Article
Understanding Drug Resistance of Wild-Type and L38HL
Insertion Mutant of HIV-1 C Protease to Saquinavir
Sankaran Venkatachalam 1,†, Nisha Murlidharan 1,†, Sowmya R. Krishnan 1, C. Ramakrishnan 1, Mpho Setshedi 2,
Ramesh Pandian 2, Debmalya Barh 3,4, Sandeep Tiwari 3,5,6, Vasco Azevedo 3, Yasien Sayed 2
and M. Michael Gromiha 1,*
Citation: Sankaran, V.;
Murlidharan, N.; Krishnan, S.R.;
Ramakrishnan, C.; Setshedi, M.;
Pandian, R.; Barh, D.; Tiwari, S.;
Azevedo, V.; Sayed, Y.; et al.
Understanding Drug Resistance of
Wild-Type and L38HL Insertion
Mutant of HIV-1 C Protease to
Saquinavir. Genes 2023, 14, 533.
https://doi.org/10.3390/genes
14020533
Academic Editor: Stefano Lonardi
Received: 29 January 2023
Revised: 16 February 2023
Accepted: 17 February 2023
Published: 20 February 2023
Copyright: © 2023 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://cre-
ativecommons.org/licenses/by/4.0/).
Genes 2023, 14, 533. https://doi.org/10.3390/genes14020533
1 Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology
Madras, Chennai 600036, India
2 Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the
Witwatersrand, Johannesburg 2000, South Africa
3 Department of Genetics, Ecology, and Evolution, Institute of Biological Sciences, Federal University of
Minas Gerais (UFMG), Belo Horizonte 31270-901, Brazil
4 Institute of Integrative Omics and Applied Biotechnology (IIOAB), Nonakuri, Purba Medinipur,
West Bengal 721172, India
5 Institute of Biology, Federal University of Bahia, Salvador 40110-909, BA, Brazil
6 Institute of Health Sciences, Federal University of Bahia, Salvador 40110-909, BA, Brazil
* Correspondence: gromiha@iitm.ac.in; Tel.: +91-44-2257-4138; Fax: +91-44-2257-4102
† These authors contributed equally to this work.
Abstract: Acquired immunodeficiency syndrome (AIDS) is one of the most challenging infectious
diseases to treat on a global scale. Understanding the mechanisms underlying the development of
drug resistance is necessary for novel therapeutics. HIV subtype C is known to harbor mutations at
critical positions of HIV aspartic protease compared to HIV subtype B, which affects the binding
affinity. Recently, a novel double-insertion mutation at codon 38 (L38HL) was characterized in HIV
subtype C protease, whose effects on the interaction with protease inhibitors are hitherto unknown.
In this study, the potential of L38HL double-insertion in HIV subtype C protease to induce a drug
resistance phenotype towards the protease inhibitor, Saquinavir (SQV), was probed using various
computational techniques, such as molecular dynamics simulations, binding free energy calcula-
tions, local conformational changes and principal component analysis. The results indicate that the
L38HL mutation exhibits an increase in flexibility at the hinge and flap regions with a decrease in
the binding affinity of SQV in comparison with wild-type HIV protease C. Further, we observed a
wide opening at the binding site in the L38HL variant due to an alteration in flap dynamics, leading
to a decrease in interactions with the binding site of the mutant protease. It is supported by an
altered direction of motion of flap residues in the L38HL variant compared with the wild-type.
These results provide deep insights into understanding the potential drug resistance phenotype in
infected individuals.
Keywords: HIV protease subtype C; L38HL double insertion; Saquinavir; drug resistance;
molecular dynamics simulations
1. Introduction
Human immunodeficiency virus (HIV) is the causative agent of acquired immuno-
deficiency syndrome (AIDS) and is responsible for millions of deaths worldwide. Partic-
ularly, the Asian and South African populations are among the most afflicted due to AIDS
infections caused by non-B HIV subtypes, whose prevalence is lower in other parts of the
globe [1–4]. As most of the anti-retroviral drugs in use today target proteins from the HIV
subtype B, their efficacy against proteins from the HIV subtype C and other non-B HIV
subtypes is quite limited [5–7]. Specifically, inhibitors of the HIV aspartic protease,
www.mdpi.com/journal/genes
Genes 2023, 14, 533 2 of 17

including lopinavir, ritonavir, saquinavir, indinavir and amprenavir, exhibit a several-

fold decrease in binding affinity to HIV subtype C in comparison with HIV subtype B
[8,9]. This has led to an increase in drug resistance phenotypes emerging in HIV subtype
C and a proportional increase in the recent prevalence of infections due to subtype C in
America and Europe [10,11]. Therefore, it is of utmost need to understand the key struc-
tural differences between proteins of subtype B and subtype C and optimize existing
drugs for repurposing and for the development of novel drugs with a broad spectrum of
activity.
The HIV aspartic protease is one of the major drug targets of interest for anti-retrovi-
ral drug development. The protease of both subtypes B and C is composed of two mono-
mers of 99 amino acids each, forming a C2-symmetric obligate homodimer [4]. The ho-
modimer formation in HIV protease is necessary for the catalytic activity of the enzyme,
as each monomer contributes to one-half of the substrate binding site. Despite the overall
high structural similarity between the HIV protease of subtypes B and C, the HIV subtype
C protease harbors eight distinct mutations in residue positions far from the substrate
binding site in comparison with the HIV subtype B protease [12]. Specifically, the M36I
mutation of HIV subtype C protease in the hinge region disrupts a critical salt bridge
formed by residues E35 and R57, thereby increasing the flexibility of the hinge region [13].
The increased flexibility of the hinge region induces an increase in flexibility of the flap
region, which indirectly affects proper substrate or inhibitor binding to the HIV protease
active site, leading to drug resistance [14]. Similarly, several other primary and secondary
drug resistance mutations have been identified in the HIV protease subtype C, which ren-
ders the existing drugs less effective in controlling the spread of AIDS infections at a global
scale [15,16]. Recently, Ledwaba et al. [17] identified an HIV protease subtype C variant
with the insertion of two residues at the 38th position (L38HL). The 3D structures of the
wild-type and L38HL variants are shown in Figure 1A,B, respectively.

Figure 1. Structure of HIV-1 protease type C. (A) Wild-type and (B) L38HL. The protein is shown in
cartoons, and the ligands are in the sticks. Insertion of residues His and Leu are represented in balls
and sticks. The flap, hinge, cantilever and fulcrum regions are colored orange, red, blue and ma-
genta, respectively. (C) 2D structure of Saquinavir (SQV).

Molecular dynamics simulations facilitate the atomistic level understanding of mo-

lecular conformations over a time period, and it is widely used in the literature [18,19]. In
this work, we performed molecular dynamics simulations for both wild-type and L38HL
Genes 2023, 14, 533 3 of 17

proteases in the presence of the protease inhibitor Saquinavir (SQV) to understand the
drug resistance phenotype due to the insertion of L38HL at an atomistic level. The struc-
ture of SQV is shown in Figure 1C. Results from the molecular dynamics trajectory re-
vealed that the L38HL variant exhibits an increase in flexibility at the hinge and flap re-
gions, similar to the M36I mutation. Detailed analysis of intermolecular interactions with
the inhibitor and binding free energy calculations showed that L38HL insertion decreases
the affinity of the protease towards the inhibitor in comparison with that of the wild-type
protease from HIV subtype C. Further, the insertion in the hinge region causes the flap tip
to narrow, leading to a reduction in the interactions available for SQV. Moreover, PCA
analysis revealed the altered direction of motion of the flap residues in the L38HL variant
compared to the wild-type. Similarly, the insertion affects the correlation of residues in
the dimer interface and flap dynamics.

2. Materials and Methods

2.1. Initial Coordinates of HIV-Protease Subtype C and the L38HL Variant
The initial coordinates of the South African wild-type HIV-protease subtype C were
obtained from PDB (PDB ID: 3U71) [12]. The structures of wild-type HIV-protease sub-
type C and L38HL variant in complex with SQV were obtained by docking using Auto-
Dock Vina [20], and this technique is commonly used in computational research [21]. In-
sights into the drug resistance mechanism due to the L38HL insertion were obtained by
comparing the structural changes observed over the course of the MD simulation in two
systems: (i) wild-type HIV-protease subtype C in complex with SQV, and (ii) L38HL var-
iant in complex with SQV.

2.2. MD Simulation Protocol

The complexed forms of HIV-protease subtype C were simulated in an explicit sol-
vent environment for 500 ns each using AMBERff14SB [22] force field available with the
AMBER18 software package [23]. Prior to production MD runs, each of the systems was
initially set by solvating the solute using TIP3P water models [24] and neutralizing the
system using counter ions (Cl−). The water box was set such that no solute atoms were
present within 10 Å from the boundary using the Particle Mesh Ewald (PME) [25] bound-
ary condition. In addition to the above steps performed using the tleap module in the
AMBER package, the protonation state of each amino acid residue was set appropriately.
The initial energy minimization was performed with 1000 cycles of steepest descent and
1500 cycles of conjugate gradient (CG) methods by restraining the protein atoms, followed
by 2500 cycles of CG-based unrestrained minimization of the whole system. In order to
attain equilibrium, each system was heated from 0 K to 300 K with 500 ps long canonical
MD using Berendsen temperature coupling [26]. The pressure of each system was then
equilibrated to approximately 1 atm. Equilibration was extended for another 500 ps to
ensure the overall equilibration process for 1 ns time period, which is essential to achieve
the equilibrium (with potential energy fluctuation of ± 200 kcal/mol) to start the produc-
tion run. All the solute atoms were restrained with a force constant of 100 kcal/mol-Å2 and
10 kcal/mol-Å2 during the temperature and pressure equilibrations, respectively. An inte-
gration time step of 2 fs was used and the snapshots were written for every 1 ps. All the
bonds involving hydrogen atoms were treated using SHAKE constraints [27]. Since the
protonation of Asp 25/Asp 25′ does not have a significant effect on the binding of the lig-
ands, they were left unprotonated [13,28,29].
Genes 2023, 14, 533 4 of 17

2.3. Trajectory Analysis

The global structural changes in each system were analyzed from the MD trajectory
using the CPPTRAJ module from the AMBER package. Root-mean-square deviation
(RMSD), root-mean-square fluctuation (RMSF) and radius of gyration (Rg) were plotted
as a function of simulation time to identify snapshots of the protein with significant con-
formational changes over the course of the simulation. Specifically, the conformational
change observed in wild-type protease in complex with SQV was compared with the
L38HL protease–SQV complex.

2.4. Dynamic Cross-Correlation

The conformational dynamics of the wild-type/L38HL protease bound to SQV were
studied using a cross-correlation analysis. This analysis yields a cross-correlation matrix,
𝐶𝑖,𝑗 , which calculates the movement of one atom with respect to the other (Equation (1)).
⟨𝛥𝑟𝑖 (𝑡). 𝛥𝑟𝑗 (𝑡)⟩𝑡
𝐶𝑖,𝑗 = ,
2 (1)
√⟨‖𝛥𝑟𝑖 (𝑡)‖2 ⟩𝑡 √⟨‖𝛥𝑟𝑗 (𝑡)‖ ⟩
𝑡

where the angular brackets indicate the time average over the simulation trajectory, 𝑟𝑖
and 𝑟𝑗 are the displacement vectors from the mean position of the α carbons in the 𝑖 and
𝑗th residues, respectively. The matrix element 𝐶𝑖,𝑗 ranges from −1.0 to 1.0. The value of
−1.0 indicates a high negative correlation between residues 𝑖 and 𝑗 and vice versa [30].

2.5. Principal Component Analysis

Principal component analysis (PCA) helps to differentiate the essential motions of
the protein residues from the local fluctuations. PCA is carried out in two steps: (i) extract-
ing the essential motions from the translational and rotational motions, which is per-
formed by fitting the trajectories to the reference frame and (ii) construction of a 3N × 3N
covariance matrix (C) as given in Equation (2).
𝐶(𝑖,𝑗) = ⟨(𝑥𝑖 − ⟨𝑥𝑖 ⟩)(𝑥𝑗 − ⟨𝑥𝑗 ⟩)⟩ (2)
The obtained matrix represents the deviation of atomic positions over the trajectory.
The matrix is then diagonalized by an orthogonal transformation, as in Equation (3) [31].
TTCT = diag (𝜆1 , 𝜆2 , 𝜆3 , 𝜆4 , 𝜆5 , … . , 𝜆𝑁 , ); 𝜆1 , > ⋯ > 𝜆𝑁 (3)
The PCA analysis was carried out using the CPPTRAJ module in the AMBER pack-
age.

2.6. Binding Free Energy Calculations Using MM/GBSA

The Molecular Mechanics Generalised Born Surface Area (MM/GBSA) [32,33]
method was used to calculate the binding free energies of SQV bound to the wild-type
and L38HL forms of HIV-1 protease subtype C. The single trajectory approach was opted
to compute the binding free energies of both systems using the MM/GBSA module in
AMBER18. For each system, a total of 1000 snapshots with an interval of 200 ps obtained
from the last 100 ns of the MD trajectories were used for energy calculations. In
MM/GBSA, the binding free energy was calculated using Equation (4). Further, the en-
thalpy component is obtained from various contributions, including the van der Waals
(𝛥𝐸𝑣𝑑𝑤 ), electrostatic (𝛥𝐸𝑒𝑙𝑒 ), polar solvation (𝛥𝐺𝑔𝑏 ) and the non-polar solvation (𝛥𝐺𝑆𝐴 ).
The polar solvation of each system is computed using the Generalised Born surface area
method, and the non-polar solvation is calculated using the empirical method based on
solvent accessible surface area.
𝛥𝐺𝑏𝑖𝑛𝑑 = 𝛥𝐺𝑐𝑜𝑚𝑝𝑙𝑒𝑥 − 𝛥𝐺𝑟𝑒𝑐𝑒𝑝𝑡𝑜𝑟 − 𝛥𝐺𝑙𝑖𝑔𝑎𝑛𝑑 , (4)
Genes 2023, 14, 533 5 of 17

𝛥𝐺𝑏𝑖𝑛𝑑 = 𝛥𝐺𝑝𝑜𝑙 + 𝛥𝐺ℎ𝑦𝑑𝑟𝑜 , (5)

𝛥𝐺𝑝𝑜𝑙 = 𝛥𝐺𝑒𝑙𝑒𝑐 + 𝛥𝐺𝑔𝑏 , (6)
𝛥𝐺ℎ𝑦𝑑𝑟𝑜 = 𝛥𝐺𝑣𝑑𝑤 + 𝛥𝐺𝑆𝐴
(7)
and
𝛥𝐺𝑆𝐴 = 𝛾𝛥𝑆𝐴𝑆𝐴 + 𝛽. (8)
For β and γ, the default values of 0.8 and 4.8 were used for empirical calculations.
𝛥𝑆𝐴𝑆𝐴 mentioned in Equation (8) was calculated using the linear combination of pairwise
overlaps (LCPO) method [34].

3. Results and Discussion

From the simulation of the two systems, trajectories were obtained with coordinates
written at 1 ps intervals for each system and aligned to their respective initial coordinates.
The global structural changes in the wild-type/L38HL protease structures bound to SQV
were analyzed using the root-mean-square deviation (RMSD), the radius of gyration (Rg)
and the root-mean-square fluctuation (RMSF).

3.1. Global Structural Stability

RMSD is primarily used to monitor fluctuations in the global structure of the protein
between every successive pair of frames in a simulation trajectory. By comparing the
RMSD variations between trajectories, the relative global structural stability can be visu-
alized, and the effect of mutation on the global structure can be quantified. The compari-
son of RMSD values for wild-type and L38HL proteases in complex with SQV is shown
in Figure 2A. Even though both the complexes were observed to maintain conformational
stability during the initial 300 ns of simulation, the wild-type protease in complex with
SQV exhibited a major fluctuation in structure between 300 ns and 400 ns of the simulation
trajectory, with the RMSD varying between 1.5 Å and 3.8 Å. After 400 ns, the wild-type
protease–SQV complex structure stabilizes with an RMSD of ~3.5 Å. On the other hand,
RMSD of the L38HL mutant in a complex with SQV was found to maintain structural
stability throughout the course of the simulation. In both wild-type and L38HL complexed
with SQV, fluctuations were observed for a period of at least 100 ns in the simulation
trajectory, indicating the structural changes in the protein induced by the presence of the
inhibitor. Hence, the initial 100 ns data was not used for further analysis. Further, the
structural changes quantified by RMSD variation seem to be more pronounced in the case
of the wild-type in complex with SQV than the L38HL variant. This can be an indication
of the reduced induction effect due to the binding of SQV with the L38HL variant, which
is insufficient to induce pronounced conformational changes in the complex, thereby po-
tentially leading to drug resistance.
Genes 2023, 14, 533 6 of 17

Figure 2. (A) RMSD plot from the MD compared the wild-type bound with SQV (black) and L38HL
bound with SQV (red). (B) Probability distribution of radius of gyration (Rg) was calculated for the
equilibrated trajectory for the wild-type (black) and L38HL variant (red).

The overall compactness of the wild-type and the L38HL variant was quantified us-
ing the radius of gyration (Rg), given as the average distance of the Cα atoms of the pro-
tease from their center of mass over the equilibrated MD trajectories. The conformational
preferences of both systems were characterized by the probability distribution of R g (Fig-
ure 2B). The L38HL variant bound to SQV explores conformational states with the Rg var-
ying between ~18 Å and ~19 Å resulting in a wider curve than the R g of the wild-type,
which varies between ~17.5 Å and 18.5 Å. The average Rg values for the wild-type and the
L38HL variant are 17.75 ± 0.16 Å and 18.33 ± 0.20 Å, respectively, which revealed that the
wild-type protease bound to SQV has a more compact structure than the L38HL variant.

3.2. Residue-Level Fluctuations in Protein Conformation

The root-mean-square fluctuation (RMSF) was calculated for all residues in each MD
simulation trajectory to explain the conformational changes observed in each domain of
the protein. Figure 3 shows the structure of the wild-type and L38HL variant colored
based on the scaled RMSF values. On comparing the RMSF values of all residues from the
wild-type and L38HL bound to SQV, the L38HL double insertion increases the conforma-
tional flexibility of the hinge residues in both monomers in comparison with the wild-type
hinge regions.

Figure 3. Root-mean-square fluctuations (RMSF) of Cα atoms of the protease. (A) wild-type bound
to SQV. (B) L38HL variant bound to SQV. The coloring scheme was scaled based on the maximum
and minimum values mentioned in the color bar. The blue color (minimum) indicates a low fluctu-
ation, whereas the red color (maximum) indicates high fluctuations.
Genes 2023, 14, 533 7 of 17

Corresponding to the increase in hinge flexibility, a mild increase in flexibility of the

flap regions of both monomers, which flank the binding site and enable inhibitor entry
and exit, was also observed in the L38HL protease, which agrees well with the previous
studies [14]. Further, increased flexibility was observed in the fulcrum region (only chain
A) and the hinge region (both chain A and chain B) of the protease.
Conclusively, the RMSF calculations of both the wild-type and the L38HL variant
show increased flexibility in the flap regions of the L38HL variant. Previous studies have
indicated that an increase in flap flexibility due to mutations in the hinge region can pre-
vent proper binding of inhibitors to the HIV protease active site, thereby potentially lead-
ing to drug resistance. Based on the RMSF values obtained from the MD simulation tra-
jectory, it can be inferred that the insertion of residues in the hinge region may lead to
drug resistance by preventing the proper binding of inhibitors, such as SQV, due to the
enhanced hinge region flexibility.

3.3. Local Conformational Changes

The local structural changes caused due to the insertion were investigated in three
directions: (i) the Val32–Val32′ Cα distances to quantify the size of the binding site, (ii) the
Tri Cα angle to understand the flap dynamics and (iii) the Hinge Cα angle to understand
the change in hinge dynamics upon insertion.

3.4. Distance between the Binding Site Residues

The change in the binding site volume has been quantified by measuring the distance
between the flap tips and the flap to active site distances [35,36]. However, the insertion
of His and Leu at the 38th position alters the coordinates of the flaps in the L38HL variant.
Hence, the change in volume of the binding site was investigated by measuring the Cα
distance between Val32 and Val32′. The probability distribution of the distance values is
given in Figure 4C. For the wild-type distributions, the distance varied between 21–26 Å
with a peak close to 23 Å, whereas for the L38HL variant, the peak lies around 25 Å and
the distances are distributed between 22–27 Å. The mean and standard deviation of the
wild-type distribution is 23.2 ± 0.85 Å, and that of the L38HL variant is 24.4 ± 0.845 Å.
Hence, the changes in the distance distribution depict the increased binding site volume
in the L38HL variant. This observation agrees with the radius of gyration analysis, where
the wild-type protease is more compact than L38HL.
Genes 2023, 14, 533 8 of 17

Figure 4. Representative snapshots showing the binding site distance measured between Val32 and
Val32′ Cα (red sphere) in the (A) wild-type and (B) L38HL variant. Protein is represented in both
the cartoon as well as surface representation. Ligand is not shown for visualization clarity. (C) Fre-
quency distribution of Cα Val32–Val32′ in SQV bound WT and L38HL.

Further, the L38HL variant maintains a wider active site, making it more solvent ex-
posed, thereby weakening the interactions of the inhibitor with the protein and signifi-
cantly impacting the drug binding. Figure 4A,B show the representative snapshots for the
average distance between Val32 and Val32′ in the wild-type and L38HL variant, respec-
tively. The surface representation of the protein clearly shows the widening of the active
site in the L38HL variant (Figure 4B).

3.5. Analysis of Flap Tri Cα Angles

The flap regions in HIV protease play a major role in the substrate/inhibitor entry
and exit, where they establish interactions with the substrate/inhibitor. Hence, the inter-
action of the flap with the substrate/inhibitor plays a key role in drug susceptibility. Fur-
ther, changes in the flap dynamics are shown to have a direct influence on drug resistance.
The Tri Cα angle of the wild-type (chain A: residues 49–51 and chain B: residues 49′–51′)
and L38HL variant (chain A: residues 51–53 and chain B: residues 51′–53′) was measured
over the equilibrated trajectory to explore the change in the flap dynamics due to the in-
sertion. The frequency distribution of the measured Cα angles is shown in Figure 5A,B.
Genes 2023, 14, 533 9 of 17

Figure 5. Frequency distribution of the Tri Cα angles in SQV-bound wild-type and L38HL com-
plexes. (A) chain A (B) chain B. Representative snapshots of average flap Tri Cα angles correspond-
ing to the wild-type (blue) and L38HL (orange) variant bound to SQV (C) Chain A (D) chain B.
Chain A and chain B are independent snapshots.

The mean and standard deviation values of the Tri Cα angle are 106.95 ± 7.4°, 97.81 ±
9.7°, 93.87 ± 8.7°, 100.0 ± 9.8°, for wild-type chain A, wild-type chain B, L38HL chain A
and L38HL chain B, respectively. From the analysis of Tri Cα angle, it can be observed
that the flap angle of chain A is narrower in the L38HL variant than in the wild-type.
However, for chain B, the angle follows a similar distribution to that of the wild-type.
Wang et al. [36] reported that the narrowing of the flap tip gives rise to conformations that
have altered protein–drug interactions, which lead to reduced drug susceptibility. The
representative snapshot showing the average curling of the flaps of chain A and chain B
in wild-type and L38HL variant bound to SQV is shown in Figure 5C,D.

3.6. Analysis of Hinge Tri Cα Angle

In order to understand the influence of the hinge dynamics on the flap, we analyzed
the hinge angle for the wild-type and the L38HL variant. Unlike the flap tip angle, inser-
tion at the 38th position did not alter the residues forming the hinge in the L38HL variant.
The angle between residues 39–41 (chain A) and residues (39′–41′) for chain B were used
to measure the hinge angle. The frequency distribution of the hinge angle is shown in
Supplementary Figure S1. The reduced frequency and the wider curve width of the L38HL
variant suggest the increase in flexibility of the hinge compared with that of the wild-type.
Further, a shift in the peak compared with the wild-type indicates the curling of hinges.
Similar to the flap angles, the narrowing of the hinge angles is observed in the L38HL
variant.

3.7. Principal Component Analysis of Wild-Type and L38HL Bound to SQV

The essential dynamics (a process of applying PCA to a protein trajectory) helps to
understand the collective molecular motion of the entire protein, which are characterized
by Eigen values and Eigen vectors of the covariance matrix 𝐶(𝑖,𝑗) (Equation (2)) and are
represented with porcupine plots (Figure 6A,B). The direction of the needles in the plot
Genes 2023, 14, 533 10 of 17

corresponds to the predominant direction of motion of the Cα atoms of residues, and the
length of the needles corresponds to the magnitude of the motion.

Figure 6. Stereo view of the porcupine plots showing the motion of the protease subunits from the
first principal component from the (A) wild-type and (B) L38HL. Dynamic cross-correlation maps
obtained of α-carbons for (C) wild-type and (D) L38HL. Residues that exhibit maximum correlation
are colored red, and the residues that exhibit maximum anti-correlations are colored black. The val-
ues range from −1.0 to 1.0, where 1.0 indicates a positive correlation (red) among residues and −1.0
indicate a negative correlation (black). Regions R1, R2 and R3 (continuous numbering of residues is
followed for clarity) represent hinge and flap regions in chain A, hinge and flap regions in chain B
and residues at the dimer interface, respectively.

The porcupine plots obtained from the first principal component (PC) show the col-
lective motion of the protease subunits of the wild-type (Figure 6A) and L38HL variant
(Figure 6B). Since the contribution from the first PC obtained over the equilibrated trajec-
tories is more than 50% (Supplementary Figure S2) for both the wild-type and L38HL var-
iant, the analyses corresponding to the first PC alone are shown.
Figure 6B shows that the L38HL insertion induces a change in the dynamics of the
protein. This is observed from the altered motions of the Cα residues corresponding to
L38HL compared with that of the wild-type. Precisely, the directionality of motion of the
residues in the flap region of the L38HL tends to move away from the binding site. In
contrast, for the wild-type, the residues of the flap regions tend to move towards the bind-
ing site. Apart from changes in the directionality of motion, the amplitude of the motion
is also affected due to insertion, especially the insertion of the amino acid residues, which
induces a mild change in the amplitude of motion in the residues lining the dimer inter-
face. Thus, from the analysis of the essential dynamics, it can be concluded that, in com-
parison with the wild-type, the insertion of the residues in the hinge region alters the di-
rectionality of motion of the residues in the flap region and the residues at the homodimer
interface.

3.8. Dynamic Cross-Correlation Analysis

Dynamic cross-correlation analysis is used to characterize the movement of atoms
with respect to one another. Due to the computational complexity of the atomistic
Genes 2023, 14, 533 11 of 17

simulations, the cross-correlation, in general, is studied between the Cα atoms. The output
of the cross-correlation analysis is an N × N heat map, where N is the number of Cα atoms
in the protein. The dynamic cross-correlation matrix plotted as a heat map for the wild-
type and L38HL protease is presented in Figure 6C,D, respectively. Regions R1 and R2 in
Figure 6C,D comprise the hinge and the flap regions in chain A andchain B, respectively,
while region R3 comprises the residues lining the dimer interface. Regions R1 and R2 ex-
hibit a strong positive correlation in the wild-type, while the strength of the correlation is
reduced in the L38HL variant. On the contrary, the residues around the active site exhibit
a mild increase in positive correlation in the L38HL variant compared to that of the wild-
type.
Interestingly, region R3 exhibits both positive and negative correlations in the wild-
type, whereas the positive correlation is lost in the L38HL variant. In addition, we ob-
served a positive correlation between the hinge of chain A and the cantilever of chain B as
well as a decrease in a negative correlation between the R1 region and the C-terminal of
chain A.
The results from PCA and the cross-correlation analysis clearly show that the inser-
tion of two residues causes a perturbation in the internal dynamics of the protease affect-
ing the directionality of motions and the correlated movements of the flap region and the
residues located at the dimer interface owing to drug resistance.

3.9. Binding Free Energy Calculations

To assess the binding affinity of SQV with wild-type and the L38HL variant,
MM/GBSA calculations were performed using the MM/GBSA module from the AMBER18
package. The binding free energy (𝛥𝐺𝑏𝑖𝑛𝑑 ) of SQV with the wild-type and L38HL variant
is −48.94 ± 4.63 kcal/mol and −45.81 ± 5.67 kcal/mol, respectively. The individual contribu-
tions from the polar and non-polar interactions are given in Table 1.

Table 1. Calculated binding free energy (kcal/mol) for wild-type and L38HL bound to SQV.

wild-type (kcal/mol) L38HL (kcal/mol)

𝛥𝐺𝑣𝑑𝑤 −57.22 ± 4.04 −58.10 ± 5.30
𝛥𝐺𝑒𝑙𝑒𝑐 −13.14 ± 12.66 35.11 ± 11.95
𝛥𝐺𝑔𝑏 29.31 ± 10.59 −15.10 ± 11.33
𝛥𝐺𝑆𝐴 −7.89 ± 0.45 −7.72 ± 0.56
𝛥𝐺𝑝𝑜𝑙 16.17 ± 11.62 20.01 ± 11.64
𝛥𝐺ℎ𝑦𝑑𝑟𝑜 −65.11 ± 8.35 −65.82 ± 2.93
𝛥𝐺𝑏𝑖𝑛𝑑 −48.94 ± 4.637 −45.81 ± 5.67
All the reported values are in kcal/mol. 𝛥𝐺𝑏𝑖𝑛𝑑 = 𝛥𝐺𝑝𝑜𝑙 + 𝛥𝐺ℎ𝑦𝑑𝑟𝑜 ; 𝛥𝐺𝑝𝑜𝑙 = 𝛥𝐺𝑒𝑙𝑒𝑐 + 𝛥𝐺𝑔𝑏 (Polar
contribution); and 𝛥𝐺ℎ𝑦𝑑𝑟𝑜 = 𝛥𝐺𝑣𝑑𝑤 + 𝛥𝐺𝑆𝐴 (Hydrophobic interactions).

From Table 1, we observe that the hydrophobic interactions comprising van der
Waals and non-polar solvation-free energies remain similar for both the wild-type and the
L38HL variant and favor the binding of SQV with the protease complexes. However, sig-
nificant changes are observed in the polar interactions. The polar interactions comprising
the electrostatic and polar solvation-free energies possess a negative effect on the binding
of SQV to the L38HL variant, thereby weakening the binding affinity. The enthalpy of
L38HL is weakened by 3.13 kcal/mol relative to that of the wild-type. Nevertheless, it was
overcompensated by the favorable polar solvation-free energy, which could be the result
of increased solvent exposure of the ligand due to the opening of the binding site. To fur-
ther understand the influence of the insertion on the protein-inhibitor interaction, a per-
residue interaction network was obtained using the MM/GBSA module from AMBER18.
Upon comparing the residue-level contributions in Figure 7A–D, it is evident that the lost
interaction of SQV with the Asp25′ plays a crucial role in reduced drug stability. Addi-
tionally, the interaction graphs clearly show the loss of interaction of SQV with chain B of
Genes 2023, 14, 533 12 of 17

the L38HL variant. The same can be observed from the hydrogen bond interactions (Fig-
ure 8) as well.

Figure 7. Per-residue interaction spectrum of (A) wild-type chain A, (B) wild-type chain B, (C)
L38HL chain A and (D) L38HL chain B with SQV.

Figure 8. Snapshots that best represent the interactions between SQV with the (A) wild-type and (B)
L38HL variant. The residues that lie within 4 Å of SQV are shown in (A) and (B). Further, the hy-
drogen bonding of SQV with the wild-type and L38HL variant is shown in (C) and (D), respectively.
The hydrogen-bonded interactions are shown in black-dotted lines. SQV is shown in balls and
sticks, and the residues forming interactions with SQV are represented as sticks.

The list of hydrogen bonds formed throughout the equilibrated trajectory is given in
Table 2. We observed that SQV establishes interactions with both the chains of the wild-
Genes 2023, 14, 533 13 of 17

type protein. However, in the L38HL variant, the interactions with chain B are lost and
make the inhibitor move toward chain A. Moreover, the number of hydrogen bonds
formed by SQV with the wild-type is more than that of the L38HL variant. The consider-
able reduction in the number of hydrogen bonds in the L38HL variant allows a wide open-
ing of the binding site.

Table 2. List of hydrogen bonds formed by SQV with wild-type and L38HL sorted based on occu-
pancy values.

Average Dis-
Acceptor Donor Angle (°) Occupancy
tance (Å)
ASP124@OD1 SQV@N5 2.74 156.17 0.628
SQV@O3 ARG8@NH1 2.81 160.26 0.51
ASP124@OD1 SQV@N6 2.85 157.83 0.47
wild-type SQV@O2 ASP128@N 2.86 162.61 0.25
ASP124@OD2 SQV@N5 2.75 157.34 0.12
ASP124@OD1 SQV@O4 2.75 162.47 0.10
GLY126@O SQV@N1 2.88 159.20 0.10

ASP25@OD2 SQV@O4 2.63 164.89 0.45

L38HL ASP25@OD1 SQV@O4 2.63 164.42 0.32
SQV@O3 GLY53@N 2.86 157.20 0.19

Overall, comparing the intermolecular interactions between the wild-type and mu-
tant protease in complex with SQV shows that there is a significant decrease in interac-
tions with SQV observed in mutant protease due to the difference in flap conformation
caused by the L38HL insertion. This can lead to the formation of a weaker protease–inhib-
itor complex, which might not be as potent as the wild-type protease–SQV complex, thus
leading to drug resistance. The three-dimensional view of the SQV–protease interactions
is also shown in Figure 8.

4. Discussion
HIV protease is one of the prominent targets for mitigating HIV infection [37,38]. To
date, there are 10 different Food and Drug Administration (FDA)-approved HIV-1 prote-
ase inhibitors, namely Saquinavir (SQV), Indinavir (IDV), Ritonavir (RTV), Nelfinavir
(NFV), Atazanavir (ATV), Tipranavir (TPV), Darunavir (DRV), Fosamprenavir (FAPV),
Amprenavir (APV) and Lopinavir (LPV), which are used to mitigate viral infections [39].
However, the selection pressure, together with the dynamic nature of viral replication,
gives rise to viral protease strains that can evade the effect of the protease inhibitors [40].
It has been reported that the HIV-1 subtype is most prevalent among the sub-Saharan
African regions, and it accounts for 70% of the HIV infections caused globally [7,41–43].
Due to this reason, many mutations/insertions have been reported in the HIV-1 protease
subtype C. Unlike mutations, insertions are rare events, even though they have become
prevalent since 1999 [44,45]. They are positively correlated with the codons associated
with resistance to protease inhibitors as well as decrease protease inhibitor susceptibility
and improve viral fitness. Recently, a new type of HIV-protease subtype C called the
L38HL variant is characterized by the insertion of His and Leu in the 38th position.
Computational analysis of the L38HL along with the wild-type HIV-1 PR type C
bound to SQV revealed that the binding of SQV is more stable in the wild-type than the
L38HL variant. The reduction in the affinity is mainly due to the wider active site confor-
mation exhibited by the L38HL compared with that of the wild-type. Further, the analysis
of flap tips showed a narrow tip region in L38HL than wild-type [36]. This narrowing of
the flap tip demonstrates the existence of an early event in the flap-opening process in the
Genes 2023, 14, 533 14 of 17

apo HIV-1 protease [46]. The increased distance between the active site residues, together
with the narrowed flap tips, leads to the loss of key interactions with the SQV [36]. This
agrees with the per-residue interaction spectrum of SQV with the wild-type and the
L38HL proteases, where it can be seen that the insertion leads to a loss of key interactions
of SQV with the chain B. Precisely, the interactions with residues Asp25′, Ala28′ and Ile82′,
which had a contribution of more than −1.0 kcal/mol in the wild-type, are lost in the L38HL
variant. As indicated above, the wider active site conformation exhibited in the L38HL
can be associated with the increased flexibility of the hinge and flap regions [35].
One reason for the increased flexibility in the flap regions could be due to the reduced
hydrogen bond lifetime between the flap residues (Supplementary Tables S1 and S2) in
the L38HL variant. Further, the frequency distribution of intermolecular hydrogen bonds
showed that the number of hydrogen bonds is more in the wild-type than in L38HL. Fur-
ther investigation of the occupancy values of inter-chain hydrogen bonds revealed that,
despite the loss of interactions in L38HL, in general, many hydrogen bonds showed in-
creased occupancy values. Specifically, the occupancy value of the main chain oxygen
atom from Leu5 that forms a hydrogen bond with the Arg186/190 side chain nitrogen
(NH1) increased from 0.58 in wild-type to 0.81 in L38HL. However, several interactions
with reduced occupancy were also observed in L38HL. Nevertheless, the overall increase
in inter-chain hydrogen bond occupancy increases the rigidness of the C-terminal and the
N-terminal regions. This agrees with our results from PCA, where the residues in the N-
terminal and the C-terminal regions had a reduced magnitude of motion. As seen from
the dynamic cross-correlation analysis, the decreased correlation could be the result of
changes in the hydrogen bond occupancy.
Conclusively, it was observed from the simulations that the insertion of residues in
the hinge region triggers conformational changes leading to increased binding site volume
and narrowing of the flap tips [36]. These conformational changes can be the result of the
sustained formation of certain hydrogen bonds together with the loss of certain hydrogen
bonds in the dimer interface, which alters the conformation, resulting in reduced motion
of residues in the N and C termini. Further, the change in hydrogen bond occupancy in-
fluences the correlated motion of residues inducing a change in the directionality of the
motion of the residues. As a result of these conformational changes, SQV loses key inter-
actions with the active site residues leading to drug resistance.

5. Conclusions
In this study, molecular dynamics simulations were used to understand and compare
the conformations of wild-type and L38HL mutant HIV protease subtype C upon interac-
tion with the protease inhibitor, Saquinavir (SQV). The structural stability and intermo-
lecular interactions of both proteases were studied during the course of the simulation to
evaluate the possibilities of the emergence of a drug-resistance phenotype due to the mu-
tation. Interestingly, the L38HL variant was found to exhibit an increase in flexibility at
the hinge and flap regions, similar to previous drug-resistant mutations reported in the
literature [13,15,16]. Further, binding free energy values for wild-type–SQV and mutant–
SQV complexes using the MM/GBSA method revealed that L38HL insertion decreased
the affinity of the protease by 3 kcal/mol towards the inhibitor in comparison with that of
the wild-type protease. Moreover, these insertions greatly influence the correlation of the
residues and the directionality of motion of the flap residues leading to an increase in flap
flexibility and a wide opening of the binding pocket. These results provide insights into
understanding the potential drug resistance phenotype in infected individuals.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/genes14020533/s1; Figure S1: Frequency distribution of
Hinge Cα Angles; Figure S2: Contributions from the first 3 principal components; Table S1: List of
hydrogen bonds formed between the homodimer in wild-type; Table S2: List of hydrogen bonds
formed between the homodimer in L38HL.
Genes 2023, 14, 533 15 of 17

Author Contributions: Methodology, S.V., N.M., C.R., M.S., D.B., V.A., Y.S. and M.M.G.; validation,
R.P. and S.T.; formal analysis, S.V., N.M., S.R.K. and M.S.; investigation, S.V., S.R.K., C.R., R.P. and
D.B.; data curation, N.M., S.R.K., R.P. and S.T.; writing—original draft, S.V.; writing—review and
editing, N.M., S.R.K., C.R., M.S., R.P., D.B., S.T., V.A., Y.S. and M.M.G.; supervision, V.A., Y.S. and
M.M.G.; project administration, M.M.G.; funding acquisition, M.M.G. All authors have read and
agreed to the published version of the manuscript.
Funding: The Department of Science and Technology (DST/ICD/BRICS/Call-3/HIV protease/2019).
Institutional Review Board Statement: Not applicable
Informed Consent Statement: Not applicable
Data Availability Statement: Data are available upon request.
Acknowledgments: We thank the Department of Biotechnology and the Indian Institute of Tech-
nology Madras for their computational facilities. This work is partially supported by the Depart-
ment of Science and Technology, Government of India, under the BRICS project
(DST/ICD/BRICS/Call-3/HIV protease/2019) to MMG, the South African National Research Founda-
tion (NRF) via the BRICS Multilateral Joint Science And Technology Research Collaboration (grant
number: 120452) to Y.S. and the Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq), Brazil (grant number: 402817/2019-2), to V.A. and S.T.
Conflicts of Interest: The authors declare no conflicts of interest.

References
1. Elangovan, R.; Jenks, M.; Yun, J.; Dickson-Tetteh, L.; Kirtley, S.; Hemelaar, J. Global and Regional Estimates for Subtype-Specific
Therapeutic and Prophylactic HIV-1 Vaccines: A Modeling Study. Front. Microbiol. 2021, 12, 690647.
https://doi.org/10.3389/fmicb.2021.690647.
2. Bbosa, N.; Kaleebu, P.; Ssemwanga, D. HIV subtype diversity worldwide. Curr. Opin. HIV AIDS 2019, 14, 153–160.
https://doi.org/10.1097/COH.0000000000000534.
3. Giovanetti, M.; Ciccozzi, M.; Parolin, C.; Borsetti, A. Molecular Epidemiology of HIV-1 in African Countries: A Comprehensive
Overview. Pathogens 2020, 9, 1072. https://doi.org/10.3390/pathogens9121072.
4. Naicker, P.; Sayed, Y. Non-B HIV-1 subtypes in sub-Saharan Africa: Impact of subtype on protease inhibitor efficacy. Biol. Chem.
2014, 395, 1151–1161. https://doi.org/10.1515/hsz-2014-0162.
5. Häggblom, A.; Svedhem, V.; Singh, K.; Sönnerborg, A.; Neogi, U. Virological failure in patients with HIV-1 subtype C receiving
antiretroviral therapy: An analysis of a prospective national cohort in Sweden. Lancet HIV 2016, 3, e166–e174.
https://doi.org/10.1016/S2352-3018(16)00023-0.
6. Velázquez-Campoy, A.; Vega, S.; Fleming, E.; Bacha, U.; Sayed, Y., Dirr HW; Freire, E. Protease inhibition in African subtypes
of HIV-1. AIDS Rev. 2003, 5, 165–171.
7. Mosebi, S.; Morris, L.; Dirr, H.W.; Sayed, Y. Active-site mutations in the South african human immunodeficiency virus type 1
subtype C protease have a significant impact on clinical inhibitor binding: Kinetic and thermodynamic study. J. Virol. 2008, 82,
11476–11479. https://doi.org/10.1128/JVI.00726-08.
8. Eche, S.; Kumar, A.; Sonela, N.; Gordon, M.L. Acquired HIV-1 Protease Conformational Flexibility Associated with Lopinavir
Failure May Shape the Outcome of Darunavir Therapy after Antiretroviral Therapy Switch. Biomolecules 2021, 11, 489.
https://doi.org/10.3390/biom11040489.
9. Shafer, R.W.; Eisen, J.A.; Merigan, T.C.; Katzenstein, D.A. Sequence and drug susceptibility of subtype C reverse transcriptase
from human immunodeficiency virus type 1 seroconverters in Zimbabwe. J. Virol. 1997, 71, 5441–5448.
https://doi.org/10.1128/JVI.71.7.5441-5448.1997.
10. Mendoza, Y.; Bello, G.; Castillo Mewa, J.; Martínez, A.A.; González, C.; García-Morales, C.; Avila-Ríos, S.; Reyes-Terán, G.;
Pascale, J.M. Molecular epidemiology of HIV-1 in Panama: Origin of non-B subtypes in samples collected from 2007 to 2013.
PLoS ONE 2014, 9, e85153. https://doi.org/10.1371/journal.pone.0085153.
11. Foster, G.M.; Ambrose, J.C.; Hué, S.; Delpech, V.C.; Fearnhill, E.; Abecasis, A.B.; Leign Brown, A.J.; Geretti, A.M. Novel HIV-1
recombinants spreading across multiple risk groups in the United Kingdom: The identification and phylogeography of
Circulating Recombinant Form (CRF) 50_A1D. PLoS ONE 2014, 9, e83337. https://doi.org/10.1371/journal.pone.0083337.
12. Naicker, P.; Achilonu, I.; Fanucchi, S.; Fernandes, M.; Ibrahim, M.A.A.; Dirr, H.W.; Soliman, M.E.S.; Sayed, Y. Structural insights
into the South African HIV-1 subtype C protease: Impact of hinge region dynamics and flap flexibility in drug resistance. J.
Biomol. Struct. Dyn. 2013, 31, 1370–1380. https://doi.org/10.1080/07391102.2012.736774.
13. Ahmed, S.M.; Kruger, H.G.; Govender, T.; Maguire, G.E.M.; Sayed, Y.; Ibrahim, M.A.A.; Naicker, P.; Soliman, M.E.S.
Comparison of the Molecular Dynamics and Calculated Binding Free Energies for Nine FDA-Approved HIV-1 PR Drugs
Against Subtype B and C-SA HIV PR. Chem. Biol. Drug. Des. 2013, 81, 208–218. https://doi.org/10.1111/cbdd.12063.
Genes 2023, 14, 533 16 of 17

14. Ragland, D.A.; Nalivaika, E.A.; Nalam, M.N.L.; Prachanronarong, K.L.; Cao, H.; Bandaranayake, R.M.; Cai, Y.; Kurt-Yilmaz, N.;
Schiffer, C.A. Drug Resistance Conferred by Mutations Outside the Active Site through Alterations in the Dynamic and
Structural Ensemble of HIV-1 Protease. J. Am. Chem. Soc. 2014, 136, 11956–11963. https://doi.org/10.1021/ja504096m.
15. Obasa, A.E.; Ambikan, A.T.; Gupta, S.; Neogi, U.; Jacobs, G.B. Increased acquired protease inhibitor drug resistance mutations
in minor HIV-1 quasispecies from infected patients suspected of failing on national second-line therapy in South Africa. BMC
Infect. Dis. 2021, 21, 214. https://doi.org/10.1186/s12879-021-05905-2.
16. Obasa, A.E.; Mikasi, S.G.; Brado, D.; Cloete, R.; Singh, K.; Neogi, U.; Jacobs, G.B. Drug Resistance Mutations Against Protease,
Reverse Transcriptase and Integrase Inhibitors in People Living With HIV-1 Receiving Boosted Protease Inhibitors in South
Africa. Front. Microbiol. 2020, 11, 438. https://doi.org/10.3389/fmicb.2020.00438.
17. Ledwaba, J.; Sayed, Y.; Pillay, V.; Morris, L.; Hunt, G. Low Frequency of Protease Inhibitor Resistance Mutations and Insertions
in HIV-1 Subtype C Protease Inhibitor-Naïve Sequences. AIDS Res. Hum. Retrovir. 2019, 35, 673–678.
https://doi.org/10.1089/AID.2019.0012.
18. Jacob, K.S.; Ganguly, S.; Kumar, P.; Poddar, R.; Kumar, A. Homology model, molecular dynamics simulation and novel pyrazole
analogs design of Candida albicans CYP450 lanosterol 14 α-demethylase, a target enzyme for antifungal therapy. J. Biomol.
Struct. Dyn. 2017, 35, 1446–1463. https://doi.org/10.1080/07391102.2016.1185380.
19. Collier, T.A.; Piggot, T.J.; Allison, J.R. Molecular Dynamics Simulation of Proteins. Methods Mol. Biol. 2020, 2073, 311–327.
https://doi.org/10.1007/978-1-4939-9869-2_17.
20. Trott, O.; Olson, A.J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient
optimization, and multithreading. J. Comput. Chem. 2010, 31, 455–461. https://doi.org/10.1002/jcc.21334.
21. Gupta, M.; Sharma, R.; Kumar, A. Docking techniques in pharmacology: How much promising? Comput. Biol. Chem. 2018, 76,
210–217. https://doi.org/10.1016/j.compbiolchem.2018.06.005.
22. Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C. Comparison of multiple Amber force fields and
development of improved protein backbone parameters. Proteins 2006, 65, 712–725. https://doi.org/10.1002/prot.21123.
23. Case, D.A.; Cheatham, T.E. III.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K.M., Jr; Onufriev, A.; Simmerling, C.; Wang, B.; Woods,
R.J. The Amber biomolecular simulation programs. J. Comput. Chem. 2005, 26, 1668–1688. https://doi.org/10.1002/jcc.20290.
24. Jorgensen, W.L. Transferable intermolecular potential functions for water, alcohols, and ethers. Application to liquid water. J.
Am. Chem. Soc. 1981, 103, 2. https://doi.org/10.1021/ja00392a016.
25. Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems. J. Chem. Phys.
1993, 98, 10089–10092. https://doi.org/10.1063/1.464397.
26. Berendsen, H.J.C.; Postma, J.P.M.; van Gunsteren, W.F.; DiNola, A.; Haak, J.R. Molecular dynamics with coupling to an external
bath. J. Chem. Phys. 1984, 81, 3684–3690. https://doi.org/10.1063/1.448118.
27. Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H.J.C. Numerical integration of the cartesian equations of motion of a system with
constraints: Molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23, 327–341. https://doi.org/10.1016/0021-9991(77)90098-5.
28. Makatini, M.M.; Petzold, K.; Sriharsha, S.N.; Ndlovu, N.; Soliman, M.E.S.; Honarparvar, B.; Parboosing, R.; Naidoo, A.;
Arvidsson, P.I.; Sayed, Y.; et al. Synthesis and structural studies of pentacycloundecane-based HIV-1 PR inhibitors: A hybrid
2D NMR and docking/QM/MM/MD approach. Eur. J. Med. Chem. 2011, 46, 3976–3985.
https://doi.org/10.1016/j.ejmech.2011.05.071.
29. Makatini, M.M.; Petzold, K.; Sriharsha, S.N.; Soliman, M.E.S.; Honarparvar, B.; Arvidsson, P.I.; Sayed, Y.; Govender, P.;
Maguire, G.E.M.; Kruger, H.G.; et al. Pentacycloundecane-based inhibitors of wild-type C-South African HIV-protease. Bioorg.
Med. Chem. Lett. 2011, 21, 2274–2277. https://doi.org/10.1016/j.bmcl.2011.02.105.
30. Kasahara, K.; Fukuda, I.; Nakamura, H. A Novel Approach of Dynamic Cross Correlation Analysis on Molecular Dynamics
Simulations and Its Application to Ets1 Dimer–DNA Complex. PLoS ONE 2014, 9, e112419.
https://doi.org/10.1371/journal.pone.0112419.
31. Ramakrishnan, V.; Jagannathan, S.; Shaikh, A.R.; Rajagopalan, R. Dynamic and Structural Changes in the Minimally
Restructuring EcoRI Bound to a Minimally Mutated DNA Chain. J. Biomol. Struct. Dyn. 2012, 29, 743–756.
https://doi.org/10.1080/073911012010525020.
32. Kollman, P.A.; Massova, I.; Reyes, C.; Kuhn, B.; Huo, S.; Chong, L.; Lee, M.; Lee, T.; Duan, Y.; Wang, W.; et al. Calculating
Structures and Free Energies of Complex Molecules:  Combining Molecular Mechanics and Continuum Models. Acc. Chem. Res.
2000, 33, 889–897. https://doi.org/10.1021/ar000033j.
33. Tsui, V.; Case, D.A. Theory and applications of the generalized Born solvation model in macromolecular simulations.
Biopolymers 2001, 56, 275–291. https://doi.org/10.1002/1097-0282(2000)56:4<275::AID-BIP10024>3.0.CO;2-E.
34. Weiser, J.; Shenkin, P.S.; Still, W.C. Approximate atomic surfaces from linear combinations of pairwise overlaps (LCPO). J.
Comput. Chem. 1999, 20, 217–230.
35. Sherry, D.; Worth, R.; Ismail, Z.S.; Sayed, Y. Cantilever-centric mechanism of cooperative non-active site mutations in HIV
protease: Implications for flap dynamics. J. Mol. Graph. Model 2021, 106, 107931. https://doi.org/10.1016/j.jmgm.2021.107931.
36. Wang, R.-G.; Zhang, H.-X.; Zheng, Q.-C. Revealing the binding and drug resistance mechanism of amprenavir, indinavir,
ritonavir, and nelfinavir complexed with HIV-1 protease due to double mutations G48T/L89M by molecular dynamics
simulations and free energy analyses. Phys. Chem. Chem. Phys. 2020, 22, 4464–4480. https://doi.org/10.1039/c9cp06657h.
37. Weber, I.T.; Wang, Y.-F.; Harrison, R.W. HIV Protease: Historical Perspective and Current Research. Viruses 2021, 13, 839.
https://doi.org/10.3390/v13050839.
Genes 2023, 14, 533 17 of 17

38. Lv, Z.; Chu, Y.; Wang, Y. HIV protease inhibitors: A review of molecular selectivity and toxicity. HIV AIDS 2015, 7, 95–104.
https://doi.org/10.2147/HIV.S79956.
39. Yu, Y.X.; Liu, W.T.; Li, H.Y.; Wang, W.; Sun, H.B.; Zhang, L.L.; Wu, S.L. Decoding molecular mechanism underlying binding of
drugs to HIV-1 protease with molecular dynamics simulations and MM-GBSA calculations. SAR QSAR Environ. Res. 2021, 32,
889–915. https://doi.org/10.1080/1062936X.2021.1979647.
40. Maseko, S.B.; Padayachee, E.; Govender, T.; Sayed, Y.; Kruger, G.; Maguire, G.E.M.; Lin, J. I36T↑T mutation in South African
subtype C (C-SA) HIV-1 protease significantly alters protease-drug interactions. Biol. Chem. 2017, 398, 1109–1117.
https://doi.org/10.1515/hsz-2017-0107.
41. Salminen, M.O.; Johansson, B.; Sönnerborg, A.; Ayehunie, S.; Gotte, D.; Leinikki, P.; Burke, D.S.; McCutchan, F.E. Full-length
sequence of an ethiopian human immunodeficiency virus type 1 (HIV-1) isolate of genetic subtype C. AIDS Res. Hum. Retrovir.
1996, 12, 1329–1339. https://doi.org/10.1089/aid.1996.12.1329.
42. McCormack, G.P.; Glynn, J.R.; Crampin, A.C.; Sibande, F.; Mulawa, D.; Bliss, L.; Broadbent, P.; Abarca, K.; Pönnighaus, J.M.;
Fine, P.E.M.; et al. Early evolution of the human immunodeficiency virus type 1 subtype C epidemic in rural Malawi. J. Virol.
2002, 76, 12890–12899. https://doi.org/10.1128/jvi.76.24.12890-12899.2002.
43. Lihana, R.W.; Ssemwanga, D.; Abimiku, A.; Ndembi, N. Update on HIV-1 diversity in Africa: A decade in review. AIDS Rev.
2012, 14, 83–100.
44. Kozísek, M.; Sasková, K.G.; Rezácová, P.; Brynda, J.; van Maarseveen, N.M.; De Jong, D.; Boucher, C.A.; Kagan, R.M.; Nijhuis,
M.; Konvalinka, J. Ninety-nine is not enough: Molecular characterization of inhibitor-resistant human immunodeficiency virus
type 1 protease mutants with insertions in the flap region. J. Virol. 2008, 82, 5869–5878. https://doi.org/10.1128/JVI.02325-07.
45. Vora, S.; Marcelin, A.-G.; Günthard, H.F.; Flandre, P.; Hirsch, H.H.; Masquelier, B.; Zinkernagel, A.; Peytavin, G.; Calvez, V.;
Perrin, L.; et al. Clinical validation of atazanavir/ritonavir genotypic resistance score in protease inhibitor-experienced patients.
AIDS 2006, 20, 35–40. https://doi.org/10.1097/01.aids.0000196179.11293.fc.
46. Yu, Y.; Wang, J.; Chen, Z.; Wang, G.; Shao, Q.; Shi, J.; Zhu, W. Structural insights into HIV-1 protease flap opening processes
and key intermediates. RSC Adv. 2017, 7, 45121–45128. https://doi.org/10.1039/C7RA09691G.

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual au-
thor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.