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\DOI{10.5802/crchim.413}
\datereceived{2025-01-01}
\daterevised{2025-05-21}
\dateaccepted{2025-07-29}
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\dateposted{2025-09-18}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{research-article}

\title{Comparative analysis of phosphodiesterase type 5 inhibitors and
usnic acid: exploring therapeutic potential in pulmonary arterial
hypertension}

\alttitle{Comparaison entre les inhibiteurs de la phosphodiest\'{e}rase
de type 5 et l'acide usnique : exploration du potentiel
th\'{e}rapeutique dans l'hypertension art\'{e}rielle pulmonaire}

\author{\firstname{Jelena} \lastname{\DJ{}orovi\'{c} Jovanovi\'{c}}\CDRorcid{0000-0003-3901-6133}}
\address{Institute for Information Technologies, University of
Kragujevac, Jovana Cviji\'{c}a bb, 34000 Kragujevac, Serbia}

\author{\firstname{Marijana} \lastname{Stanojevi\'{c} Pirkovi\'{c}}\CDRorcid{0000-0001-8769-5682}}
\address{University of Kragujevac, Faculty of Medical Sciences,
Department of Medical Biochemistry, Svetozara Markovi\'{c}a 69, 34000
Kragujevac, Serbia}

\author{\firstname{\v{Z}iko} \lastname{Milanovi\'{c}}\CDRorcid{0000-0003-1819-2808}\IsCorresp}
\addressSameAs{1}{Institute for Information Technologies, University of
Kragujevac, Jovana Cviji\'{c}a bb, 34000 Kragujevac, Serbia}
\email[\v{Z}. Milanovi\'{c}]{ziko.milanovic@uni.kg.ac.rs}

\shortrunauthors

\thanks{Ministry of Science, Technological Development and Innovation
of the Republic of Serbia and the Ministry of Education of the Republic
of Serbia (Agreements No. 451-03-66/2024-03/200378)}

\keywords{\kwd{Pulmonary arterial hypertension}\kwd{PDE5
inhibitors}\kwd{Usnic acid}\kwd{ADMET analysis}\kwd{Molecular 
docking}\kwd{Molecular dynamics}}

\altkeywords{\kwd{Hypertension art\'{e}rielle
pulmonaire}\kwd{Inhibiteurs de la PDE5}\kwd{Acide usnique}\kwd{Analyse
ADMET}\kwd{Amarrage mol\'{e}culaire}\kwd{Dynamique mol\'{e}culaire}}

\begin{abstract}
Pulmonary arterial hypertension (PAH) is a progressive disorder
characterized by increased pressure in the pulmonary arteries,
frequently resulting in right ventricular hypertrophy and heart
failure. Phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil
(\textbf{SIL}), tadalafil (\textbf{TAD}), and avanafil (\textbf{AVA}), have demonstrated
efficacy in the treatment of pulmonary arterial hypertension (PAH). The
growing interest in natural compounds, like usnic acid (\textbf{UA}), a
dibenzofuran derived from lichens, stems from their promising potential
as complementary therapies in various medical conditions. This study
investigates the potential of \textbf{UA} as a PDE5 inhibitor compared to
\textbf{SIL}, \textbf{TAD}, and \textbf{AVA} through ADMET analysis, molecular docking, and
molecular dynamic simulations. The ADMET analysis suggests significant
plasma protein binding and an extended half-life for \textbf{UA}, perhaps
improving retention relative to synthetic inhibitors. Molecular docking
analyses indicate that \textbf{UA} establishes stable interactions with PDE5,
exhibiting binding energies similar to those of synthetic inhibitors.
Molecular dynamic simulations validate the stability of these
interactions and suggest that \textbf{UA} reduces the flexibility of PDE5,
enhancing its inhibitory potential. The results demonstrate that \textbf{UA}
may have a potential synergistic effect with PDE5 inhibitors in
treating PAH, offering a favorable toxicity profile, particularly
regarding cardiotoxicity and neurotoxicity.
\end{abstract}

\begin{altabstract}
L'hypertension art\'{e}rielle pulmonaire (PAH) est une maladie
\'{e}volutive caract\'{e}ris\'{e}e par une augmentation de la pression
dans les art\`{e}res pulmonaires, entra\^{i}nant fr\'{e}quemment une
hypertrophie ventriculaire droite et une insuffisance cardiaque. Les
inhibiteurs de la phosphodiest\'{e}rase de type 5 (PDE5), tels que le
sild\'{e}nafil (\textbf{SIL}), le tadalafil (\textbf{TAD}) et l'avanafil (\textbf{AVA}), ont
d\'{e}montr\'{e} leur efficacit\'{e} dans le traitement de
l'hypertension art\'{e}rielle pulmonaire (PAH). L'int\'{e}r\^{e}t
croissant pour les compos\'{e}s naturels, tels que l'acide usnique
(\textbf{UA}), un dibenzofurane d\'{e}riv\'{e} des lichens, d\'{e}coule de leur
potentiel prometteur en tant que th\'{e}rapies compl\'{e}mentaires dans
diverses affections m\'{e}dicales. Cette \'{e}tude examine le potentiel
de l'\textbf{UA} en tant qu'inhibiteur de la PDE5 par rapport au \textbf{SIL}, au \textbf{TAD} et
\`{a} l'\textbf{AVA} par analyse ADMET, amarrage mol\'{e}culaire et simulations
de dynamique mol\'{e}culaire. L'analyse ADMET sugg\`{e}re que l'\textbf{UA}
pr\'{e}sente une liaison significative aux prot\'{e}ines plasmatiques
et une demi-vie prolong\'{e}e, ce qui pourrait am\'{e}liorer sa
r\'{e}tention par rapport aux inhibiteurs synth\'{e}tiques. Les
analyses d'amarrage mol\'{e}culaire indiquent que l'\textbf{UA} \'{e}tablit des
interactions stables avec la PDE5, pr\'{e}sentant des \'{e}nergies de
liaison similaires \`{a} celles des inhibiteurs synth\'{e}tiques. Les
simulations de dynamique mol\'{e}culaire valident la stabilit\'{e} de
ces interactions et sugg\`{e}rent que l'\textbf{UA} r\'{e}duit la
flexibilit\'{e} de la PDE5, renfor\c{c}ant ainsi son potentiel
inhibiteur. Les r\'{e}sultats d\'{e}montrent que l'\textbf{UA} pourrait avoir un
effet synergique avec les inhibiteurs de la PDE5 dans le traitement de
la PAH, offrant un profil de toxicit\'{e} favorable, en particulier en
ce qui concerne la cardiotoxicit\'{e} et la neurotoxicit\'{e}.
\end{altabstract}

\maketitle

\twocolumngrid

\end{noXML}

\section{Introduction} \label{sec1}

Pulmonary arterial hypertension (PAH) is a serious and progressive
disorder marked by increased pressure in the pulmonary arteries,
potentially resulting in right ventricular hypertrophy and ultimately
heart failure~\cite{1,2,3}. The condition may be categorized as
idiopathic, lacking an identifiable underlying cause, or associated
with other diseases, \mbox{including} chronic obstructive pulmonary disease
(COPD), congenital heart defects, and connective tissue
disorders~\cite{4,5}.  The precise etiology of PAH is intricate,
frequently encompassing a blend of genetic predispositions and
environmental influences that lead to endothelial dysfunction,
inflammation, and vascular remodeling~\cite{6,7,8}.

\begin{figure*}
{\vspace*{1pt}}
\includegraphics{fig01}
\caption{\label{fig1}Chemical structures of the investigated
compounds.}
{\vspace*{3pt}}
\end{figure*}

The pathophysiological characteristics of PAH involve significant
alterations in the pulmonary vasculature. Endothelial dysfunction is a
critical characteristic that disrupts the equilibrium between
\mbox{vasodilatory} and vasoconstrictive factors, resulting in the excessive
proliferation of vascular smooth muscle cells~\cite{1,2,3}.
Inflammation exacerbates this process, playing a central role in
disease progression, alongside a reduction in programmed cell death
(apoptosis) and the subsequent vascular remodeling that characterizes
advanced disease stages. These processes result in the constriction of
the pulmonary arteries, heightened pulmonary vascular resistance, and
increased pressures in the pulmonary circulation. The right ventricle
exerts progressively greater effort to circulate blood through the
lungs, resulting in hypertrophy and potential heart failure if not
addressed~\cite{9,10}.

Recent advances in the management of PAH have led to the development of
various therapeutic strategies designed to alleviate symptoms, enhance
functional capacity, and decelerate disease progression.
Phosphodiesterase type 5 (PDE5) inhibitors represent a significant
advancement in the therapy for PAH. Sildenafil (\textbf{SIL}), tadalafil
(\textbf{TAD}) and avanafil (\textbf{AVA}) (Figure~\ref{fig1}) are pharmacological
agents acting on the nitric oxide (NO)--cyclic guanosine monophosphate
(cGMP) pathway, a crucial vascular tone regulator~\cite{11,12,13,14}.
These drugs inhibit PDE5, an enzyme responsible for the degradation of
cGMP, leading to elevated intracellular cGMP levels. This elevation
results in the relaxation of pulmonary artery smooth muscle cells and a
decrease in pulmonary vascular resistance~\cite{15}. First PDE5
inhibitor approved for PAH treatment, \textbf{SIL} has demonstrated
significant clinical efficacy, though its relatively short half-life
necessitates frequent dosing to maintain therapeutic effects~\cite{11}.
Another PDE5 inhibitor, \textbf{TAD}, has a longer half-life, which allows
less frequent dosing and makes it easier for patients to follow their
treatment regimen~\cite{12}. More recently, \textbf{AVA} has been introduced
as a highly selective PDE5 inhibitor, offering greater selectivity and
a more favorable side-effect profile compared to earlier drugs, which
reduces the risk of cardiovascular complications~\cite{13}.\looseness=1

In addition to the synthetic PDE5 inhibitors, there has been growing
interest in the potential of naturally occurring compounds as
alternative or complementary therapies for PAH. Usnic acid (\textbf{UA},
Figure~\ref{fig1}), a lichen-derived dibenzofuran, has emerged as a
compound of interest due to its diverse biological
activities~\cite{16,17,18}, including antioxidant~\cite{19,20},
\mbox{anti-inflammatory}~\cite{21}, and antimicrobial properties~\cite{22}.
Although \textbf{UA} has not been directly investigated for its inhibitory
activity on PDE5, its mechanisms of action in other biological
contexts---such as reducing oxidative stress and inflammation---suggest
that it warrants further exploration as a potential treatment option
for PAH. Given that oxidative stress and inflammation are critical
processes implicated in the pathogenesis of PAH, \textbf{UA} may still offer
therapeutic potential through these pathways, possibly complementing
existing treatments targeting the \mbox{NO--cGMP} axis~\cite{15}. This
investigation into \textbf{UA} activity adds to the growing body of research
that seeks to identify natural compounds with pharmacological effects
that may rival or complement synthetic drugs. Its role as a potential
PDE5 inhibitor, or as a compound influencing alternative pathways
involved in pulmonary vasodilation, makes it a subject of considerable
scientific interest.

This study aims to evaluate the inhibitory potential of usnic acid
(\textbf{UA}) in comparison to clinically used PDE5 inhibitors \textbf{AVA}, \textbf{SIL}
and \textbf{TAD} through a comprehensive in silico approach. ADMET profiling
was performed to assess \textbf{UA}'s pharmacokinetic and toxicity-related
properties relative to reference compounds. Molecular docking and
molecular dynamic simulations explored the binding affinity,
interaction patterns, and stability of enzyme--ligand complexes within
the PDE5 active site. This integrative analysis seeks to elucidate the
molecular basis of \textbf{UA}'s activity and its potential as a novel
therapeutic candidate for the treatment of PAH.

\section{Materials and methods}\label{sec2}
\subsection{ADMET analysis}\label{ssec21}

The ADMETlab 3.0 web server~\cite{23} facilitated a thorough assessment
of the toxicity profiles and pharmacokinetic properties of compounds. A
comprehensive ADMET analysis (Absorption, Distribution, Metabolism,
Excretion, and Toxicity) is crucial for assessing the safety and
biological efficacy of compounds. The analysis presented essential data
regarding absorption, tissue distribution, metabolic stability,
elimination pathways, and potential toxicities, facilitating
predictions about behavior within a biological system.

\subsection{DFT method}\label{ssec22}

Computational analyses were performed using the Gaussian16 software
suite~\cite{24}, while molecular visualizations were executed with
GaussView 6.0.16~\cite{25}. Quantum chemical calculations were
conducted to optimize molecular geometries using density functional
theory (DFT) with the B3LYP-D3BJ functional and the 6-311${{+}{+}}$G(d,p)
basis set~\cite{26,27}. This combination utilizes polarization and
diffuse functions to improve computational accuracy. The selected
theoretical model was based on its proven capacity to closely match
experimental geometrical parameters, as evidenced by multiple
studies~\cite{28,29,30}.

\subsection{Molecular docking simulation}\label{ssec23}

The computational analysis of PDE5 inhibition involved molecular
docking simulations to evaluate the binding affinities and interactions
of \textbf{SIL}, \textbf{TAD}, \textbf{AVA}, and \textbf{UA}. The docking studies utilized AutoDock
4.2 software~\cite{31}, employing the Lamarckian genetic
algorithm~\cite{32} to optimize binding poses and assess interaction
energies. In this study, parameters were defined to ensure precise and
dependable predictions: a maximum of 250~000 energy evaluations were
established, and 27~000 generations were permitted for the evolutionary
search. The mutation rate was established at 0.02, while the crossover
rate was determined to be 0.8. These parameters maintain a balance
between computational efficiency and comprehensive exploration of
potential ligand conformations. Kollmann partial charges were assigned
to the ligands, and polar hydrogens were added using AutoDockTools to
enhance the accuracy of electrostatic interaction predictions in the
docking simulations. In the docking procedure, ligands, whose
structures were optimized according to the previously described method,
were treated as flexible entities, enabling them to assume multiple
conformations within the PDE5 active site, whereas the protein
structure was maintained in a rigid state. This method facilitates the
investigation of ligand flexibility, crucial for comprehending the
dynamic characteristics of ligand--protein interactions. The protein
structure of PDE5 was sourced from the Protein Data Bank (PDB code:
6L6E, accessed on 18.12.2023)~\cite{33}. Preprocessing was conducted
using Discovery Studio 4.0, during which co-crystallized water
molecules and ligands were eliminated to facilitate the preparation of
the binding site. The docking grid box was centered at coordinates
${-}2.479 \times {-}26.083 \times {-}27.632$, featuring a grid
size of $53 \times 69 \times 56$ points and a grid spacing of
0.375~\AA{}. The selected settings accurately define the active site of
PDE5, ensuring comprehensive coverage of the region involved in ligand\break
\mbox{binding.}

\subsection{Molecular dynamic simulation}\label{ssec24}

All molecular dynamics simulations were conducted utilizing the AMBER22
software suite~\cite{34}, while AmberTools23 was used for system
preparation~\cite{35}. Protein--ligand complexes were constructed
utilizing the tleap module, employing the ff19SB force field for the
protein and GAFF2 (General Amber Force Field 2) for the
ligands~\cite{36}. The initial protein--ligand complexes were obtained
from previous molecular docking simulations. Each complex was
solubilized in a truncated octahedral box of TIP3P water molecules,
maintaining a 12~\AA{} buffer surrounding the solute. Counterions were
added to neutralize the charge of the system. Energy minimization
occurred in two phases: the initial phase involved the application of
500 kcal/mol/\AA{}$^{2}$ restraints to the solute, while the solvent
and ions were minimized for 5000 steps to resolve potential steric
clashes. During the second phase, all restraints were lifted, and the
complete system underwent an additional 10~000 steps of full
minimization to achieve structural relaxation. After minimization, the
system was incrementally heated from 0 to 300 K in 100 ps, utilizing a
Langevin thermostat with a collision frequency of 2 ps$^{-1}$. In this
phase, solute atoms were constrained using a force constant of 10
kcal/mol/\AA{}$^{2}$. A 1 ns equilibration run was conducted at a
constant pressure of 1 atm and a temperature of 300 K following
heating, during which the restraints were gradually diminished. The
molecular dynamics simulations, lasting 100 ns, were performed
utilizing the CUDA-enabled pmemd engine in AMBER22, facilitating
efficient GPU acceleration~\cite{34,35,36}. These simulations employed
the NPT ensemble, ensuring a constant number of particles, pressure,
and temperature, while applying periodic boundary conditions. The SHAKE
algorithm~\cite{37} constrained hydrogen-involved bonds, facilitating
the application of a 2 fs time step. Long-range electrostatic
interactions were handled using the particle mesh Ewald (PME)
method~\cite{38}, with a 10~\AA{} cutoff applied for nonbonded\break
interactions.

\subsubsection{Molecular mechanics generalized Born surface area}
\label{sssec241}

The binding energies of the protein--ligand complexes were estimated
using the molecular mechanics generalized Born surface area (MM-GBSA),
as implemented in the MMPBSA.py module of AMBER~\cite{35,39}. A total
of 1000 snapshots, derived from the final 20 ns of the 100 ns molecular
dynamics simulations, were utilized to confirm system equilibrium. The
binding free energy ($\Delta G_{\mathrm{bind}}$) is
determined by the difference between the free energy of the complex and
the sum of free energies of the receptor and ligand in their unbound
states (Equation~(\ref{eq1})):
{\begin{equation}\label{eq1}
\Delta G_{\mathrm{bind}} = G_{\mathrm{complex}} - 
(G_{\mathrm{receptor}} + G_{\mathrm{ligand}})
\end{equation}}\unskip
where $G_{\mathrm{complex}}$, $G_{\mathrm{receptor}}$, and
$G_{\mathrm{ligand}}$ represent the total free energies of the complex,
receptor, and ligand, respectively~\cite{39}. 

The free energy of each system was partitioned into gas-phase and
solvation components. The gas-phase energy ($\Delta
G_{\mathrm{gas}}$) was calculated by summing the van der Waals and
electrostatic interactions between protein and ligand:
{\begin{equation}\label{eq2}
\Delta G_{\mathrm{gas}} = E_{\mathrm{VDW}} + E_{\mathrm{ele}}
\end{equation}}\unskip
where $E_{\mathrm{VDW}}$ represents the van der Waals interactions,
calculated using the Lennard-Jones potential, and $E_{\mathrm{ele}}$
denotes the electrostatic interactions, derived from Coulomb's law.
Both of these components were obtained directly from the molecular
mechanics force field~\cite{39}.

The solvation free energy ($G_{\mathrm{solv}}$) was divided into polar
and nonpolar components. In MM-GBSA, the polar solvation energy
($G_{\mathrm{polar}}$) is determined using the generalized Born model,
which approximates the electrostatic component of solvation. The
nonpolar solvation energy ($G_{\mathrm{nonpolar}}$) is estimated based
on the solvent-accessible surface area (SASA) utilizing the linear
combination of pairwise overlap (LCPO) method. The nonpolar solvation
energy was calculated as follows:
{\begin{equation}\label{eq3}
G_{\mathrm{nonpolar}} = \gamma \times \mathrm{SASA} + b
\end{equation}}\unskip
where $\gamma$ is the surface tension constant and $b$ is an
offset term that accounts for the nonpolar contribution to
solvation~\cite{39}.

The total solvation free energy ($\Delta G_{\mathrm{solv}}$) was then
determined as the sum of the polar and nonpolar components:
{\begin{equation}\label{eq4}
\Delta G_{\mathrm{solv}} = G_{\mathrm{polar}} +
G_{\mathrm{nonpolar}}.
\end{equation}}\unskip
Finally, the $ \Delta G_{\mathrm{bind}}$ was obtained by summing the
gas-phase energy and the solvation free energy~\cite{39}:
{\begin{equation}\label{eq5}
\Delta G_{\mathrm{bind}} = \Delta G_{\mathrm{gas}} + 
\Delta G_{\mathrm{solv}}.
\end{equation}}\unskip

The decomposing of binding energy into van der Waals, electrostatic,
and solvation components facilitated a thorough examination of the
factors influencing ligand binding to the PDE5 enzyme. The evaluation
of these contributions facilitated a comprehensive understanding of the
factors affecting the stability and affinity of the ligand--protein
complex.

\section{Results and discussion}\label{sec3}
\subsection{ADMET analysis of investigated compounds}\label{ssec31}

Standing for ``Absorption, Distribution, Metabolism, Elimination,
Toxicity'', ADMET analysis is essential in drug discovery, evaluating
compounds for their pharmacokinetic and pharmacodynamic suitability.
The bioavailability radar plots in Figure~\ref{fig2} depict 13
physicochemical parameters for \textbf{SIL}, \textbf{TAD}, \textbf{AVA}, and \textbf{UA}, with the
{yellow} zone representing optimal bioavailability ranges.
Comparatively, \textbf{UA} has a lower molecular weight than \textbf{SIL}, \textbf{TAD}, and
\textbf{AVA}, which can enhance membrane permeability and systemic absorption.
Its moderate lipophilicity (logP) balances solubility and distribution,
reducing the risk of tissue accumulation. {The} higher topological
polar surface area (TPSA) of \textbf{UA} suggests limited central nervous
system (CNS) penetration, potentially lowering CNS side effects
compared to PDE5 inhibitors.

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Bioavailability radar chart of (a) \textbf{SIL}, (b)
\textbf{TAD}, (c) \textbf{AVA} and (d) \textbf{UA} with the following parameters: molecular
weight (MW), number of rigid bonds (nRig), formal charge (fChar),
number of heteroatoms (nHet), number of atoms in the biggest ring
(MaxRing), number of rings (nRing), number of rotable bonds (nRot),
topological polar surface area (TPSA), number of hydrogen bond donors
(nHD), number of hydrogen bond acceptors (nHA), log of the
octanol/water partition coefficient (logP), logP at physiological pH
(logD), and log of aqueous solubility (logS).}
\end{figure*}

Regarding hydrogen bonding, \textbf{UA} shows increased H-bond donors and
acceptors, which may enhance water solubility and gastrointestinal
absorption. The rigidity of \textbf{UA}'s structure, with fewer rotatable
bonds, may improve stability in binding but limits conformational
flexibility. Finally, \textbf{UA}'s favorable water solubility (logS) supports
effective oral absorption, while structural heteroatoms and a mild
formal charge promote stable binding in diverse environments. Overall,
\textbf{UA}'s unique pharmacokinetic profile---characterized by favorable
solubility, balanced lipophilicity, and reduced CNS impact---presents a
promising therapeutic profile compared to synthetic PDE5 inhibitors.

Absorption of the potential drug implies the transition into the
organism's systemic circulation. The evaluated parameters encompass
Caco-2 permeability (a model for intestinal absorption using human
colon carcinoma cells)~\cite{40,41}, MDCK permeability (Madin--Darby
canine kidney cells used to assess renal absorption)~\cite{42}, human
intestinal \mbox{absorption} (HIA)~\cite{43}, PAMPA permeability (parallel
artificial membrane permeability assay, a model for passive diffusion
across lipid membranes)~\cite{44}, the biopharmaceutical absorption
model ($F_{20\%}$, representing the fraction of drug
absorbed)~\cite{45}, plasma protein binding (PPB)~\cite{46},
the~fraction of unbound drugs (\textit{F}u\%, indicating the portion of the drug
that remains unbound in the plasma)~\cite{47}, volume of distribution
(VD, representing the extent of drug distribution into
tissues)~\cite{48}, and interactions with OATP1B1 and OATP1B3
transporters (organic anion transporting polypeptides, important for
drug transport into liver cells)~\cite{49} (Table~\ref{tab1}).

%tab1
\begin{table*}
\caption{\label{tab1}Pharmacokinetic properties of investigated
compounds based on ADMET analysis: absorption and distribution
parameters of investigated compounds}
\begin{tabular}{@{}cccccccccc@{}}
\thead
\xmorerows{1}{Comp.} & \multicolumn{5}{c}{Absorption} &
\multicolumn{4}{c}{Distribution} \\ \cline{2-6}\cline{7-10}
& \parbox[t]{3pc}{\centering Caco-2p (cm$\cdot$s$^{-1}$)} & 
\parbox[t]{3pc}{\centering MDCK (cm$\cdot$s$^{-1}$)} & HIA & PAMPA
& $F_{20\%}$ & PPB\% & \textit{F}u\% & \parbox[t]{3pc}{\centering VD (L$\cdot$kg$^{-1}$)} & 
OATP1B1/OATP1B3\vspace*{2pt} \\
\endthead
\textbf{SIL} & ${-}$4.812 & ${-}$4.771 & 0.003 & 0.171 & 0.049 & 96.845 & 2.745 & \mn0.153 & 0.017/0.005 \\ 
\textbf{TAD} & ${-}$5.138 & ${-}$4.401 & 0.000 & 0.007 & 1.000 & 96.596 & 2.968 & ${-}$0.084 & 0.092/0.000 \\ 
\textbf{AVA} & ${-}$5.453 & ${-}$5.054 & 0.001 & 0.787 & 0.013 & 96.077 & 3.136 & ${-}$0.130 & 0.998/0.998 \\ 
\textbf{UA}  & ${-}$5.197 & ${-}$4.787 & 0.003 & 0.899 & 0.008 & 97.162 & 3.164 & ${-}$0.465 & 0.724/0.499
\botline
\end{tabular}
\end{table*}

The absorption parameters indicate distinct profiles for the compounds
under investigation. The Caco-2 and MDCK models indicate low
permeability for \textbf{UA} (${-}$5.197 and ${-}$4.787 cm/s), aligning with the
values for \textbf{TAD} and \textbf{AVA}, whereas \textbf{SIL} demonstrates marginally
improved permeability. The PAMPA model demonstrates that \textbf{UA} exhibits
significantly higher permeability (0.899) than \textbf{SIL}, \textbf{TAD}, and \textbf{AVA},
suggesting enhanced passive diffusion across lipid membranes for \textbf{UA}.
The HIA parameter for \textbf{UA} (0.003) aligns closely with \textbf{SIL} and \textbf{AVA},
indicating restricted absorption via the human intestinal system. The
absorption fraction, denoted as $F_{20\%}$, shows nearly complete
absorption for \textbf{TAD} (1.000), whereas \textbf{UA} exhibits a markedly lower
value (0.008). The results indicate that, while \textbf{UA} demonstrates
significant permeability in the PAMPA model, its overall absorption is
restricted relative to other compounds, especially in comparison to
\textbf{TAD}.

Plasma protein binding (PPB) indicates that \textbf{UA} exhibits a high
binding rate of 97.162\%, consistent with the values observed for other
drugs. Although this may decrease the availability of the unbound drug
in plasma, high binding may also extend the half-life in circulation.
The unbound drug fraction (\textit{F}u\%) for \textbf{UA} is 3.164\%, comparable to the
values for \textbf{AVA}. In contrast, \textbf{SIL} and \textbf{TAD} exhibit slightly lower
values, suggesting that \textbf{UA} may persist in its unbound form in plasma
for a longer duration than \textbf{SIL}. The volume of distribution (VD) for
\textbf{UA} (3.164 L/kg) exceeds that of \textbf{SIL} (2.745 L/kg) while being
comparable to \textbf{AVA}, suggesting considerable tissue distribution
(Table~\ref{tab1}). This parameter indicates that \textbf{UA} may be widely
distributed in tissues, which could have implications for its
therapeutic efficacy and potential toxicity. 

%tab2
\begin{table*}
\caption{\label{tab2}Pharmacokinetic properties of investigated
compounds based on ADMET analysis: metabolism and elimination
parameters of investigated compounds\vspace*{-2pt}}
\begin{tabular}{@{}cccccccccc@{}}
\thead
\xmorerows{2}{Compounds} & \multicolumn{7}{c}{Metabolism; P450} & 
\multicolumn{2}{c}{Elimination} \\ \cline{2-8}\cline{9-10}
& \multicolumn{2}{c}{1A2} & \multicolumn{2}{c}{2D6} & 
\multicolumn{2}{c}{2C9} & \xmorerows{1}{HLM} & 
\xmorerows{1}{$T_{1/2}$ (h)} &  
\xmorerows{1}{CL (mL$\cdot$min$^{-1}\cdot$kg$^{-1}$)} \\
\cline{2-3}\cline{4-5}\cline{6-7}
& $I$ & $S$ & $I$ & $S$ & $I$ & $S$ & & & \\ 
\endthead
\textbf{SIL} & 0.000 & 0.167 & 0.000 & 0.000 & 0.003 & 0.999 & 0.999 & 0.400 & 8.757 \\ 
\textbf{TAD} & 0.000 & 1.000 & 0.000 & 0.000 & 0.948 & 0.000 & 0.008 & 1.023 & 2.930 \\ 
\textbf{AVA} & 0.966 & 0.374 & 0.045 & 0.200 & 0.791 & 0.984 & 0.641 & 0.515 & 7.078 \\ 
\textbf{UA}  & 0.479 & 0.998 & 0.000 & 0.000 & 0.006 & 0.001 & 0.258 & 1.446 & 1.413 
\botline
\end{tabular}
\vspace*{-2pt}
\end{table*}

The interaction between OATP1B1 and OATP1B3 transporters is essential
for the hepatic distribution of drugs. The significant interaction of
\textbf{UA} with OATP1B1/B3 (0.724/0.499) markedly exceeds the interactions
observed with \textbf{SIL} and \textbf{TAD}. This may suggest enhanced uptake of \textbf{UA}
by hepatocytes, potentially influencing its distribution and
elimination. Minimal interaction occurs between \textbf{SIL} and OATP
transporters, indicating a reduced level of hepatic uptake.

These results indicate that \textbf{UA} exhibits distribution characteristics
akin to those of \textbf{AVA}, yet it possesses a unique absorption profile.
Although \textbf{UA} exhibits significant permeability across lipid membranes
(PAMPA), its overall absorption remains constrained, potentially
impacting its bioavailability. The interaction with OATP1B1/B3
transporters indicates a potential for enhanced hepatic uptake, which
could affect its metabolism and elimination.

Table~\ref{tab2} presents the estimated parameters of metabolism and
elimination for the investigated compounds in the ADMET analysis,
providing key insights. The metabolism of the compounds is assessed
through interactions with cytochrome P450 enzymes (CYP1A2, CYP2D6, and
CYP2C9), with values indicating their potential as inhibitors ($I$) or
substrates ($S$) of these enzymes~\cite{50}. Elimination is evaluated by
measuring the half-life ($T_{1/2}$), which indicates how long the
compound remains active in the body~\cite{51}, and clearance (CL),
representing the rate at which the compound is removed from the
bloodstream~\cite{52}.\looseness=1 

Regarding interaction with the 1A2 enzyme, \textbf{AVA} exhibits the highest
level of inhibition ($I=0.966$), whereas \textbf{UA} displays moderate
inhibition ($I=0.479$). \textbf{SIL} and \textbf{TAD} exhibit no inhibitory
effects on this enzyme, whereas \textbf{TAD} serves as the primary substrate
($S=1.000$) for 1A2. There is considerable potential for \textbf{UA} as a
substrate for this enzyme ($S=0.998$), highlighting the significant
involvement of 1A2 in its metabolism. In the analysis of interactions
with the 2D6 enzyme, \textbf{SIL} and \textbf{UA} exhibit low or negligible
inhibition. They do not serve as substrates for this enzyme, whereas
\textbf{AVA} exhibits low inhibition ($I=0.045$) and moderate substrate
activity ($S=0.200$). The 2C9 enzyme should be significantly
inhibited by \textbf{AVA} ($I=0.791$), whereas \textbf{UA} shows minimal inhibition
($I=0.006$). The values indicate distinct metabolic pathways for
these \mbox{compounds} within the body. The elimination parameters indicate
that \textbf{UA} possesses the longest half-life ($T_{1/2}=1.446$ h), which is
markedly longer than that of \textbf{SIL} ($T_{1/2}=0.400$~h) and \textbf{AVA}
($T_{1/2}=0.515$ h). This suggests that \textbf{UA} has an extended duration
in the body, which may influence its therapeutic profile and cumulative
effects. The clearance (CL) for \textbf{UA} is the lowest at 1.413 mL/min/kg,
indicating a slower elimination rate compared to other compounds
studied, particularly \textbf{SIL} at 8.757 mL/min/kg and \textbf{AVA} at 
7.078~mL/min/kg. \textbf{TAD} exhibits a relatively low clearance rate of 2.930
mL/min/kg, although it remains faster than \textbf{UA}. The findings indicate
that \textbf{UA} demonstrates distinct metabolic and elimination properties,
exhibiting significant inhibition and substrate activities for the 1A2
enzyme, while displaying limited interaction with 2D6 and 2C9. The
longer half-life and slower clearance of \textbf{UA} relative to other
compounds suggests its potential for prolonged presence in the body,
which may be significant for its pharmacokinetic and pharmacodynamic
characterization.

%tab3
\begin{table*}
\caption{\label{tab3}Toxicological parameters of investigated compounds
based on ADMET analysis}
\tabcolsep=3pt
\fontsize{9.3}{12}\selectfont
\begin{tabular}{@{}ccccccccc@{}}
\thead
Compounds & hERG & DILI & 
\parbox[t]{5pc}{\centering Human hepatotoxicity}\vspace*{2pt} & AMES & 
Hematotoxicity & Carcinogenicity & Nephrotoxicity & Neurotoxicity \\ 
\endthead
\textbf{SIL} & 0.570 & 1.000 & 0.966 & 0.507 & 0.720 & 0.894 & 0.968 & 0.935 \\ 
\textbf{TAD} & 0.372 & 0.995 & 0.984 & 0.125 & 0.776 & 0.231 & 0.988 & 0.979 \\ 
\textbf{AVA} & 0.821 & 0.985 & 0.979 & 0.562 & 0.183 & 0.691 & 0.946 & 0.970 \\ 
\textbf{UA}  & 0.003 & 0.739 & 0.339 & 0.252 & 0.675 & 0.340 & 0.071 & 0.099
\botline
\end{tabular}
\end{table*}

Table~\ref{tab3} provides estimated toxicity parameters from the ADMET
analysis of the investigated compounds, offering key insights into
potential toxic effects. The analysis included parameters such as hERG
channel inhibition (assessing the risk of cardiotoxicity by evaluating
the potential to block the human \textit{ether-\`{a}-go-go}-related
gene potassium channel)~\cite{53,54}, drug-induced liver injury
(DILI)~\cite{55}, human hepatotoxicity (the potential to cause liver
damage)~\cite{56}, the AMES test (evaluating mutagenicity or the
potential to cause genetic mutations)~\cite{57,58}, hematotoxicity
(toxicity to blood cells), carcinogenicity (potential to cause cancer),
nephrotoxicity (toxicity to the kidneys), and neurotoxicity (toxicity
to the nervous system) for \textbf{SIL}, \textbf{TAD}, \textbf{AVA}, and \textbf{UA}. 

The lowest hERG channel inhibition value is observed for \textbf{UA} at 0.003,
indicating a reduced risk of cardiotoxicity in comparison to \textbf{SIL}
(0.570) and \textbf{AVA} (0.821), which exhibit a greater risk of channel
inhibition and are potentially associated with cardiovascular
complications. About the DILI parameter, \textbf{UA} exhibits a lower value of
0.739 in contrast to \textbf{SIL} at 1.000 and \textbf{TAD} at 0.995, suggesting a
reduced risk of drug-induced liver injury. The human hepatotoxicity
value for \textbf{UA} is 0.339, categorizing it as medium risk (yellow
category). In contrast, \textbf{SIL} (0.966), \textbf{TAD} (0.984), and \textbf{AVA} (0.979)
are classified as high risk for hepatotoxicity (red category).

The Ames test for mutagenicity indicates a low value for \textbf{UA} (0.252),
comparable to \textbf{TAD} (0.125), while \textbf{AVA} and \textbf{SIL} exhibit higher risks
than \textbf{UA} (0.562 and 0.507, respectively). The hematotoxicity of \textbf{UA}
(0.675) is comparable to that of \textbf{SIL}, whereas \textbf{AVA} demonstrates
markedly lower hematotoxicity (0.183). The carcinogenicity of \textbf{UA} is
0.340, which is lower than that of \textbf{SIL} at 0.894 and \textbf{AVA} at 0.691,
but higher than \textbf{TAD}, which has a value of 0.231. Nephrotoxicity for
\textbf{UA} (0.071) is significantly lower than that of the other compounds,
indicating a diminished risk of kidney damage. The neurotoxicity of
\textbf{UA} (0.099) is significantly lower than that of \textbf{SIL} (0.935), \textbf{TAD}
(0.979), and \textbf{AVA} (0.970), suggesting a reduced risk of neurological
complications. The data indicate that \textbf{UA} presents a low risk of
cardiotoxicity and neurotoxicity, a medium risk of hepatotoxicity, and
a reduced risk of nephrotoxicity, which may be pertinent to its
long-term use.

\subsection{Inhibitory activity toward PDE5 enzyme---molecular docking
study}\label{ssec32}

Before analyzing the docking results, the optimized geometries of the
compounds---\textbf{SIL}, \textbf{TAD}, \textbf{AVA}, and \textbf{UA}---were determined using DFT
methods (Figure~\ref{fig3}). The rigid structure of \textbf{UA} is stabilized
by intramolecular hydrogen bonds and van der Waals interactions, with
key bond distances (1.605 and 1.709~\AA{}) contributing to its
stability. In contrast, PDE5 inhibitors such as \textbf{SIL} and \textbf{TAD} exhibit
flexibility in their aromatic rings, enhancing their adaptability.
\textbf{AVA}, with hydrogen bond lengths of 1.838 and 2.614~\AA{}, is further
stabilized through these key hydrogen bonding interactions,
contributing to the overall stability of the molecule.

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Optimized geometries of investigated compounds at
the B3LYP-D3BJ/6-311${{+}{+}}$G(d,p) level of theory. Legend: grey---carbon
atoms, red---oxygen atoms, blue---nitrogen atoms, white---hydrogen
atoms, yellow---sulfur atom (in \textbf{SIL}), green---chlorine atom (in
\textbf{AVA}). The dashed line represents a hydrogen bond and the number
indicates the bond length~(\AA{}).}
\end{figure*}

%tab4
\begin{table*}
\caption{\label{tab4}Important thermodynamic parameters ($\Delta
G_{\mathrm{bind}}$: binding free energy, $K_{\mathrm{i}}$: inhibition constant,
$\Delta G_{\mathrm{total}}$: total internal energy, $\Delta
G_{\mathrm{tor}}$: torsional free energy, $\Delta G_{\mathrm{unb}}$:
energy of the unbound system, $\Delta G_{\mathrm{elec}}$:
electrostatic energy, and  $\Delta G_{\mathrm{vdw} + \mathrm{hbond} +
\mathrm{desolv}}$: sum of van der Waals interactions, hydrogen bonds,
and desolvation energies, in kcal/mol) for the most stable
conformations of the investigated compounds within the PDE5 enzyme
active site, obtained through molecular docking simulations}
\begin{tabular}{@{}ccccccccc@{}}
\thead
Compound & 
$\Delta G_{\mathrm{bind}}$ & $K_{\mathrm{i}}$ ($\upmu $M) & 
$\Delta G_{\mathrm{inter}}$ & 
$\Delta G_{\mathrm{vdw} + \mathrm{hbond} + \mathrm{desolv}}$ & 
$\Delta G_{\mathrm{elec}}$ & $\Delta G_{\mathrm{total}}$ & 
$\Delta G_{\mathrm{tor}}$ & $\Delta G_{\mathrm{unb}}$ \\ 
\endthead
\textbf{PDE5--SIL} & ${-}$11.01 & $8.5\times 10^{-3}$ & ${-}$12.36 & ${-}$11.94 & ${-}$0.42 & ${-}$1.92 & 2.09 & ${-}$1.18 \\
\textbf{PDE5--TAD} & ${-}$10.36 & $2.5\times 10^{-2}$ & ${-}$10.83 & ${-}$10.77 & ${-}$0.06 & ${-}$0.61 & 0.30 & ${-}$0.78 \\
\textbf{PDE5--AVA} & ${-}$11.39 & $4.5\times 10^{-3}$ & ${-}$13.34 & ${-}$12.19 & ${-}$1.15 & ${-}$1.86 & 2.68 & ${-}$1.13 \\
\textbf{PDE5--UA}  & ${-}$10.12 & $3.8\times 10^{-2}$ & \0${-}$9.79& \0${-}$9.69& ${-}$0.10 & ${-}$2.03 & 1.49 & ${-}$0.21
\botline
\end{tabular}
{\vspace*{-2pt}}
\end{table*}

The molecular docking study detailed in Table~\ref{tab4} elucidates the
thermodynamic parameters associated with the binding of investigated
compounds to PDE5. While not a traditional PDE5 inhibitor, \textbf{UA} is
highlighted as a natural compound that demonstrates considerable
potential relative to established synthetic inhibitors. The binding
free energy ($\Delta G_{\mathrm{bind}}$) of \textbf{UA} at ${-}$10.12 kcal/mol
is marginally higher than that of \textbf{AVA} (${-}$11.39 kcal/mol) and \textbf{SIL}
(${-}$11.01 kcal/mol) yet still signifies a stable interaction with PDE5.
The $K_{\mathrm{i}}$ value for \textbf{UA} is $3.8\times 10^{-2}~\upmu $M, which
exceeds that of \textbf{AVA} ($4.5\times 10^{-3}~\upmu $M) and \textbf{SIL}
($8.5\times 10^{-3}~\upmu$M), suggesting that a greater concentration
of \textbf{UA} is necessary to attain half-maximal enzyme inhibition. The
natural origin of \textbf{UA} enhances its importance, as natural compounds
typically exhibit favorable safety profiles and possess additional
biological effects, including antioxidant and anti-inflammatory
properties. The energetic components of \textbf{UA} binding, including
$\Delta G_{\mathrm{vdw} + \mathrm{hbond} + \mathrm{desolv}}$ 
(${-}$9.69~kcal/mol) and  $\Delta G_{\mathrm{elec}}$ (${-}$0.10 kcal/mol),
indicate stable intermolecular interactions. However, its $\Delta
G_{\mathrm{total}}$ energy (${-}$2.03 kcal/mol) is relatively lower than
that of the other inhibitors. The findings indicate the potential of
\textbf{UA} in the therapy of PAH, functioning as a PDE5 inhibitor and
possessing natural properties that may aid in reducing oxidative stress
and vascular remodeling, which are critical factors in the disease's
pathogenesis.

Figure~\ref{fig4} presents the crystal structure of the PDE5 enzyme in
a~complex with \textbf{AVA} (PDB: 6L6E), which features a well-defined active
site~\cite{31}. This active site served as the basis for subsequent
studies, where other investigated compounds, including \textbf{SIL}, \textbf{TAD},
and \textbf{UA}, were also examined. The structure of the active site allowed
for a detailed analysis of the binding modes of various inhibitors,
providing insights into the key factors influencing their selectivity
and inhibitory potential.

\begin{figure}
\includegraphics{fig04}
{\vspace*{-3pt}}
\caption{\label{fig4}Crystal structure of PDE5 enzyme in complex with
\textbf{AVA} (PDB: 6L6E). The red ${\upalpha}$-helices represent the helical
regions of the PDE5 enzyme, contributing to the overall stability and
structural framework of the protein. The green loops and
${\upbeta}$-turns depict flexible regions of the protein, which allow
conformational changes critical for ligand binding. The ligand \textbf{AVA}
(avanafil) is located in the active site of PDE5 and is displayed near
the center of the image. Carbon atoms are grey, nitrogen atoms blue,
oxygen atoms red, hydrogen atoms white, and chlorine atoms in green.
Water molecules (shown as small red dots) play a role in the
stabilization of the ligand and protein structure.}
{\vspace*{-3pt}}
\end{figure}

Comparing the intermolecular interactions between inhibitors and the
PDE5 enzyme is essential for elucidating their inhibitory potency and
selectivity. The interactions determine the binding affinity of an
inhibitor to the enzyme, thereby affecting its \mbox{therapeutic} efficacy.
Figure~\ref{fig5} presents the most stable conformations of the
investigated compounds within the active site of the protein, while
Figure~\ref{fig6} highlights the key intermolecular interactions between
the investigated compounds and amino acid residues in the active site.

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig05}
{\vspace*{2pt}}
\caption{\label{fig5}The most stable conformations of the investigated
compounds: \textbf{SIL} (a), \textbf{TAD} (b), \textbf{AVA} (c) and \textbf{UA} (d) in the active
site of the PDE5 enzyme. The red ${\upalpha}$-helices represent the
helical regions of the PDE5 enzyme, contributing to the overall
stability and structural framework of the protein. The green loops and
${\upbeta}$-turns depict flexible regions of the protein, which allow
conformational changes critical for ligand binding. Carbon atoms are
grey, nitrogen atoms blue, oxygen atoms red, hydrogen atoms white, and
chlorine atoms green.} 
{\vspace*{2pt}}
\end{figure*}

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig06}
{\vspace*{2pt}}
\caption{\label{fig6}Two-dimensional representation of significant
intermolecular interactions between the PDE5 enzyme (PDB: 6L6E) and the
inhibitors \textbf{SIL}, \textbf{TAD}, \textbf{AVA}, and \textbf{UA}, displaying interatomic
distances (\AA{}) obtained after molecular docking studies. Different
colors represent various types of interactions, as detailed in the
legend.}
{\vspace*{2pt}}
\end{figure*}

The analysis of intermolecular interactions between the inhibitors and
the PDE5 enzyme indicates the presence of significant hydrogen bonds,
${\uppi}$--${\uppi}$ interactions, and alkyl and ${\uppi}$--cation
interactions. These interactions collectively enhance the stabilization
of the complexes and suggest potential inhibition (Figure~\ref{fig6}).

In protein--ligand complexes, conventional hydrogen bonds are crucial
for the proper orientation of inhibitor molecules, facilitating optimal
positioning within the enzyme's binding pocket. A conventional hydrogen
bond is formed between \textbf{SIL} and A:TYR 612 through its carbonyl oxygen,
facilitating its anchoring in the active site. A hydrogen bond is
formed between \textbf{TAD} and A:GLN 817 through the --NH-- group of its
indole ring, contributing to the molecule's stabilization. Hydrogen
bonds are formed between \textbf{AVA} and amino acids such as A:TYR 612, A:THR
723, and A:LEU 725, thereby ensuring stable binding within the enzyme's
active site and enhancing its affinity for PDE5. A conventional
hydrogen bond is established between \textbf{UA} and A:LEU 725, a critical
interaction that facilitates the correct positioning and stabilization
of \textbf{UA} within the active site.

Besides hydrogen bonds, ${\uppi}$--${\uppi}$ interactions significantly
contribute to the stabilization of the inhibitors. All studied
compounds engage in ${\uppi}$--${\uppi}$ interactions with the aromatic
amino acid residue A:PHE 820, which plays a significant role in
stabilizing the ligand within the binding pocket of the PDE5 enzyme.
The \textbf{TAD} and \textbf{AVA} molecules also form additional ${\uppi}$--${\uppi}$
interactions with A:PHE 786, which further anchors them in the
hydrophobic region of the active site, thus increasing the inhibitors'
affinity for the enzyme.

Furthermore, the ${\uppi}$--cation interactions observed in inhibitors
such as \textbf{AVA} and \textbf{SIL}, particularly with A:HIS 613, highlight the
critical role of electrostatic interactions in stabilizing the
complexes. These interactions between positively charged residues and
the ligand's ${\uppi}$-system significantly strengthen the inhibitors'
binding affinity to the PDE5 enzyme.

For the stabilization of complexes, ${\uppi}$--Sigma contacts are
important. All compounds established ${\uppi}$--sigma interactions with
the A:VAL 782 residue, thereby enhancing the stability of the ligand
within the enzyme's binding site. Furthermore, alkyl/${\uppi}$--alkyl
interactions and carbon--hydrogen bonds/${\uppi}$-donor hydrogen bonds
between the inhibitors and residues including A:LEU 765, A:ALA 767,
A:PHE 786, A:LEU 804, A:ILE 813, A:MET 816, and A:GLN 817 play a
crucial role in enhancing the stability of the complexes. The results
are supported by thermodynamic parameters, with $\Delta
G_{\mathrm{inter}}$ and $\Delta G_{\mathrm{vdw} + \mathrm{hbond} +
\mathrm{desolv}}$ indicating the contributions of hydrophobic, van der
Waals, and solvation interactions to the stability of the complexes
(Table~\ref{tab4}). The lowest values for these parameters are obtained
for \textbf{AVA}, suggesting strong hydrophobic stabilization due to a high
number of ${\uppi}$--${\uppi}$, ${\uppi}$--sigma, and
alkyl/${\uppi}$--alkyl interactions. These intermolecular interactions
collectively enhance the stability of the inhibitor complexes with the
PDE5 enzyme, allowing each inhibitor to leverage a unique combination
of interactions to achieve high inhibitory potency.

\subsection{Inhibitory activity toward PDE5 enzyme---molecular dynamic
study}\label{ssec33}

This study utilized molecular dynamics (MD) simulations to examine the
conformational changes and stability of a receptor in the presence of
various ligands. Key parameters, including root mean square deviation
(RMSD), root mean square fluctuation (RMSF), and radius of gyration
(Rg), were analyzed to evaluate the impact of ligands on the stability
and flexibility of the protein structure. Special emphasis was placed
on \textbf{UA} and its inhibitory potential compared to other tested ligands,
including \textbf{AVA}, \textbf{SIL}, and \textbf{TAD}. 

Analysis of RMSD indicated that all systems attained stable
conformations throughout the \mbox{simulations} (Figure~\ref{fig7}a). The
receptor devoid of a ligand exhibited an average RMSD value of 5.631
nm, whereas the systems with ligands \textbf{AVA}, \textbf{SIL}, \textbf{TAD}, and \textbf{UA} fell
within a narrow range of 5.625--5.711 nm. Despite the absence of
significant deviations between the systems, \textbf{UA} demonstrated a stable
RMSD value of 5.668 nm, comparable to \textbf{TAD}'s 5.625 nm, suggesting that
\textbf{UA} does not cause notable conformational changes in the protein.

\begin{figure*}
\includegraphics{fig07}
{\vspace*{3pt}}
\caption{\label{fig7}Comparative analysis of 100 ns molecular dynamics
(MD) simulations showing (a) RMSD (root mean square deviation) of the
C--C${\upalpha}$--N backbone, (b) Rg (radius of gyration), and (c) RMSF
(root mean square fluctuation) for the PDE5 enzyme with and without
various ligands (\textbf{AVA}, \textbf{SIL}, \textbf{TAD}, and \textbf{UA}).}
{\vspace*{3pt}}
\end{figure*}

The Rg values, indicating the compactness of the protein structure,
exhibited minimal variations across the systems (Figure~\ref{fig7}b).
The receptor in the absence of a ligand exhibited an average Rg value
of 4.003 nm, and the presence of ligands did not result in notable
alterations in the protein's overall compactness. The lowest Rg values
recorded were for \textbf{SIL} at 3.989 nm and \textbf{TAD} at 3.991 nm, whereas \textbf{UA}
exhibited an Rg value of 3.997 nm. Despite the similarity in values,
\textbf{UA} exhibits a stable, compact structure that corresponds with its
inhibitory potential. The compactness of the protein in the presence of
\textbf{UA} may enhance structural stabilization and limit access to active
sites, which is essential for inhibitor efficacy. The lack of notable
variations in Rg values indicates that \textbf{UA}, similar to \textbf{TAD} and
\textbf{SIL}, preserves a stable protein conformation with minimal changes in
compactness, thereby reinforcing its inhibitory potential.\looseness=1

The values of RMSF revealed notable differences in the fluctuations of
the proteins in the presence of the ligands (Figure~\ref{fig7}c). The
receptor without a ligand exhibited the highest average RMSF value
(1.454 nm), indicating greater flexibility. The ligands \textbf{SIL} and \textbf{UA}
significantly reduced protein fluctuations (0.973~nm and 0.829 nm,
respectively), suggesting that these ligands stabilize key parts of the
protein structure. The ligand that most effectively diminishes protein
flexibility is \textbf{UA}, exhibiting the lowest RMSF value of 0.829 nm. This
indicates that \textbf{UA} establishes more stable interactions with the
protein than the other \mbox{ligands}, specifically \textbf{AVA} (1.403 nm) and \textbf{SIL}
(0.973~nm), which is essential for inhibiting protein function. The
diminished flexibility of protein chains in the presence of \textbf{UA} may
hinder the dynamic alterations required for protein activity,
significantly increasing its inhibitory potential.

In comparison to other ligands, \textbf{UA} demonstrates significant efficacy
as a protein stabilizer, as indicated by its lowest RMSF values and
stable RMSD. This combination suggests that \textbf{UA} reduces protein
flexibility while preserving a stable conformation, characterized by
minimal fluctuations and a~lack of destabilization. Based on these
characteristics, it can be concluded that \textbf{UA} exhibits significant
potential as an inhibitor, especially when compared to \textbf{AVA} and \textbf{SIL},
which demonstrate greater fluctuations and relatively less stable RMSD
values. Additionally, \textbf{UA} exhibits comparable Rg values and stability
in conformational behavior when contrasted with \textbf{TAD}. The \textbf{UA} ligand
demonstrates a marginally superior capacity to stabilize the protein,
positioning it as a more viable candidate for subsequent research
regarding inhibitory potential.

The energy analysis of the binding of \textbf{SIL}, \textbf{TAD}, \textbf{AVA}, and \textbf{UA} to
the PDE5 enzyme reveals important trends in complex stability and the
contributions of different energetic components. Van der Waals
interactions play a pivotal role in stabilizing all complexes. The
strongest van der Waals interactions ($E_{\mathrm{VDW}}$) are obtained
for \textbf{TAD} and \textbf{AVA}, with values of ${-}$48.23 and ${-}$48.61 kcal/mol,
respectively. In comparison, \textbf{SIL} and \textbf{UA} demonstrate favorable, but
less pronounced, interactions of ${-}$38.96 and ${-}$22.18 kcal/mol,
respectively (Table~\ref{tab5}). These results suggest that, although
somewhat less potent, \textbf{UA} still effectively interacts with the
enzyme's hydrophobic pocket.

%tab5
\begin{table*}
\caption{\label{tab5}Free energy decomposition analysis (MM-GBSA) for
PDE5 enzyme complexed with different ligands (\textbf{SIL}, \textbf{TAD}, \textbf{AVA}, and
\textbf{UA}): van der Waals ($E_{\mathrm{VDW}}$), electrostatic
($E_{\mathrm{ele}}$), polar and nonpolar solvation energies
($G_{\mathrm{polar}}$ and $G_{\mathrm{nonpolar}}$), gas-phase binding
energy ($\Delta G_{\mathrm{gas}}$), solvation free energy difference
($\Delta G_{\mathrm{solv}}$), and binding free energy ($\Delta
G_{\mathrm{bind}}$) in kcal/mol}
\begin{tabular}{@{}cccccccc@{}}
\thead
Compound & $E_{\mathrm{VDW}}$ & $E_{\mathrm{ele}}$ & 
$G_{\mathrm{polar}}$ & $G_{\mathrm{nonpolar}}$ &  $\Delta
G_{\mathrm{gas}}$ & $\Delta G_{\mathrm{solv}}$ & $\Delta
G_{\mathrm{bind}}$ \\
\endthead
\textbf{PDE5-SIL} & ${-}$38.96 & ${-}$18.89 & 51.78 & ${-}$5.49 & ${-}$57.85 & 46.29 & ${-}$11.56 \\ 
\textbf{PDE5-TAD} & ${-}$48.23 & ${-}$12.71 & 35.84 & ${-}$6.01 & ${-}$60.95 & 29.82 & ${-}$31.12 \\ 
\textbf{PDE5-AVA} & ${-}$48.61 & \0${-}$9.76  & 50.05 & ${-}$6.79 & ${-}$58.36 & 43.25 & ${-}$15.11 \\ 
\textbf{PDE5-UA}  & ${-}$22.18 & \mn\03.69  & 10.75 & ${-}$3.27 & ${-}$18.49 & \07.48& ${-}$11.01 
\botline
\end{tabular}
{\vspace*{-3pt}}
\end{table*}

Electrostatic interactions further contribute to the overall stability
of the complexes. The most favorable negative energy electrostatic
values ($E_{\mathrm{ele}}$) are obtained for \textbf{SIL} and \textbf{TAD},
indicating strong electrostatic interactions with amino acids in the
active site. In contrast, \textbf{UA} shows a positive electrostatic energy
value of +3.69 kcal/mol, suggesting an alternative interaction
mechanism that relies less on electrostatic stabilization and more on
other interaction types within the complex. All compounds \mbox{display}
expected unfavorable polar solvation energies ($G_{\mathrm{polar}}$),
attributed to the exposure of polar groups to the solvent during
complex formation. Polar solvation energies are similar for \textbf{AVA} and
\textbf{SIL}, around 50 kcal/mol, while \textbf{TAD} exhibits a more favorable value
of 35.84 kcal/mol. Notably, \textbf{UA} shows a much lower polar solvation
energy (10.75 kcal/mol), suggesting its binding is less impacted by
solvent exposure compared to the other compounds (Table~\ref{tab5}).
This implies that, despite lacking strong electrostatic interactions,
\textbf{UA}'s interaction with the solvent enhances its binding affinity.

The total binding energies ($\Delta G_{\mathrm{bind}}$) provide a
comprehensive evaluation of the stability of each complex. The lowest
binding energy is obtained for \textbf{TAD} (${-}$31.12 kcal/mol), indicating
the strongest interaction with PDE5. In comparison, \textbf{AVA} and \textbf{SIL}
exhibit slightly higher binding energies of ${-}$15.11 and ${-}$11.56
kcal/mol, respectively. The \textbf{UA} ligand shows a competitive binding
energy of ${-}$11.01 kcal/mol, suggesting that, despite its unique
interaction profile, it remains an effective PDE5 inhibitor relative to
established pharmaceutical compounds. The ability of \textbf{UA} to form a
stable complex with PDE5 highlights its potential for further
exploration, particularly in terms of modifications that could enhance
its inhibitory effectiveness (Table~\ref{tab5}).

\section{Conclusion}\label{sec4}

This study presents an analysis of the inhibitory potential of usnic
acid (\textbf{UA}) in comparison to established PDE5 inhibitors, including
sildenafil (\textbf{SIL}), tadalafil (\textbf{TAD}), and avanafil (\textbf{AVA}), for the
treatment of pulmonary arterial hypertension (PAH). The ADMET analysis
indicated that \textbf{UA} possesses a distinct pharmacokinetic profile
characterized by high plasma protein binding and an extended half-life,
potentially facilitating its prolonged retention in the body. Its
absorption is limited relative to synthetic inhibitors, yet it exhibits
considerable permeability across lipid membranes. Crucially for its
potential therapeutic application, \textbf{UA} demonstrated low toxicity,
exhibiting minimal risks of cardiotoxicity and neurotoxicity.

The combination of natural compounds such as \textbf{UA} with synthetic
PDE5 inhibitors offers a promising approach for achieving synergistic
effects in the treatment of PAH. This combination may improve the
effectiveness of standard PDE5 inhibitors and potentially lower the
necessary dosage of synthetic drugs, thereby reducing side effects. The
antioxidant properties exhibited by UA may address oxidative stress and
inflammation, both of which are crucial in the progression of PAH,
thereby offering a multi-targeted therapeutic strategy. Exploring the
synergistic potential of \textbf{UA} in conjunction with \textbf{SIL}
or \textbf{TAD} may lead to the development of novel combination
therapies that enhance both safety and\break efficacy.

Molecular docking results indicate that \textbf{UA} establishes stable
interactions with the PDE5 enzyme, exhibiting a binding free energy of
${-}$10.12 kcal/mol, which is comparable to \textbf{SIL} at ${-}$11.01 kcal/mol
and \textbf{AVA} at ${-}$11.39 kcal/mol. Significant intermolecular hydrogen
bonds and ${\uppi}$--${\uppi}$ interactions are established by \textbf{UA},
which are essential for the stabilization of the complex. Molecular
dynamics simulations indicated that \textbf{UA} decreases the flexibility of
the PDE5 \mbox{enzyme,} as evidenced by low RMSF values, suggesting its
\mbox{potential} role as an enzyme stabilizer.

The MM-GBSA analysis revealed further details regarding the binding
energy, indicating that \textbf{UA} exhibited a total binding energy of
${-}$11.01 kcal/mol, which is comparable to that of \textbf{SIL} and
\textbf{AVA}. Van der Waals interactions were crucial for the stability
of the complex with \textbf{UA}, and favorable solvation interactions
further enhanced this stability. While electrostatic interactions were
less significant than those of synthetic inhibitors, \textbf{UA}
demonstrated considerable potential for stable interaction with PDE5
through alternative mechanisms.

The findings indicate that \textbf{UA} possesses significant potential as a
natural PDE5 inhibitor, along with antioxidant and anti-inflammatory
properties that may enhance its therapeutic application in PAH. The
results of this study indicate that \textbf{UA} may serve as a complementary
or adjunctive therapy in the management of PAH. Future research must
prioritize in vivo studies to evaluate the bioavailability, efficacy,
and safety of \textbf{UA} in clinical contexts. Long-term studies are
essential to assess the potential for chronic toxicity and interactions
with current PAH medications. Furthermore, clinical trials that
integrate \textbf{UA} with synthetic PDE5 inhibitors could investigate the
potential of \textbf{UA}'s antioxidant and anti-inflammatory properties to
enhance treatment outcomes and improve patients' quality of life.

\section*{Acknowledgement}

The authors wish to acknowledge the Ministry of Science, Technological
Development and Innovation of the Republic of Serbia and the Ministry
of Education of the Republic of Serbia (Agreements No.
451-03-66/2024-03/200378) for financial support.

\CDRGrant[MoSTDI]{451-03-66/2024-03/200378}

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Supplementary materials}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-413-suppl.pdf}}

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