Biology Reference
In-Depth Information
Discovery of Potential Inhibitors of the
S. pneumoniae
VicK HK by Virtual
Screening
The target site for high throughput virtual screening (HTVS) was the ATP-binding
pocket of the VicK HATPase_c model of
S. pneumoniae
, which consisted of residues
within a radius of 4 Ǻ around the ATP site. In the primary screening, the database
SPECS containing about 200,000 molecules was searched for potential binders using
the program DOCK4.0 [30, 31]. Subsequently, structures ranked in the first 10,000
were re-scored by using the Autodock 3.05 program [32]. As a result, about 200 mol-
ecules were filtered out by these highly selective methods. Finally, we manually se-
lected 105 molecules according to their molecular diversity, shape complementarities,
and the potential to form hydrogen bonds and hydrophobic interactions in the binding
pocket of the VicK HATPase_c domain.
Inhibition of the VicK' Protein ATPase Activity
In Vitro
In order to confirm the interaction of the potential VicK inhibitors with their puta-
tive target protein, we expressed and purified His-tagged VicK' protein by using the
pET28a plasmid in BL21(DE3) as shown in Figure 3A. The kinase activity of VicK'
protein was measured by quantifying the amount ATP remained in solution after the
enzymatic reaction (Figure 3B). These results indicated that the purified VicK' pro-
tein possessed the ATPase activity, which can hydrolyze ATP
in vitro
. Using the puri-
fied active VicK, we obtained 23 compounds from the 105 candidate inhibitors which
could decrease the ATPase activity of VicK' protein by more than 50%, indicating
these compounds may also be potential VicK inhibitors in
S. pneumoniae
.
Figure 3.
(A) SDS-PAGE analysis of VicK' purification (B) Identification of kinase activity of VicK'
protein
in vitro
. Variant amounts of VicK' proteins were added into reaction systems containing a
constant ATP concentration (5 μM). Each assay was performed in quadruplicate and repeated three
times. Luminescent output is inversely correlated with the concentration of the kinase.
Antimicrobial Activities of Potential Vick' Inhibitor and Cytotoxicity of the
Antimicrobial Compounds
In
Vitro
We investigated the bactericidal activity of these 23 compounds against
S. pneumoni
ae
using a standard minimal bactericidal concentration assay (MIC) (Table 1). Six
