STRUCTU RE-BASED PHARMACOPHORE

GEN ERATION AN D
VIRTUAL SCREENING FOR POTENTIAL INHIBITORS OF
PANTOTHENATE
SYNTHETASE (ps)
oF M. tubercurosis
by
VIVIEN CHERIE C. UY
An Undergraduate Thesis
Submitted to
the Department of Physical Sciences and Mathematics
University of the Philippines Manila
In partial
fulfillment of the requirements
for the Degree of
Bachelor of Science in Biochemistry
April 2011
The thesis entitled
"Structure-based
Pharmacophore Generation and
Virtual Screening for Potential lnhibitors of Pantothenate Synthetase (PS) of M.
tuberculosrs", prepared and submitted by Vivien Cherie C. Uy in partial fulfillment
of the requirements for the degree of Bachelor of Science and Biochemistry is
hereby accepted.
hp"'l 6 ,
htl
Date
Accepted as partial fulfillment of the requirements for the degree of
Bachelor of Science in Biochemistry^
Department of Physical Sciences and Mathematics
Reynaldo H. lmperial, Ph.D
Dean
College of Arts and Sciences
Junie Billones, Ph. D.
Adviser
Chair
ACKNOWLEDGEMENT
To God, thank you for engineering my whole thesis life, from the
conceptualization of the proposal
to the book-binding of the manuscript. Thank
you for giving me the people
around me, my thesis advisor, my family, my
classmates who gave me support and the people who helped me both directly
and indirectly. Thank you for giving me the good luck in downloading the
database which had the compound which I was searching for. And thank you for
giving me the inspiration to have finished this stage of my life.
To Dr. Billones, thank you for everything. Thank you for suggesting this
topic when I needed to change topics. Thank you for being supportive especially
during the time when I thought that I would be unable to finish this. Thank you for
being approachable and allotting time for us even when you were very busy.
Thank you for all the advice, the constructive criticisms and the explanations to
both my methods and my manuscript. And even with all of my weakness, thank
you for still guiding me. Thank you very much, sir!
To Accelrys lnc, thank you for the software which I have used for the all
the computations required. Also, thank you for the tutorial, incorporated in the
software, which guided me throughout my thesis.
To my family, and friends, thank you for praying for me and being
supportive. Thank you to my parents who were my major inspiration in finishing
this thesis before graduation. Thank you for my friends for lending me their ears,
and for the advice they gave. Thank you to my sisters and cousins for being the
helping hands in front of me, and the supportive, but at the same time, pushing
hands behind me. Thank you for waking me up at night when I had something to
do. Thank you for rejoicing with me upon finishing both the defense and
manuscript.
To my fellow classmates, who are also doing computational studies, thank
you for the companionship, advice, comparison of methods and sharing of
resources. Thank you for the knowing the feeling of doing computational studies
and sharing your
own experiences. Special thanks to Kuya Tristan who has been
very accommodating in answering my questions about the use of Accelrys.
To Ate Catrina Yang, thank you for being my model in the use of
Pantothenate Synthetase my protein target.
To my teachers for the past four years and the years before that, thank
you for building up the foundations which I needed to do and interpret my thesis
and understand the reason for it. Without a background about proteins, inhibitors,
amino acids, the different interactions, and other things, I would have been very
lost during the thesis.
To my classmates, thank you for being helping me through class, and for
just
being there. Thank you for being accepting, being nice, and being fun.
Especially to the class representatives, thank you for informing me of all the
announcements and requirements. Thank you for answering the questions I had,
even if it's stupid.
To everyone who has helped me, directly or indirectly, thank you! Thank
you! Thank you very much!
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
I INTRODUCTION
Background of the Study
Significance of the Study
Objectives
Scope and Limitation
II. REVIEW OF RELATED LITERATURE
III. MATERIALS AND METHODS
IV. RESULTS AND DISCUSSION
V, CONCLUSION AND RECOMMENDATION
VI. REFERENCES
APPENDIX
A. Structures and Binding Energies of Ligands
B. First Screening Compounds and their Fit Values from
Complete Screening
C. First Screening Compounds and their Fit Values from
Hitfinder
D. First Screening Compounds and their Fit Values from
TOSLab
E. First Screening Compounds and their Fit Values from
Natx
F. First Screening Compounds and their Fit Values from
NCI
G. Second Screening Compounds, Fit Values, and their
Binding Energies
H. Interaction of LIGAND and 1N2H receptor
I
2
2
3
4
7
ll
23
24
27
aa
af
34
35
36
37
38
39
LIST OF TABLES
Table L Rigid and Flexible Fit Value, and Initial and Final Docking Binding Energy of
compounds which were Final Docked 15
Table 2. The number of polar, hydrophobic, H-bonding, charged and pi interactions of
dockedcompounds 15
Table 3. Binding Energy from the Docking of the Modification of NSC_125296 19
Table 4. The number of polar, hydrophobic, h-bonding, charged and pi interactions of
modified compounds 19
LIST OF FIGURES
Figure 1. (left) Superimposition of original (blue) and prepared (orange) PS. (right)
Superimposition of instance of PA (blue) and docked PA (orange). 12
Figure 2. Violet residues have polar interactions; green residues have vdw interactions;
dashed blue and green lines have H-bonding interactions; and orange line represents pi
interaction (top) diagram of native PA. (bottom) interaction diagram of docked PA 13
Figure 3. (left) The site sphere (red) used in all the calculations has a radius of 10.6151.
(right) The clustered pharmacophore generated is composed of 20 features: acceptor
features (green), donor features (violet) and hydrophobe features (blue) 14
Figure 4. Interaction diagram of Nafronyl Oxalate 17
Figure 5. Interaction diagram of NSC_125296 17
Figure 6. Interaction diagram of NSC_167356 18
Figure T.Interaction diagram of modified compound, K. 20
Figure 8. Interaction diagram of modified compound, L. 2A
Abstract
Tuberculosis (TB) is a bacterial infection which is affecting the people around the
world, especially those in South East Asia. Great concern is arising because of the
emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB
POR-TB).
A structure-based pharmacophore was generated based on the binding site of
the native PA in the original protein. The pharmacophore was used to screen five
compound libraries, NCI, Complete Screening, TOSLab, HitFinder and NATx. Among
the library compounds, NSC_I25296 was determined to have the highest binding energy
of -356.418 kcal/mol. On the other hand, the substrate intermediate pantoyl adenylate
(PA) had -482.0A2 kcal/mol while nafronyl oxalate
O{O)
(a known inhibitor) had -
98.5778 kcal/mol. NSC_125296 was modified, and five of the modifications were found
to have a greater binding energy than PA. In particular, a modified structure K in which a
hydroxyl group was substituted in each of the naphthalene ring has binding energy that is
twice gteater than that of PA. Generally, the addition of a hydroxyl group to aromatic
ring of NSC_125296 resulted to the formation of charged interactions with ArgI32, Arg
198 and Arg298 and increased the binding energy. Since K, C, F, D and L had a higher
binding energy than PA, they are potential competitive inhibitors.
I.INTRODUCTION
Background ofthe Study
Tuberculosis (TB), which is caused by the bacterium, Iufycobacterium tuberculosis,
is an infectious disease usually transmitted through droplets in the air (Tuberculosis,
n.d.). Although the incidence of TB is dropping in countries like America and Europe, it
is still a major concern in South-East Asian Countries (Tuberculosis, 2010.). Varaine,
Henkens & Gauzard (2010) enumerates that the common drugs against TB are Isoniazid
and Rifampicin; however, with the use and misuse of these drugs, multidrug-resistant
tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis
Q(DR-TB)
develop
(XDR-TB, n.d.). These resistances lead to the need for new drugs.
Pantothenate synthetase (PS) is an enzyme which catalyzes the formation of
pantothenate (Vitamin 85) from the condensation pantoate and
B-alanine.
Humans,
unlike bacteria, do not require the PS because they obtain Vitamin 85 through the diet.
Because the enzyme is not present in humans, PS is a good target for drugs against TB
(White, Southworth & Ross, 2007).
According to Irfan (n.d.), structure based drug design uses the known 3D structure
of known binding sites. A pharmacophore is a framework of features which are important
for a better receptor-ligand binding. After creation, the pharmacophore can be used to
virtually screen a database of small molecules for compounds which may be active.
Molecular docking can then evaluate the protein-ligand fit and the complex formed
(Accelrys Software Inc., 2001-2009).
Significance of the Study
TB is a worldwide pandemic. Furthermore, the emergence of MDR-TB and )OR-
TB makes TB harder to cure; thus, there is a need to develop new drugs to combat TB.
Structure-based pharmacophore screening allows the screening of a larger set of
compounds; thus, allowing the exploration and evaluation of a greater variety of
compounds. Screening could mark compounds which are likely to be drug candidates;
thus they prioritize compounds which are to be evaluated in the laboratory. This
prioritization saves both money and time in that it avoids the randomized testing of
compounds, but rather, focuses on compounds which have already been evaluated.
Objectives
General Objectives
To identifu compounds which have higher binding energies to PS compared to the
reaction intermediate, pantoyl adenylate, and the known inhibitor, nafronyl oxalate.
Spectfic Objectives
1. To generate a pharmacophore based on the binding site
2. To screen compounds from compound databases using the generated
pharmacophore
3. To dock the screened compounds and determine the binding energy
4. To modify the compound with the highest binding energy
5. To dock the modified compounds and determine the binding energy
Scope and Limitation
All computational methods were done using the software, Discovery Studio 2.5.5.
The binding site of the reaction intermediate, pantoyl adenylate, was the basis for the
pharmacophore generation and the docking. During the docking process, the protein was
held rigid while the ligands were allowed to have different conformations. The pdb file of
the PS used was 'Crystal Structure of a Pantothenate Synthetase from M. tuberculosis in
complex with a reaction intermediate, pantotyl adenylate', while the compound libraries
were NCI-Open_09-03, Complete Screening January 201L, TOSlab_Collection,
Hitfi nder*V I 0, and NATx_$elease_ 1 00 9 I 5
_All
20 67 4_cpds.
IL REVMW OF LITERATURE
Tuberculosis
Tuberculosis (TB) is an infectious disease, which is caused by M. tuberculosis, and
is usually transmitted by coughing, sneezing, talking, or singing (Tuberculosis(TB), n.d.).
Varaine, Henkens & Gouzard(2010) believes that the aerobic quality and slow-growth of
the M. tuberculosisas accounts for the slow evolution of TB and the often infection of the
lungs. Howeover, unless the infected person's immune system is compromised, they do
not necessarily get sick (Tuberculosis, 2010).
The two common drugs against TB are Isoniazid and Rifampicin; other firstline
drugs include Streptomycin, Pyrazinamide, and Ethambutol (Varaine, Henkens &'
Gouzard,2010). Johnson, et.al. (2006) claims that the use of drugs not only leads to the
killing of the pathogen, but also to an increased number of resistant strains, by allowrng
these strains to live. Resistance also arise from the non-compliance in the usage of drugs
in either taking less of the dose
Q(DR-TB,
n.d.) or taking only one of the combination
drugs (Davies, 1999). The misuse of TB drugs results to MDR-TB and )OR-TB. MDR-
TB is resistant to first-line drugs while XDR-TB is resistant to second-line drugs
OOR-
TB, n.d.).
WHO estimates that M. tuberculosis has infected about 33o/o of the world's
population. The organization also estimated that about 9.4M TB cases emerged in 2009
and about 1.3M people (excluding those with HIV) died. Among the 6 WHO regions,
Africa, the Americas, Eastern Mediterranean, Europe, South-East Asia and Western
4
pacific,
South-East Asia had the highest TB incidence in 2009. In this same year, the
philippines
was considered the 9ft country most afflicted with TB (Philippines, n.d.).
Pantothenate Synthetase
Pantothenate synthetase
(PS) is an enzyme which uses ATP to catalyze the
formation of pantothenate (Vitamin 85) from pantoate and
B-alanine.
Pantothenate is
important in bacteria because it is necessary for the "biosynthesis of Coenzyme A (CoA)
and Acyl Carrier Protein (ACP)". Humans, unlike bacteria, do not have the PS; instead
they obtain Vitamin 85 through the diet. Because the enzy'rne is not present in humans,
PS is a good target for drugs against TB (White, Southworth & Ross, 2007).
One of the crystal structures of PS is "Crystal Structure of a Pantothenate
Synthetase
from
M. tuberculosis in complex with a reaction intermediate, pantoyl
adenylate" (1N2H). This structure was derived by Wang and Eisenberg (2003) from an x-
ray diffraction experiment at a resolution of 2A.The protein crystallized with 3 sulfate
anions, 2 ethanal,l glycerol, 1 manganese and 2 pantoyl adenylate (one on each domain).
Pantoyl adenylate is the reactive intermediate of the condensation of pantoate and
p-
alanine with the help of ATP. It is produced by the reaction of pantoate to ATP. The
formation of pantoyl adenylate is important in the binding of
p-alanine to the enzyme and
subsequently, the continuation of the condensation reaction (Wang & Eisenberg,2003).
Zheng, et al (2004) published in their paper that substituting the H44, H47, N69,
Q72,
and Kl60, and to a lesser extent
Q164,
by alanine results in reduction of the abiltty
of PS to form the reaction intermediate pantoyl adenylate; thus, H44,H47, N69,
Q72,
K160 and
Q164
are the residues responsible for the catalytic activity of PS.
Nafronyl Oxalate was identified as a competitive inhibitor of PS by White,
Southworth, & Ross (2007). They also determined that Nafronyl Oxalate has a Ki 1/5ft of
PA's substrate's Km. Furthermore, a maximum dosage of l0ug/kg-body weight/ day of
Nafronyl Oxalate is allowed for humans.
Structure-Based Drug Design
Irfan (n.d.) summarized that the structure-based drug design uses the known 3D
structure of the known binding site of the protein. These proteins are usually important in
signaling pathways or are in involved with a disease as enzymes of involved reactions.
A pharmacophore describes the features and the location of these features, which are
important for a better receptor-ligand binding. After creation, the pharmacophore can be
used as a skeleton to screen a database of small molecules for compounds which may be
active (Accelrys Software Inc., 2001-2009).
Virtual screening, which follows the pharmacophore generation, is the screening of
large collections of databases for possible drug candidates and prioritizes which
compounds are to be synthesizedand assayed. (Waszkowycz,et. a1.,2001).
Molecular docking can then evaluate the protein-ligand fit and the complex formed
by the protein and ligand (Accelrys Software Inc., 2001-2009).
Itr. MATERIALS AND METHODS
All computational methods were done using Discovery Studio 2.5.5.
Preparation of Protein
The pdb file for the 'Crystal
Structure of a Pantothenate Synthetase from r1.4
tuberculosls in complex wrth a reaction intermediate, pantoyl adenylate' was obtained
from RCSB Protein Data Bank. The protein was then cleaned using the Clean Protein
tool, prepared using the Prepare Protein protocol and minimized using the Minimization
protocol with the CHARMm forcefield and an RMS of 0.1. The main chain atoms of the
minimized protein and the original protein are compared by the Align and Superimpose
Proteins protocol.
Because PS is a homodimer, only the domain A was used in the computational
processes. The pantoyl adenylate bound in domain A was used to determine the site
sphere by the Define Sphere
from
Selection tool. After which, all ligands bound in the
protein were removed, and then the protein was defined as the receptor using the Define
Selected Molecule as Receptor tool.
Generation of Pharmacophore
The pharmacophore
was generated using the Interaction Generation tool. The
resulting pharmacophore
was trimmed using the Cluster Curuent Feature and. Keep Only
Cluster centers tools for lc ceptor, Donor, and Hydrophobe Features.
Preparation of Screening Libraries
The compound libraries NCI-Open_09-03 (260,071), Complete Screening January
2011 (42,7A9), TOSlab_Collection (16,657), Hitfinder-V10 (14,400), and
NATx_Release_l00915*AllJ0674_cpds (20,674), were'obtained as SDF from NCI,
Ryan Scientific, Toslab, Ryan Scientific, and AnalytiCon-Discovery, respectively. The
reaction intermediate, pantoyl adenylate, was downloaded from RCSB; nafronyl oxalate,
on the other hand- was sketched.
The compounds were then prepared using the Prepare Ligands protocol allowing
ionization, generation of tautomers and isomers, and the Lipinski Filter (except for
pantoyl adenylate because it is not a candidate drug). Then, the compounds were built
into databases using the Build 3D Databctse, and the number of conformations to be
generated was set to 0.
Screening of Libraries
Pharmacophore screening was done on the databases using the Screen Library
protocol. For the first screening, a rigid fitting method was used. Compounds with a fit
value greater than 3.35 were used for the second screening.
For the second screening, the compounds (from the original file) were reprepared
and rebuilt as databases (similar to the preparation of screening libraries). This second set
of databases was then screened with a flexible fitting method. Compounds with a fit
values greater than 4.0 were used for the initial docking. Pantoyl adenylate and nafronyl
oxalate were rescreened regardless of the fit value.
Initial Docking
The compounds were selected from the flexible screening result. These compounds
were docked using the Dock Ligands (CDOCKER) protocol and the top hits based on the
-CDOCKER energy was set to 3. All docking protocols were done with the CHARMm
forcefield, the final minimization with Full Potential, and the generation of 10 Random
Conformations. The binding energies were then calculated using Calculate Binding
Energies protocol. Compounds with binding energy greater than -200kcal/mol, pantoyl
adenylate and nafronyl oxalate were used for the final docking.
Final Docking
The compounds were obtained from the raw file and prepared. The prepared
compounds were then minimized using the Ligand Minimization tool with an RMS of 0.1
and CHARMm forcefield. The compounds were then docked. However, this time, the top
hits were set to 10.
The docked ligands were once again minimized with the Ligand Minimization tool
with an RMS of 0.1 and using the docked protein as the receptor. Finally, the binding
energy was calculated.
The docked pantoyl adenylate with the highest binding energy was superimposed by
tether to the instance of pantoyl adenylate in the original protein. The RMSD of heavy
atoms was calculated.
Modification
The ligand with the highest binding energy was modified. The final docking steps
were then repeated using the modified compounds.
10
IV. RESULTS AND DISCUSSION
The PS protein is a homodimer; thus, only one of the identical domains (domain A),
was used in all the computational processes. This is because the results of the
computation executed in one domain are expected to be the same in the other domain.
Superimposition
The original and minimized proteins were superimposed to determine the extent of
the modification of the protein made by the program. This determines whether the protein
was relaxed to an extent of destroying its native conformation, and thus, failing to
simulate nature. The superimposed proteins in Figure 1 (left) show that there wasn't
much change in the main-chain atoms. This observation is supported by the calculated
RMSD, 0.814, which is lower than the upper limit of the acceptable protein RMSD of
1.54, as reported by (Dias & de Azevedo, 2008). Thus, the minimized protein is a usable
model of the protein.
Likewise, the native PA and the docked PA were superimposed, as shown in Figure
I (right), and an RMSD of 0.93A was calculated. This RMSD is also within the
acceptable limit which is 2.0A for ligands as reported by Costakes (2005). This step is
also important to determine if the docking protocol manages to duplicate nature.
Therefore, even if this is not an absolute test of docking protocol, it is one of the
validation steps.
t7
Figure 1. (left) Superimposition of original (blue) and prepared (orange) PS. (right)
Superimposition of instance of PA (blue) and docked PA (orange).
Moreover, looking at the interactions of the native and the docked protocol, shown
in Figure 2, the interactions are almost the same. The interacting residues which were
retained in both the native and docked PA are Pro38, Thr39, Met40, His44, Gly46,His47,
Leu50, Asn69, GIn72, Tyr82, Val139, Yall42, Yal143, Phel57, Gly158, Lys160,
Asp161, Gln164, Pro185, Thr186, Val187, and Met195. However, for some of the
residues, the interaction changed from nonpolar to polar (i.e. Thr39), new H-bonds were
formed (i.e. His47), and new interactions were formed. PA, being the reaction
intermediate, binds to the active site of PS. These interactions are characteristic of PA in
pS,
and some of these interacting residues have been identified as the catalyic residues.
Zheng, et. al. (2004) observed that changing His44, His 47, Asn69, Gln72, Lys160, or
Gln164 into alanine resulted to a decrease in PS activity. They therefore concluded that
these residues are in PA formation.
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Figure 2. Violet residues have polar interactions, green residues have vdw interactions;
dashed blue and green lines have H-bonding interactions; and orange line represents pi
interaction (top) diagram of native PA. (bottom) interaction diagram of docked PA
13
Pharmacophore Generation, Library Screening and Initial Docking
Figure 3. (left) The site sphere (red) used in all the calculations has a radius of 10.6151.
(right) The clustered pharmacophore generated is composed of 20 featwes: acceptor
features (green), donor features (violet) and hydrophobe features (blue)
The native PA was used to define the site sphere for the pharmacophore generation
and docking. This site sphere, shown in Figure 3 (left), has a radius of 10.6151A and an
x,y,z coordinate of (3T.792,34.104,43.017). The use of the native PA for site definition
means that the ligands which are being screened are patterned to fit the active site, and
would be docked to the active site; thus, they are potential competitive inhibitors.
The pharmacophore generated, shown in Figure 3 (right), consists of 20 features.
This pharmacophore has first been used to screen the databases rigidly, then flexibly. The
fit values from these screening are tabulated in Appendix B-F and G respectively.
Compounds which passed the flexible test were screened with initial docking. Likewise
the binding energies are tabulated in Appendix G.
Final Docking
Compounds, which had a binding energy greater than -200kca1lmol, PA and
Nafronyl Oxalate were evaluated in the final docking. These compounds are
NSC_I25296, NSC_I44248, NSC_618496, and NSC_I67356 from NCI, and ER748740
14
from TOSLab. The structures of these compounds are found in Appendix A. On the other
hand, the fit values for both rigid and flexible screening, and the binding energies of both
initial and final docking, of these compounds are tabulated in Table 1.
Table 1. Rigid and Flexible Fit Value, and Initial and Final Docking Binding Energy of
unds which Final Docked
PA was observed to have the highest binding energy of -482.002kcaVmal, while
Nafronyl Oxalate was observed to have the lowest binding energy of -98.5778kca1lmol.
Among the library compounds, NSC_125296 had the highest binding energy of -
356.4l8kcal/mol.
com lon wglg rura
Compound Fit Value
(Rigid)
Fit Value
(Flexible)
Initial Docking
Binding Energy
(kcal/mo1)
Final Docking
Binding Energy
(kcal/mol)
PA 2.76 3.31
-236.90 -482.00
NO
-98.58
NSC 125296 )-J I 4.r2 -362.ss -356.42
NSC 167356 3.t I 4.26 -20t.46 -301.93
NSC 144248 3.39 4.08
-248.68 -287.81
F,F(748740 3.35 4.05 -207.38 -252.08
NSC 618496 4.04 4.33 -209.13 -221.87
Table 2. The number of polar, hydrophobic, H-bonding, charged and pi interactions of
docked com
Compound Polar Hydrophobic H-Bond Charged Pi
PA
-
orieinal 1l t1 10 0 I
PA
-
docked T9 1l 10 0 I
NO 6 20 0 0 0
NSC 12s296
F,
l7 2 0 5
NSC 167356 6 10 4 2 I
NSC 144248 9 10 2 2 2
F,p(748740 9 t2 5 0 0
NSC 618496 7 l0 2 ^
L 0
The magnitude of the binding energy
interactions which the compound participates
is accounted for by
in. Table 2 tabulates
the strenglh o[ the
the number of polar,
15
van der waals (vdw), H-bonding, charged, and pi interactions which the final docked
compounds participated in.
PA (Figure 2, bottom) has a very high binding energy corresponding to the high
number of H-bonds formed with the residues Th39, Met40, His47, Gln72, Glnl58,
Glnl64, Va1187, and Met195. Furthermore, a cation-pi interaction is formed with
Lys160, and there are a lot of polar and hydrophobic interactions formed.
On the other hand, Nafronyl Oxalate (Figure 4) has a very low energy. This may be
accounted for by the lack of H-bonds, charged interactions and pi interactions, and the
little polar interactions formed. The interacting residues are Yal37, Pro38, Thr39, Met40,
His44, Gly46, His47, Leu50, Phe67, Asn69, GIn72, Tyr82, His135, Vall39, Yal742,
Va1143, Leu146, Phe157, Gly158, Glu159, Lys160, Asp161, G1n164, Met195, Ser196,
and Serl97. It can be observed that Nafronyl Oxalate, like PA, binds to the important
catalytic residues, which are identified by Zheng, et al (2004) They also wrote that
nafronyl oxalate is expected to have H-bonds with His44 and His47 at a distance of
-3.54;
however, the parameters used to view H-bonding only includes those within a
distance of 2.5A;thus, the expected H-bonds are not observed.
16
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Figure 4. Interaction diagram of Nafronyl Oxalate
t
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Figure 5.Interaction diagram of NSC_125296
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Figure 6. Interaction diagram of NSC_167356
NSC_125296 (Figure 5), was found to have the highest binding energy among
library compounds. Compared to NSC_167356 (Figure 6), it was observed to have fewer
charged and H-bonding interactions. However, its higher binding energy may be the
contribution of the high number of cation-pi interactions and van der waals interactions.
Lys160 have cation-pi interactions with 3 ofNSC_125296's aromatic rings, while His135
with 2 of rings. Cation-pi interactions are interactions between an electron pi system and
a cation; these interactions are considered to be as strong as H-bonding interactions in
Cation-Pi Interactions (n.d.). Despite the fact that vdw forces are one of the weakest
forms of interparticle forces of attraction, they still contribute to the binding energy;
many vdw interactions can contribute a lot for the binding. Furthermore, the binding is
not only dictated by one type of interaction; it is contributed to by all the interactions in
different extent. Other interactions of NSC 1225296 include H-bondins interactions with
18
His44 and Tyr82, polar interactions with His44, Tyr82, Asp161, Gln164, Met195,
Ser196, and Ser197, and vdw interactions with Va137, Pro38, Tk39, Met40, Gly46,
His47, Leu50, Yalt43, Phe156, Phel57, Glyl58, Glu159, Lys160, Thr186, Vall87,
Arg198, and Leu280.
Meanwhile, the interaction diagrams of NSC
_144248,8R748740
and NSC_618496
can be found in the Appendix H. It can be observed that many interacting residues are
common to most, if not all compounds. Included in these residues are Thr39, Met40,
His44, His47, Leu50, Lys160, Asp161, Glnl64, Met195, and His135.
Modification
NSC_125296, which is the highest ranking library compound in terms of final
docking binding energy, was modified. Sixteen modifications (A-P) were made, the
structures of which are shown in Appendix A, Five of sixteen had a greater binding
energy than PA and are tabulated in Table 3.
able 3. Bindine Energv from the Dockins of the Modification of NSC 125296
Modification Bindine Enersy
(kcal/mol)
K -721.76
C -7t2.79
F -706.23
D -649.47
L -488.07
comoou
Compound Polar Hvdroohobic H-Bond Chareed Pi
K 12 9 I 6 I
C t4 10 I 8 4
F 11 t2 I 5 0
D I4 9
a
J 6 2
L 10 10
-
2 2
Table 4. The
modified com
number of polar, hydrophobic, h-bonding, charged and pi interactions of
nds
19
a
o
I
a
lo
Q'*'
*int$
"=.'.
3
t
-"*
ll
.".-'l ,,
L--
,',*. -o
e-..-la"),
''
,i
Ja
**"-i.',i^.
i1}
;
g:'|v'r
o
ol
.
'il?
I
!*r
,,#A't
f
-'-lt--,t-
t
;t-"
o*,1
"'{
'?.
,-
I
I
Figure 7. Interaction diagram of modified compound, K.
a
t
I
Figure 8. Interaction diagram of modified compound, L.
C
c
I
2A
Among the modifications, K had the highest binding energy of -72l.764kcaVmol,
closely followed by C, F and D. L, on the other hand, l:mid a binding energy of -488.058,
which is very near to the binding energy of PA. The number of interactions of K, C, F, D,
and L are tabulated in Table 4. Meanwhile, the interaction diagram of K and L is shown
in Figure 7 and Figure 8 respectively. On the other hand, the interaction diagrams of F, D,
and L are shown in Appendix H.
K has polar interactions with His44, Argl32, His135, Gly158, Lys160, Asp161,
Gln164, Met195, Ser196, Ser197, Argl98, arrd Arg278, and vdw interactions wrth Th39,
Met40, His47, Leu50, Tyr82, Glu128, Phe137, Leu280, and Phe157. Furthermore, it has
formed an H-bond with His135, which has not been found in the parent molecule,
NSC_125296.
It can be observed that for K, C, F, and D, the addition of the hydroxyl substituent
resulted to the formation of charged interactions. Among these interactions, the charged
interaction with Arg132, Argl98, and Argp78 to the added oxygen seemed to be the
common factor. This interaction may also be the main reason for the huge increase in
binding energy compared to NSC_125296, since it accounts for 3 of the charged
interactions.
On the other hand, L was unable to form charged interaction with Arg132, and
Arg278. This may be caused by an intamolecular interaction between the adjacent
hydroxyl groups which were substituted into the aromatic ring. The intermolecular
interaction resulted to the sequestering of the electrons of the anionic oxygen. Thus, the
oxygen anion was no longer able to form charged interactions.
l
l
I
I
l
-l
21
K, C, F, D, and L had a greater binding energy than the reactive intermediate PA.
Thus, they are potential competitive inhibitors to PA, especially since they also interact
with four of the six important residues, His44, His47, Lysl60, and Gln164.
22
VI. CONCLUSION AND RECOMMENDATION
Pharmacophore generation and library screening gave five compounds with a
relatively high binding energy. Among these compounds, NSC_125296 had the highest
binding energy of -356.42kcaVmol. This high affinity to the active site of the protein was
accounted to the high number of cation-pi interactions between Lys160 and His135
residues to the ligand. Addition of hydroxyl groups to the naphthalene rings of
NSC_125296 yielded five compounds which have a binding energy greater than the
reaction intermediate PA. Four of these modified compounds, K, C, F, and D have a
binding energy ranging from 1.5 to 2 times that of PA, while L had almost similar
binding energy to PA. The high binding energy of K, C, F, and D are accounted to the
positioning of the hydroxyl group in a way that allows charged interactions with Argl32,
Arg198, and Argp98 residues. Because these compounds have a higher binding energy
than PA, they are potential drug candidates.
It is recommended that more compound libraries be screened and that parameters be
changed to improve accuracy
-
For example, flexible docking could be done given more
time or the top hits and the conformation generated for the docking process could be
increased. De novo ligand design could also be done. The toxicity of the compounds
could be evaluated, and the lead compound and its modifications could be synthesized
and tested with the PS assav.
23
References
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[software]
AnalyticCon Discovery (n.d.). NATx_Release_L0091 5.zip Retrieved from
http://www. ac-discovery. com/downloads,A{ATx_Release* 1 009 1 5.zip
Cation-Pi Interactions (n.d.). Retrieved April 4,2011 from Protein Interactions and
Protein Explorer: http:/lpipe. ibibiosolutions. com/Cation-Pi. html
Costakes, G. (2005). Overview of current docking programs. MMG 115 eJournal, I: 4
http://ejournal.vudat.msu.edl/index.php/mmg445larticle/viewFile/1A4l5l
Crystal structures of a pantothenate synthetase
from
M. tuberculosis and its complexes
with substrqtes and a reaction intermediate (2003). Retrieved April 4, 201 I from
RCSB Protein Data Bank:
http://www.pdb. org/pdb/explore/explore. do?structureld: 1N2H
Davies, D.O. (1999). Multi-Drug Resistant Tuberculosis. Retrieved from
http'. I I priory. com/cmol/TBMultid. htm
Dias, R. & de Azevedo, W.F. Jr. (2008). Molecular Docking Algorithms. Current Drug
Targets,9: fi40-I047
Irfan, N. (n.d.).
]SAR,
Pharmacophore and Docking Studies on Human PLA2 Inhibitors.
Retrieved from http:/iwww.scribd.com/doc|46502546126385082-QSAR-
Pharmacophore-and-Docking-Studies-on-Human-Phaspholipase-a2-Inhibitors
Johnson, R., Streicher, E.M., Louw, G.E., Warren, W., van Helden, P.D., & Victor, T.C.
(2006). Drug Resistance in Mycobacterium tuberculosis. Mol Biol,8:97-112
National Cancer Institute (nd.). 260,071 structures in SDF
format.
Retrieved from
http. I I cactus. nci. nih. govidownload/nciA{CI-Open_09-03. sdf. gz
Pantoyl Adenylate. (n.d.).Retrieved April 4, 2011 from RCSB Protein Data Bank:
http://www. pdb. org/pdb/explorelexplore. do?structureid: 1N2H
Philippines (n.d.). Retrieved April 4,2011 from USAID from the American People:
http://www.usaid.gov/our_worlCglobal_health/idltuberculosis/countries/asia/phili
ppinesgofile.html
Ryan Scientific, Inc. (n.d.) Complete Screening
(HTS) Database, ISIS SD
format,
ZIPed.
Retreived from http' I lwww.
ryansci.
com/downloads/biohts.zip
Ryan Scientific, Inc. (n.d.) HitFinder Collection,lS/S SD
file,
ZIPed. Retrieved from
http://www.
ryansci.
com/downloads/HitFinder_V 1 O_sdf. zip
24
Technology for organic Synthesis Laboratory
(n.d.). TosLab collection of compounds'
Retrieved
from
http://www.toslab.com/Download/Collection-Compounds/TOSlab-Collection-s
df.zip
Tuberculosis
(2010). Retrieved April 4, 20ll from World Health Organization:
http : //www. who. intlmediacentre/factsheets/fs
10 4 I enl index. html
Tuberculosis
(TB) (n.d.).Retrieved April 4, 2011 from center for Disease control and
Prevention: http:l/www. cdc. gov/tbltopic/basics/default.
htn
Varaine, F., Henkens, M., & Gouzard, V. (editors).(2010). Tuberculosrs'-
(5ft ed.).
http://www.refbooks.msf.orgAvlSF_Docs/En/Tuberculosis/Tuberculosis-en.pdf
Wang, S., & Eisenberg, D. (2003). Crystal structures of a pantothenate. synthetase from
M. tuberculosi and'its complexes with substrates and a reaction intermediate.
Prot e in S ci ence, 12:1097
*1
108
waszkowycz,8.,
Perkins, T.D.J., Skyes, R.A., & Li, J. (2001). Large-scale virtual
scieening for discovering leads in the postgenomic era. IBM Systems Journal,
a}Q):360-376
white, E.L, Southworth, K., & Ross, L. et al. (2007). A Novel Inhibitor of
'
lt6ycobacterium
tuberculosis Pantothenate Synthetase. J Biomol Screen
nDR-TB (n.d.) Retrieved April 4, zan from world Health organization:
http://www. who. int/tblchallenges
I xdr I enl index' html
zheng,R., Dam, T.K., Brewer, c.F., & Blanchafd, J.s. (2004). Active site residues in
-
Mycobacterium tuberculosis
pantothenate slmthetase required in the formation
and stabilization of the adenylate intermediate. Biochemistry,43(22):7171'8
25
APPENDD(
26
Appendix A. Structures and Binding Energies of Ligands
Legend:
Carbon
-
black
Oxygen- red
Phosphorus
-
green
Sulfur
-
v*ilrr*'
Nitrogen
-
i:lue
LieandName Lisand Structwe Bindine Enersv
(kcaVmol)
NSC 12s296 -3s6.42
NSC 144248 -287.81
NSC 618496 -221.87
27
NSC 167356
-301.93
8F(748740
-252.48
NO
-98.58
PA
-482.00
28
A
-437.50
B
-4r3.12
C
-712.79
D
-649.47
I
ra
E
-347.13
F
-706.23
G
-409.17
H
-354.54
lo
M -351.39
N -345.98
o -374.7s
P -4tt.02
i
I
t
I
32
Appendix B. First Screening Compounds and their Fit Values from
Complete Screening
MFCDO3730872 3.4992
MFCDO1314799 3.4847
MFCD097577t2 3.4784
MFCDOs669957 3.4745
MFCD0097s569 3.4706
MFCD05669566
3.4693
MFCD013 r5r78
3.4683
MFCDOO2I57O7
3.4676
MFCD00664788 3.4s68
MFCDOO793045 3.453s
MFCD00245130 3 4522
MFCDOO140613 3.44s5
MFCDOO171827 3.4159
MFCD06496206 3.413s
MFCD05668924 3.4T29
MFCD00169612 3.4114
MFCD00202789 3.4107
MFCDOO170616 3.4053
MFCD00202s09 3.3975
MFCDO330530l 3.3938
MFCDOO140146 3.3918
MFCD09757767 3.3870
MFCDO1789093
3.3846
MFCDOO17293s
1 a1na
J.)t tJ
MFCD09757761 3.3722
MFCD0 t3t6154 3.3695
MFCDOO172928 3.3612
MFCDO3787094 3.3606
MFCD00793577 3.3604
MFCD00955352 3.3600
MFCD00202861 3.3507
ID Fit Value
MFCD02571570
4.0274
MFCD03617426
3.9849
MFCD013l5588
3.9548
MFCD01315590
3.95Is
MFCD0266t762
3.9173
MFCD00955A97 3.8902
MFCD00244789
3.8433
MFCDOO172043
3.8392
MFCD05670491
3.8103
MFCD018l5ls2 3.8031
MFCDO3697903 3.7326
MFCD01623198
3.7262
MFCDO3697896
3.7r93
MFCD00243486
3.6740
MFCDOO173328 3.6444
MFCDO4149071 3.6402
MFCD00232669
3.6379
MFCD0266176l 3.6233
MFCDOs1s5527
3.62t9
MFCDO1315589
3.6052
MFCD02571181
3.5943
MFCDO1315587
3.5930
MFCDOO127515
3.s822
MFCD00794262
3.5767
MFCD0257t726
3.s7s9
MFCD02082059
3.s714
MFCD00975399
3.5421
MFCD00975409
3.5395
MFCD00793558
3.53 15
MFCDO1443663
3.s297
33
!
Appendix C. First Screening Compounds and their Fit Values from HitFinder
ID Fit Value
cD03630
4.t725
8T808060
4.1 089
sEwo1332
3.9642
cD09014
3.7832
HTS04l40
3.7739
cD10054
3,6999
scR01457
3.6654
KMl0785
3,6230
HTS05233
3 5924
RF04517 3.5873
8T808067
3.5251
HT504553
3.4886
BTB,04792
3.4477
RFO2302
3.4313
8T807136
3,4118
KM03141
3.4A96
NR803740 3.3912
sP807392
3.3800
DFP00338
3.3776
HTS03687
3.3751
sEw01764
3.3612
HTSO3090
3.3568
RJFO1162 3.3531
34
Appendix E. First Screening Compounds and their Fit Values from Natx
ID Fit Value
NAT2-343853 4.0570
NAT2-252250 3.9s59
NAT2-343996 3.7444
NATl7-347338 3.7241
NATS-258563 3.6824
NATS-304562 3.6482
NATl7-347339 3.6339
NATS-258616 3.6447
NAT3-177883 3.6022
NATS-264017 3.5511
NAT2-18408 3.5t99
NAT2-2s2125 3.5005
NAT3-21119s 3.4949
NAT2-252099 3.4301
NAT9-32292s 3.3651
NAT6-319244 3.3637
NAT2-252078 3.3591
c_:a
36
I
kja
Appendix F. First Screening Compounds and their Fit Values from NCI
ID Fit value
NSe 679259 3.5633
NSC 681161 3.545r
NSC 683894 3.3767
NSC 690194 3.4664
NSC 693103 3.4879
NSC 694482 33974
NSC 695316 3.4582
NSC 700643 3.3s3t
NSC 710505 3.3587
NSC 711300 3.6036
NSC 718137 3.3663
NSC 721546 3.4095
NSC 721858 3.5662
NSC 90774 3.3838
NSC 95389 3.6s23
NSC 98619 3.4060
NSC 9905 3.3626
37
q7--
Appendix G. Second Screening Compounds, Fit Values, and their Binding Energies
ID Fit Value
Binding Energy
(kcaVmot)
NSC_125296 4.1243 -362.55
NSC 144248 4.0832 -248.68
NSC 618496 4.3287 -209.13
EP-748740 4.0459 -207.38
NSC r673s6 4.3580 -201.46
sE859136 4.4059 -141.65
NAT2-252125 4.0809 -139.80
NSC*667559 4.1771 -136.98
NAT3-177883
+.JZZJ -129.61
MFCDO1315588
4.4472 -128.71
sEw01332 4.2105 -126.07
sE859185
4.0531 -r25.59
NSC 718137 4.2496 -r22.29
NSC s25339 4.0531 -r21.37
HTS04I40
|
q.oztt
-r20.9a
sE8sel84
| +. rsro 119.60
sE8593s7
4.4530
-115 74
MFCD00793558 .+.
z. zJ L -113.51
NSC_628978
4.3717
r12.79
8T808060
4.5767 r l 1.86
s8859137
+.J I lJ
11 1.84
ll"{FCD09757712
4.0650 -lll 18
sE859l 55 4.4260 -r04.73
MFCDOl315587
4.2214 -143.12
NSC 263220
4.3474 -100.01
ER778384
+.1 /oJ
-95.40
MFCD02082059
4.t609 -93.29
MFCD00955097
4.1989
-91.52
wcD02661761
4.0778
-90 97
P1^720311
4.1427 -90.17
wcD0266r762
4.2446
-88 68
8T808067
4.6237
-87.78
MFCD06496206
4.0392 -86.61
s8859208
4.0332
-86.30
s8878 I 65 4.3454
-83.98
MFCD01789093
4.1378 -83.84
NSC 274538
4.2197 -80.82
NSC 634582 4.4201 -77.48
NAT2-252250 4.2471
-73.58
sE859350 4.t291 -69.61
PR.725764 4.4309 -67.89
MFCD00243486 4.3694 -67.37
sE8s9187 4.0442 -66.86
MFCDOO173328 4.4665 -66.32
cD03630 4.2625 -66.32
MFCDOs669566
4.0571 -64.77
NAT8-258616 4.4597 -63.46
NAT2-18408 4.0310 -59.80
MFCD01315s90 4.3522 -59. t6
MFCDOO171827
4.1359 -56.58
NSC_45207 4.2012 -) 1.4)
MFCDo1315589 4.2170 -5r.32
sH874619 4.5529 -49.40
MFCDO257l570
4.0446 -48.49
NSC 45189 4.1933 -45.88
MFCD00975399
4.3372 -44.63
P/.798057
4.1975 -42.88
NSC_144469 4.0613 -40.37
NSC_I38663 4.4123 -35.44
sH865497 4.2724 -29.70
MFCD03697896
4.0597 -28.42
MFCDO3697903
4.ttt3 -26.31
MFCD00244789
4.4946 -?5
?O
NAT8-264017
4.3573 -25.02
DE823446
4.13s9 -22 81
NAT8-304562
4.2306
18.74
NSC 672294 4 0316
I 1.89
NSC_546s7
4.1933 -J.))
sHS79451 4. l8 10 t.14
NSC_146519
4.0182 5.09
NSC_658912
4.2923 tl.77
NAT2-343853
4.3985
19.40
cD09014
4.0119
22.36
NAT17-347338
4,0896
35.67
NATS-258563
4.0406
36 39
NSC 281790 4.0308 69.27
38
Legend:
Interaction
Display style
van der Waals
Gteen
Masenta
Polar
Charse
fvfase"tu dashed line with arrow at both ends
ftvOroeen bond with amino acid sdqcha:n
Blue dashed line with arrow at H-boq4 acceplgl-
HyArogen bond with amino acid main chain Gte"t dashed line with arrow at H-bond acceptor
Black dashed line with arrow at H-bond acceptor
Hydrogen bond with non amino acrd
Appendix H.Interaction
of Ligand and 1N2H receptor
Docked NSC-144248
(binding energy = -287'81kcaUmol)
o
r@
,&,
ffi
w
rt
lt
t.l
I
o.
g.
s\.
to-
o
@
@
I
.o
@
o
o@
A
-
-
39
J
Docked NSC_618496 (binding energy = -221.87 kcaVmol)
t6
.o-
s
I
o\
:
l
I
:o
3iF
-
*
t
s
?-'t
-.x"
Docked ER148740 (binding energy = -252.08 kcaVmol)
s
s
t
,r)
*o-
I
i&
_s
fl,
r.r
-.0
I
","t
rel
40
lcjC.
Docked C ( binding energy = -712.79 kcaUmol)
t
o
r {,,-}
I
t
,=.=l a
.,,.r\-.17
!
!\f.f
I ls--'':"
t
Docked D (binding energy = -649.47 kcaVmol)
o
I I
o t
a
a
I
I
t
I
o
o
o
I
C
I
o
o
4t
Docked F (binding
energy
= -706.23 kcaUmol) _
I
I
I
oo
]:-io:;f
i-:;
-r
*L"
o
I
at
o
I
o
eO
lO
-
42