH018 ) in comparison with the known inhibitors of DENV protein targets: E protein at kl loop and Y site (FN5Y [ 11 ]), NS2B-3 pro at allosteric site (compound 9 [ 25 ]), NS5 MTase at SAM binding site (Sinefungin [ 27 ]), and NS5 Pol at the active site (NITD-107 [ 26 ]), predicted by molecular docking method using AutoDock VinaXB. …”

“…Besides, the binding affinity of TH011 and TH002 at both kl loop and Y site of the DENV E protein were relatively lower than that of the potent flavanone FN5Y [11] in consistent with no fusion inhibition observed (Supplementary Figure S5). [11]), NS2B-3 pro at allosteric site (compound 9 [25]), NS5 MTase at SAM binding site (Sinefungin [27]), and NS5 Pol at the active site (NITD-107 [26]), predicted by molecular docking method using AutoDock VinaXB.…”

Lichen-Derived Diffractaic Acid Inhibited Dengue Virus Replication in a Cell-Based System

“…The native inhibitors of NS2/3 protease (compound 9 [ 18 ]), NS3 helicase (ST-610 [ 19 ]), and NS5 RdRp (NITD-107 [ 20 ]) were superior to diffractaic acid. On the contrary, the binding energy of the native inhibitor (sinefungin, −8.9 kcal/mol [ 21 ]) overlapped with diffractaic acid in the range of −7.8 to −9.4 kcal/mol. The SAM binding of NS5 MTase is mainly contributed by hydrophobic interaction, involving alkyl–π (K105, H110, F133, I147, R160, and R163), van der Waals (E111, G148, and K180), and anion–π (E149) interactions due to the methyl group and aromatic structure of diffractaic acid ( Figure 4 B).…”