PDBsum entry 5k5m

Hydrolase

PDB id: 5k5m

Contents

Protein chain

563 a.a.

Ligands

68T

MES

Metals

_ZN ×2

Waters ×429

PDB id:

5k5m

Name:

Hydrolase

Title:

Co-crystal structure of dengue virus serotype 2 RNA dependent RNA polymerase with compound 27

Structure:

RNA dependent RNA polymerase. Chain: a. Fragment: unp residues 2757-3391. Engineered: yes

Source:

Dengue virus 2. Organism_taxid: 11060. Expressed in: escherichia coli. Expression_system_taxid: 511693. Expression_system_variant: de3

Resolution:

2.01Å R-factor: 0.180 R-free: 0.216

Authors:

R.Arora,T.E.Benson

Key ref:

S.P.Lim et al. (2016). Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling. Plos Pathog, 12, e1005737. PubMed id: 27500641 DOI: 10.1371/journal.ppat.1005737

Date:

23-May-16 Release date: 24-Aug-16

Protein chain

A0A097PIE2  (A0A097PIE2_9FLAV) -  Genome polyprotein from dengue virus type 2

Seq: 3391 a.a.

Struc: 563 a.a.

Enzyme reactions

• Enzyme class 1: E.C.3.4.21.91 - flavivirin.
• Reaction: Selective hydrolysis of Xaa-Xaa-|-Xbb bonds in which each of the Xaa can be either Arg or Lys and Xbb can be either Ser or Ala.
• Enzyme class 2: E.C.3.6.1.15 - nucleoside-triphosphate phosphatase.
• Reaction: a ribonucleoside 5'-triphosphate + H2O = a ribonucleoside 5'-diphosphate + phosphate + H⁺
• Enzyme class 3: E.C.3.6.4.13 - Rna helicase.
• Reaction: ATP + H2O = ADP + phosphate + H⁺

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1371/journal.ppat.1005737

Plos Pathog 12:e1005737 (2016)

PubMed id: 27500641

Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.

S.P.Lim, C.G.Noble, C.C.Seh, T.S.Soh, A.El Sahili, G.K.Chan, J.Lescar, R.Arora, T.Benson, S.Nilar, U.Manjunatha, K.F.Wan, H.Dong, X.Xie, P.Y.Shi, F.Yokokawa.

ABSTRACT

Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket"). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.