Withania somnifera extracts induced attenuation of HIV-1: a mechanistic approach to restrict viral infection

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In vitro cytotoxicity of Withania somnifera extracts on TZM-bl cell line and PBMCs

WSAQ and WSHA extracts were initially examined for their cellular viability on TZM-bl cell lines and PBMCs using MTT quantitative colorimetric assay. Increasing the concentrations directly attributed to the increase in the cytotoxicity for WSHA and WSAQ extracts in the TZM-bl cells (Fig. 1A and B), as well as in the PBMCs (Fig. 1C and D). The CC₅₀ values of 0.269 mg/ml and 2.136 mg/ml were determined for WSHA and WSAQ extracts, respectively (Fig. 1E). A similar pattern was observed in PBMCs as well, where CC₅₀ values were obtained as 0.252 mg/ml and 1.987 mg/ml, respectively, for the hydroalcoholic and aqueous extracts of Withania somnifera (Fig. 1E).

Anti-HIV-1 activities of Withania somnifera extracts

The activity of WSHA and WSAQ against two clades of HIV-1 strains was tested in the TZM-bl cell line and later the same was validated in PBMCs. The concentration below the cytotoxic levels in accordance with CC₅₀ values was used for anti-HIV-1 screening. The potential of extracts to inhibit virus replication through cell-associated (CA) as well as cell-free (CF) assays was evaluated subsequently. Azidothymidine (AZT: 0.49 µM) and Dextran sulfate (DS: 15 µg/ml) were used as positive controls in CA and CF assays, respectively.

Inhibition of HIV-1 infection by the extracts in TZM-bl cells

In the cell-associated assays, the pre-infected TZM-bl cells were treated with the extracts of Withania somnifera, with a range of multiple concentrations (WSHA: 0.016–0.250 mg/ml, and WSAQ: 0.125–2.000 mg/ml) below the CC₅₀ values, showed a clear dose-dependent inhibition of HIV-1 infection for both clades, HIV-1_VB028 and HIV-1_UG070 (Fig. 2A and B). While, the cell-free assay, where the virus was introduced to the different concentrations of the extracts before the administration to the cells, showed similar dose-dependent anti-HIV-1 activity in the presence of WSHA (0.016–0.250 mg/ml) and WSAQ (0.125–2.000 mg/ml) (Fig. 2C and D).

The activity of WSHA extract showed excellent inhibition of HIV-1 infection even at the lower dosages, revealing the half maximal effective concentration or EC₅₀ values of 0.029 mg/ml and 0.020 mg/ml in CA assays, and 0.022 mg/ml and 0.001 mg/ml in CF assays, for the HIV-1_VB028 and HIV-1_UG070, respectively (Fig. 2E and F). Whereas, the EC₅₀ values of WSAQ extract were identified as 0.378 mg/ml and 0.273 mg/ml in CA and 0.130 mg/ml and 0.239 mg/ml in CF for the inhibition of HIV-1_VB028 and HIV-1_UG070 strains, respectively (Fig. 2E and F).

Inhibition of HIV-1 infection in peripheral blood mononuclear cells (PBMCs)

Consequently, the CA and CF confirmatory assays using the primary isolate HIV-1_VB028 infected PBMCs, and WSHA extracts at different concentrations (0.016–0.250 mg/ml) showed a dose-dependent inhibition of HIV-1 p24 protein (Fig. 3A). The EC₅₀ values for both the CA and CF assays were obtained as 0.098 mg/ml and 0.017 mg/ml, whereas the EC₈₀ values as 0.123 mg/ml and 0.047 mg/ml for the WSHA extract in PBMCs (Fig. 3B).

Similarly, the WSAQ extracts also showed a comparable pattern of dose-dependent (0.031–1.000 mg/ml) inhibition of HIV-1 p24 protein in both CA and CF assays (Fig. 3C). However, both the EC₅₀ and EC₈₀ values of WSAQ in PBMCs were observed to be 0.059 mg/ml and 0.294 mg/ml in the CA assay, and 0.059 mg/ml and 0.307 mg/ml in the CF assay (Fig. 3D).

FDA-approved standard drugs, AZT and DS, showed 100% inhibition of HIV-1 both in the TZM-bl and PBMCs in the in vitro CA or CF assays and confirmatory studies (data not shown).

Elucidation of the mechanism of action of extracts by TOA assay

The time-of-addition assay (TOA) was conducted for the Withania somnifera extracts, WSHA (0.029 mg/ml) and WSAQ (0.378 mg/ml), with known antiretrovirals to determine the targets of these extracts. Final concentrations of drugs and extracts were introduced at various time intervals before and/or after the HIV-1 infection (0, 1, 2, 4, 6, 8, 12, 16, and 24 h). The infection percentage (RLU) was calculated accordingly. It was noted that the extract of WSAQ’s inhibition began to reduce at 16 hpi, but the inhibition of WSHA activity was initiated at 8 hpi. According to the findings, WSHA followed an inhibition pattern similar to the known HIV-1 Integrase inhibitor RAL, whereas, the WSAQ exhibited a pattern similar to the HIV-1 Protease inhibitor RTV (Fig. 4). Hence, it can be predicted that the modus operandi of WSHA and WSAQ might be similar to the known Protease and Integrase inhibitors of HIV-1 inhibition, respectively.

In vitro inhibition of HIV-1 Integrase by Withania somnifera extracts

To interrogate potential anti-HIV-1 mechanisms of Withania somnifera extracts, in vitro HIV-1 Integrase assay was conducted with the different concentrations of WSHA (0.002–0.200 mg/ml) and WSAQ (0.016–2.000 mg/ml). Whereas WSHA showed maximum inhibition of 86.18% at a concentration of 0.200 mg/ml (Fig. 5A), 93.98% inhibition was observed at the highest sub-cytotoxic concentration (2.000 mg/ml) of WSAQ (Fig. 5B). Based on the obtained results of dose-dependent inhibition of HIV-1 Integrase, the EC₅₀ values were calculated as 0.010 mg/ml for WSHA and 0.070 mg/ml for WSAQ. The kit supplied positive control, Sodium Azide (1%), exhibited 95.99% inhibition, whereas, Raltegravir (0.48 µM) was taken as an additional inhibitor of HIV-1 Integrase, showed 100.0% inhibition in support of the assay validation.

In vitro inhibition of HIV-1 Protease by Withania somnifera extracts

Furthermore, we examined the activity of WSHA and WSAQ extracts in HIV-1 Protease inhibition through the in vitro kit-based assay with the different concentrations of WSHA (0.006–0.200 mg/ml) and WSAQ (0.063–2.000 mg/ml). The result revealed 91.776% inhibition of HIV-1 Protease activity at 0.200 mg/ml for WSHA extract (Fig. 5C), while 84.840% inhibition for WSAQ at 2.000 mg/ml (Fig. 5D). Based on the obtained results of dose-dependent inhibition of HIV-1 protease, the EC₅₀ values were calculated as 0.024 mg/ml and 0.337 mg/ml for WSHA and WSAQ, respectively. The result was compared with the known HIV-1 Protease inhibitor Ritonavir (10 µM) as a positive control with 77.65% inhibition of Protease activity, as well. Further, the assay was validated with the kit-provided Inhibitor control, Pepstatin (1 mM) and Enzyme control (EC).

In vitro inhibition of HIV-1 Reverse transcriptase by Withania somnifera extracts

The Withania Somnifera extracts were evaluated for their potential to inhibit HIV Reverse transcriptase as described previously [12]. The WSHA showed 38.46–76.82% inhibition of HIV-1 RT activity in a dose-dependent (0.006–0.200 mg/ml) kinetics assay (Fig. 5E). However, the multiple sub-cytotoxic concentrations of WSAQ extract (0.063–2.000 mg/ml) revealed not-so-promising inhibition of HIV-1 RT activity with a range of 24.63–58.53% only (Fig. 5F). Based on the obtained results, the EC₅₀ values were found to be 0.016 mg/ml and 1.286 mg/ml for WSHA and WSAQ, respectively. The results were compared with the known RT inhibitor AZT (0.49 µM) with more than 90% inhibition of the HIV-1 RT enzyme.

Molecular docking simulations of phytomolecules against HIV-1 proteins

The phytomolecules from natural sources have significantly impacted human health due to their medicinal potential according to existing literature. Most of these phytomolecules might be good leads as druggable pharmacophores with lesser or even no toxic side effects due to their natural occurrence. During the HIV infection cycle, HIV-1 Integrase, HIV-1 Protease, and HIV-1 Reverse transcriptase proteins are expressed and well-documented in the literature as causative agents of HIV infection. The phytomolecules or the active metabolites, characterized from the root extracts of Withania somnifera prepared in hydroalcoholic and aqueous solvent using UHPLC-PDA and mass spectrometry studies, were used for molecular docking simulations [31]. AutoDoc v4.2 was used to understand the inter- as well as intra-molecular interactions between the HIV-1 proteins and the bioactive phytomolecules. The binding energy scores were used to assess the strength of HIV-1 protein-phytomolecule interactions. In docking simulations, all parameters were set to default, however, the protein structures were made rigid and bioactive ligands provided flexibility to interact efficiently. Stable protein–inhibitor complexes were obtained after molecular docking simulations due to molecular interactions between the phytomolecule and residues of the protein. The molecular interactions profile of HIV-1 proteins and the active metabolites are presented in Table 1.

Molecular interaction between HIV-1 Integrase and 12-Deoxywithastramonolide & 27-Hydroxywithanone

HIV-1 Integrase is one of the essential enzymes involved in the retrovirus’ replication cycle and it stimulates the integration of viral DNA that has undergone Reverse transcription into chromosomal DNA. Such integration of HIV-1 DNA ensures the stability of the viral genome, hence, the virus continues to exist in the host [37]. In the present study, the molecular docking analysis of HIV-1 Integrase (PDB: 1QS4) with 12-Deoxywithastramonolide and 27-Hydroxywithanone revealed low binding energies of -7.83 Kcal/mol and -7.75 Kcal/mol, respectively, for molecular conformations induced by the phytomolecules (Table 1). 12-Deoxywithastramonolide established two hydrogen bonds with THR66, and ASN155 within 4Å cut-off and nine hydrophobic interactions with ASP64, HIS67, GLU92, ASP116, GLY118, SER119, ASN120, GLU152, and LYS159 (Fig. 6A), whereas, 27-Hydroxywithanone established three hydrogen bonds with GLU92, ASN155, and LYS159 residues within 4Å cut-off and nine hydrophobic interactions with ASP64, THR66, HIS67, ASP116, GLY118, SER119, ASN120, GLU152, and LYS156 residues during molecular docking simulations in the vicinity of the active site binding pocket responsible for the catalytic activity of HIV-1 Integrase (Fig. 6B).

The molecular docking simulations interaction analyses for the known FDA approved HIV-1 Integrase inhibitors, viz. Cabotegravir, Dolutegravir and Raltegravir were presented in Table 2. Cabotegravir was able to establish only five hydrogen bond with CYS65, HIS67, GLU92, SER119, and LYS159 within 4Å cut-off and eight hydrophobic interactions with ASP64, THR66, ASP116, GLY118, ASN120, GLU152, ASN155, and LYS156 in the active binding pocket residues of HIV-1 Integrase (Fig. 7A), however, Dolutegravir established three hydrogen bonds with HIS67, LYS159, and ASN155 within 4Å cut-off and six hydrophobic interactions with ASP64, CYS65, THR66, ASP116, ILE151, and LYS156 (Fig. 7B). Furthermore, Raltegravir established three hydrogen bonds with ASP64, ASP116, and LYS159 within 4Å cut-off and ten hydrophobic interactions with CYS65, HIS67, GLU92, GLY118, SER119, PHE121, GLU152, ASN155, and LYS156 (Fig. 7C). Interestingly, the lower binding energy of -7.83 Kcal/mol for 12-Deoxywithastramonolide and  -7.75 Kcal/mol for 27-Hydroxywithanone emphasizes strong binding capabilities to inhibit HIV-1 Integrase activity and higher than the FDA approved drugs (Table 2).

In silico analysis also elucidated that phytomolecules from Withania somnifera, such as Withanolide B (-6.93Kcal/mol), Withanone (-6.77 Kcal/mol), Withacoagin (-6.49 Kcal/mol), Withaferin A (-7.29 Kcal/mol), and Withanolide A (-6.16 Kcal/mol) could be responsible for inhibition of HIV-1 Integrase precisely, as evidenced from the binding energy analysis, hydrogen bond, and hydrophobic bond interactions (Table 1 and Figure S1).

Molecular interaction between HIV-1 Protease and Ashwagandhanolide & Withacoagin

HIV-1 Protease is crucial for HIV replication and maturation into an infectious form. Therefore, one of the main focus areas for HIV-1 drug development efforts is primarily the inhibition of this enzyme [38]. The HIV-1 Protease (PDB:5KR0) activity may also be inhibited by the phytomolecules of Withania somnifera as identified through the in silico molecular interactions in the present docking simulations study. Out of the ten such phytomolecules, Ashwagandhanolide (-11.53 Kcal/mol) and Withacoagin (-10.79 Kcal/mol) were identified as the lead molecules, however, others were also found effective by binding in the active binding pocket of HIV-1 Protease. Ashwagandhanolide established three hydrogen bonds with LEU24, ASN25, and THR26 within 4Å cut-off and thirteen hydrophobic bond interactions with PRO9, GLY27, ASP29, ASP30, VAL32, GLY48, GLY49, GLY52, PHE53, ILE54, THR80, PRO81, VAL82 and three Pi-alkyl bonds with LEU23, ILE47, and ILE50 residues present in the active binding pocket (Table 1; Fig. 8A). Additionally, Withacoagin established molecular interactions with HIV-1 Protease residues via five hydrogen bonds with ASP29, GLY48, GLY49, ILE50, and ILE54 within 4Å cut-off and nine hydrophobic interactions with GLY27, ASP30, VAL32, LYS45, MET46, GLY51, GLY52, PHE53, ILE84 residues available in binding pocket (Table 1; Fig. 8B). The in silico analysis was also performed on FDA approved HIV-1 Protease inhibitor Ritonavir. Interestingly, Ritonavir was able to establish only two hydrogen bonds with ASN25 and ILE50 within 4Å cut-off and ten hydrophobic interactions with LEU24, THR26, GLY27, VAL32, GLY48, PHE53, PRO79, THR80, VAL82, and ILE84 residues of HIV-1 Protease active binding pocket (Table 2; Fig. 8C). The molecular docking simulations were further extended to evaluate other phytomolecules of Withania somnifera against HIV-1 Protease. The phytomolecules viz., 27-Hydroxywithanone (-7.97 Kcal/mol), Withanolide A (-10.75 Kcal/mol), 12-Hydroxywithastramonolide (-10.72), Withanolide B (-10.55 Kcal/mol), and Withaferin A (-9.65 Kcal/mol) interacted with HIV-1 Protease showing promising binding energies, forming many hydrogen bonds within 4Å cut-off and hydrophobic bond interactions with active binding site pocket residues (Table 1 and Figure S2). All the above-mentioned residues occupied by these phytomolecules are responsible for the activation of HIV-1 Protease, however, this occupancy renders the inhibitory effect on HIV replication.

Molecular interaction between HIV-1 Reverse transcriptase and Ashwagandhanolide & Withanolide B

The HIV-1 Reverse transcriptase or RT is a unique and essential enzyme that cooperates in the reverse transcription step of HIV-1 replication. The HIV-1 RT (PDB:3QIP) activity may also be inhibited by the phytomolecules Ashwagandhanolide and Withanolide B, as they were ranked the best among all the tested phytomolecules of Withania somnifera. Ashwagandhanolide exhibited  -11.16 Kcal/mol binding energies interacting with HIV-1 Reverse transcriptase via forming five hydrogen bonds with HIS96, ILE94, THR165, ARG172, and GLN182 residues within a 4 Å cut-off and seven hydrophobic bond interactions with VAL90, GLY93, GLY99, HIS101, GLN161, MET184, and VAL381 residues and six Pi-alkyl bonds with PRO95, LEU100, VAL179, THR181, TYR183, and ILE382 residues found in the active site (Table 1; Fig. 9A). Whereas, Withanolide B found molecular interactions with HIV-1 RT via forming three hydrogen bonds with ARG172, ILE180, and TYR181 within a 4 Å cut-off along with four hydrophobic interactions with LYS101, TYR188, VQL189, and GLY190 residues. Moreover, three Pi-alkyl interactions with LEU100, VAL106, and VAL179 were also established with the binding pocket residues of HIV-1 RT by Withanolide B (Table 1; Fig. 9B). Withanolide B was found to be the top ranking phytomolecule that exhibited the least binding energy of -11.94 Kcal/mol in comparison to any other phytomolecules found during this study (Table 1).

These residues are part of the active binding pocket of HIV-1 RT and are responsible for the catalytic mechanism. The occupancy of these important residues by Ashwagandhanolide and Withanolide B might hinder the opportunity for HIV-1 viral protein to replicate. The HIV-1 RT activity governed by these important residues might also be impeded as they were occupied by many phytomolecules, for example Withanolide A (-10.33 Kcal/mol), Withanoside V (-10.13 Kcal/mol), Withaferin A (-9.48 Kcal/mol), Withacoagin (-9.47 Kcal/mol), Withanone (-7.8 Kcal/mol), 27-Hydroxywithanone (-7.53 Kcal/mol), and 12-Deoxywithastramonolide (-7.37 Kcal/mol) (Table 1 and Figure S3). These phytomolecules successfully established hydrogen and hydrophobic bond interactions with many important residues found in the active binding pocket of HIV-1 RT and might be responsible for the inhibition of HIV-1 replication, therefore restricting the viral infection.

A control in silico experiment with FDA approved HIV-1 Reverse transcriptase inhibitor, Zidouvdine, was performed for molecular interaction comparison. It is quite interesting to note that Zidouvdine bound with HIV-1 RT with a binding energy of -7.23 Kcal/mol which was still far less than most of the phytomolecules of Withania somnifera (Table 2; Fig. 9C). Molecular interaction analysis also revealed that Zidouvdine established only two hydrogen bonds with LYS101 and HIS235 within a 4Å cut-off and only seven hydrophobic interactions with LYS102, VAL179, GLY190, PHE227, PRO236, ASP237, and LYS238 residues (Fig. 9C).

In silico prediction of ADMET properties of phytomolecules

The pharmacokinetics and drug-likeliness potential of the ten phytomolecules were predicted using the Swiss-ADME, cheminformatics platform. Bioavailability RADAR analysis showed that Withacoagin, Withaferin A, Withanone, Withanolide A, and Withanolide B are orally bioavailable except Ashwagandhanolide, Withanoside IV, and Withanoside V (Table 3). The ADMET properties also reveal that the phytomolecules selected in this study will be the substrate of the P-glycoprotein transporter (Table 3). P-glycoprotein is a part of the ATP-binding cassette (ABC) transporter, therefore, the answer “yes” for the P-glycoprotein substrate denotes that all phytomolecules are predicted to be transported across the cell membrane by the ABC transporter. However, none of the molecules has shown an ability to penetrate the blood-brain barrier (BBB). The measured skin permeation coefficient (Log Kp cm/s) for all phytomolecules with a value of ≤ -2.5 suggests poor skin permeability due to high molecular size and lipophilicity. However, all the phytomolecules can be well absorbed into the body of the patients.

The results of the docking interaction of phytomolecules with the HIV-1 proteins (i.e., Integrase, Protease and Reverse transcriptase) revealed low binding energies, which means the selected phytoconstituents might establish good atomic interactions with the active binding pocket residues. Furthermore, the structural properties of these selected phytomolecules of Withania Somnifera are presented in Table 4.

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