The HCV NS5B RNA dependent RNA polymerase plays an essential role in viral replication. The discovery of a novel class of inhibitors based on an N,N-disubstituted phenylalanine scaffold and structure-activity relationships studies to improve potency are described.
"Nonnucleoside inhibitors are a class of small organic compounds of hydrophobic nature that have been known to be potent and effective therapeutics with great specificity against HIV-1. Similar inhibitors targeting HCV RdRp are currently under development (Chan et al., 2003; Dhanak et al., 2002; Love et al., 2003). These inhibitors act kinetically in a non-competitive manner with respect to dNTP or rNTP substrates. "
[Show abstract][Hide abstract] ABSTRACT: The RNA-dependent RNA polymerase (RdRp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV RdRp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-RdRp was found to cleave into three main fragments: an N-terminal p12 fragment, a middle p30 fragment, and a C-terminal p64 fragment comprising the catalytic domain, presumably due to bacterial proteases. Biochemical assays show that the full-length GST-RdRp has RdRp activity and the p64 and p12 fragments form a complex that exhibits comparable RdRp activity, whereas the GST-p64 protein has no activity, suggesting that the p12 domain is required for polymerase activity possibly via involvement in template-primer binding. Nonnucleoside HIV-1 RT inhibitors are shown to have no evident inhibitory effect on SARS-CoV RdRp activity. This work provides a basis for biochemical and structural studies of SARS-CoV RdRp and for development of anti-SARS drugs.
[Show abstract][Hide abstract] ABSTRACT: Herein, we describe the structure-activity relationship (SAR) of N,N-disubstituted phenylalanine series of NS5B polymerase inhibitors of hepatitis C. The NS5B polymerase inhibitory activity of the most active compound exhibited an IC(50) of 2.7 microM.
[Show abstract][Hide abstract] ABSTRACT: The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS‐CoV. The RNA‐dependent
RNA polymerase (RdRp) of SARS‐CoV plays a pivotal role in viral replication and is a potential target for anti‐SARS therapy.
There is a lack of structural or biochemical data on any coronavirus polymerase. To provide insights into the structure and
function of SARS‐CoV RdRp, we have located its conserved motifs that are shared by all RdRps, and built a three‐dimensional
model of the catalytic domain. The structural model permits us to discuss the potential functional roles of the conserved
motifs and residues in replication and their potential interactions with inhibitors of related enzymes. We predict important
structural attributes of potential anti‐SARS‐CoV RdRp nucleotide analog inhibitors: hydrogen‐bonding capability for the 2′
and 3′ groups of the sugar ring and C3′ endo sugar puckering, and the absence of a hydrophobic binding pocket for non‐nucleoside analog inhibitors similar to those observed
in hepatitis C virus RdRp and human immunodeficiency virus type 1 reverse transcriptase. We propose that the clinically observed
resistance of SARS to ribavirin is probably due to perturbation of the conserved motif A that controls rNTP binding and fidelity
of polymerization. Our results suggest that designing anti‐SARS therapies can benefit from successful experiences in design
of other antiviral drugs. This work should also provide guidance for future biochemical experiments.
Nucleic Acids Research 12/2003; 31(24):7117-7130. DOI:10.1093/nar/gkg916 · 9.11 Impact Factor
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