1. Using a computational approach to identify potential inhibitors of the
Leishmania donovani lipase, LdLIP3
Stephanie Beels, Dr. Steven Symington, and Dr. Alison Shakarian
Department of Biology and Biomedical Sciences
Salve Regina University Newport, RI 02840
Leishmaniasis is a disease caused by a parasite transmitted by the sand fly
in areas such as Africa, the Middle East, and Latin America. Leishmania
donovani is one species of a single-celled parasite that causes
Leishmaniasis, and can often result in a fatal visceral disease. LdLIP3 is a
specific secreted lipase found in the supernatant of Leismania donovani
that is involved with energy utilization. The long-term goal of this project is
to identify efficient natural substrates of LdLIP3 which will facilitate the
discovery of possible inhibitors for the enzyme that can be utilized for
Leishmaniasis treatment. To that end, we utilized open source
computational tools to model the substrate binding site of LdLIP3 and
identify the potential inhibitors of lipase activity. A previously crystallized
control lipase from Rhizomucor meihei (3TGL), was modeled and docked
with two natural substrates (palmitate and stearate) and potential inhibitors.
Autodock was utilized for the molecular docking experiments to quantify
the various hydrogen bonds, electrostatic interactions, binding affinities and
energy minimizations associated with enzyme-substrate relationships. It
was found that palmitate is a more efficient natural substrate than stearate
for lipase from Rhizomucor meihei. We also evaluated the binding
characteristics of three potential competitive inhibitors from the terpenoid
class of drugs (citral, menthol, and THC) on the Rhizomucor meihei lipase.
Each of the competitive inhibitors were analyzed for binding affinities and
energy minimization. The computational data indicate that THC is a
stronger inhibitor than citral and menthol. Collectively, the results from the
competitive inhibitor study using the control lipase, strongly suggest that
THC could be a potential inhibitor for the Leishmania donovani lipase,
LdLIP3. Future studies will evaluate the results obtained from the
computational approach by determining the effect of the terpenoid drugs of
LdLIP3 purified from Leishmania donovani.
Abstract!
Conclusions!
Acknowledgements!
Potential LdLIP3 Competitive Inhibitors!
Palmitate’s and Stearate’s predicted binding energies are statistically equivalent.
THC has a ~100% better binding affinity than Citral, and a ~50% better binding
affinity than Menthol. Menthol has a higher binding affinity for 3TGL than Citral
by ~35%. The significance of these findings suggest the exploration of THC as
a potential competitive inhibitor for LdLIP3 may be warranted in future studies.
The Control Enzyme and Potential LdLIP3 Natural Substrates!
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Project Goal!
By finding efficient natural substrates of LdLIPS, we will then be able to
deduce possible inhibitors for the enzyme. By inhibiting LdLIP3, we may
be able to identity new drug targets for this important human disease.
Lipase Background!
Experimental Procedure!
Modeling
Preparation
Input small
molecule .mol2 and
macromolecule .pd
bqt files found in
Protein Data Bank
Fix small
molecule
into .pdbqt and
macromolecule
into rigid residue
AutoGrid
Setup
Convert .pdbqt files
into .gpf files
Run AutoGrid
for .glg file output
AutoDock
Setup
Convert .pdbqt files
into .dpf files
Run AutoDock
for .dlg file output
Evaluation
View and sort poses
of top ten
conformations for
each run
Calculate residue
distances from
small molecule
Figure 4. Summary of THC Predicted Dockings.
Roughly sixty percent of the fifty predicted
conformations share the same top pose. The average
binding energy for these poses is -9.18 kcal/mol. This
graph demonstrates the average binding energy for
each of the eight different poses for THC, compared to
the frequency of each pose.
Figure 6. Summary of Citral Predicted Dockings.
Roughly sixty percent of the fifty predicted
conformations same the same two top poses. The
average binding energy for these poses is -4.59 (Pose
7) and -4.65 kcal/mol (Pose 5). This graph
demonstrates the average binding energy for each of
the eight different poses for Citral, compared to the
frequency of each pose.
Figure 8. Summary of Menthol Predicted Dockings.
Roughly seventy-five percent of the fifty predicted
conformations shame the same top pose. The average
binding energy for these poses is -6.29 kcal/mol. This
graph demonstrates the average binding energy for
each of the eight different poses for Menthol, compared
to the frequency of each pose.
THC
Citral
Menthol
Figure 5. Structure (A) and Predicted Binding Site THC
of Pose 2 (B). THC binds to the following residues:
ILE89, TRP88, LEU92, SER82, SER144, VAL205,
PRO209, PHE215, PHE94, PHE111, and HIS108.
Figure 7. Structure (A) and Predicted Binding Site of
Citral of Pose 7 (B). Citral binds to the following
residues: ARG178, PRO209, PHE215, LEU92, PHE94,
PHE111, and HIS108.
Figure 9. Structure (A) and Predicted Binding Site of
Menthol of Pose 1 (B). Menthol interacts with the
following residues: LEU208, ARG178, PRO209, PRO177,
PHE213, PHE215, PHE94, PHE111, and HIS108
Stearate
Palmitate
Control Lipase
Figure 1. Summary of Control Lipase. 3TGL is
known to be secreted by Rhizomucor meihei,
similar to the way LdLip3 is secreted by
Leishmania donovani. Residues 83-94 are
known to act as a lid over the active site,
serving a kinetic and mechanical purpose in the
activation of the enzyme.
Figure 2. Palmitate Predicted Binding Site. The average
binding energy for palmitate when bound to 3TGL for
five dock runs and fifty conformations is -4.31 kcal/
mol. The top conformation for palmitate interacts with
the following residues: PHE111, HIS108, PHE94,
THR93, LEU92, TRP88, ALA90, ILE89, ILE204,
VAL205, LEU208, PRO208, ARG178, and PHE215.
Figure 3. Stearate Predicted Binding Site. The
average binding energy for stearate when
bound to 3TGL for five dock runs and fifty
conformations is -4.15 kcal/mol. The top
conformation for stearate interacts with the
following residues: PHE94, PHE111, THR93,
LEU92, PRO209, SER144, VAL205, LEU268,
PRO209, and ILE204.
Lipases catalyze the hydrolysis of lipids at the ester linkages. Acting at a
specific position on the glycerol backbone of lipids, lipases break down
lipids into monoglycerides and two fatty acid chains. These enzymes play
a crucial role in the growth, development, survival, and virulence in several
pathogenic organisms. Applying what we know about lipases, we can
hypothesize that LdLIP3 may play in large role in its survival in a human
host.
Supported by RI-INBRE grant #P20RR016457
Printing services provided by Salve Regina University
The contents of this poster are solely the responsibility of the author and do not
represent the professional views of the NCRR or NIH.
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• Shakarian AM, McGugan GC, Joshi MB, Stromberg M, Bowers L, Swyer DM, et al.
(2010) Identification, characterization, and expression of a unique secretory lipase
from the human pathogen Leishmania donovani. Mol Cell Biochem 341:17-31.
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