Virtual Screening for RNA-Interacting Small Molecules - Biophysical Approaches to Translational Control of Gene Expression - page 237

The results suggested that TAR NMR-MD improved the accuracy of docking compared
with TAR MD . From this result TAR NMR-MD was chosen for further virtual screening to
identify small molecule TAR-Tat inhibitors.

12.4.1.3 Virtual Screening Against TAR Dynamic Ensemble

A chemical database consisting of 49,166 compounds was obtained from the Center
for Chemical Genomics at the University of Michigan and 2,060 compounds from
the author's in-house library. Based on the ICM Score, the top 57 commercially
available hit compounds were selected and their binding activities were tested by
fluorescence-based assays. This identified six small molecules that bound to TAR
with high affinity ( K d = 55 nM to 122 mM) and inhibited TAR interaction with Tat
( K i = 710 nM to 169 mM) in vitro. Among them, netilmicin ( K d = ~1.4 m M) bound to
HIV-1 TAR with the highest selectivity over HIV-2 TAR. Netilmicin repressed Tat-
mediated transactivation of the HIV-1 promoter through its interaction with TAR in
live human T-cells and inhibited HIV-1 replication in the HIV-1 indicator cell line
TZM-bl.

12.4.2 Case 2: RNA Pseudoknots

Ribosomal −1 frameshifting (−1 FS) is an essential event during translation for the
synthesis of two or more proteins encoded by overlapping reading frames on a single
mRNA (Dinman and Berry 2007 ) in many RNA viruses such as retroviruses, corona-
viruses, yeast, plant virus, and even bacteria (Jacks and Varmus 1985 ; Brierley et al.
1991 ; Chamorro et al. 1992 ; Tzeng et al. 1992 ; ten Dam et al. 1994 ; Kang and Tinoco
1997 ; Jacobs et al. 2007 ) . Two cis -acting elements are required to regulate −1 FS.
One is a slippery sequence where ribosome-associated tRNAs slip, and the other is
RNA secondary structure such as a hairpin or pseudoknot that promotes ribosome
pausing. Thermodynamic or kinetic control of RNA secondary structure folding
may be important in regulating the efficiency of −1 FS. Human immunodeficiency
virus type 1 (HIV-1) utilizes −1 FS to regulate the expression ratio of Gag to Gag-
Pol, which is critical for the production of infectious virion particles (Paulus et al.
1999 ). The RNA stem-loop sequence that is involved in −1 FS of HIV-1 is highly
conserved in the main subtypes of HIV-1 (Gareiss and Miller 2009 ). Mutations of
this sequence reduce −1 FS and decrease viral infectivity and replication (Baril et al.
2003 ; Dulude et al. 2006 ). In severe acute respiratory syndrome coronavirus (SARS-
CoV), replicase genes mainly encode two large replicative polyproteins (pp1a and
pp1ab) which are expressed by two partially overlapped open reading frames ORF
1a and ORF 1b. As ORF 1b has no independent translation initiation site, polypro-
tein pp1ab encoded by ORF 1b is only translated as a fused protein form with ORF
1a through −1 FS. As pp1ab includes RNA-dependent RNA polymerase (RdRp)
and other replication components which are important proteins for viral replication,