A Conserved Pocket in the Dengue Virus Polymerase Identified through Fragment-based Screening

Abstract

We performed a fragment screen on the dengue virus serotype 3 RNA-dependent RNA polymerase using x-ray crystallography. A screen of 1,400 fragments in pools of eight identified a single hit that bound in a novel pocket in the protein. This pocket is located in the polymerase palm subdomain and conserved across the four serotypes of dengue virus. The compound binds to the polymerase in solution as evidenced by surface plasmon resonance and isothermal titration calorimetry analyses. Related compounds where a phenyl is replaced by a thiophene show higher affinity binding, indicating the potential for rational design. Importantly, inhibition of enzyme activity correlated with the binding affinity, showing that the pocket is functionally important for polymerase activity. This fragment is an excellent starting point for optimization through rational structurebased design.Dengue virus (DENV) 3 is the most widespread mosquitoborne viral infection. Disease symptoms of DENV-infected patients range from a mild fever to severe plasma leakage and hemorrhagic shock (1). There are four serotypes of DENV (DENV-1 to -4) that concurrently circulate around the world in tropical and subtropical regions. The vast majority of clinical cases are not reported, and there are estimated to be approximately 390 million human cases of dengue worldwide per year (2). There is currently no licensed vaccine or antiviral to treat DENV infection, underlining the urgency for the development of safe therapeutics (3). DENV is a member of the Flavivirus genus that also includes other viruses that are pathogenic to humans such as West Nile virus (WNV), yellow fever virus, Japanese encephalitis virus (JEV), and tick-borne encephalitis virus.One of the most attractive antiviral targets is the DENV RNA-dependent RNA polymerase (RdRp) because (i) viral polymerases are clinically proven therapeutic targets and (ii) the RdRp is the most conserved viral protein among the four serotypes of DENV so that the likelihood of a single compound with pan-serotype activity is higher than compounds targeting other viral proteins. The DENV RdRp activity resides in the C-terminal two-thirds of the viral nonstructural protein 5 (NS5) (reviewed in Ref. 4), whereas the N-terminal one-third of DENV NS5 encodes a methyltransferase (5).Crystal structures of the RdRp domain and full-length NS5 have been determined (6, 7). The overall architecture of the polymerase resembles a right hand with fingers, palm, and thumb subdomains. DENV RdRp catalyzes de novo initiation, as well as elongation. Like other RdRps that perform de novo RNA synthesis, DENV RdRp has a fully encircled active site (8). These polymerases undergo a conformational change from a closed to an open conformation during the transition from de novo initiation to elongation. During this process, the initiation loop (also known as the priming loop) is thought to move out of the active site, in a manner similar to the hepatitis C virus polymerase (9).A number of in vitro enzyme assays have been developed for DENV Rd...

The crystal structure of Zika virus NS 5 reveals conserved drug targets

“…The priming loop locates between α19 and α20 in the subthumb domain, which is deployed inside the catalytic chamber to promote formation of the initiation complex. This loop is well‐ordered and inserting into a narrow RNA binding tunnel in our structure, implying that our ZIKV RdRp adopts a “closed” pre‐initiation state conformation (Noble et al , , ; Lim et al , ).…”

“…Previously, two conserved inhibitor‐binding sites have been identified for DENV RdRp (Noble et al , , ; Lim et al , ; Yokokawa et al , ). One is targeting the RNA template entry tunnel (Noble et al , ), and the other pocket, called as the “N pocket”, located in the thumb subdomain, close to the enzyme active site (Lim et al , ; Noble et al , ; Yokokawa et al , ). By targeting the RNA tunnel, the compound NITD107 was first identified by enzyme activity‐based screening and the binding site was defined by complex structure (Noble et al , ).…”

“… Superimposition of ZIKV RdRp with DENV3 RdRp apo structure (gray) (PDB: 4HHJ) or DENV3 RdRp (light pink) bound to NTTD107 molecule (purple) (PDB: 3VWS) (Niyomrattanakit et al , ; Noble et al , ). The detailed interactions are shown in the panel on the right side. Superimposition of ZIKV RdRp with DENV3 RdRp (gray) bound to JF‐31‐MG46 molecule (orange) (PDB: 5F3T) (Noble et al , ). The detailed interactions are shown in the panel on the right side. Superimposition of ZIKV RdRp with DENV3 RdRp (gray) bound to 29 compound (gold) (PDB: 5I3Q) (Lim et al , ; Yokokawa et al , ).…”

Identification of allosteric binding sites for PI3Kα oncogenic mutant specific inhibitor design

“…31,32 Fragments are small molecular weight compounds (MW < 300 Da) that are frequently used for the discovery of novel drugs. They generally possess good drug-like properties, high ligand efficiency (a measure of affinity corrected by size), and good coverage of available chemical space.…”

Repurposing an HIV Drug for Zika Virus Therapy

“…The structural information could be used for rational design, as previously shown for dengue RdRp inhibitors. 13 In addition, compounds with improved potency could also be tested in a pregnant mouse model for prevention of maternal-to-fetal viral transmission. 14 Collectively, the study by Sariyer et al 2 reinforces the potential of drug repurposing for therapeutic development against emerging and re-emerging pathogens.…”