JOURNAL OF IQRO – ЖУРНАЛ ИҚРО – IQRO JURNALI – volume 16, issue 02, 2025
ISSN: 2181-4341, IMPACT FACTOR ( RESEARCH BIB ) – 7,245, SJIF – 5,431
ILMIY METODIK JURNAL
Abduxaliqov Fayozbek, Shukurullayeva Farangiz, Jalilov Mirjalol, Toshov Hamza
National University of Uzbekistan
IN SILICO DOCKING STUDY OF MYCOBACTERIUM TUBERCULOSIS RV1250
PROTEIN USING ISONIAZID AND A NOVEL MODELED COMPOUND
Abstract :
Tuberculosis (TB) remains a major global health threat, largely due to the emergence
of drug-resistant strains of Mycobacterium tuberculosis. Identifying novel molecular targets is
critical for the development of new therapeutic agents. In this study, the Rv1250 efflux protein of
M. tuberculosis was selected as a potential drug target. Molecular docking analysis was
conducted using AutoDock Vina to evaluate the binding affinity of two ligands: isoniazid, a
known anti-TB drug, and a newly designed in silico compound. The docking results revealed that
the novel compound exhibited a binding affinity of –8.0 kcal/mol, which is more favorable than
that of isoniazid (–6.1 kcal/mol). The new ligand formed stronger hydrogen bonds and
hydrophobic interactions within the active site of the Rv1250 protein. These findings suggest that
the novel molecule may serve as a potential inhibitor of Rv1250 and warrants further biological
investigation.
Keywords:
Mycobacterium tuberculosis, Rv1250 protein, isoniazid, molecular docking,
AutoDock Vina, in silico design
1. Introduction
Tuberculosis (TB) is a highly infectious disease that poses a serious global health threat,
affecting more than 10 million people annually. The primary causative agent, Mycobacterium
tuberculosis, has become increasingly difficult to treat due to the widespread emergence of
antibiotic-resistant strains. According to the World Health Organization (WHO), TB was
responsible for over 1.6 million deaths in 2021 alone. Isoniazid, a first-line anti-TB drug, acts by
inhibiting the biosynthesis of mycolic acids, which are essential components of the bacterial cell
wall. However, the growing incidence of isoniazid resistance has created an urgent need for
novel bioactive compounds. In silico approaches, particularly molecular docking, are widely
used to identify new drug candidates and to overcome microbial resistance. These computational
methods help predict the binding interactions between small molecules and target proteins. The
Rv1250 protein of M. tuberculosis, a member of the major facilitator superfamily (MFS), is
considered a potential drug target due to its role in efflux-mediated antibiotic resistance. In the
present study, we performed molecular docking using AutoDock Vina to analyze the interactions
of the Rv1250 protein with two ligands: isoniazid, a known anti-TB agent, and a newly modeled
compound. The binding affinities and interaction patterns with key amino acid residues were
examined to evaluate the potential of the novel ligand as a TB therapeutic candidate.
2. Materials and Methods
2.1. Preparation of Target Protein
The target protein used in this study was the Mycobacterium tuberculosis Rv1250 protein, an
efflux transporter belonging to the Major Facilitator Superfamily (MFS). Its crystallographic
structure was retrieved from the Protein Data Bank (PDB ID: 6G9X) in .pdb format. To prepare
the protein for docking simulations, the structure was cleaned using AutoDock Tools (ADT)
version 1.5.6. This included the removal of non-essential components such as crystallographic
JOURNAL OF IQRO – ЖУРНАЛ ИҚРО – IQRO JURNALI – volume 16, issue 02, 2025
ISSN: 2181-4341, IMPACT FACTOR ( RESEARCH BIB ) – 7,245, SJIF – 5,431
ILMIY METODIK JURNAL
water molecules, metal ions, and co-crystallized ligands, which may interfere with the docking
process or create artifacts. Hydrogen atoms were added to all polar residues to stabilize hydrogen
bonding networks, and Kollman United Atom charges were applied. In addition, torsional
flexibility was kept restricted on the protein side, assuming the receptor behaves as a rigid div
during docking. The processed protein structure was saved in PDBQT format, which is
compatible with AutoDock Vina. Prior to docking, the active site was visually inspected to
confirm accessibility and the presence of key binding residues.
2.2. Ligand Preparation
Two ligand molecules were selected for comparative docking:
1. Isoniazid – a well-established first-line anti-tuberculosis drug used as a reference compound.
2. A novel compound – computationally designed and not yet synthesized, serving as a
theoretical inhibitor.
Both molecules were drawn in Avogadro molecular editor (version 1.2.0), and their geometries
were optimized using the Universal Force Field (UFF) with the steepest descent algorithm.
Optimization was conducted until the energy gradient converged below the threshold of 10⁻⁷
kcal/mol/Å. Following energy minimization, the ligands were saved in .mol and converted
to .pdb format using Open Babel. In AutoDock Tools, rotatable bonds were detected and defined,
Gasteiger charges were added, and torsion trees were generated. The final ligands were exported
in PDBQT format, allowing flexible docking around rotatable bonds.
2.3. Docking Procedure
Molecular docking was performed using AutoDock Vina (version 1.1.2), a well-established
open-source tool known for its speed and accuracy in predicting binding affinities. The docking
grid box was defined to encompass the predicted active site of the Rv1250 protein, ensuring
coverage of the key binding pocket. The center of the grid was set to X = 83.911, Y = 69.166, Z
= 77.416, with a grid size of 54 × 58 × 58 Å and a default grid spacing of 0.375 Å. These
dimensions were selected to include both the known binding cavity and adjacent potential
allosteric regions. The exhaustiveness parameter was set to 8, balancing computational demand
and conformational space sampling. For each ligand, Vina generated nine different binding poses,
ranked according to predicted binding affinity in kcal/mol. The conformation with the lowest
(most negative) binding energy was selected for further structural and interaction analysis.
2.4. Visualization and Interaction Analysis
Protein–ligand complexes obtained from docking were analyzed using both PyMOL (version 2.5)
for three-dimensional visualization and BIOVIA Discovery Studio Visualizer for detailed
interaction profiling. Key interactions such as hydrogen bonds, hydrophobic contacts, van der
Waals forces, π–π stacking, and electrostatic interactions were identified and documented.
Residues in close contact with the ligands were highlighted, and interaction diagrams were
generated for comparative assessment of binding modes between isoniazid and the novel
compound. These analyses provided insight into the binding orientation, anchoring residues, and
potential pharmacophoric features responsible for high-affinity interactions with the Rv1250
protein.
3. Results
JOURNAL OF IQRO – ЖУРНАЛ ИҚРО – IQRO JURNALI – volume 16, issue 02, 2025
ISSN: 2181-4341, IMPACT FACTOR ( RESEARCH BIB ) – 7,245, SJIF – 5,431
ILMIY METODIK JURNAL
Molecular docking simulations were performed to evaluate the binding affinity and interaction
profiles of two ligands—isoniazid, a well-known anti-tuberculosis drug, and a novel in silico-
designed compound—against the Mycobacterium tuberculosis Rv1250 efflux protein. The
docking process was executed using AutoDock Vina, which ranked binding poses based on
predicted Gibbs free energy of binding (ΔG) in kcal/mol. The docking results demonstrated that
the novel compound exhibited a significantly stronger binding affinity (–8.0 kcal/mol) in
comparison to isoniazid (–6.1 kcal/mol). This difference in energy values suggests that the novel
ligand may form a more stable and thermodynamically favorable complex with the Rv1250
protein.
Figure 1.
2D interaction diagrams of isoniazid (left) and the novel in silico compound (right)
docked with the Rv1250 protein (PDB ID: 6G9X). Hydrogen bonds, hydrophobic contacts, and
π–π interactions with key residues such as CYS, PHE, GLY, and LEU are illustrated. The
binding affinities were –6.1 kcal/mol and –8.0 kcal/mol, respectively.
3.1. Interaction Analysis of the Novel Compound
The top-ranked pose of the novel compound revealed multiple stabilizing interactions within the
active site of Rv1250. Key residues involved in these interactions included:
CYS-114 and GLY-117: formed conventional hydrogen bonds with the ligand’s polar
groups, helping to anchor it within the cavity.
PHE-211: participated in π–π stacking interactions with the aromatic ring system of the
compound, contributing to its orientation.
LEU-89 and VAL-121: established hydrophobic contacts, providing additional van der
Waals stability.
The ligand was deeply embedded within the binding pocket and showed a complementary
surface fit, indicating high spatial compatibility with the receptor's topology. The three-
dimensional visualization confirmed a snug and oriented positioning within the core of the active
site, avoiding solvent exposure and enhancing affinity.
3.2. Interaction Analysis of Isoniazid
In contrast, the docking of isoniazid yielded a less negative binding energy, indicating a weaker
interaction profile with the target. Isoniazid formed fewer hydrogen bonds, primarily with SER-
JOURNAL OF IQRO – ЖУРНАЛ ИҚРО – IQRO JURNALI – volume 16, issue 02, 2025
ISSN: 2181-4341, IMPACT FACTOR ( RESEARCH BIB ) – 7,245, SJIF – 5,431
ILMIY METODIK JURNAL
118 and ASP-92, and showed limited hydrophobic contacts. The small molecular size and
reduced aromatic surface area may have limited its ability to engage in extensive non-covalent
interactions within the active site. Moreover, the binding pose of isoniazid was slightly offset
from the geometric center of the cavity, indicating suboptimal anchoring and potentially lower
residence time. These observations are consistent with its lower docking score.
3.3. Comparative Visualization and Binding Mode Analysis
Superposition of both ligand–protein complexes revealed distinct binding orientations. The novel
compound penetrated deeper into the hydrophobic core and established a more extensive
interaction network, while isoniazid remained relatively peripheral and loosely associated. Two-
dimensional interaction maps generated using Discovery Studio Visualizer illustrated the ligand–
residue contacts, confirming that the novel compound engaged in more than six key interactions,
compared to two to three for isoniazid. These findings suggest that the novel compound not only
binds with greater affinity, but also targets a broader set of pharmacologically relevant residues,
which may enhance its efficacy as a potential inhibitor of the Rv1250 efflux pump.
4. Conclusion
The Rv1250 efflux protein of Mycobacterium tuberculosis has emerged as a compelling
molecular target in the pursuit of new therapeutic strategies to combat tuberculosis, especially in
the context of rising drug resistance. This study employed a structure-based drug design
approach to investigate the molecular interactions between Rv1250 and two ligands: the widely
used anti-TB drug isoniazid, and a novel compound designed via in silico modeling. Using
AutoDock Vina, molecular docking simulations revealed that the novel compound exhibited a
significantly higher binding affinity (–8.0 kcal/mol) compared to isoniazid (–6.1 kcal/mol),
suggesting a more stable and energetically favorable interaction. Detailed interaction profiling
demonstrated that the novel ligand formed a diverse set of non-covalent interactions, including
multiple hydrogen bonds and hydrophobic contacts with essential active site residues such as
CYS, GLY, LEU, and PHE. In contrast, isoniazid showed a relatively limited interaction profile,
which may account for its weaker binding energy in this model. These computational findings
strongly support the hypothesis that the in silico-designed compound could serve as a potential
lead candidate for the development of novel anti-TB agents. Its superior binding characteristics
warrant further in vitro and in vivo validation to assess biological activity, pharmacokinetics,
toxicity, and resistance profiles. In conclusion, this study demonstrates the power of
computational drug discovery approaches in identifying promising inhibitors of M. tuberculosis
targets. The novel ligand modeled here lays the groundwork for the next steps in structure-
guided anti-TB drug development, addressing a critical need in global health.
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JOURNAL OF IQRO – ЖУРНАЛ ИҚРО – IQRO JURNALI – volume 16, issue 02, 2025
ISSN: 2181-4341, IMPACT FACTOR ( RESEARCH BIB ) – 7,245, SJIF – 5,431
ILMIY METODIK JURNAL
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