Introduction

The Congo viral fever, also known as the Crimean Congo Hemorrhagic Fever (CCHF) virus, is well-recognized as a tick-borne virus. It is one of the most fatal diseases ever discovered in human history, having a fatality rate of up to 40% and varies geographically. It is mainly found in Asian countries along with Africa, the Middle East and Europe1. The discovery of CCHFV took place in the late 20th century though it is mentioned in the literature that a physician from Iran named Jurjani discussed this kind of virus in one of his works in the medical field Zakhire Kharazmshahi2. CCHFV is known to belong to the Nairoirus genus from the Bunyaviridae family. CCHFV was first recognized in the south of present-day Ukraine in the area named as Crimean Peninsula in the 1940s. It was first isolated in 1956 in the Democratic Republic of Congo, and that’s how it was named as Congo virus3.

CCHFV is found to be transmitted by arthropods like ticks, mosquitos, and flies, etc. The secondary source of CCHFV transmission is having contact with infected blood or tissues. The incubation period of CCHFV is believed to be 3–9 days in case of a tick bite while in case of contact with infected blood, the incubation period usually lies up to 6–13 days4. The symptoms include diarrhea, abdominal pain and sore throat along with sharp mood swings. After prevailing for some days CCHF causes sleeplessness, depression and a disease called hepatomegaly which is the enlargement of the liver5. Some evidence depicts the chances of hepatitis, kidney diseases and liver failure6. The gemome of CCHFV is found to have three segments with distinct functions. The small segment is found to encode the nucleoprotein. The medium segment is identified as the center of information on glycoproteins that are known as Glycoprotein N and Glycoprotein C. The last and largest segment encodes the RNA-dependent RNA polymerase (RDRP)7. RDRP enzyme plays a role in the replication of viral RNA and transcription which signifies its importance of it8,9,10.

The nucleoprotein of CCHFV envelops the RNA of the virus, as observed in the other negative viruses, forms the complex named ribonucleoprotein particle which is considered to be responsible for the protection of RNA genome counter to degradation by the host cell nucleases and is found to help in the packaging of the recently constructed RNA to form the virions11,12. The nucleoprotein contains two domains, in the regular three-dimensional structure, named head and stalk domains13,14. The head ___domain mainly comprises of residues like Lys339, Lys343, Lys346, Arg384, Lys411, His453, Gln457, Arg134, Arg140 and Gln468 whereas stalk ___domain is found to have residues like Arg195, His197, Lys222, and Arg282, Arg28613,15. These regions are found to serve as potential sites for the attachment of the motifs for the genomic RNA. With a greater affinity for the negative viral RNA than the positive sense RNA, the CCHFV nucleoprotein exhibits differential affinities for the attachment of negative and positive sense RNA strands16,17. CCHF being a global issue has no specific therapy for its prevention. The drug named Ribavirin was suggested in the past by WHO for its treatment18. One of the studies mentions the treatment of CCHF by the use of FDA drugs doxycycline and minocycline with relative affinities of -9.1 and − 8.5 kcal/mol respectively. It has been mentioned that the 4AQG protein which is the nucleoprotein of CCHFV has an active site named Lys411 (Lysine) being the potential source of nucleoprotein mutation hence controlling CCHFV19.

Some of the recent studies also have mentioned the vaccine for the control of CCHF. Bulgaria is one of them but is not approved by the US FDA and European Medicine Agency due to which it is inactive regarding its use for the treatment of CCHF20. CCHF_GN728 is also another suggested vaccine for the treatment of CCHF with good efficiency to control the CCHF virus but it still needs to be approved21. Apart from synthetic drugs, natural drugs are also being developed using computational chemistry. Computational chemistry is being used in this modern era for the development of drugs helping medicinal chemistry. Many useful and effective drugs have been developed using modern techniques in computational chemistry22,23. The use of phytochemicals in drug development utilizing computational chemistry against CCHF can be an effective way to control CCHF.

Phytochemicals are chemicals that are naturally produced by plants and have potential resistance against several disease-causing agents. Phytochemicals can be used against viral diseases24. With the advancement in silico approaches are attaining attention universally for their effectiveness regarding medical treatments. The use of phytochemicals is primarily cost-effective and can be used for drug design. CCHF being a global issue has still no specific disease control method. There is a much need to develop a drug or some anti-CCHF agent that can be the source of controlling CCHF. The development of a new molecule will require a lot of expenses for its development and its approval. Using the existing FDA-approved drugs and phytochemicals is the alternative way of controlling the CCHF disease.

This study aims to propose the use of phytochemicals in the control of CCHF viral disease by analyzing their interactions with the viral nucleoprotein. For this particular task binding site of nucleoprotein was analyzed and phytochemicals were docked with nucleoprotein over this potential site and different results were obtained showing good interactions with the nucleoprotein.

Materials and methods

The CCHFV nucleoprotein and several phytochemicals were used in this study’s series of studies to find possible inhibitors. In Fig. 1, the flow chart is shown below.

Obtaining phytochemicals

Although, the database had several phytochemicals about 200 phytochemicals were obtained from the database as they were reported to have potential inhibiting capability and pharmacological properties against the CCHF virus and other hemorrhage viruses and low toxicity. The three-dimensional Structures (3D) of the phytochemicals were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov). The properties involving pharmacology and pharmacokinetic behavior based on the Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) were assessed using the SwissADME webserver and PreADMET server being reported in the past25,26] and [27,28,29. The ADME properties of the phytochemicals were evaluated using SwissADME30. On the other hand, the toxicity level of the drugs was assessed by using PreADMET. The predictions were done using SDF structural file. Figure 1 shows the flow chart of the methodology used in this study.

Fig. 1
figure 1

Flow chart illustrating methodology.

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Also, to evaluate the properties of these phytochemicals being drugs Lipinski’s rule of five was employed. Different lenses were used to analyze the drug-like characteristics of the phytochemicals by setting Lipinski’s violation to 0, solubility to high, GI absorption was made high, blood-brain barrier penetrability set to no, and toxicity to zero.

Obtaining protein of CCHFV

The 3D x-ray crystallographic structure of the protein was available on RCSB under PDB ID: 4AQG. The protein has chains A and B [auth A (If the two PDB chain IDs (label_asym_id: assigned by the PDB) and auth _asym_id (selected by the author) do not coincide then the chain ID is presented as “label_asym_id [auth, auth _asym_id].)]. Chain A was found to sequence length of 483 without any mutation while B [auth A] contained a residue group in the form of SO4 group. This SO4 group was removed using UCSF Chimera tools before the docking of the protein with phytochemicals. Also, the water molecules and ligands were removed using Chimera tools.

Analysis of molecular docking and binding

The docking approach was used to ascertain the phytochemicals that exist naturally and their capacity to inhibit the specific protein. The molecular docking of the protein and ligands was done using Vina AutoDock tools (version 1.5.7)31. The binding energies were evaluated, and binding interactions were analyzed. Ki values were calculated using binding energies resulting from the docking analysis by Eq. 1 given below:

$${\varvec{K}}_{\varvec{i}}={\varvec{e}}^{^(-}\frac{\varDelta\:\varvec{G}}{\varvec{R}\varvec{T}}$$
(1)

Equation 1: Equation for calculating Ki values.

Binding energy is denoted by ΔG, the temperature is represented by T which is about 298.15 K and R illustrates the gas constant having a value of 1.98 kcal/mol. The binding affinities of the inhibitors were analyzed using docking analysis. Vina-Auto Dock tools were used to generate a grid box having x, y, and z dimensions. The grid box values are given below in Table 1.

Table 1 Grid box dimensions.
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The docking analysis was performed using the size of 40 along x, y, and z as mentioned in Table 1, and the exhaustiveness used was 8. Discovery Studio was used to get 2D and 3D pictures of the binding based on the docking analysis output files32. Groningen Machine for Chemical Simulations (GROMACS) v 5.0 was used to run MD simulations at a constant temperature to investigate the stability of phytochemical binding to proteins33. Only those complexes with high binding energies for phytochemicals were included in the analysis. For each protein-ligand combination, the optimal potential was used for liquid simulation, and the system was solved using spc216 water molecules. By introducing Na + and Cl-ions, the water-containing system was neutralized. With the limit set at 50,000, the system was designed to minimize energy using the most cutting-edge technique. Then, at 300 K in temperature and 1 atm in pressure, constant number volume and temperature (NTV) and constant number pressure and temperature equilibrations were carried out. All the simulations were made to run at a specific pH of 7.0 and pH was made 7 by manual protonation of residues. The simulations were kept following the human biological system. Particle Mesh Ewald (PME) was the force field that was used in the equilibrations with a three-dimensional cubic interpolation and the duration was 10 ns34. Linear Constraint Solver (LINCS) was used to readjust the hydrogen bonds. Using the same approach as equilibrations, the final MD simulation was set to run for 50 ns35.

DFT studies of reactivity

The reactivity of ligands with nucleoprotein of CCHFV was investigated using density functional theory (DFT) analysis. For research purposes, the energies for Highest Occupied Molecular Orbitals (HOMO) and Lowest Unoccupied Molecular Orbitals (LUMO) were calculated. The HOMO and LUMO energy gap provide insights into the reactivity and stability of a molecule. A lower energy gap indicates high reactivity while a higher energy gap indicates lower reactivity. The expression ELUMO - EHOMO was used to calculate the band energy gap or E. These descriptors were created using the Object-oriented Real-time Control and Acquisition (ORCA) software which has been based on quantum physics and related calculations36. The calculations were made using hybrid exchange-correlation functional B3LYP, which is an exchange-correlation functional. Typically, the Hartree-Fock exact exchange function and any other density function are combined to form the hybrid exchange correlation. But the B3LYP, or targeted correlation, is defined as:

$$E_{{xc}} ^{{{\text{B3LYP}}}} = {\text{E}}_{{\text{x}}} ^{{{\text{LDA}}}} + {\text{a}}_{{\text{o}}} {\text{ }}\left( {{\text{E}}_{{\text{x}}} ^{{{\text{HF}}}} {-}{\text{ E}}_{{\text{x}}} ^{{{\text{LDA}}}} } \right){\text{ }} + {\text{a}}_{{\text{x}}} \left( {{\text{E}}_{{\text{x}}} ^{{{\text{GGA}}}} {-}{\text{E}}_{{\text{x}}} ^{{{\text{LDA}}}} } \right){\text{ }} + {\text{E}}_{{\text{c}}} ^{{{\text{LDA}}}} + {\text{a}}_{{\text{c}}} \left( {{\text{E}}_{{\text{c}}} ^{{{\text{GGA}}}} {-}{\text{E}}_{{\text{c}}} ^{{{\text{LDA}}}} } \right)$$

where a0 = 0:20, ax = 0:72, and ac = 0:81. EGGA x is the generalized gradient approximation for the Becke 88 functional while the EGGAc reflects the correlation functional of Lee-Yang-Parr. With this hybrid functional, local density approximation is added in the form of ELDAc37.

Results and discussion

Crimean Congo Virus is one of the deadliest viruses ever found in human history but there is still no vaccine for the prevention of Crimean Congo fever. There is an innate need to design a drug for the treatment of CCHF. In recent studies, the inhibitors of the nucleoprotein of CCHFV are being found. This study presents natural inhibitors in the form of phytochemicals having drug-like properties for the inhibition of nucleoprotein of CCHFV (Fig. 2).

Evaluation of protein structure

The possible binding sites of nucleoprotein that are reported in the literature in the head ___domain and stalk ___domain such as the head ___domain mainly comprises residues like Lys339, Lys343, Lys346, Arg384, Lys411, His453, Gln457, Arg134, Arg140 and Gln468 whereas the stalk ___domain is found to have residues like Arg195, His197, Lys222, and Arg282, Arg28638. After analyzing these sites phytochemicals were docked with the proteins and results are given in tabular forms.

Molecular docking and binding analysis of phytochemicals

The obtained phytochemicals having good properties with low toxicity were docked with nucleoprotein of the Crimean Congo Virus and results are shown in Figs. 2, 3, 4, 5, 6 and 7; Table 2.

Fig. 2
figure 2

Nucleoprotein of CCHFV (https://discover.3ds.com/discovery-studio-visualizer-download).

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Fig. 3
figure 3

2D model representing interaction of Skullcapflavone I with nucleoprotein of CCHFV.

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Fig. 4
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2D model representing Interaction of Niazirin with nucleoprotein of CCHFV.

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Fig. 5
figure 5

2D model representing interaction of Quercetin with nucleoprotein of CCHFV.

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Fig. 6
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2D model representing Interaction of Luteolin with Nucleoprotein of CCHFV.

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Fig. 7
figure 7

2D model representing Interaction of Withanolide E with Nucleoprotein of CCHFV.

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Table 2 Results of the interaction of phytochemicals with nucleoprotein of CCHF.
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The nucleoprotein was docked first with Skullcapflavone I. The binding energy was obtained as −8 kcal/mol. The 2D interaction of Skullcapflavone I is shown in Table 2 in Fig. 3 and the 3D interaction is shown below in Fig. 8. The unfavorable Donor-Donor interaction is shown with red dotted lines. The presence of pi (π) bonds is illustrated using yellow dotted lines. Grey-colored dotted lines are used to depict the π-donor hydrogen bonds. π-π interaction is highlighted using a dark pink color. The interaction between π electrons and alkyl groups is shown by a light pink color.

The docking of Niazirin with the nucleoprotein released energy of about − 8.3 kcal/mol and the respective interaction is shown in Table 2 in Figs. 3d and 4 interaction is shown below in Fig. 9. The interaction of Quercetin resulted in a binding energy of about − 8 kcal/mol and the interaction is shown in Table 2 in Fig. 5. The conventional hydrogen bond is represented by a green dotted line whereas the presence of π-cation interaction is illustrated using yellow dotted lines. The unfavorable Donor-Donor interaction is shown with red dotted lines. π-σ electronic interaction is highlighted using a dark pink color. The interaction between π-electrons and alkyl groups is shown by a light pink color. The grey color highlights the Carbon-Hydrogen interaction. The nucleoprotein of CCHFV was docked with Luteolin and the binding energy of -8.1 kcal/mol was observed. The interaction of phytochemicals as ligands with nucleoproteins is shown in Table 2 in Fig. 6. The conventional hydrogen bond is represented by a green dotted line whereas the presence of π-cation interaction is illustrated using yellow dotted lines. The unfavorable Donor-Donor interaction is shown with red dotted lines. π-σ electronic interaction is highlighted using a dark pink color. The interaction between π-electrons and alkyl groups is shown by a light pink color. The grey color highlights the Carbon-Hydrogen interaction.

Withanolide E was docked over with nucleoprotein of CCHF and it resulted in the binding energy of -9.4 kcal/mol and the interaction is shown in Table 2 in Figs. 3D and 7 interaction is shown below in Fig. 10. The conventional hydrogen bond is represented by a green dotted line whereas the grey dotted line highlights the π-Donor hydrogen bond. The presence of alkyl groups is shown by a light pink color.

Fig. 8
figure 8

Interaction of Skullcapflavone I with nucleoprotein (https://discover.3ds.com/discovery-studio-visualizer-download).

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Fig. 9
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Interaction of Niazirin with nucleoprotein (https://discover.3ds.com/discovery-studio-visualizer-download).

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Fig. 10
figure 10

Interaction of Withanolide E with nucleoprotein (https://discover.3ds.com/discovery-studio-visualizer-download).

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Complexity and stability analysis using MD simulations

Once the interactions of the compounds with receptors were examined, MD simulations were used to explore the stability of the complexes and the ligand binding to the proteins. The stability of the binding nucleoprotein of screened ligands was ascertained using these MD simulations. To look at the stability of complexes, the radius of gyrations, or Rg, was plotted to analyze the stability of complexes. The radius of gyration varied less in Fig. 11. These values demonstrate strong protein tertiary structure stability, compactness, and stable folding in addition to stability in protein-ligand complexes. All complexes were subject to changes and fluctuations, not just one complex. Because these compounds were not strongly bound, the stability and compactness of the complexes with lower binding affinity decreases as evident from preceding trends39.

Fig. 11
figure 11

Graph showing the radius of gyration.

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Reactivity studies for complexes of targeted proteins and phytochemicals

The reactivity of the Phytochemicals and the nucleoprotein of CCHFV was computed using DFT (Density functional) computations. The findings demonstrated that both proteins were well-bound by phytochemicals with a wide range of chemical diversity. For the nucleoprotein reactivity analysis, different compounds were chosen namely Withanolide E, Niazirin, Luteolin, Skullcapflavone I and Quercetin. Table 3 displays the results of computing the band energy gap for the study of reactivity using molecular orbital energy descriptors.

Thus, the results shown in Table 3 demonstrated the high reactivity of phytochemicals with specific receptors. There is strong reactivity at lower band energy gaps. The results showed that the band energy gaps were small and had high reactivity qualities. The band energy gap values were 0.227 kcal/mol, 0.219 kcal/mol, 0.273 kcal/mol, 0.324 kcal/mol, and 0.307 kcal/mol. To investigate the molecular orbital energies, the B3LYP function from DFT was used. It is well known in the literature that compounds with a smaller band energy gap are more reactive. The explanation is that molecular descriptors which are used to compute band energy gaps are also in charge of transferring charges during chemical reactions. These energies can describe a substance’s nucleophilic or electrophilic nature of the compound. Hence, the screened phytochemicals showed high reactivity as reported previously as well. In one of the previous studies, it has been reported that FDA-approved drugs can be used in the treatment of CCHF being inhibitors of the nucleoprotein of Crimean Congo fever virus40. Although FDA-approved drugs are found to be effective being inhibitors of the nucleoprotein of CCHFV they have side effects as well. In the ongoing study, natural inhibitors of CCHFV have been found which can inhibit the nucleoprotein of CCHFV and eventually can help in the treatment of CCHF.

Table 3 Table showing DFT reactivity studies.
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The phytochemicals are found to be less likely to cause a disease being a side effect but on the other hand, FDA-approved drugs have some side effects as well. The adverse reactions that a medication may cause are known as side effects and are typically described as unpleasant responses. The severity of side effects could vary from minor issues like a runny nose to potentially deadly circumstances like liver damage or a heart attack. Drowsiness, dry mouth, and upset stomach are typical side effects. Serious side effects include those that cause hospitalization, permanent impairment, death, life-threatening conditions, exposure before conception, or exposure during pregnancy. On the contrary, phytochemicals are not found to cause all of these side effects as mentioned above. So, it is better to use naturally produced drug-like chemicals rather than using synthetic drugs41,42,43,44,45,46,47. However, there are a few limitations to using phytochemicals as drugs including bioavailability, and complex structures as they are natural sources of drugs that react with other drugs used in combination and also, they are difficult to standardize. These limitations can be overcome using advanced isolation and characterization techniques and by use of Nano-capsulation.

Conclusions

Crimean Congo fever is a worldwide disease mostly found in Africa and Asia, having a high ability to cause fatal issues and a significant mortality rate. It is declared endemic by the World Health Organization. However, there is no specific medication, vaccine, or drug for the treatment of this disease is available. This study highlights the use of phytochemicals to inhibit nucleoprotein of CCHFV which is an essential part of CCHFV. Through the analysis of phytochemicals by docking them against the nucleoprotein of the CCHF virus, it is concluded that Skullcapflavone I, Niazirin, and Withanolide E have a good ability to inhibit nucleoprotein of CCHF virus based on the pharmacological properties, stability, reactivity, and binding affinities while Withanolide E is the best of among all of them concerning its binding affinity with the nucleoprotein of CCHFV. It can be considered as an inhibitor for targeting nucleoprotein as a drug with proper formulations after being examined in vitro and in vivo tests which include cytotoxicity, mechanism of action, and bio-marker analysis, to monitor treatment response and predict outcomes and going through advanced screening and isolation techniques along with clinical trials.