Int J Pharm Pharm Sci, Vol 12, Issue 8, 30-35Original Article

PHYTOCHEMICAL TO INTERACT WITH NLS BINDING SITE ON IMA3 TO INHIBIT IMPORTIN Α/Β1 MEDIATED NUCLEAR IMPORT OF SARS-COV-2 CARGO

BHARATH B. R.1*, HRISHIKESH DAMLE2, SHIBAN GANJU2, LATHA DAMLE1

1Atrimed Biotech LLP, BBC, Electronics City Phase 1, Electronic City, Bengaluru, Karnataka 560100, 2Atrimed Pharmaceuticals Pvt. Ltd. 14th Floor no 29, Prestige Meridian Tower 2, 30, Mahatma Gandhi Rd, KG Halli, D' Souza Layout, Ashok Nagar, Bengaluru, Karnataka 560001
Email: bharath.br@atrimed.com

Received: 06 May 2020, Revised and Accepted: 09 Jun 2020

ABSTRACT

Objective: Ivermectin is an FDA-approved, broad-spectrum anti-parasitic agent. It was originally identified as an inhibitor of interaction between the human 29 immunodeficiency virus-1 (HIV-1) integrase protein (IN) and the Importin (IMP) α/β1 30 heterodimers, which are responsible for IN nuclear import. Recent studies demonstrate that ivermectin is worthy of further consideration as a possible SARS-CoV-2 antiviral.

Methods: We built the pathogen-host interactome and analyzed it using PHISTO. We compared Ivermectin and plant molecules for their interaction with Importin α3 (IMA3) using molecular docking studies.

Results: A phytochemical ATRI001 with the lowest binding energy-7.290 Kcal/mol was found to be superior to Ivermectin with binding energy-4.946 Kcal/mol.

Conclusion: ATRI001 may be a potential anti-SARS-CoV-2 agent; however, it requires clinical evaluation.

Keywords: Ivermectin, SARS-CoV-2, IMA3, Phytochemical and Molecular docking

© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2020v12i8.38184. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps.

Speedy peer review was done as the subject of the manuscript was related with pandemic.

INTRODUCTION

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, is a single-stranded positive-sense RNA virus, which is closely related to earlier SARS-CoV [1]. Available reports on SARS-CoV proteins have demonstrated a potential role of IMPα/βduring infection in the signal-dependent nucleocytoplasmic shutting of the SARS-CoV Nucleocapsid protein [2-5], that may cause significant impact on host cell division [6, 7]. Additionally, the SARS-CoV accessory protein ORF6 has been shown to antagonize the antiviral activity of the STAT1 transcription factor by sequestering IMP α/β1 on the rough ER/Golgi membrane [8]. These reports suggest that nuclear transport inhibitory activity of ivermectin may be effective against SARS-CoV-2.

Seven human isoforms of IMA mediate nuclear import of cargo into the tissue in an isoform-specific manner. Active transport of proteins from the cytoplasm to the nucleus is mediated by a family of nuclear transport receptors known as importins (or karyopherins), together with several ancillary proteins, including nucleoporins and Ran [8-10]. The classical nuclear import pathway is initiated by a unique NLS on importin α [11]. The cargo-IMA complex gets transported through the nuclear pore by building a heterotrimer complex with importin β (IMB), necessitating interactions with FG repeat regions on nucleoporin proteins [12, 13].

Once the complex traverse the nuclear envelope, the RanGTP dissociates the complex, and the import receptors get recycled back to the cytoplasm for the next rounds of transport [14-17]. IMA includes importin β-binding (IBB) domain at its N-terminal and NLS binding domain towards C-terminal featuring ten armadillo (ARM)-repeat motifs [18]. Most commonly, the cargo NLS binds on the concave site of the ARM repeats and involves interactions at either the major site through ARM repeats 2–4 or minor site ARM repeats 6–8. The classical monopartite NLSs (e. g., SV40T-ag [19]) are known to interact with the major site, while, human phospholipid scramblase [20] and TPX2 [21] with the minor site. The classical bipartite NLSs like nucleoplasmin interact with both the major and minor sites [19]. Although this process has been well characterized for the importin α1 adaptor protein, many nuclear proteins exhibit specificity for other importin α isoforms.

For example, both RCC1 [22], HIV-1 integrase [23], W protein of Nipah virus (NiV) [24, 25], avian influenza PB2 viral polymerase subunit [26] and SARS-CoV2 bind specifically to importin α3. Whereas, STAT1, a signaling molecule in the innate immune system response, binds specifically to the convex C-terminal surface of importin α5, α6, and α7 [27, 28].

A wide variety of active phytochemicals have been found to have therapeutic applications against viruses. The antiviral mechanism of these agents may be explained by their antioxidant activities, scavenging capacities, inhibition of DNA, RNA synthesis, or blocking of viral reproduction. Epidemiological and experimental studies have revealed that a large number of phytochemicals have promising antiviral activities [29].

More than 220 Phyto-compounds evaluated by others for activity against anti-severe acute respiratory syndrome-associated coronavirus (SARS-CoV) using a cell-based assay measuring the SARS-CoV-induced cytopathogenic effect on Vero E6 cells and compounds [30–32] showed excellent activities [33, 34]. The bioactive compounds with anti-SARS-CoV activity in the mmol range included abietane and labdane-type diterpenes sesquiterpenes and lupane-type triterpenes [34].

The current study is aimed to screen a library of plant small molecules library against IMA3 using molecular docking studies. The plant small molecule library developed in-house, consists of 4,08,000 small molecules, which are classified using physicochemical parameters as major classifiers.

MATERIALS AND METHODS

Topological analysis of pathogen-host interactome (PHI)

The drug target identification and validation were carried out using a network-based topological analysis method using a web-based application Pathogen-Host Interaction Search Tool (PHISTO) available at the URL: http://www.phisto.org/browse.xhtml#. PHISTO is the most comprehensive pathogen-human protein-protein interaction database on the web. It is used to explore molecular connectivities between the pathways in SARS-CoV and Human interaction through topological analysis [35, 36]. The information over the interaction of human IMA3 (Uniprot ID: O00629) was retrieved from PHISTO.

Protein preparation

The crystal structure of IMA3 was pre-processed for docking studies using the Protein Preparation Wizard [37] available in Schrödinger suite 2019-2. Crystallographic water molecules (water molecules without 3 H bonds) were deleted and hydrogen bonds corresponding to pH 7.0 were added, considering the appropriate ionization states for both the acidic and basic amino acid residues. Missing side-chain atoms were added, and breaks present in the structure were built using Prime v4.0, Schrodinger 2019-2 [38]. Using the OPLS_2005 force field [39] energy of the modeled structure was minimized.

Binding site prediction

The crystal structure of Hendra virus W protein C-terminus in complex with IMA3 crystal form 2 (PDB ID: 6BWA) and IMA3 in cargo free state (PDB ID: 6BVZ) was superimposed to understand the conformational differences between cargo bound state and free state. The protein structure alignment and superimposition was performed concerning backbone atoms using Schrodinger package Maestro ver9.3.

Ligand preparation

The three-dimensional conformers of Ivermectin and 4,08,000 small plant molecules in our library were subjected for ligand minimization using the Ligprip application provided in Schrödinger Maestro [39]. The ligand minimization was performed by assigning force field OPLS_2005 and stereoisomers were calculated retaining specific chiralities. The ADME (absorption, distribution, metabolism, and excretion) predictions were done for all ligands using the QikProp package (version 4.6 Schrodinger, LLC, New York, NY, 2015) [40].

Molecular docking

The crystal structure of human IMA3, an adaptor protein involved in the transport of viral protein from the cytoplasmic compartment of an infected cell into a nuclear compartment through NPCs, was prepared using the Protein Preparation Wizard [41]. The major NLS binding site (137–229) was defined with a 10 Å radius around the selected residues (Asn141, Ser144, Trp179, Asn183, and Asn219) present in the crystal structure which are identified as key residues in SARS_nCoV-2 protein and host IMA3 complex formation and a grid box 20X20X20 Å was generated at the centroid of the active site for docking. The molecular docking of prepared small molecules over IMA3 was performed using Grid-Based Ligand Docking with Energetics Glide v7.8, Schrodinger 2019-2 [42] in ‘High Throughput Virtual Screening’ HTVS mode without applying any constraints. Considering the glide score 13,000, molecules were shortlisted and subjected for docking in ‘standard precision’ SP mode. The top molecules with high glide score and Ivermectin were further screened using in ‘extra precision’ mode. The final best-docked structure was selected using a Glide score function, Glide energy, and Glide E model energy. Finally, the lowest-energy docked complex of three plant molecules and ivermectin were interpreted to derive the conclusion.

RESULTS AND DISCUSSION

Topological analysis of pathogen-host interactome and drug target identification

A thorough analysis of virus-host interactomes may reveal insights into viral infection and pathogenic strategies [43, 44]. In the current study, IMA3 centric Virus-Human interactome was built by screening domain interactions between Virus-Human protein-protein interactions (PPIs), as shown in table 1. The list of human viruses, including SARS-CoV is reported to transport their cargo protein through IMA3 (Uniprot ID: O00629) mediated nuclear transportation mechanism. Due to the lack of experimental interaction data on SARS-CoV-2, the significant identity between SARS-CoV (Taxonomy ID: 227859) and SARS-CoV-2 (Taxonomy ID: 2697049) proteomes has encouraged us to considered and the SARS-CoV-Human interactome. Through PHI analysis, it is understood that the transportation of SARS-CoV-2 cargo protein NS6 from the cytoplasmic compartment to the nuclear compartment of the infected host cell is mediated by IMA3 [2,3]. Hence, IMA3 is identified as a potential target and molecular docking was performed.

Binding site prediction

The human IMA3 has its significant function in nuclear protein import as an adapter protein for nuclear receptor KPNB1. IMA3 binds specifically and directly to substrates containing either a simple or bipartite NLS motif [45]. Docking of the importin/substrate complex to the nuclear pore complex (NPC) is mediated by KPNB1 through binding to nucleoporin FxFG repeats and the complex is subsequently translocated through the pore by an energy-requiring, Ran-dependent mechanism. At the nucleoplasmic side of the NPC, Ran binds to IMB, and the three components separate to get re-exported from the nucleus to the cytoplasm where GTP hydrolysis releases Ran from importin [46]. Hence, the NLS site on IMA3 is very important in nuclear protein import.

As tabulated in table 2, IMA3 Consists of an N-terminal hydrophilic region, a hydrophobic central region composed of 10 ARM repeats, and a short hydrophilic C-terminus. The N-terminal hydrophilic region contains the IBB domain, which is sufficient for binding IMB and essential for nuclear protein import [47]. The IBB domain is thought to act as an intragastric autoregulatory sequence by interacting with the internal autoinhibitory NLS as shown in fig. 1.

Fig. 1: Simplified representation of IMA3 and nuclear pore complex of cargo containing a classical NLS occurs via importin-α/importin-β heterodimer. As noted IMA3 has major and minor NLS binding sites specific to cargo type

Table 1: Interaction of human importin α3 (Uniprot_ID: O00629) with diverse pathogen proteins

Pathogen Taxonomy ID Uniprot ID Pathogen protein Experimental method Pubmed ID
Bovine papillomavirus TYPE 1 (BPV 1) 10559 P03116 VE1_BPV1 CIP 17192311
Deltapapillomavirus 4 337052 P03116 VE1_BPV1 CIP 17192311
Hendra virus ISOLATE HORSE/AUSTRALIA/HENDRA/1994 928303 P0C1C6 W_HENDH TAP 22810585
Human herpesvirus 8 37296 O40917 O40917_HHV8 Other Methods 25544563
Human herpesvirus 8 STRAIN GK18 868565 Q2HRC8 ORF11_HHV8P ACT 25544563
Human immunodeficiency virus 1 (HIV1) 11676 B9A2Q4 B9A2Q4_9HIV1 Y2H, CIP, NMR 8659115, 9548947, 8105392, 9282826, 9918876
HIV1 11676 Q79822 Q79822_9HIV1 Y2H, CIP, NMR 8659115, 9548947
HIV1 11676 Q71B33 Q71B33_9HIV1 Y2H, CIP, NMR 8659115, 9548947
HIV1 11676 Q72874 Q72874_9HIV1 FT, imaging technique 10366569, 12414950
HIV1 11676 Q77YF8 Q77YF8_9HIV1 pull down 22174317
HIV1 ISOLATE BRU 11686 P04620 REV_HV1BR pull down 22174317
HIV1 ISOLATE HXB2 11706 P04618 REV_HV1H2 ACT 22174317
HIV1 ISOLATE HXB2 11706 P04585 POL_HV1H2 ATC, pull down 20554775
HIV1 ISOLATE HXB2 11706 P69726 VPR_HV1H2 ATC 20554775
Influenza A virus STRAIN A/AICHI/2/1968 (H3N2) 387139 I6TAH8 I6TAH8_I68A0 ATC 28169297
Influenza A virus STRAIN A/PUERTO RICO/8/1934 (H1N1) 211044 P03466 NCAP_I34A1 pull down, ATC 12740372, 28169297
Influenza A virus STRAIN A/PUERTO RICO/8/1934 (H1N1) 211044 P03428 PB2_I34A1 ATC 28169297
Influenza A virus STRAIN A/PUERTO RICO/8/1934 (H1N1) 211044 P03433 PA_I34A1 ABC 26789921
Influenza A virus STRAIN A/UDORN/1972 (H3N2) 385599 Q20MD0 Q20MD0_9INFA Other Methods 17376915
Influenza A virus STRAIN A/VICTORIA/3/1975 (H3N2) 392809 H9XIJ5 H9XIJ5_I75A3 Molecular sieving, pull down, x-ray crystallography 25599645
Influenza A virus STRAIN A/Wilson-Smith/1933 (H1N1) 381518 P03427 PB2_I33A0 CIP 25464832
Influenza A virus STRAIN A/Wilson-Smith/1933 (H1N1) 381518 P15682 NCAP_I33A0 CIP 25464832
Influenza A virus STRAIN A/Wilson-Smith/1933 (H1N1) 381518 P03470 NRAM_I33A0 CIP 25464832
Influenza A virus STRAIN A/Wilson-Smith/1933 (H1N1) 381518 B4URF7 B4URF7_9INFA ACT, ATC 26651948, 28169297
JC polyomavirus 10632 Q9DUG7 Q9DUG7_POVJC TAP 22810586
Macaca mulatta polyomavirus 1 1891767 P03070 LT_SV40 ELISA, TAP, pull down 9168958, 22810586, 20701745
Macaca mulatta polyomavirus 1 1891767 Q9DH70 Q9DH70_SV40 Y2H 9168958, 12740372
Merkel cell polyomavirus 493803 B8ZX42 B8ZX42_9POLY TAP 22810586
Murid herpesvirus 4 (Murine herpesvirus 68) 33708 O41946 O41946_MHV68 22028648
Nipah virus 121791 Q997F2 V_NIPAV TAP 22810585
Nipah virus 121791 P0C1C7 W_NIPAV TAP 22810585
Plasmodium yoelii yoelii 73239 P06914 CSP_PLAYO pull down 17981117
Severe acute respiratory syndrome (SARS) coronavirus 227859 P59634 NS6_CVHSA Y2H 17596301
Yersinia pestis 632 Q8D1P8 Q8D1P8_YERPE Y2H, Pooling approach 20711500

Table 2: List of molecular features and functional sites on human IMA3

Feature key Description Position(s) on O00629
Domain IBB 2–58
Repeat ARM 1, Truncated 66–106
Repeat ARM 2 107–149
Repeat ARM 3 150–194
Repeat ARM 4 195–233
Repeat ARM 5 234–278
Repeat ARM 6 279–318
Repeat ARM 7 319–360
Repeat ARM 8 361–400
Repeat ARM 9 401–443
Repeat ARM 10; Atypical 447–485
Region NLS binding site (major) 137–229
Region NLS binding site (minor) 306–394
Motif Nuclear localization signal 43–52

Binding of KPNB1 probably overlaps the internal NLS and contributes to a high affinity for cytoplasmic NLS-containing cargo substrates [48]. After dissociation of the importin/substrate complex in the nucleus the internal autoinhibitory NLS contributes to a low affinity for nuclear NLS-containing proteins. The major and minor NLS binding sites are mainly involved in recognition of simple or bipartite NLS motifs. As described by Elena et al., in 1998 [49], structurally located within a helical surface groove, they contain several conserved Trp and Asn residues of the corresponding third helices (H3) of ARM repeats which mainly contribute to binding as shown in fig. 1. The secondary structure superimposition of the two IMA3 structures in cargo bounded and Free State reveals a global root-mean-square deviation (RMSD) of 1.27 Å. And the major RMSD contribution was observed at the ARM2-3 position because of the remarkable conformational change in the loop at the NLS site, as shown in fig. 2A-2C. Hence the amino acids at the ARM2-3 region (Asn141, Ser144, Trp179, Asn183, and Asn219) were identified as a key binding site residue.

Fig. 2: A: The crystal structure of IMA3 in cargo free state with an open loop at the NLS Major Site (PDB ID: 6BVZ). B: The crystal structure of Hendra virus W protein C-terminus (the loop in golden color) in complex with IMA3 crystal form 2 rendering closed loop at NLS Major Site (PDB ID: 6BWA). C: The superimposed conformers of cargo bounded and free structures

A B
C

Fig. 3: Molecular interaction of ligands with at NLS major binding site on human IMA3 (PDB ID: 6BWA). A: Two-Dimensional (2D) representation of ATRI001 interaction with IMA3 facilitated by 6 H-bonds shown in pink arrow. B: Three dimensional (3D) illustration of ATRI001 interaction with IMA3 facilitated by 6 H-bonds shown in yellow dotted lines. C: Three dimensional (3D) illustration of Ivermectin interaction with IMA3 facilitated phobic enclosures but no H-Bonds

Molecular docking

The molecular docking study was performed to understand the molecular interaction of plant molecules with human IMA3. Initial HTVS screening suggested 13,000 molecules with reasonable interaction with IMA3, and further, the shortlisted molecules were docked in the standard precision mode where the accuracy of prediction is better than the HTVS mode [50].

The docking in SP mode has suggested 20 top molecules as lead molecules. As ivermectin is reported to inhibit IMA3 mediated cargo nuclear import, it was also subjected to subsequent docking in extra precision (XP) mode. All 20 plant molecules showed better interaction than ivermectin with IMA3. However, the lowest energy ligand-bound conformers are always energetically favorable [51]. Hence, the plant molecule ATRI001 with the lowest binding energy-7.290 Kcal/mol was identified as a potent inhibitor of IMA3 by displaying better interactions with NLS site on human IMA3 by forming 6 H-bonds with Asp102, Asn141, Ser144, Trp179, Asn183 and Asn219 as shown in fig. 3A and 3B.

ATRI001 is a glycoconjugate having (2R,3R,4S,5S,6R)-6-Ethyloxane-2,3,4,5-tetrol as a monosaccharide sugar group and connected to phyto-moiety (1R,2R,4S)-1-[(3R)-3-Hydroxybut-1-enyl]-2,6,6-trimethylcyclohexane-1,2,4-triol through glycosidic linkage. The interaction of sugar group with IMA3 is facilitated by 3 H-bonds, two of them were formed by donating electrons to side-chain atoms of Asn141 and Ser144, one of 3 H-bonds was formed by accepting the electron from backbone atoms of Asp102. Whereas interaction between photo-moiety and IMA3 was established by means of 3 H-bonds, two of them were formed by donating electrons to side-chain atoms of Asn183 and Asn219, remaining H-bond found formed by accepting the electron from backbone atoms of Asp102. Ivermectin, a known IMA3 inhibitor, showed its lowest biding energy-4.946 Kcal/mol at its lowest energy conformation without any hydrogen bonds, as shown in fig. 3C. Hence, the interaction of ATRI001 was found better than ivermectin by using three parameters: fitting at NLS binding site, low interaction penalties, and a good number of bonded interactions.

CONCLUSION

The Comparative analysis to evaluate the IMA3 inhibition activity of Ivermectin and plant small molecules using in silico approaches suggested that a plant molecule ATRI001 is superior to Ivermectin. Our in silico experiment shows ATRI001 can block the nuclear import of SARS-Cov-2 cargo. These predictions, however, require further investigations.

FUNDING

Nil

AUTHORS CONTRIBUTION

All the authors have contributed equally.

CONFLICT OF INTERESTS

The study was funded by Atrimed Pharmaceuticals, Bangalore. Latha Damle is the founder of Atrimed Biotech LLP and holds equity in Atrimed Pharmaceuticals. Shiban Ganju and Hrishikesh Damle hold equity in Atrimed Pharmaceuticals. Bharath BR is an employee of Atrimed Biotech LLP.

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