Identification of Inhibitors for the DEDDh Family of Exonucleases and a Unique Inhibition Mechanism by Crystal Structure Analysis of CRN-4 Bound with 2-Morpholin-4-ylethanesulfonate (MES)

Abstract

The DEDDh family of exonucleases plays essential roles in DNA and RNA metabolism in all kingdoms of life. Several viral and human DEDDh exonucleases can serve as antiviral drug targets due to their critical roles in virus replication. Here using RNase T and CRN-4 as the model systems, we identify potential inhibitors for DEDDh exonucleases. We further show that two of the inhibitors, ATA and PV6R, indeed inhibit the exonuclease activity of the viral protein NP exonuclease of Lassa fever virus in vitro. Moreover, we determine the crystal structure of CRN-4 in complex with MES that reveals a unique inhibition mechanism by inducing the general base His179 to shift out of the active site. Our results not only provide the structural basis for the inhibition mechanism but also suggest potential lead inhibitors for the DEDDh exonucleases that may pave the way for designing nuclease inhibitors for biochemical and biomedical applications.

Nucleoside analogues for the treatment of coronavirus infections

“…Classes of candidate companion compounds include NIs, helicase inhibitors, protease inhibitors, monoclonal antibodies, viral entry inhibitors, and DEDDh family exoribonuclease inhibitors with potential activity against ExoN [46,47 ,48], the latter deserving special interest. DEDDh inhibitors aurintricarboxylic acid and pontacyl violet 6R effectively inhibited Lassa virus NP exonuclease in a proof-of-concept biochemical assay [48]. The structural and functional conservation across CoV family members and the absence of redundant functions elsewhere in the genome makes ExoN a potential Achilles' heel.…”

Computational Characterizations of the Interactions Between the Pontacyl Violet 6R and Exoribonuclease as a Potential Drug Target Against SARS-CoV-2

“…Results were analyzed using Autodock Tools and Chimera packages (Forli et al, 2016;Anil, 2019). Blind dock studies were carried out using previously identified three DEDDh/DEEDh subfamily nuclease inhibitors, pontacyl Violet 6R (PV6R), aurintricarboxylic acid (ATA) and 5,5′-dithiobis (2nitrobenzoic acid) (DTNB) (Huang et al, 2016). The homology model of the SARS-CoV-2 nsp14 was used as the macromolecule.…”

“…Further, structural studies of SARS-CoV nsp14 showed the presence of five catalytic residues. These features reveal that nsp14 is a member of DEDDh/DEEDh subfamily (Huang et al, 2016;Ogando et al, 2019). Interestingly, nsp14 in SARS-CoV exhibits both the (N7-guanine)-methyltransferase (N7-MTase) activity and ExoN activity.…”

“…A recent report argued that an effective anti-viral drug design has to consider the balancing act between nsp12 and nsp14 (Shannon et al, 2020). Despite being a potential drug target, few compounds have been identified to inhibit the nsp14 activity in vitro (Huang et al, 2016). Simultaneous attack of RdRp and nsp14 using a combination of a nucleoside analog and a specific nsp14 inhibitor may enhance lethal mutagenesis in SARS-CoV-2 (Ogando et al, 2019).…”

Cryo-electron microscopy and X-ray crystallography: complementary approaches to structural biology and drug discovery

“…They represent an immense step in understanding molecular pharmacology at the closest level, and have made it possible for medicinal chemists to gain information leading them to rationally synthesize series of molecules designed to become drug candidates. Such successful examples are scattered throughout the medicinal chemistry literature and can be found easily (see, for example, Huang et al, 2016;Gustafsson et al, 2017;Hazel et al, 2017;Mills-Davies et al, 2017).…”