Therapeutic potential of Andrographis paniculata against Monkeypox virus targets—a computational insight

Abstract

Monkeypox, a viral zoonotic disease caused by the monkeypox virus (MPXV), has recently become a significant global health threat due to increased human-to-human transmission. As of November 2023, the Mpox outbreak had impacted 116 countries worldwide, with a total of 92,783 confirmed cases and 171 reported deaths. With limited antiviral options available, natural plant-based compounds like Andrographis paniculata have shown promise for their antimicrobial and antiviral properties. This study explores the potential of bioactive compounds from A. paniculata against monkeypox viral targets through computational methods. Protein structures 4E90, 8B07, 8C9K, 8IZT and 8J8G were obtained from the Protein Data Bank (PDB), and molecular dynamic simulation studies were conducted using the Schrödinger Suite. Furthermore, the trajectory analysis assessed the stability and binding affinity of the ligand-protein complexes. The compounds andrographolide, luteolin, and andrographidin-A demonstrated strong binding interactions and stable protein-ligand complexes, along with favorable thermodynamic profiles, supporting their potential as promising therapeutic candidates for monkeypox treatment.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40203-025-00423-4.

Introduction

Monkeypox is a viral zoonotic disease caused by the monkeypox virus (MPXV), a member of the Orthopoxvirus genus within the Poxviridae family. As a member of the poxvirus family, it has a characteristic brick-like shape, encased in a lipoprotein envelope, and contains a linear double-stranded DNA genome. (Kugelman et al. 2014) Poxviruses are unique in that, while they depend on the host's ribosomes for mRNA translation, their genome encodes all the proteins required for replication, transcription, assembly, and viral release. (Walsh 2017) Traditionally endemic to Central and West Africa, monkeypox has recently emerged as a global health concern, with outbreaks affecting multiple countries.(Bunge et al. 2022) As of November 2023, the Mpox outbreak had impacted 116 countries worldwide, with a total of 92,783 confirmed cases and 171 reported deaths. (World Health Organization 2023) Historically, Mpox was primarily a zoonotic infection, transmitted from animals to humans. However, it now spreads predominantly through human-to-human transmission, facilitated by direct contact, respiratory droplets, infected lesions, and contaminated surfaces. Recent studies have indicated elevated viral loads in various bodily fluids further highlighting the disease's potential for widespread transmission. (Alakunle et al. 2020) The disease manifests with symRptoms such as fever, rash, and lymphadenopathy, and severe cases can lead to complications, particularly in immunocompromised individuals (Thornhill et al. 2022).

Tecovirimat which was earlier used for the management of smallpox, amidst viral outbreak European Medicines Agency (EMA) approved it as first treatment against monkey pox. Being promising antiviral against smallpox and monkey pox it remains a challenge in immunocompromised subjects. (Almehmadi et al. 2022) A study reported tecovirimat resistance in immunocompromised patients.(Fortier et al. 2024).

Due to the limited availability of effective antiviral treatments, there is growing interest in exploring alternative therapeutic options, including natural plant-based compounds. Andrographis paniculata (Burm. f.) Wall. ex Nees., a widely used herb in traditional medicine, has been recognized for its antimicrobial, anti-inflammatory, and antiviral properties.(Chauhan et al. 2013) Various studies have reported the antiviral potential of Andrographis paniculata (AP) against several viruses, including HIV(Feng et al. 2012), chikungunya (Jain et al. 2020), dengue, hepatitis (Lee et al. 2014), influenza (Cai et al. 2015) and SARS-CoV-2.(Sa-Ngiamsuntorn et al. 2021).

In this study we have taken protein receptors 4E90, 8B07, 8C9K, 8IZT and 8J8G of monkey pox virus. 4E9O is a carbonic anhydrase-like protein from Vaccinia virus, structurally homologous to MPXV proteins. It comprises two domains—an ectodomain (residues 1–235) involved in host adhesion and a helical domain (residues 249–304) for structural support—linked by 13 residues. Though catalytically inactive, it likely facilitates viral entry via binding to host glycosaminoglycans like chondroitin sulfate. (Matho et al. 2012) PDB ID 8B07 corresponds to the 2′‑O‑RNA methyltransferase (VP39) from Monkeypox virus, crystallized in complex with the pan‑MTase inhibitor sinefungin at a resolution of 2.05 Å. This enzyme is essential for viral mRNA cap‑1 formation, akin to nsp14 of SARS‑CoV‑2, underscoring its potential as a broad‑spectrum antiviral target.(Silhan et al. 2023) 8C9K, The mpox poxin protein consists of two domains an N-terminal protease-like domain (gold), comprising twelve β-strands forming five β-sheets, and a C-terminal domain (cyan) with one α-helix and six β-strands forming two β-sheets. The functional protein forms a homodimer with a heart-like surface morphology, stabilized by hydrogen bonds between antiparallel β-strands of both subunits. The cGAMP binding site lies at the dimer interface, with His17 from one monomer and Tyr138 and Lys142 from the other mediating catalytic cleavage of cGAMP. The mpox poxin shares 91% sequence identity with the Vaccinia virus (VACV) poxin. (Duchoslav and Boura 2023) 8IZT, The MPXV protein A7N121 is made up of five α-helices and three 3₁₀ helices, arranged in the sequence α1–α2–α3–η1–η2–η3–α4–α5. Among these, α1, α2, and α5 are noticeably longer than the others. Two amino acids, Glu58 and Cys69, show double side chain conformations, indicating some flexibility. Overall, the 3D structure of A7N121 in MPXV is highly like the A6N protein of Vaccinia virus (VACV), suggesting a conserved structure and possibly similar function. (Ni et al. 2024) PDB ID 8J8G having resolution 2.79 Å reveals the DNA replication holoenzyme of Monkeypox virus, composed of three subunits F8 (DNA polymerase), A22 (processivity factor), and E4 (uracil-DNA glycosylase), assembled as a functional heterotrimer (A1:B1:C1) in complex with a double-stranded DNA. (Xu et al. 2024).

Computational studies have become an important tool to identify and predict the phytochemical potential to tackle the disease and understanding its pharmacodynamics/pharmacokinetics. We have assessed binding affinity of bioactive compounds from Andrographis paniculata against viral targets, offering promising insights into the development of novel treatments for monkeypox (Sahoo et al. 2021) These computational approaches provide a rapid and cost-effective method for drug discovery, potentially accelerating the identification of therapeutic agents for emerging infectious diseases like monkeypox.

Material and method

Screening active ingredients of Andrographis panniculata

The phytoconstituents of AP were collected through scientific literature available on PUBMED and IMPPAT. Subsequently, the 3D sdf structures of the phytochemicals were retrieved from PUBCHEM and subjected to drug-likeness and ADMET profiling using Qik Prop.(QikProp and LLC 2021) The screening criteria included Molecular weight, Bioavailability, Solubility, GI absorption, Hydrophilicity, Lipophilicity, Rule of 5, and Rule of 3. Further toxicity assessment was done via utilizing ProTox 3.0 (Banerjee et al. 2024) Screened phytochemicals were used as ligands for further computational work.

Protein retrival and preparation

The term “monkeypox” was searched on the PDB, and all available receptors were evaluated based on their structure, resolution, mutations, and the number of associated ligands. The appropriate receptor was selected for subsequent molecular docking and molecular dynamics simulation studies. Crystallographic structures of the target proteins 4QWO (A42R Profilin-like Protein from Monkeypox Virus), 8C9K (Structure of Mpox virus poxin), 8IZT (Crystal structure of the N-terminal domain (residues 1-121) of MPXV A7), 8J8G (Monkeypox virus DNA replication holoenzyme F8, A22 and E4 in complex with a DNA duplex and cidofovir diphosphate), 8JC6 (Crystal structure of Mpox virus A41L protein), 8OIV (Monkeypox virus VP39 in complex with SAH and cap0), 8ORV (Crystal structure of monkeypox virus poxin in complex with the STING agonist MD1203), 8P44 (Crystal structure of monkeypox virus poxin in complex with the STING agonist MD1202D), 8XIG (The crystal structure of the AEP domain of MPXV E5), 4E9O(Vaccinia D8L ectodomain structure) and 8B07(Crystal structure of monkeypox virus methyltransferase VP39 in complex with sinefungin) were sourced from the Protein Data Bank (PDB). The receptor binding domains (RBDs) were isolated and prepared using the “Protein Preparation Wizard” tool within the Schrödinger Suite 2021-4 (Protein Preparation Wizard 2021) (License: 27008@172.25.31.158 (/opt/schrodinger). This preparation process included removing water molecules and cofactors, correcting any mislabeled atoms, adding hydrogen atoms, assigning bond orders, optimizing hydrogen bonding, and performing restrained energy minimization using the OPLS4 force field. The prepared proteins were then used to generate receptor grids through the “Receptor Grid Generation” panel of the Glide module within the Schrödinger suite.

Ligand preparation

Ligand preparation involved downloading 3D Spatial Data File (SDF) structures of the selected compounds using PubChem. These structures were processed using the LigPrep module (Release 2021a). The LigPrep process included optimizing the phytochemicals at a pH of 7 ± 2 with the Epik tool. The optimized structures were then subjected to energy minimization using the OPLS4 force field to ensure accurate molecular conformations for docking studies. The resulting ligand library was subsequently used in molecular docking and dynamics simulations.

Molecular docking

Molecular docking studies were conducted using the Glide module in the Schrödinger Suite (Release 2021b). The standard precision (SP) mode was applied to evaluate binding interactions between the ligand and target protein. Flexible ligand docking was performed, allowing the ligand’s conformational flexibility to be considered, thereby enabling the exploration of multiple potential binding modes. The resulting data were analyzed to identify the most favorable binding conformations and interactions.

Molecular dynamics simulation

Molecular dynamics (MD) simulations were performed using the Desmond module in the Schrödinger Suite (Release 2021c). Protein–ligand complex obtained from docking studies were chosen. The system was solvated in an orthorhombic simulation box with explicit water molecules following the TIP4P model. To neutralize the system, appropriate counterions (Na  or Cl−) were added, with an additional ionic strength adjustment to 0.15 M by adding more ions. The system was then minimized and equilibrated in multiple stages to resolve any steric clashes or unfavorable interactions. MD simulations were carried out under periodic boundary conditions with the OPLS4 force field, maintaining a temperature of 300 K using a Nosé-Hoover thermostat and a pressure of 1.01325 bar via a Martyna-Tobias-Klein barostat. The simulations ran for 100 ns (ns) with a timestep of 2 femtoseconds (fs), recording trajectory data every 10 picoseconds (ps). The resulting trajectory was analyzed to assess the stability of the protein–ligand complex, focusing on parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), and hydrogen bonding interactions over the simulation time.

Gibbs free energy

MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) binding energy calculations were performed using the Prime MM/GBSA module in Schrödinger. Snapshots from the MD simulation was extracted at regular intervals (every 10 ns) to account for dynamic conformational states. The calculations employed the OPLS4 force field and the Generalized Born (GB) with surface area (SA) implicit solvation model. The binding free energy The binding free energy was computed using the formula ΔGbind = Ecomplex −(Eprotein + Eligand ).

Results

ADME qualified candidates from Andrographis paniculata

Notable phytochemicals include Andrographidine C, Bisandrographolide, Neoandrographolide, Andrographolactone, Andrographolide, 14-Deoxy-12-hydroxyandrographolide, Andrographiside, Andrographidin A, 8,17-Epoxy-14-deoxyandrographolide, Luteolin, Andrograpanin, 14-Deoxyandrographolide, 14-Deoxy-11-hydroxyandrographolide, Apigenin shown in Tables 1 and 2.

Table 1.

Phytochemicals and their ADME properties

Sr. No	PCID	Phytochemical	Mol. Wt.	Rotor	accptHB	QPlogS	QPlogBB	QPlogKhsa	Human oral absorption	Percent Human Oral Absorption	Rule of five	Rule of three
1	3899	Leflunomide	270.06	2	4.000	− 3.957	− 0.157	− 0.047	3	100.000	0	0
2	188,316	5-Hydroxy-7,8-dimethoxyflavone	298.08	3	3.750	− 3.782	− 0.430	0.162	3	100.000	0	0
3	5,280,443	Apigenin	270.05	3	3.750	− 3.317	− 1.411	− 0.043	3	73.955	0	0
4	5,280,445	Luteolin	286.05	4	4.500	− 3.039	− 1.910	− 0.205	3	62.050	0	0
5	5,318,484	Andrographidine C	460.14	9	13.250	− 3.051	− 1.627	− 0.655	3	74.143	0	0
6	5,318,517	Andrographolide	350.21	7	8.100	− 2.837	− 1.222	− 0.212	3	79.068	0	0
7	5,708,351	14− Deoxy-11,12-didehydroandrographolide	332.2	5	6.400	− 3.641	− 0.890	0.082	3	89.337	0	0
8	9,848,024	Neoandrographolide	480.27	11	13.200	− 3.848	− 2.299	− 0.382	2	68.808	0	1
9	11,624,161	14-Deoxyandrographolide	350.21	6	6.400	− 3.303	− 0.860	0.037	3	90.524	0	0
10	11,666,871	Andrograpanin	318.22	5	4.700	− 3.818	− 0.483	0.336	3	100.000	0	0
11	12,000,062	Bisandrographolide	664.4	12	12.800	− 6.753	− 2.657	0.746	1	70.723	1	2
12	13,963,762	Andrographidin A	462.15	9	13.500	-4.104	− 2.262	-0.538	2	67.921	0	1
13	12,041,831	(2 S)-5-Hydroxy-6,7-dimethoxy-2-phenyl-2,3-dihydrochromen-4-one	300.1	3	4.000	− 4.828	− 0.556	0.301	3	100.000	0	0
14	38,350,572	14-Deoxy-12-hydroxyandrographolide	350.21	7	7.100	− 3.053	− 1.110	− 0.052	3	84.842	0	0
15	44,206,466	Andrographolactone	296.18	3	3.000	− 4.916	− 0.081	0.642	3	100.000	0	1
16	44,575,263	8,17-Epoxy-14-deoxyandrographolide	350.4	6	8.400	− 2.462	− 0.703	− 0.269	3	88.435	0	0
17	44,593,583	Andrographiside	512.26	13	16.600	− 2.850	− 3.178	− 0.858	2	18.442	2	2
18	91,884,987	14-Deoxy-11-hydroxyandrographolide	350.21	7	8.100	− 2.730	− 0.938	− 0.212	3	85.812	0	1
19	101,243,415	Isoandrographolide	350.21	3	7.150	− 3.359	− 0.606	0.039	3	89.381	0	0

Table 2.

Toxicity assessment of the phytochemicals

Phytochemical	Hepatotoxicity	Neurotoxicity	Nephrotoxicity	Respiratory toxicity	Cardiotoxicity	Carcinogenicity	Mutagenicity	Cytotoxicity
Leflunomide	Active	Active	Inactive	Active	Inactive	Active	Inactive	Inactive
5-Hydroxy-7,8-dimethoxyflavone	Inactive	Inactive	Active	Active	Inactive	Inactive	Inactive	Inactive
Apigenin	Inactive	Inactive	Active	Active	Inactive	Inactive	Inactive	Inactive
Luteolin	Inactive	Inactive	Active	Active	Inactive	Active	Active	Inactive
Andrographidine C	Inactive	Inactive	Active	Active	Active	Inactive	Inactive	Active
Andrographolide	Inactive	Inactive	Inactive	Active	Active	Inactive	Inactive	Inactive
14-Deoxy-11,12-didehydroandrographolide	Inactive	Inactive	Active	Active	Inactive	Inactive	Inactive	Inactive
Neoandrographolide	Inactive	Inactive	Active	Inactive	Active	Inactive	Inactive	Active
14-Deoxyandrographolide	Inactive	Inactive	Active	Active	Active	Inactive	Inactive	Inactive
Andrograpanin	Inactive	Inactive	Inactive	Inactive	Active	Inactive	Inactive	Inactive
Bisandrographolide	Inactive	Inactive	Active	Active	Active	Inactive	Inactive	Active
Andrographidin A	Inactive	Inactive	Active	Active	Active	Inactive	Inactive	Active
(2 S)-5-Hydroxy-6,7-dimethoxy-2-phenyl-2,3-dihydrochromen-4-one	Inactive	Inactive	Active	Active	Active	Inactive	Inactive	Inactive
14-Deoxy-12-hydroxyandrographolide	Inactive	Inactive	Active	Active	Active	Inactive	Inactive	Inactive
Andrographolactone	Active	Active	Inactive	Active	Inactive	Inactive	Inactive	Inactive
Andrographiside	Inactive	Inactive	Active	Active	Active	Inactive	Inactive	Active
14-Deoxy-11-hydroxyandrographolide	Inactive	Inactive	Active	Active	Active	Inactive	Inactive	Inactive
Isoandrographolide	Inactive	Inactive	Active	Active	Inactive	Inactive	Inactive	Active

The docking analysis reveals varying binding affinities and interaction profiles of compounds with the target protein. Leflunomide-8B07 complex forms three hydrogen bonds (one each with ASP95, VAL116, and ARG140) and a π-π stacking interaction with PHE115, yielding a docking score of − 4.484 kcal/mol. (5E)-5-{[1-(2,4-dimethylphenyl)-2,5-dimethyl-1 H-pyrrol-3-yl]methylidene}-3-(prop-2-yn-1-yl)-1,3-thiazolidine-2,4-dione (1883166)-8B07 complex exhibits one hydrogen bond with VAL116 and two π-π stacking interactions with PHE115, achieving the highest binding affinity of − 6.144 kcal/mol. Compound Apigenin (5280443)- 8B07 complex shows one π-π stacking interaction with PHE115 and three hydrogen bonds (one each with VAL116, ARG140, and SER141) with a binding energy of − 5.711 kcal/mol. Luteolin (5280445)-8B07 interacts through one π-π stacking with PHE115 and three hydrogen bonds (GLY68, VAL116, ARG140), scoring − 5.541 kcal/mol. Andrographidine C-8B07 complex forms three hydrogen bonds (two with ASP138 and one with ARG140), with a binding energy of -3.643 kcal/mol. Andrographolide (5318517)-8B07 engages two hydrogen bonds (ASP95 and ASP138) with a binding energy of − 6.094 kcal/mol. 14-Deoxy-11,12-didehydroandrographolide-8B07 complex forms two hydrogen bonds (GLN39 and ARG140), scoring − 5.243 kcal/mol. Neoandrographolide-8B07 complex has four hydrogen bonds (two with GLU207 and one each with LYS41 and ARG140), resulting in -3.741 kcal/mol. 14-Deoxyandrographolide − 8B07 complex forms four hydrogen bonds (two with ASP95 and ARG97, one each with ALA70 and VAL116) and has a binding energy of − 5.470 kcal/mol.11666871-8B07 complex shows no direct bonding interactions with a binding energy of − 4.834 kcal/mol. (2 S)-5-Hydroxy-6,7-dimethoxy-2-phenyl-2,3-dihydrochromen-4-one-8B07 complex interacts via one π-π stacking with PHE115 and binds with − 5.012 kcal/mol. Andrographidin A- 8B07 complex exhibits one π-π stacking with PHE115 and three hydrogen bonds (two with ASP138 and one with LYS175), scoring − 3.642 kcal/mol. 14-Deoxy-12-hydroxyandrographolide-8B07 complex forms a single hydrogen bond with ASP95 and achieves a binding energy of − 6.170 kcal/mol. 4-[2-(1,3,6-trimethyl-8,9-dihydro-7 H-benzo[7]annulen-2-yl)ethyl]-2 H-furan-5-one − 8B07 complex interacts through one hydrogen bond with ASN156 with a binding energy of − 4.564 kcal/mol. 4-[2-[(1 S,2R,4aS,5R,6R,8aR)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethylspiro[3,4,4a,6,7,8-hexahydro-1 H-naphthalene-2,2’-oxirane]-1-yl]ethyl]-2 H-furan-5-one − 8B07 complex forms one hydrogen bond with ASP138 and two water-bridged hydrogen bonds, scoring − 5.886 kcal/mol. 14-Deoxy-11-hydroxyandrographolide − 8B07 complex engages two hydrogen bonds (one each with ASP95 and ASP138) and two water-bridged hydrogen bonds, yielding − 6.355 kcal/mol. Isoandrographolide-8B07 complex forms two hydrogen bonds (one each with GLN39 and ARG97) and two water-bridged hydrogen bonds with a binding energy of − 4.193 kcal/mol. Tecovirimat-8B07 complex forms one hydrogen bond with ASP138 with a binding energy of − 4.125 kcal/mol. These findings highlight diverse interaction patterns and affinities of 8B07, with (5E)-5-{[1-(2,4-dimethylphenyl)-2,5-dimethyl-1 H-pyrrol-3-yl]methylidene}-3-(prop-2-yn-1-yl)-1,3-thiazolidine-2,4-dione and 14-Deoxy-11-hydroxyandrographolide showing the strongest binding. Figure 1 and Table 3.

Fig. 1.

2D Interaction Diagram of Protein and Ligand Complexes

Table 3.

The phytochemical binding profile of Andrographis panniculata against Mpox protein with Docking score

Protein	LIGANDS	Bonding Interaction	Non-Bonding Interaction	Docking Score
8B07	5-Hydroxy-7,8-dimethoxyflavone (1883166)	One hydrogen bond with VAL 116 and two pi-pi stacking with PHE115	ILE67, GLY68, ILE94, ASP95, GLY96, ARG97, ASP117, VAL139, ARG140, SER141, SER155, ASN156, LEU159	− 6.144
	Andrographolide (5318517)	Two hydrogen bond one each with ASP95 and ASP138	LEU42, ILE67, GLY68, SER69, ALA70, PRO71, GLY96, ARG97, PHE115, VAL116, VAL139, ARG140, SER141, ASN156, LEU159, LYS175	− 6.094
	Andrographidin A (38350572)	One hydrogen bond with ASP95	ILE67, GLY68, GLY96, ARG97, VAL116, PHE115, VAL116, ASP138, VAL139, ARG140, SER141, ASN156, LEU159	− 6.170
	14-Deoxy-11-hydroxyandrographolide (91884987)	Two hydrogen bond one each with ASP95 and ASP138 and two hydrogen bond with water molecules	LEU42, ILE67, GLY68, SER69, ALA70, PRO71, GLY96, ARG97, ARG114, PHE115, VAL116, VAL139, ARG140, SER141, ASN156, LEU159, LYS175	− 6.355
	Tecovirimat (16124688)	One hydrogen bond with ASP138	GLY38, GLN39, LYS41, LEU42, GLY68, SER69, ALA70, PRO71, GLY72, ASP95, ARG97, PHE115, VAL139, ARG140, SER141	− 4.125
8C9K	Luteolin (5280445)	Three hydrogen bond one each with LEU75, ARG95, PRO192, two pi-pi stacking with TYR94	TYR74, GLN76, ILE77, VAL92, THR93, PRO192, ILE193, NMA194A, PRO194	− 6.000
	Tecovirimat (16124688)	One pi-pi stacking with PHE171 and one hydrogen bond with GLY458	MET175, VAL174, PHE290, HIS293, GLY294, TYR297,CYS302, CSO303, ILE304, TYR457, VAL459, VAL460,	− 7.714
8J8G	Luteolin (5280445)	One metallic bond with CA1104, one hydrogen bond with ASN665, two hydrogen bonds with ASP751	ASP549, TYR550, PRO550, ASN551, SER552, LEU553, TYR554, THR752, ASP753, SER754	− 5.722
	(2 S)-5-Hydroxy-6,7-dimethoxy-2-phenyl-2,3-dihydrochromen-4-one (12041831)	One metallic bond with CA1104	ASP549, TYR550, SER552, LEU553, TYR554, PRO555, ASN665, TYR668, ASP751, THR752, ASP753, SER754	− 5.562
	Tecovirimat (16124688)	One metallic bond with CA1104, one pi cation with LYS826 and one hydrogen bond with ASP753	ILE547, ASP549, TYR550, SER552, LEU553, TYR554, PRO555, LYS661, THR752, SER754, PHE756, GLU792, TYR795, TYR806	− 5.443
4E90	Luteolin (5280445)	Four hydrogen bond two with ASP111 and one each with THR168 and GLU230, one pi-pi stacking with TYR232	PHE56, ASP112, TYR169, LEU170, THR229, VAL231	− 6.764
	Andrographidine C (5318484)	Six hydrogen bond two with PRO2, one hydrogen bond each with Asp228 and GLU230 and one intra molecular hydrogen bond	GLN3, GLN4, LEU21, PHE56, THR168, TYR169, LEU170, VAL181, ASN226, TYR232	− 6.555
	Andrographidin A (13963762)	Five hydrogen bond, two hydrogen bond with PRO2 and one hydrogen bond each with ASP228, THR229, GLU230	GLN3, GLN4, LEU21, PHE56, ASP111, THR168, TYR169, LEU170, VAL181, ASN226, TYR232	− 6.418
	Tecovirimat (16124688)	Two hydrogen bond one each with LEU170 and GLU230	PRO2, GLN4, LEU21, PHE56, THR168, TYR169, VAL181, ASN226, ASP228, THR229, TYR232	− 5.762
8IZT	Andrographidine C (5318484)	One metallic bond with ZN202	ASP31, ASP33, PHE34, LEU36, ASN37, MET40, VAL111, LEU114	− 6.610
	Tecovirimat (16124688)	One hydrogen bond with ASN118	MET1, ASP33, PHE34, LEU36, ASN37, MET40, VAL111, LEU114, LEU117	− 5.481

The docking analysis of compounds with the target protein 8C9K demonstrates diverse interaction patterns and binding affinities. Leflunomide-8C9K complex forms two hydrogen bonds with ARG95 and one π-π stacking interaction with TYR94, interacting with residues like ASN54, TYR74, and TYR100 with docking score − 5.614. (5E)-5-{[1-(2,4-dimethylphenyl)-2,5-dimethyl-1 H-pyrrol-3-yl]methylidene}-3-(prop-2-yn-1-yl)-1,3-thiazolidine-2,4-dione-8C9K complex shows three π-π stacking interactions with TYR94 and one hydrogen bond with ASN54 with docking score − 5.548. Apigenin − 8C9K complex interacts via one hydrogen bond with ASN54 and two π-π stacking interactions with TYR94 and docking score − 5.537. Compound forms three hydrogen bonds (LEU75, ARG95, PRO192) and two π-π stacking interactions with TYR94 with docking score − 6.000. Andrographidine C-8C9K complex exhibits two π-π stacking interactions with TYR94 and three hydrogen bonds (TYR74, THR93, NMA194A) and docking score − 5.346. Andrographolide-8C9K complex has two hydrogen bonds with THR93 and ARG95 and docking score − 4.582. 14-Deoxy-11,12-didehydroandrographolide – 8C9K complex forms one hydrogen bond with ARG95 and docking score − 4.815. Neoandrographolide-8C9K complex interacts through three hydrogen bonds (two with HIS121 and one with ARG95) and docking score − 4.875. 14-Deoxyandrographolide-8C9K complex forms one hydrogen bond with ARG95. Andrograpanin-8C9K complex exhibits two hydrogen bonds, one each with GLN56 and PRO192. (2 S)-5-Hydroxy-6,7-dimethoxy-2-phenyl-2,3-dihydrochromen-4-one-8C9K complex shows one π-π stacking interaction with TYR94. Compound 13,963,762 forms two π-π stacking interactions (TYR94, HIS121) and two hydrogen bonds (TYR74, NMA194A). 14-Deoxy-12-hydroxyandrographolide-8C9K complex interacts via two hydrogen bonds (ARG95, NMA194A). 4-[2-(1,3,6-trimethyl-8,9-dihydro-7 H-benzo[7]annulen-2-yl)ethyl]-2 H-furan-5-one-8C9K shows one π-π stacking interaction with TYR94. Compound 44,575,263 forms two hydrogen bonds (ARG95, NMA194A). 14-Deoxy-11-hydroxyandrographolide-8C9K complex has three hydrogen bonds (TYR74, LEU75, NMA194A). Compound forms two hydrogen bonds (ASN54, HIS121).Tecovirimat shows one π-π stacking interaction with PHE171 and one hydrogen bond with GLY458. These interactions highlight significant binding properties, with (5E)-5-{[1-(2,4-dimethylphenyl)-2,5-dimethyl-1 H-pyrrol-3-yl]methylidene}-3-(prop-2-yn-1-yl)-1,3-thiazolidine-2,4-dione and Andrographidine C showing strong π-π stacking and hydrogen bonding, contributing to high binding affinities. Figure 1 and Table 3.

The target protein 8J8G reveals various binding interactions and docking scores. Leflunomide-8J8G complex forms one hydrogen bond with TYR554, interacting with residues like TYR550, SER552, and ASP753 and docking score − 4.716 kcal/mol. (5E)-5-{[1-(2,4-dimethylphenyl)-2,5-dimethyl-1 H-pyrrol-3-yl]methylidene}-3-(prop-2-yn-1-yl)-1,3-thiazolidine-2,4-dione-8J8G complex exhibits a metallic bond with CA1104, a hydrogen bond with ASP549, and a π-π stacking with TYR554 and docking score − 5.145 kcal/mol. Apigenin-8J8G complex forms two hydrogen bonds one each with TYR554, ASP751 and two π-cation interactions with LYS661 and docking score − 4.968 kcal/mol. Luteolin-8J8G complex binds through a metallic bond with CA1104 and three hydrogen bonds (ASN665, ASP751, ASP753) and docking score − 5.722 kcal/mol. Andrographidine C-8J8G complex interacts via two metallic bonds with CA1104 and a hydrogen bond with ASN551 and docking score − 5.098 kcal/mol. Andrographolide-8J8G complex forms four hydrogen bonds (LEU553, ASP753, GLY531, LYS804) and docking score − 4.513 kcal/mol. 14-Deoxy-11,12-didehydroandrographolide-8J8G complex shows one metallic bond with CA1104 and a hydrogen bond with LYS804 and docking score − 4.649 kcal/mol. Neoandrographolide-8J8G complex forms a metallic bond with CA1104 and two hydrogen bonds (ASP753, LYS804) and docking score − 4.894 kcal/mol. 14-Deoxyandrographolide-8J8G complex binds through a metallic bond with CA1104 and two hydrogen bonds with LYS804 and docking score − 4.675 kcal/mol. Andrograpanin-8J8G complex exhibits a metallic bond with CA1104 and two hydrogen bonds each with LEU553 and TYR806, docking score − 4.612 kcal/mol. (2 S)-5-Hydroxy-6,7-dimethoxy-2-phenyl-2,3-dihydrochromen-4-one-8J8G complex shows one metallic bond with CA1104 and docking score − 5.562 kcal/mol. Andrographidin A-8J8G complex forms a metallic bond, with two hydrogen bonds (ASN551, GLU792) and docking score − 5.249 kcal/mol. 14-Deoxy-12-hydroxyandrographolide-8J8G complex interacts via a metallic bond with CA1104 and two hydrogen bonds (LEU553, TYR806) and docking score − 5.015 kcal/mol. 4-[2-(1,3,6-trimethyl-8,9-dihydro-7 H-benzo[7]annulen-2-yl)ethyl]-2 H-furan-5-one-8J8G complex shows a metallic bond with CA1104 and a π-cation bond with LYS661 and docking score − 4.935 kcal/mol. 4-[2-[(1 S,2R,4aS,5R,6R,8aR)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethylspiro[3,4,4a,6,7,8-hexahydro-1 H-naphthalene-2,2’-oxirane]-1-yl]ethyl]-2 H-furan-5-one-8J8G complex forms a metallic bond with CA1104 and a hydrogen bond with LYS804 and docking score − 3.809 kcal/mol. 14-Deoxy-11-hydroxyandrographolide-8J8G complex binds through a metallic bond with CA1104 and a hydrogen bond with ASN665 and docking score − 4.317 kcal/mol. Isoandrographolide-8J8G complex exhibits no bonding interactions and docking score − 3.888 kcal/mol. Tecovirimat demonstrates strong interactions, including a metallic bond with CA1104, a π-cation with LYS826, and a hydrogen bond with ASP753 and docking score − 5.443 kcal/mol. These interactions highlight Luteolin and (2 S)-5-Hydroxy-6,7-dimethoxy-2-phenyl-2,3-dihydrochromen-4-one as the most stable compounds based on binding energies and interaction diversity. Figure 1 and Table 3.

The docking analysis of compounds with the 4E90 target protein reveals a range of binding interactions and binding energies. Leflunomide-4E90 complex forms a single hydrogen bond with LEU170 and has a docking score of − 5.190 kcal/mol. (5E)-5-{[1-(2,4-dimethylphenyl)-2,5-dimethyl-1 H-pyrrol-3-yl]methylidene}-3-(prop-2-yn-1-yl)-1,3-thiazolidine-2,4-dione-4E90 complex creates two hydrogen bonds, one each with LEU170 and GLU230, with a docking score of − 5.087 kcal/mol. Apigenin-4E90 complex shows two hydrogen bonds (ASN18, ASP111) and a π-π stacking interaction with TYR232, achieving a docking score of − 5.484 kcal/mol. Luteolin-4E90 complex demonstrates strong binding, forming four hydrogen bonds (two with ASP111, one with THR168, and one with GLU230), along with a π-π stacking interaction with TYR232, and a docking score of − 6.764 kcal/mol. Andrographidine C-4E90 complex forms six hydrogen bonds, including two with PRO2 and one each with ASP228 and GLU230, with a docking score of − 6.555 kcal/mol. Andrographolide-4E90 complex forms three hydrogen bonds (PHE56, LEU170, GLU230), resulting in a docking score of − 5.532 kcal/mol. 14-Deoxy-11,12-didehydroandrographolide-4E90 complex binds with one hydrogen bond to LEU170, achieving a docking score of − 4.612 kcal/mol. Neoandrographolide-4E90 complex forms four hydrogen bonds (two with GLU230, and one each with LEU170 and ARG20), with a docking score of − 4.462 kcal/mol. 14-Deoxyandrographolide-4E90 complex and Andrograpanin-4E90 complex each form three hydrogen bonds (PHE56, LEU170, GLU230), with docking scores of − 5.153 kcal/mol and − 5.307 kcal/mol, respectively. (2 S)-5-Hydroxy-6,7-dimethoxy-2-phenyl-2,3-dihydrochromen-4-one-4E90 complex interacts through two hydrogen bonds (LEU170, GLU230), with a docking score of − 5.065 kcal/mol. Andrographidin A-4E90 complex forms five hydrogen bonds (two with PRO2, and one each with ASP228, THR229, and GLU230), resulting in a docking score of − 6.418 kcal/mol. 14-Deoxy-12-hydroxyandrographolide-4E90 complex exhibits two hydrogen bonds (LEU170, TYR232) and a docking score of − 5.416 kcal/mol. 4-[2-(1,3,6-trimethyl-8,9-dihydro-7 H-benzo[7]annulen-2-yl)ethyl]-2 H-furan-5-one-4E90 complex binds via two hydrogen bonds (LEU170, GLU230), with a docking score of − 5.583 kcal/mol. 4-[2-[(1 S,2R,4aS,5R,6R,8aR)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethylspiro[3,4,4a,6,7,8-hexahydro-1 H-naphthalene-2,2’-oxirane]-1-yl]ethyl]-2 H-furan-5-one-4E90 complex forms two hydrogen bonds (PHE56, GLU230) and has a docking score of − 5.250 kcal/mol. 14-Deoxy-11-hydroxyandrographolide-4E90 complex forms two hydrogen bonds (ASN18, ARG20) but has a weaker binding energy of -2.920 kcal/mol. Isoandrographolide-4E90 complex form two hydrogen bonds (LEU170, GLU230), with docking scores of − 4.815 kcal/mol. Tecovirimat binds through two hydrogen bonds (LEU170, GLU230) and several other interactions, achieving a docking score of − 5.762 kcal/mol. Among all the compounds, Luteolin and Andrographidine C exhibit the most promising binding energies and extensive interactions. Figure 1 and Table 3.

The docking analysis of compounds with the 8IZT target protein reveals a range of interactions and binding energies. Leflunomide, (5E)-5-{[1-(2,4-dimethylphenyl)-2,5-dimethyl-1 H-pyrrol-3-yl]methylidene}-3-(prop-2-yn-1-yl)-1,3-thiazolidine-2,4-dione, Andrograpanin, (2 S)-5-Hydroxy-6,7-dimethoxy-2-phenyl-2,3-dihydrochromen-4-one, 4-[2-(1,3,6-trimethyl-8,9-dihydro-7 H-benzo[7]annulen-2-yl)ethyl]-2 H-furan-5-one, 4-[2-[(1 S,2R,4aS,5R,6R,8aR)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethylspiro[3,4,4a,6,7,8-hexahydro-1 H-naphthalene-2,2’-oxirane]-1-yl]ethyl]-2 H-furan-5-one, and 14-Deoxy-11-hydroxyandrographolide with 8IZT complex show no bonding interactions, with binding energies between − 3.386 kcal/mol and − 5.184 kcal/mol. Apigenin-8IZT complex forms three hydrogen bonds (two with ASN118 and one with LEU36) and a binding energy of − 5.186 kcal/mol, while Luteolin-8IZT complex creates one hydrogen bond with ASP33 and a docking score of − 5.249 kcal/mol. Andrographidine C-8IZT complex exhibits a metallic bond with ZN202, demonstrating the strongest binding energy at − 6.610 kcal/mol. Andrographolide-8IZT complex forms two hydrogen bonds (with ASP33 and ASN118) and a weaker binding energy of -3.433 kcal/mol. 14-Deoxy-11,12-didehydroandrographolide-8IZT complex binds with ASN118 via one hydrogen bond, achieving a docking score of − 4.610 kcal/mol, while Neoandrographolide-8IZT complex forms one hydrogen bond with ASN37 and a binding energy of -3.586 kcal/mol. 14-Deoxyandrographolide-8IZT complex also forms two hydrogen bonds (with LEU36 and ASN118) and a binding energy of -3.505 kcal/mol. Andrographidin A-8IZT complex shows a metallic bond with ZN202 and a docking score of − 5.144 kcal/mol. 14-Deoxy-12-hydroxyandrographolide-8IZT complex forms two hydrogen bonds (with ASP33 and ASN118) and a binding energy of -3.964 kcal/mol. Isoandrographolide-8IZT complex form one hydrogen bond with ASN118, with binding energies of − 4.172 kcal/mol. Tecovirimat binds with one hydrogen bond to ASN118, achieving a binding energy of − 5.481 kcal/mol. Among these compounds, Andrographidine C, Luteolin, and Tecovirimat exhibit the most promising binding affinities and interactions. Figure 1 and Table 3.

Molecular dynamic simulations

The protein-ligand complexes with the highest docking scores were further analyzed through molecular dynamics simulations for 100 ns, using poses generated via a flexible ligand docking approach. Protein RMSD (Root Mean Square Deviation) was employed to assess the stability of the protein structure during thermodynamic movements. In this study, RMSD was specifically utilized to evaluate the interactions of various MP-related proteins with phytochemicals derived from Andrographis panniculata.

14-Deoxy-12-hydroxyandrographolide-8B07.

14-Deoxy-12-hydroxyandrographolide demonstrated three hydrogen bonds: two with ARG A:140 (58%, 53%) and one with VAL A:116 (88%), along with one intramolecular hydrogen bond (31%). The highest protein-ligand contact was observed with ARG140, which included both water bridges and hydrogen bonds. The highest interaction fraction reported was 1.2 for water bridges. The protein RMSD increased rapidly during the first ~ 10 ns and then stabilized around 1.8–2.0 Å, indicating structural equilibration. The ligand RMSD, plotted on the secondary y-axis, followed a similar trend and stabilized around 9–10 Å, suggesting that the ligand reached a stable conformation relative to the protein. Both RMSD values remained stable, with only minor fluctuations, indicating a steady system throughout the simulation. The secondary structure of the protein consists of 35.68% helix, 16.70% strands, and 52.38% total SSE. Ligand properties, including Radius of Gyration (3.92–4.16 Å), Solvent Accessible Surface Area (100–250 Ų), and Polar Surface Area (152–176 Ų) further indicate the compactness and structural consistency of the ligand. Figure 2 and Table 4.

Fig. 2.

Molecular Dynamic Simulation analysis of 14-Deoxy-12-hydroxyandrographolide-8B07

Table 4.

MMGBSA analysis of bioactives from Andrographis panniculata with Mpox protein

Protein-Ligand complex	MMGBSA dG bind	Prime energy	MMGBSA dG bind (NS)	MMGBSA dG bind (NS) Coulomb	MMGBSA dG bind (NS) Covalent	MMGBSA dG bind (NS) vdW
14-Deoxy-12-hydroxyandrographolide -8B07	− 244.83	− 8344.01	− 287.63	− 459.49	− 0.00	− 166.15
Andrographolide -8B07	− 238.08	− 8200.97	− 283.15	− 430.82	0.00	− 194.21
Andrographidin A-4E9O	− 328.97	− 7422.15	− 375.31	− 126.26	− 0.00	− 225.67
Luteolin-4E9O	− 329.08	− 7822.53	− 366.60	− 136.65	0.00	− 209.35
Luteolin-8C9K	− 5.31	− 5813.58	− 27.57	− 27.57	− 0.00	− 28.44
Andrographidine C-8IZT	− 26.87	− 3986.91	− 29.00	− 1.90	0.00	− 27.13

Andrographolide-8B07

Andrographolide formed two hydrogen bonds, one with ARG A:140 (81%) and the other with GLY A:96 (73%), along with one water-bridged hydrogen bond with ASP A:95 (41%) and an intramolecular hydrogen bond (34%). ARG140 exhibited the highest number of protein-ligand contacts, including both hydrogen bonds and water bridges. The highest interaction fraction of 1.2 was reported for water bridges. The protein RMSD stabilized at 1.5 Å and remained stable throughout the simulation. The ligand started at 0.9 Å, and both the protein and ligand remained stable throughout the entire simulation. The protein’s secondary structure consisted of 35.60% helix, 16.02% strand, and 51.62% total SSE. Key ligand parameters such as Radius of Gyration (3.75–4.20 Å), Solvent Accessible Surface Area (180–360 Ų), and Polar Surface Area (170–190 Ų) supports the ligand’s compact and stable conformation. Figure 3 and Table 4.

Fig. 3.

Molecular Dynamic Simulation analysis of Andrographolide − 8B07

Luteolin-8C9K

Luteolin demonstrated one pi-pi stacking with TYR A: 94 (54%), three hydrogen bond one with ALA A:8 (34%), and two hydrogen bonds with ARG A:95 (36%, 33%), and one water bridged hydrogen bond with THR A:93(55%). ARG 95 showed highest number of Protein-Ligand contact which included hydrogen bonds and water bridges. The highest interaction fraction noted was 1.4 for water bridges. The protein RMSD, stabilizes around 1.5 Å after an initial equilibration period (around 10–15 ns), indicating that the protein structure achieves stability over the remainder of the simulation. The ligand RMSD, starts at 0.75 Å both protein and ligand remain almost stable throughout the simulation. The secondary structure of protein have 4.47% helix, 38.65% strand and 43.11% total SSE. Ligand-specific parameters such as Radius of Gyration (3.9–4.08 Å), Solvent Accessible Surface Area (50–200 Ų), and Polar Surface Area (236–244 Ų) further confirm the compact and stable nature of the ligand during the simulation. Figure 4 and Table 4.

Fig. 4.

Molecular dynamic simulation analysis of andrographidine A-4E9O

Andrographidine A-4E9O

Andrographidin A demonstrated significant hydrogen bond interactions with GLU X:230 (69%, 46%), two water-bridged hydrogen bonds with GLU X:230 (70%, 30%), one water-bridged hydrogen bond with LEU X:170, one hydrogen bond with ASP X:111, and two pi-pi stacking interactions with PHE X:56 (38%) and TYR X:232b (70%). GLU X:230 showed the highest number of protein-ligand contacts, including hydrogen bonds, ionic interactions, and water bridges, throughout the simulations. The highest interaction fraction of 3.0 was reported for water bridges. The protein RMSD stabilized around 1.5 Å after an initial equilibration period of approximately 10–15 ns, indicating that the protein structure achieved stability for the remainder of the simulation. The ligand RMSD started at a low value (~ 0.5 Å) and exhibited a sharp rise around 10 ns before stabilizing at 5.6 Å, suggesting that the ligand underwent significant rearrangement within the binding pocket but eventually stabilized, maintaining its position relative to the protein. The secondary structure of the protein consists of 7.92% helix, 30.51% strand, and 38.42% total secondary structure elements (SSE). Key ligand properties such as Radius of Gyration (4.25–5.00 Å), Solvent Accessible Surface Area (150–300 Ų), and Polar Surface Area (200–240 Ų) further indicate that the ligand maintained a stable and compact conformation during the simulation. Figure 5 and Table 4.

Fig. 5.

Molecular dynamic simulation analysis of Luteolin-4E9O

Luteolin_4E9O

Luteolin (5280445) showed one pi-pi stacking interaction with TYR X:232 (32%), one hydrogen bond with THR X:168 (36%), and one intramolecular hydrogen bond (97%). TYR X:232 exhibited the highest protein-ligand contact, which included hydrophobic interactions and water bridges. The highest interaction fraction of 0.8 was reported for water bridges. The protein RMSD gradually increased, stabilizing around 1.5 Å, indicating structural equilibration. The ligand RMSD exhibited initial fluctuations before stabilizing around 9.0 Å after approximately 20 ns, suggesting that the ligand reached a relatively stable conformation within the binding pocket after an initial adjustment phase. Both systems remained stable throughout the simulation. The secondary structure of the protein consisted of 8.16% helix, 30.75% strand, and 38.91% total SSE. Additional ligand parameters, including Radius of Gyration (3.92–4.08 Å), Solvent Accessible Surface Area (100–250 Ų), and Polar Surface Area (235–245 Ų), further affirm its compact and stable nature. Figure 6 and Table 4.

Fig. 6.

Molecular dynamic simulation analysis of Luteolin-8C9K

Andrographidine C-8IZT

Andrographidine C (5318484) demonstrated one water-bridged hydrogen bond with ASP A:33 and two metallic bonds with ASP A:33 (100%, 100%) and ASP A:31 (63%, 100%), along with one intramolecular hydrogen bond (31%). ASP A:33 showed the highest protein-ligand contacts, including hydrogen bonds, ionic bonds, and water bridges. The highest interaction fraction of 2.5 was observed for water bridges. The protein RMSD stabilized around 2.8 Å after an initial rise at approximately 20 ns, indicating that the protein achieved structural stability. The ligand RMSD sharply increased around 20 ns and fluctuated between 10 and 12 Å for most of the simulation, with a sharp rise near 90 ns followed by a drop toward the end, suggesting that the ligand underwent significant movement and conformational changes during the simulation while the protein remained relatively stable. The secondary structure of the protein consisted of 55.71% helix, 0.01% strand, and 55.72% total SSE. Additional ligand properties such as Radius of Gyration (4.8–5.25 Å), Solvent Accessible Surface Area (300–600 Ų), and Polar Surface Area (195–240 Ų) further support its structural stability. Figure 7 and Table 4.

Fig. 7.

Molecular dynamic simulation analysis of Andrographidine C-8IZT

Gibbs free energy – MMGBSA

The MMGBSA analysis of various protein-ligand simulation complexes revealed diverse binding affinities and interaction profiles. The 14-Deoxy-12-hydroxyandrographolide-8B07 complex exhibited the highest binding energy of − 178.1 kcal/mol and the most favorable ΔG binding energy of − 244.83 kcal/mol, driven primarily by strong Coulombic interactions (− 459.49 kcal/mol) and van der Waals (vdW) forces (− 166.15 kcal/mol). Similarly, the Andrographolide-8B07 complex showed a maximum ΔG binding energy of − 174.5 kcal/mol and a minimum of − 238.08 kcal/mol, with significant contributions from vdW (− 194.21 kcal/mol) and Coulomb (− 430.82 kcal/mol) interactions.

Among the 4E9O complexes, the Andrographidin A-4E9O complex displayed ΔG binding energies ranging from − 234.3 kcal/mol to -328.97 kcal/mol, dominated by vdW interactions (-225.67 kcal/mol). The Luteolin-4E9O complex exhibited comparable values, with maximum and minimum ΔG binding energies of − 228.9 kcal/mol and − 329.08 kcal/mol, respectively, also driven by vdW contributions (− 209.35 kcal/mol). Notably, Luteolin-8C9K demonstrated similar binding energy values to the Luteolin-4E9O complex, indicating a consistent interaction profile.

In contrast, the Andrographidine C-8IZT complex showed the weakest binding affinity, with a ΔG binding energy of – 26.87 kcal/mol, minimal Coulombic contributions (− 1.90 kcal/mol), and predominant vdW interactions (− 27.13 kcal/mol), reflecting its comparatively lower interaction strength. These findings underscore the variability in binding energies and interaction patterns across the analyzed protein-ligand complexes. Figure 8.

Fig. 8.

MMGBSA and Prime energy of the Protein-Ligand Complexes

Principal component analysis

A detailed molecular dynamics trajectory analysis utilizing Principal Component Analysis (PCA) of the protein to identify the key interactions within the protein-ligand complex. The study emphasized the two principal modes governing the collective motion of the complex during molecular simulations. Pronounced anticorrelated fluctuations were observed, particularly in the loop regions of the binding site residues, as illustrated by the vectors in the corresponding Fig. 9. Principal Component Analysis (PCA) was performed on the 8B07 protein complexed with Deoxy-12-hydroxyandrographolide and Andrographolide to investigate the dominant conformational motions during molecular dynamics simulations. The PCA free energy landscape plots illustrate the distribution of conformations along the first two principal components (PCA1 and PCA2), revealing significant energy minima indicative of stable states. The Deoxy-12-hydroxyandrographolide complex showed two energy minima ranged from (− 10, 10)kcal/mol shows a more localized clustering, suggesting restricted conformational flexibility, whereas the Andrographolide complex demonstrated 3 energy minima (– 20, 10)kcal/mol exhibits a broader distribution, indicating greater flexibility and multiple metastable states. The cross-correlation heatmaps further depict the dynamic residue fluctuations, highlighting correlated and anti-correlated motions, which provide insights into ligand-induced structural variations. 4E9O complexed with Andrographidine A exhibits an energy minima around (− 10, 5) kcal/mol, suggesting a stable conformation with limited conformational shifts. In contrast, the 4E9O Luteolin complex shows broader energy distribution with three minima around (– 10, 15) kcal/mol. The differences in energy landscapes and residue fluctuations suggest that Andrographidine A stabilizes the protein more rigidly, whereas Luteolin allows greater conformational adaptability, which may influence functional activity and binding efficiency. 8C9K-Luteolin exhibits three energy minima around (− 15, 10) kcal/mol, indicating a moderately stable conformation with notable flexibility while 8IZT-Andrographidine C complex shows three energy minima near (– 25, 5) kcal/mol.

Fig. 9.

Post molecular dynamics structural analysis of the protein-ligand complex

Discussion

The emergence of Monkeypox virus (MPXV) as a global public health concern, especially post-2022, has highlighted significant challenges in viral surveillance, containment, and therapeutic management. While MPXV was historically confined to endemic regions of Central and West Africa, recent outbreaks across non-endemic countries underscore its potential for wider transmission. Current management therapeutics includes Tecovirimat and Brincidofovir, which come with concerns of accessibility, resistance, and side effects [27]. In this context, Ayurvedic herb Andrographis paniculata (Kalmegha), may offer promising alternatives. It exhibits antiviral, immunomodulatory, and anti-inflammatory effects, validated by in vitro studies. In this study we have taken the Monkey Pox Virus proteins namely 4E90, 8B07, 8C9K, 8IZT and 8J8G. 4E9O showed highest binding affinity with Luteolin (− 6.7764) followed by Andrographidine C and Andrographidin A (− 6.555 and − 6.418) while Tecovirimat demonstrated the docking score of − 5.762 all phytochemicals demonstrated hydrogen bond with GLU230. 8B07 showed highest affinity with 14-Deoxy-11-hydroxyandrgrapholide (− 6.335) while with Tecovirimat it showed relatively low affinity (− 4.125) exhibiting similar interaction with ASP138. 8C9K showed high binding affinity with Tecovirimat (− 7.714) while low affinity with Luteolin (− 6.000) although they didn’t show similar bonding interactions. 8IZT demonstrated different interaction with Andrographidine C (− 6.610) and Tecovirimat (− 5.481). 8J8G interacted with the phytochemicals each with different amino acid residues and exhibited docking score − 5.722, − 5.562 and − 5.443 with Luteolin, (2 S)-5-Hydroxy-6,7-dimethoxy-2-phenyl-2,3-dihydrochromen-4-one and Tecovirimat. Previous studies by Nikitha et al., 2024 reported binding energy − 5.46kacl/mol interactions via hydrogen bonds at amino acid residues PHE188, TYR189, LYS33, GLN37, GLN38, GLY38, GLY96, ARG97, PHE115, PRO202 and SER203 with 8B07-Fucoxanthin complex [28].

The binding free energy calculations using the MM-GBSA approach provide valuable insight into the thermodynamic stability of the protein-ligand complexes and help in ranking the ligands based on their binding affinities [29]. Among the compounds studied, Luteolin-4E9O and Andrographidin A-4E9O exhibited the most favorable binding free energies, with MM-GBSA ΔG_bind values of − 329.08 kcal/mol and − 328.97 kcal/mol, respectively. These values suggest strong binding affinities and stable interactions with their respective targets, likely driven by significant van der Waals (vdW) and Coulombic contributions. Specifically, Andrographidin A showed a substantial vdW component (− 225.67 kcal/mol) and Coulombic energy (− 126.26 kcal/mol), indicating balanced non-covalent interactions. Andrographidin A-4E9O and Luteolin-4E9O again ranked highest with ΔG_bind (NS) values of − 375.31 kcal/mol and − 366.60 kcal/mol, indicating sustained binding throughout the simulation trajectory. In contrast, Luteolin-8C9K and Andrographidine C-8IZT retained lower binding energies during simulations, suggesting comparatively less stable interactions in dynamic conditions. MM-GBSA analysis reveals that Andrographidin A and Luteolin (4E9O complex) exhibit the most favorable binding thermodynamics among the tested compounds, making them promising candidates for further investigation. Based on interaction profiles and molecular docking at the catalytic sites, we propose that selected compounds may inhibit viral proteins by disrupting key interactions critical for enzymatic function, potentially including DNA polymerase inhibition.

In silico studies often fall short in accurately reflecting the dynamic conformational changes that proteins undergo during ligand binding. In biological systems, the complexity and variability of protein-ligand interactions can lead to outcomes that differ significantly from those predicted through computational techniques. Moreover, computational approaches may overlook crucial aspects of the cellular environment, which play a key role in determining the true pharmacological behavior of a compound. Given the theoretical nature of these simulations, experimental validation is essential to confirm their findings.

Conclusion

The study underscores the predictive therapeutic potential of Andrographis panniculata bioactive in Monkey Pox related proteins. The protein-ligand complexes have strong binding interactions, stable dynamics, structural stability and energy minima highlights the ability of these compounds to form stable and significant interactions within protein binding pockets. Notably, phytochemicals like andrographolide, luteolin, and andrographidin-A shows diverse secondary structural elements and steady thermodynamic profiles, validate the potential of these phytochemicals as promising candidates for therapeutic applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

PKG, RM and AK conceptualized the study, PKG planned, supervised, and edited the study, AKM and SKR supervised and edited the manuscript, RS performed the computational work and constructed the manuscript.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Artificial intelligence utility declaration

No AI tool is used for Manuscript writing.