FUNCTIONALIZED FULLERENE CONJUGATED NANOMOLECULES FOR THE INHIBITION OF NIPAH VIRUS INFECTIONS

Abstract

The present invention relates to functionalized fullerene conjugated nanomolecules for the inhibition of Nipah virus infections having formula (I) and formula (II). Formula (I) comprises C20 Fullerene nanostructure and formula (II) comprises C60 Fullerene nanostructure with ‘R1’ group as functionalized diterpene lactone and functionalized phytosterol covalently conjugated with C20 and C60 Fullerene nanostructures through an amide bond. The nano-sized molecules can also self-assemble to form further complex of functionalizable nanostructures. The present invention also relates to the method of preparation of the phytochemicals-conjugated modified C20 and C60 Fullerenes using different functional groups of plant metabolites and phytosterols. The said phytochemicals and the fullerenes are basically a) functionalized Andrographolide-conjugated modified C20 or C60 fullerene nanomolecules and b) functionalized Stigmasterol-conjugated modified C20- or C60- fullerene nanomolecules. The working mechanisms include simultaneous inhibition of RNA-dependent RNA polymerase (RdRp) and the viral chaperone activity in Nipah virus (NiV) phosphoprotein and nucleoprotein.

Specification

Description:FIELD OF THE INVENTION

The present invention relates to the field of antiviral and covalently conjugated pharmaceutics, including nanoconjugates. More particularly, the present invention relates to the development of functionalized phytochemicals-conjugated modified fullerene molecules that can also intrinsically result in nanostructures for the effective inhibition of Nipah virus infections.

BACKGROUND OF THE INVENTION

Despite the initial outbreak in 1998 and a staggering mortality rate of 40-70%, no drug has been approved by any drug agencies in the past 25 years to treat Nipah virus disease. The alarming gap in inhibition options has driven the researchers to invent potential agents to combat such lethal diseases. In order to effectively tackle the Nipah virus infections, it is essential to simultaneously target multiple critical molecular machineries of the virus. A multi-targeted approach is believed to be more effective than targeting a single stage of Nipah virus (NiV) infections as it would make it more difficult for the virus to develop resistance to the new developed inhibition methods.
Screening of the existing literatures shows that the research in this field mainly focussed on inhibiting the entry or/and replication of Nipah virus. Specifically, there is no mention of multitargeted anti Nipah virus (NiV) nanomedicine formulations particularly of those that can simultaneously usurp the virus activation, replication and propagation. In none of the prior art disclosures, there is mention of antiviral nanomedicine formulations that can simultaneously inhibit the Nipah (NiV) viral chaperone activity or the virus ribonucleic acid (RNA) synthesis and its recruitment by specific multitargeted approaches [such as targeting Nipah virus chaperone proteins and ribonucleic acid (RNA) dependent RNA polymerase (RdRp) Nipah virus (NiV) combinedly] or about any nanomedicines equipped with molecular architectures of functionalized Andrographolide/Stigmasterol-conjugated modified fullerene molecules or their nanostructures) to combat Nipah virus (NiV) infections.
In the present invention by amalgamating high throughput virtual screening, molecular docking, knowledge- and target-based in silico drug designing approaches, an attempt is made to develop rationally designed and optimized nanomedicines to achieve an efficient target protein binding in the chaperone-governing sites of Nipah virus (NiV) nucleoprotein and/or phosphoprotein and viral ribonucleic acid (RNA) dependent RNA polymerase (RdRp), simultaneously. Moreover, present invention discloses the concept of rationally designed, molecularly tailored nanomedicines that operates by their multitargeted approach and can significantly improve the prognosis in Nipah virus (NiV) infected patients. Hence, the present invention focuses on both the working mechanism (that is the method of working) as well as the novel molecular architectures of the disclosed nanomedicines (chemical structures). The sequel or consequence of these chemical modifications leads to an unobvious modified physicochemical and synergistic antiviral activity and hence such structures become potential candidate materials for such inhibition studies.

OBJECTIVES OF THE INVENTION

An objective of the present invention is to develop multitargeted, antiviral molecules that can also form nanostructures or multifunctional nanopharmaceutic covalent conjugates that are molecularly targeted toward certain key proteins and enzymes involved in the life cycle of Nipah virus (NiV).
Another objective of the present invention is to develop a method of inhibition of viral infections caused by Nipah virus (NiV) by simultaneously targeting the viral chaperone activity in Nipah virus (NiV) phosphoprotein, NiV nucleoprotein and the ribonucleic acid dependent RNA polymerase (RdRp).
Another objective of the present invention is to develop a method of preparation for various functionalized modified fullerene molecules that can form nanostructures using different substituents of plant metabolites and phytosterols in conjugation with carboxylated C20 Fullerene or carboxylated C60 Fullerene structures or their derivatives.
Another objective of the present invention is to simultaneously inhibit the Nipah virus (NiV) replication and propagation using such multitargeted nanomedicine conjugates by simultaneously interfering the viral RNA synthesis, recruitment, packaging and assembly.
Another objective of the present invention is to lay down a pathway to develop many such multifunctional molecules, their related nanostructures and conjugates by substituting the C20 Fullerenes and C60 Fullerenes with other endohedral fullerenes, or other water dispersible or water soluble fullerene derivatives, or fullerene adducts such as peptide-linked fullerenes.
Additional objectives and features of the invention will be outlined in the part of description below. The foregoing facts and the following descriptions hereafter outlines some of the pertinent objectives of the invention. The afore mentioned objectives should not be construed to be merely illustrative of some of the more prominent features and applications of the intended invention. Many other beneficial results can also be obtained by applying the disclosure of the invention in a different or modified manner within the scope of disclosure.

SUMMARY OF THE INVENTION

A summary is provided to facilitate an understanding of the innovative characteristics unique to the disclosed embodiments of the invention and is not intended for the full description of the invention. In accordance, a full appreciation of the various aspects of the preferred embodiments disclosed herein can be understood in debt by taking the entire specification, claims, drawings and abstract as a whole.
An aspect of the present invention discloses Functionalized fullerene conjugated molecules that can also form nanostrcutures for inhibition of Nipah virus infections having formula (I).




(I)

and formula (II)






(II)

wherein formula (I) comprises C20 Fullerene structure and formula (II) comprises C60 Fullerene structure, wherein 'R1' group is a functionalized diterpene lactone and a functionalized phytosterol that is covalently conjugated with the C20 and C60 Fullerene structures through an amide bond.
Another aspect of the present invention is to develop a method of preparation of functionalized Andrographolide Conjugated Fullerene Molecule (ACFN) and functionalized Stigmasterol Conjugated Fullerene Molecule (SCFN) by conjugation of C20 and C60 Fullenere nanostructures using functionalized substituents of plant-based metabolites and phytosterols such that the formed ACFN and SCFN molecules also intrinsically form nanostructures.
Yet another aspect of the present invention include the analysis of the working mechanisms of the functionalized fullerene conjugated molecules for inhibition the treatment of Nipah Virus.
REFERENCE NUMERALS FOR THE DEVICE AND THE PARTS THEREIN
C20-ACFN Functionalized Andrographolide-conjugated C20 Fullerene nano molecules/nanostructures

C60-ACFN Functionalized Andrographolide-conjugated C60 Fullerene nano molecules/ nanostructures

ADME Absorption, distribution, metabolism and excretion
ADMT Absorption, distribution, metabolism and toxicity
BBB Blood-brain barrier
CACNG4 Calcium Voltage-Gated Channel Auxiliary Subunit Gamma 4 gene
CHRNE Cholinergic receptor nicotinic epsilon subunit gene
CTD Carboxy-terminal domain
FOXO4 Forkhead Box O4
GI Gastrointestinal tract
GRAS Generally regarded as safe
MR Molar Refractivity
NiV Nipah virus
PDB Protein data bank
PV Portal vein
QSAR Quantitative structure-activity relationship
RLR Rig-I-like receptors
SFRP1 Secreted frizzled-related protein 1
SC Systemic circulation
C20-SCFN Functionalized Stigmasterol-conjugated C20 Fullerene nano molecules/nanostructures

C60-SCFN Functionalized Stigmasterol-conjugated C60 Fullerene nano molecules/ nanostructures

SMI Small molecule inhibitors
SQLE Squalene Epoxidase gene
TLR Toll-like receptor
TPSA Topological polar surface area
VDR Vitamin-D receptor gene.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention that is described herein and the related drawings provided herein are to serve only for illustrative purposes so as to relate well with the preferred embodiments of the invention; and they are not intended to limit the scope of the present disclosure. The invention is best understood by its reference to the detailed description, which follows in conjunction with the below mentioned details of the drawings:
FIG. 1 shows the following: (a) Schematic of Nipah virus (NiV) ribonucleic acid (RNA) sandwiched between nucleoprotein (N-protein) and phosphoprotein (P-protein), (b) Crystal structure of Nipah virus (NiV) ribonucleic acid (RNA) free nucleoprotein-phosphoprotein complex (P-protein: yellow, N-protein: green), Nipah virus (NiV) chaperone-governing interface residues in P-protein (magenta) and N-protein (red) is based on Protein data bank record (PDB entry: 4CO6), (c) Residues of P-protein and N-protein that are involved in Nipah virus (NiV) chaperone activity, (d) Potential drug-binding sites in the viral chaperone-governing residues of P-protein, (e) chaperone-governing residues in N-protein, (f) Hydrogen bond formation, (g) hydrophobic interaction, (h) electrostatically interacting residues within the chaperone sites of N-protein and (i) Potential drug-binding sites in N-protein, with arrows denoting the drug-binding pockets lying in the proximity of the chaperone-governing sites.
FIG. 2 shows (a) Active sites in Nipah virus (NiV) P-protein, (b) Top-10 Nipah virus (NiV) P-protein inhibitors following the virtual screening (n=72), (c) Quantitative structure-activity relationship (QSAR) studies on Andrographolide, (d) Molecular docked structure of Andrographolide-Nipah virus (NiV) P-protein complex, (e) Closely-interacting active site residues of Andrographolide-Nipah virus (NiV) P-protein complex (< 4 Å distance) and (f) Top-9 drug binding modes of Andrographolide in Nipah virus (NiV) P-protein. Bottom Inset: Crystal structure of Nipah virus (NiV) P-protein (yellow) showing its viral chaperone-governing sites (green; protein data bank (PDB) entry: 4CO6), (g) Venn diagram showing the viral chaperone-involved residues in Nipah virus (NiV) P-protein (blue) and the residues targeted by Andrographolide shown in green color (< 4 Å distance). The chaperone-involved residues targeted by Andrographolide as shown at the centre in bluish-green color. Simulation of Andrographolide absorption in the (h) human gastrointestinal tract, (i) blood (plasma) and the (j) drug distribution in the portal vein (PV) and systemic circulation (SC) using Gastroplus tool.
FIG. 3 shows (a) Active sites in Nipah virus (NiV) N-protein, (b) Top-10 Nipah virus (NiV) N-protein inhibitors following the virtual screening (n=72), (c) Quantitative structure-activity relationship (QSAR) studies on Stigmasterol, (d) Molecular docked structure of stigmasterol-Nipah virus (NiV) N-protein complex, (e) Closely-interacting active site residues of stigmasterol Nipah virus (NiV) N-protein complex (< 4 Å distance), (f) Top-9 drug-binding modes of stigmasterol in Nipah virus (NiV) N-protein, (g) Venn diagram shows the viral chaperone-involved residues in Nipah virus (NiV) N-protein (blue) and the residues targeted by stigmasterol shown in green color (< 4 Å distance). The chaperone-involved residues targeted by stigmasterol as shown at the centre in bluish-green color. Simulation of stigmasterol absorption in the (h) human gastrointestinal tract, (i) blood (plasma) and the (j) drug distribution in the portal vein (PV) and systemic circulation (SC) using Gastroplus tool.
FIG. 4 shows the in silico measured sizes of Andrographolide-conjugated C20 Fullerene nanostructure and Andrographolide-conjugated C60 Fullerene nanostructure.
FIG. 5 shows the in silico measured sizes of Stigmasterol-conjugated C20 Fullerene nanostructure and Stigmasterol-conjugated C60 Fullerene nanostructure.
FIG. 6 shows (a) Homology model of Nipah virus RNA-dependent RNA polymerase (NiV RdRp), (b) Superimposition of Nipah virus RNA-dependent RNA polymerase (NiV RdRp) structures homology modeled in normal mode (Green) and in intensive modes (Blue), (c) Potential drug binding sites, (d) Modeled Andrographolide-conjugated C20 fullerene (C20-ACFN) (arrows denote PO4; oxygen in red), (e) Andrographolide-conjugated C20 fullerene nanostructure-Nipah virus RNA-dependent RNA polymerase docked complex (C20-ACFN-NiV-RdRp), (f) Andrographolide-conjugated C20 fullerene nanostructure docked with NiV phosphoprotein (C20-ACFN -NiV P-protein), (g) modeled Stigmasterol-conjugated C20 fullerene (C20-SCFN ), with arrows denoting diphosphate and carboxylate group; oxygen in red, (h) Stigmasterol-conjugated C20 fullerene nanostructure docked with RNA-dependent RNA polymerase (C20-SCFN-NiV RdRp) and (i) Stigmasterol-conjugated C20 fullerene-Nipah virus nucleoprotein docked nanocomplex (C20-SCFN-NiV N-protein).
FIG. 7 shows the non-covalent interactions of andrographolide-conjugated C20 Fullerene-(C20-ACFN) with (a) Nipah virus RNA-dependent RNA polymerase (NiV RdRp) and (b) Nipah virus phosphoprotein (NiV P). Non-covalent interactions of stigmasterol-conjugated C20 Fullerene (C20-SCFN) with (c) Nipah virus ribonucleic acid dependent RNA polymerase (NiV RdRp) and (d) Nipah virus (NiV) nucleoprotein, covalent interactions of C20-ACFN with (e) (NiV RdRp) and (f) Nipah virus (NiV) phosphoprotein.
FIG. 8 shows the (A1, B1) Modeled C60-ACFN, (A2) NiV Phosphoprotein, (A3) C60-ACFN - NiV Phosphoprotein docked complex, (A4) C60-ACFN - NiV phosphoprotein interacting residues. (B2) Homology modeled NiV RdRp, (B3) C60-ACFN - NiV RdRp docked complex, (B4) C60-ACFN - NiV RdRp interacting residues. (C1, D1) Modeled C60-SCFN, (C2) NiV Nucleoprotein, (C3) C60-SCFN-NiV Nucleoprotein docked complex, (C4) C60-SCFN - NiV Nucleoprotein interacting residues, (D2) Homology modeled NiV RdRp, (D3) C60-SCFN - NiV RdRp docked complex, (D4) C60-SCFN - NiV RdRp interacting residues. The table shows the closely interacting residues of C60-based ACFN and SCFN with their respective targets along with the binding energies (Residues involved in hydrogen bonding are shown in bold).
FIG. 9 shows the Closely interacting ligand-target residues (< 4 Å distance) between (A) C60-ACFN-NiV phosphoprotein, (B) C60-ACFN-NiV RdRp, (C) C60-SCFN- NiV nucleoprotein and (D) C60-SCFN-NiV RdRp.

DETAILED DESCRIPTION OF THE INVENTION

The preferred embodiments of the present invention are provided so that the disclosure will be thorough and fully convey the scope to those who are skilled in the art. Various specific details are set as specific components to provide an overall understanding of the preferred embodiments of the present disclosure. It will be apparent to those skilled in the art that the specific details need not be employed and the preferred embodiments may be embodied in many different forms and the steps followed do not limit the scope of the disclosure. The present invention will be illustrated with the help of the following experiments are not intended to limit the scope of the invention and any such modification therein falls within the scope of the invention. It is to be understood that both the foregoing general description and the detailed description are exemplary only and are intended to provide further explanation of the subject matter.
An embodiment of the present invention is to prepare various phytochemical-conjugated fullerene molecules that can also form nanostructures, or pharmaceutic conjugates including nanoconjugates using different substituents of plant-based functionalized metabolites and phytosterols in conjugation with modified structures of C20 Fullerene or C60 Fullerene or their chemical derivatives.
An embodiment of the present invention discloses chemical structures of the functionalized therapeutics containing multitargeted fullerene molecules which can also form nanostructures and their covalent conjugates (involving functionalized Andrographolides, functionalized Stigmasterols, carboxylated C20 Fullerene or C60 Fullerene) designed by employing virtual screening, molecular docking, quantitative structure activity, pharmaceutical regulatory organization used physiology based pharmacokinetic modeling, Absorption, distribution, metabolism, excretion (ADME) simulations, gene expression annotation and toxicity studies to combat Nipah virus (NiV) infections.
Another embodiment of the present invention relates to two in silico designed antiviral and multitargeted phytochemical-conjugated fullerene molecules that can also form nanostructures namely andrographolide-conjugated fullerene (C20-ACFN ) and stigmasterol-conjugated fullerene (C20-SCFN) and study of their physicochemical characteristics, drug like characteristic properties, ligand target specificity, binding affinity, drug-target binding residue characterization and plasma drug concentration, along with the mobility of the ligands in gastrointestinal compartments, and the ligand dissolution characteristics during the blood circulation.
Both the above-mentioned pharmaceutic covalent structures have the potential to work individually and independently combat Nipah virus (NiV) infections; however, they can synergistically work if given in combination either by one of following common mechanisms of working that involves the inhibition of Nipah virus (NiV) and RNA-dependent RNA polymerase (RdRp) or by following distinct working mechanisms wherein andrographolide-conjugated fullerene (C20-ACFN/C60-ACFN) works by inhibiting RNA-dependent RNA polymerase (RdRp) and NiV phosphoprotein and stigmasterol-conjugated fullerene nanostructures (C20-SCFN/C60-ACFN) works by inhibiting RNA-dependent RNA polymerase (RdRp) and Nipah virus (NiV) nucleoprotein effectively at the viral chaperone-governing sites. Moreover, for the effective therapeutic management of Nipah virus (NiV), it is better to simultaneously target the virus RNA-dependent RNA polymerase (RdRp), chaperone-governing phosphoprotein residues and chaperone-governing nucleoprotein residues.
Another embodiment of the present invention relates to the chemical structures of both the conjugates (C20-ACFN and C20-SCFN) and their associated descriptions related to the development of functionalized phytochemicals C20 Fullerene nanoconjugates and functionalized phytochemicals C60 Fullerene nanoconjugates. The individual atoms have been numbered based on IUPAC standards and the generic structures are described below for C20 Fullerene based phytochemical nanoconjugates, i.e., for C20 ACFN and C20 SCFN alone. In the case of C60 Fullerene based phytochemicals conjugates, the below mentioned generic structures and their synthesis procedures remain the same, only the C20 Fullerene moieties will be replaced by C60 Fullerene moieties to obtain C60 Fullerene based phytochemical nanoconjugates, i.e., C60-ACFN and C60-SCFN.
1.Functionalized Andrographolide-conjugated modified fullerene nanomolecules (C20-ACFN):







Andrographolide is a herbal compound. The decahydronaphthalene ring of Andrographolide methylated at 2- and 6-positions are further functionalized with two phosphate groups. Additionally, a methyl group in the decahydronaphthalene ring at the 3rd position functionalized with a primary amine group covalently conjugated with a carboxylated C20 Fullerene or with a carboxylated C60 Fullerene by an amide linkage through condensation reactions. These strategic chemical modifications lead to the formation of a novel structures of either functionalized Andrographolide C20 Fullerene conjugates or functionalized Andrographolide C60 Fullerene nanoconjugates for combating the Nipah virus (NiV) infections are non-obvious for a skilled researcher trained in the art. Further, tailored modifications of the developed structures lead to the formation of multifunctional phytochemical carboxylated fullerene conjugates to simultaneously inhibit the key Nipah virus (NiV) chaperone-governing residues in the viral phosphoprotein and ribonucleic acid dependent RNA polymerase (RdRp). The resulting structures followed by sequel of chemical modifications and the idea of inhibiting the Nipah virus (NiV) chaperone-governing sites in phosphoprotein and RNA-dependent RNA polymerase (RdRp) by rationally designed Andrographolide-conjugated fullerene (C20-ACFN/C60-ACFN) is new and not apparent or obvious. Owing to the substantial chemical modifications, the Andrographolide-conjugated fullerene (C20-ACFN/C60-ACFN ) is expected to behave as a novel molecule and as a novel nanostructure both in terms of its physiochemical characteristics and the biological fate. For example, its aqueous solubility at the physiological pH, the octanol-water coefficient and the number of hydrogen bond donors and acceptors of the developed the Andrographolide-conjugated fullerene has been found to be distinct from the unmodified andrographolide or C20 Fullerene or C60 Fullerene. Consequently, the cellular uptake and host cell receptor binding property and the intracellular drug uptake and retention of such Andrographolide-conjugated fullerenes would significantly vary in vitro. Furthermore, the viral target protein binding characteristic, drug absorption, distribution, metabolism, excretion, toxicity and pharmacodynamic profiles (in vivo) of Andrographolide conjugated C20- and C60- fullerene molecules and the resulting nanostructures are distinct from that of the unmodified Andrographolide or unmodified or pristine C20 Fullerene or C60 Fullerenes render a strong scope for protecting such structures.
2. Functionalized Stigmasterol-conjugated modified Fullerene nanomolecules (C20-SCFN):






Stigmasterol is another herbal compound. Methylation followed by diphosphate functionalization of Stigmasterol at the 4-position in cyclopentaphenanthrene ring and the carboxylic functionalization of the same ring at the 8-position followed by primary amine functionalization (1st position) and finally covalently conjugating with a carboxyl functionalized C20 Fullerene or a carboxyl functionalized C60- Fullerene through amide linkages to obtain Stigmasterol conjugated Fullerene molcules (C20-SCFN or C60-SCFN ). It has been observed that Stigmasterol-conjugated Fullerene structures have the potential to inhibit the Nipah virus (NiV) nucleoprotein residues involved in viral chaperone activity and viral RNA-dependent RNA polymerase (RdRp).
Similar to Andrographolide-conjugated C20 Fullerene or C60 Fullerene (C20-ACFN or C60-ACFN) and owing to the rational tailoring and functionalization, the physicochemical properties, the target viral protein binding, the cellular uptake, the host receptor recognition, pharmacodynamic and pharmacokinetic profiles of Stigmasterol-conjugated C20 Fullerene molecules and the resulting nanostructures (C20-SCFN) and Stigmasterol-conjugated C60 Fullerene molecules (C60-SCFN) are distinct from the unmodified Stigmasterol or the pristine C20- or C60- Fullerene structures.
Basically, the naturally existing phytochemicals (andrographolide, stigmasterol) and the known fullerene forms (C20 and C60 fullerenes) are chemically, structurally and functionally different from that of the in silico designed phytochemical conjugated fullerene nanomolecules/nanostructures disclosed herein. Andrographolide-conjugated fullerene (C20-ACFN/C60-ACFN) and Stigmasterol-conjugated fullerene (C20-SCFN/C60-SCFN) are in their uniquely modified, functionalized and combined in their rationally tailored conjugated forms such that the physicochemical characteristics and biological activity of C20-/C60-ACFN and C20/C60-SCFN are significantly different from that of the properties of their unmodified or individually existing parental compounds. The modifications have the potential to strongly inhibit certain key proteins in Nipah viruses (NiV) to synergistically combat the viral infections. While the parental structures, Andrographolide and Stigmasterol are hydrophobic in nature, the in silico designed molecules of Andrographolide-conjugated C20 Fullerene (C20-ACFN), Andrographolide-conjugated C60 Fullerene (C60-ACFN), Stigmasterol-conjugated C20 Fullerene (C20-SCFN) and Stigmasterol-conjugated C60- Fullerene nanomolecules (C60-SCFN) have been found to be relatively hydrophilic in silico. Further, the in vivo absorption, distribution, metabolism and excretion profiles influences the biological activity of the Andrographolide- and Stigmasterol-fullerene conjugates were found to be different from that of the unmodified and individual parent molecules of Andrographolide, Stigmasterol, C20 Fullerene or C60 Fullerenes. Since the type of chemical modifications (functionalization) and conjugation employed for the development of C20-ACFN and C20-SCFN remains the same as that of their C60 counterparts (C60-ACFN, C60-SCFN), the sequel or consequence of the chemical modifications from the viewpoint of their biological activities remains very similar between C20-based ACFN or SCFN and C60-based ACFN or SCFN. Hence, for the present invention, the carboxylated C60 fullerenes may be substituted in the place of carboxylated C20 fullerenes for the conjugation purpose with the functionalized phytochemicals, to anticipate similar inhibitory, antiviral and other biological effects as mentioned in this disclosure.
Another embodiment of the present invention relates to the method of preparation of the Andrographolide-conjugated C20 Fullerene and C60 Fullerene (C20-ACFN/C60-ACFN) and Stigmasterol-conjugated C20 Fullerene and C60 Fullerene (C20-SCFN/C60-SCFN).
EXPERIMENTS:
1. Method of preparation of Andrographolide-conjugated Fullerene nanomolecules (C20-ACFNor C60-ACFN)
The Fullerenes may be carboxylated by 'Prato' reaction using sodium hydride (NaH base), toluene (solvent) and bromomalonate (carboxylation reagent). Briefly, sodium hydride mediated deprotonation occurs at the alpha (α)-carbon of bromomalonate for the generation of a carbanion intermediate which attacks the electrophilic carbon atom of fullerenes through nucleophilic addition reactions to form a carbon-carbon (C-C) bond thereby leading to the formation of fullerene-malonate covalent complex, which on hydrolysis yield carboxyl-functionalized fullerenes. In a typical reaction, known concentrations of sodium hydride and fullerenes are dispersed in toluene and dark red solution obtained is allowed to interact with bromomalonate at 25° C for 10 hours under inert gas atmosphere. The residue obtained is eluted by toluene followed by the addition of excess sodium hydride and the resulting solution is heated to 75° C for 8 hours to 11 hours. Following the incubation, the reaction is terminated by the addition of a suitable (acid-primary alcohol) mixture. The resulting precipitate is centrifuged, washed repeatedly using methanol, hydrochloride (HCl) and deionized water using column chromatography. The purity of the product is studied using mass spectrometry (MS) and Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy measurements. Further, the above-mentioned procedure is illustrated by the following below-mentioned flow chart.
(IA) Preparation of carboxylated C20 Fullerene or C60 Fullerene:

Dissolve sodium hydride (NaH) and C20 Fullerene or C60 Fullerene in toluene
↓
Dark red colour solution
↓
Mixing of the red colour solution with bromomalonate
25° C for 10 h ↓ inert gas atmosphere
Residue elution using toluene
↓
Addition of excess sodium hydride (NaH)
↓
Solution heated to 75° C for 8 to11 hours
↓
Reaction termination using acid-alcohol mixture
↓
Resulting precipitate centrifuged
↓
Pellet washed repeatedly with methanol, HCl
and deionized water
↓
Purified using column chromatography
↓
Analysis
(Mass spectrometry (MS), Infrared spectroscopy (FT-IR))


In a separate experiment, known concentrations of andrographolide, methyl iodide, phosphoryl chloride or phosphorous trichloride are dissolved slowly in a suitable mixture of base (for methylation) and other solvent co-solvent mixtures for phosphorylation under inert gas conditions and stirred at 400-600 rpm and incubated at low temperatures. The temperature of the reaction mixture is gradually increased to 25° C and incubated at 800-1000 rpm for additional 12 hours. The excess of reagents is quenched and resulting product is purified using a column chromatography and subjected to mass spectrometry and Nuclear Magnetic Resonance (NMR) studies for structural elucidation. The resulting product is aminated using combination of suitable amine reagents such as primary or secondary amines, a reducing agent such as sodium cyanoborohydride in presence of a catalyst of acetic acid in suitable organic solvent such as methanol or dichloromethane (DCM). The product is then washed and purified using suitable chromatographic technique followed by further characterization using high performance liquid chromatography and mass spectrometry (HPLC-MS) technique. The aminated product is covalently conjugated with carboxyl functionalized C20- or C60-fullerenes using 1-ethyl-3-3-dimethylaminopropyl (EDC) mediated coupling chemistry to obtain the functionalized Andrographolide conjugated C20- or C60- fullerene nanomolecules/nanostructures (C20-ACFN or C60-ACFN) . For clarity, the procedure is illustrated by the following below mentioned flow chart.
(IIA) Functionalization of Andrographolide and covalent conjugation with carboxylated C20 Fullerene or C60 Fullerene:

Methyl iodide (CH3I) + Andrographolide + Phosphorous oxychloride-Phosphorus iodide (POCl3/PCl3)
Inert gas ↓ Sodium hydroxide (NaOH, solvent, co-solvent)
Stirred at 400-600 rpm at 4 °C, 12hours
↓
Gradual temperature increase and stirring at 25 °C
at 800-1000 rpm, 12 hours
↓
Quenching of excess reagents
↓
Product extraction, purification (Column chromatography)
↓
Product analysis with MS and NMR studies
↓ 1° / 2° amine, Sodium cyanoborohydride (NaBH3CN), Acetic acid (CH3COOH), Methyl chloride (CH2Cl2)
Product centrifuged, washed, and purified
using chromatography
↓
Characterized using HPLC-MS technique
↓
Conjugation of aminated product with carboxylate functionalized
C20 Fullerene or C60 Fullerene using 1-ethyl-3-3-dimethylaminopropyl (EDC)
↓
Conjugated product washed and purified
↓
(C20 ACFN or C60 ACFN)

3. Method of preparation of Stigmasterol-conjugated fullerene nanostructures C20-SCFN or C60-SCFN):
Methylation of Stigmasterol is done using suitable methylating agents such as methyl iodide (CH3I) and bases such as potassium carbonate (K2CO3) or sodium hydroxide (NaOH), and dissolving the components slowly in a suitable organic solvent/co-solvent mixture. The resulting methylated product obtained after the acid neutralization of the excess reagents is purified using column chromatography. The product is characterized using Nuclear Magnetic Resonance (NMR) and mass spectrometrometric (MS) techniques to confirm the successful methylation. The obtained methylated product is dispersed in an appropriate solvent and phosphorus oxychloride to form an intermediate phosphate ester which upon addition of a suitable nucleophile such as ethylene glycol lead to the formation of a diphosphate group. After extracting the desired product from the reaction mixture, it is purified and characterized using Nuclear Magnetic Resonance (NMR) and mass spectrometry (MS) to confirm successful diphosphorylation. Finally, the amination and covalent conjugation using 1-ethyl-3-3-dimethylaminopropyl (EDC) mediated coupling chemistry is performed to obtain the functionalized Stigmasterol-conjugated fullerene molecules (SCFN) that can also form nanostructures.
(IIB) Functionalization of Stigmasterol and covalent conjugation with the carboxylated C20 Fullerene or C60 Fullerene
Methylation of stigmasterol using Methyl iodide (CH3I) + Potassium carbonate-sodium hydroxide (K2CO3/NaOH)
↓
Dissolved in a suitable organic solvent/ co-solvent mixture
↓
acid neutralization
↓
Product purified using column chromatography
↓
NMR and MS characterization for methylation confirmation
↓
Disperse the product in appropriate solvent phosphorus oxy chloride (POCl3)
↓
Addition of nucleophile donor (ethylene glycol)
↓
Product extraction, purification
(column chromatography/HPLC)
↓
Resulting product subjected to MS, NMR
↓ 1° / 2° amine, Sodium cyanoborohydride (NaBH3CN), Acetic acid (CH3COOH), dichloromethane (DCM)
Product centrifuged, washed, and purified using chromatography
↓
Characterized using HPLC-MS
↓
Conjugation of aminated product with COOH-functionalized
C20 Fullerene or C60 Fullerenes using 1-ethyl-3-3-dimethylaminopropyl (EDC)
↓
Conjugated product washed and purified
↓
(C20-SCFN or C60-SCFN)

Another embodiment of the present invention is based on the working mechanisms of the developed conjugates (ACFN and SCFN).
4. Working mechanism of the conjugate structures:
(I) By inhibiting the ribonucleic acid (RNA) dependent RNA polymerase (RdRp) of Nipah virus (NiV):

Inhibiting the RNA dependent RNA polymerase (RdRp) can help disrupt the viral replication process and prevent Nipah virus (NiV) usurpation of the host cells. RNA-dependent RNA polymerase is a crucial enzyme required for the replication of genome of NiV. It catalyses the synthesis of new viral ribonucleic acid (RNA) molecules using the template by inhibiting RdRp activity, the synthesis of viral RNA is impaired leading to a reduction in Nipah virus (NiV) replication and generation of new viral particles. RNA dependent RNA polymerase inhibition also prevent the accurate and efficient replication of the viral genome. The disruption halts the production of new viral RNA molecules necessary for the synthesis of viral proteins and the asse
mbly of new virions. Without functional RdRp, the virus cannot complete its replication cycle, effectively limiting the virus propagation within the host cells. Further, the RdRp inhibition impedes the production of infectious viral particles, thereby limiting viral invasion to the neighboring uninfected host cells. Therefore , Andrographolide-conjugated fullerenes and Stigmasterol-conjugated fullerenes has the potential to reduce the viral load and its ability to develop systemic infections. When the ACFN is combined with the SCFN, the combination therapy synergistically targets multiple stages of the viral life cycle providing a more comprehensive strategy for controlling the Nipah virus (NiV) infection and mitigating the drug resistance. Capacity to inhibit the NiV RdRp, makes the C20- or C60-ACFN and C20- or C60-SCFN potential functionalized conjugates to disrupt viral replication, impair the Nipah virus (NiV) ribonucleic acid (RNA) synthesis and to hamper the virus spread and inhibit the ability of Nipah virus to usurp the host cells.
(II). By inhibiting the viral chaperone activity of Nipah virus (NiV) phosphoprotein and nucleoprotein
Conjugates disclosed in the present invention can also work by inhibiting the Nipah virus (NiV) chaperone activity governed by certain specific residues of phosphoprotein and nucleoprotein. It has been found that Nipah phosphoprotein acts as viral chaperone for the nucleoprotein to ensure an open, active state monomeric conformation so as to grasp ribonucleic acid (RNA) molecule for the formation of new virus particles. Molecular docking studies indicated that ACFN and SCFN can selectively or specifically recognize and bind strongly to residues implicating chaperone activity and thereby interfering the nucleoprotein-phosphoprotein interactions. The chaperone activity is essential for various events associated with the life cycle of the virus. By inhibiting the chaperone activity, NiV ribonucleic acid synthesis, recruitment, packaging and assembly disrupt the viral usurpation of the host cells. Viral chaperones play essential roles in facilitating the assembly and maturation of viral particles by assisting the folding and assembly of viral proteins and nucleic acids. Therefore, inhibition can disrupt the proper folding and assembly of viral components leading to the formation of non-infectious or defective viral particles; without the functional chaperones, Nipah virus (NiV) can barely package its ribonucleic (RNA) genome and assemble new virions efficiently impairing its ability to usurp the host cells. Finally, the combinatorial inhibition mediated by the co-delivery of ACFN and SCFN can simultaneously inhibit NiV RdRp and viral chaperone-governing proteins such that the net effect can effectively disrupt the viral replication cycle and NiV infection in the host cells due to the synergistic antiviral activity of the conjugated molecules and the resulting nanostructures.
Another embodiment of the present invention relates to the results of analysis, virtual screening of P-protein (P) and N-Protein interacting sites and drug binding pockets of Nipah virus.
RESULTS:
1. Analyses of Nipah virus (N-protein (N) and P-protein (P)) interacting sites and drug binding pockets:
Prior to screening of Nipah virus N-protein (N) and P-protein (P) inhibitors virtually, the crucial interactions essential for Nipah virus (NiV) chaperone activity at the phosphoprotein-nucleoprotein interface vital for new virus particle formation and replication were investigated. (Fig. 1, A) illustrates the Nipah (NiV) ribonucleic acid (RNA) enclosed by nucleocapsid shielded from host cell nucleases with the phosphoprotein safeguarding viral ribonucleic acid (RNA) by interacting with nucleoprotein forming a critical (P-N) complex activating viral RNA polymerase for RNA synthesis, replication and virion budding. The complex was reconstructed based on two experimentally resolved Protein data bank (PDB) structures (PDB entry: 4CO6 and 7NT5). (Fig. 1, B) displays the crystal structure of RNA-free NiV phosphoprotein-nucleoprotein complex regulating viral chaperone activity (PDB:4CO6). Interface residue analysis showed stabilized P-N interactions through hydrophobic, Van der Waals forces, salt bridges and hydrogen bonds with a binding energy of -7.8 kcal/mol (Fig. 1, C). Phosphoprotein chaperone-governing residues found to act as potential drug binding sites (Sites 1-6) (Fig. 1, D) allowing inhibition of P-protein via small molecule inhibitors (SMIs) to disrupt Nipah virus (NiV) chaperone activity. In silico analyses on the nucleoprotein identified the chaperone-governing sites primarily within its carboxy terminal domain (CTD) as shown in (Fig. 1, E) with stability of P-N complex primarily mediated by hydrophobic interactions (Fig. 1, G), followed by hydrogen bonds (Fig. 1, F) and electrostatic interactions (Fig. 1, H). The study indicated that the ligands interacting through hydrophobic, electrostatic and hydrogen bond combinations at specific N-protein sites can effectively disrupt the P-protein and N-protein (P-N) complex formation in the Nipah virus (NiV). Further, many potential drug binding sites were found in chaperone-governing sites of nucleoprotein (Fig. 1, I). Thus, virtual screening helped to identify the potential phosphoprotein inhibitors to disrupt the Nipah virus (NiV) phosphoprotein and nucleoprotein-mediated chaperone activity.
2. Virtual screening, Quantitative structure-activity relationship (QSAR) and pharmacokinetic modeling of Nipah virus (NiV) phosphoprotein inhibitors
Previously identified drug binding sites on phosphoprotein designated as active sites (Fig. 2, A). Seventy two molecules from active pharmaceutical ingredients of selected herbs showing promising antiviral activity served as ligands. Andrographolide, a natural diterpene from Andrographis paniculata exhibited most favorable binding energy (-6.0 kcal/mol) against Njpah virus (NiV) (Fig. 2, B), targeting precisely the chaperone governing residues in the phosphoprotein (Fig. 2, D). Notably, the Andrographolide closely targeted residues (Ile 13, Ile 17, Gln 21, Ile 24, Gln 25, Tyr 28, Arg 30, Ser 31; < 4 Å distance) crucial for Nipah virus (NiV) phosphoprotein chaperone activity (Fig. 2, E). Quantitative structure-activity relationship (QSAR) studies suggested andrographolide's drug like characteristics suitable for oral delivery, satisfying Lipinski's rule of five (Fig. 2, C). Fig. 2, F illustrates top-9 drug-binding modes with Andrographolide specifically targeting NiV phosphoprotein chaperone-governing residues. Interestingly, 80% (11/14) of the phosphoprotein residues targeted by andrographolide typically resided in chaperone-governing sites (Fig.2, G Blue: Chaperone-governing residues, Green: andrographolide-targeting residues). Molar refractivity (MR >20) and topological polar surface area study (TPSA <140 Å2) indicated potential for efficient brain penetration and good oral bioavailability, despite its inherent hydrophobicity. Simulation of the gastrointestinal tract (GI) absorption (Gastroplus) showed 95% cumulative absorption in the duodenum, jejunum, and ileum compartments within 50 minutes of drug administration (Fig. 2, H). Moreover, Plasma simulation over 24 hours indicated steady-state increase in drug concentration within 10 minutes post administration with peak concentration in 2 hours and 50% decrease in 6 hours (Fig. 2, I). Andrographolide demonstrated rapid dissolution (>99%) and (~90%) absorption in portal vein within two hours post-administration with ~80% circulating systemically within three hours (Fig. 2, J). Collectively, simulation data on virtual screening, Quantitative structure-activity relationship (QSAR), docking and pharmacokinetic modeling suggested andrographolide's specific protein binding to Nipah virus (NiV) chaperone governing sites in the phosphoprotein and a significant aqueous phase dissolution, enhanced GI absorption and oral bioavailability.
3. Virtual screening, Quantitative structure-activity relationship (QSAR), and pharmacokinetic modeling of Nipah virus (NiV) nucleoprotein inhibitors:

Following that, virtual screening experiments were conducted to identify potential Nipah virus (NiV) nucleoprotein inhibitors to hinder its chaperone-governing sites. Unlike, Nipah virus (NiV) phosphoprotein (6 drug-binding sites) the nucleoprotein was found to have many potential drug-binding sites (>10). Recognizing the therapeutic efficacy of co-inhibition of nucleoprotein with phosphoprotein, screening encompassed two active site settings: a) inclusive of all potential drug-binding sites, including chaperone-governing sites (Fig. 3, A) and b) confined to NiV chaperone-governing sites in Carboxy-terminal domain (CTD) (residues 259-371, colored red in Fig. 1, E). Among 72 ligands screened, stigmasterol exhibited strong binding affinity (-9.2 kcal/mol) (Fig. 3, B) and Quantitative structure-activity relationship (QSAR) studies revealed stigmasterol's relatively higher lipophilicity compared to andrographolide as evidenced by Log Poctanol/water (6.97), MR (132.75), TPSA (~20 Å2) and limited aqueous solubility as shown in (Fig. 3, C). towards Nipah virus (NiV) nucleoprotein near chaperone-governing sites (Fig. 3, D, E) suggesting potential competitive inhibition against phosphoprotein (-7.8 kcal/mol). Even with active site residues restricted to (259-371), stigmasterol displayed considerable binding affinity toward NiV nucleoprotein (-7.0 kcal/mol) with Top-9 drug binding poses of stigmasterol with Nipah (NiV) nucleoprotein (Fig. 3, F) and viral chaperone-governing residues targeted by stigmasterol are illustrated (Fig. 3, G; Blue: Chaperone-governing residues in NiV nucleoprotein, Green: stigmasterol-targeting residues) indicating strong potential for effective inhibition at nucleoprotein chaperone-governing sites. Gastrointestinal tract (GI) absorption simulation showed stigmasterol's limited intestinal absorption (2% in 50 minutes) (Fig. 3, H) leading to significantly reduced plasma drug concentration (0.025 µg/ml absorption in 24 hours) with marginal improvement even after extending simulation to 240 hours (0.030 µg/ml) (Fig. 3, I). However, it is worthwhile to recognize the potential accumulation of highly lipophilic molecules in muscle and fat/adipose tissues acting as controlled drug releasing depots into plasma over time. Further, albumin in plasma may hinder tissue extraction of lipophilic drugs from blood. Dissolution and systemic circulation of stigmasterol were lower compared to andrographolide possibly due to its high lipophilicity toward plasma proteins (Fig. 3, J).

Further, the dimensions of the functionalized fullerene conjugated nanomolecules have been measured by in silico studies. FIG. 4 shows the in silico measured sizes of Andrographolide-conjugated C20- and C60- Fullerene nanostructures and FIG. 5 shows the Stigmasterol-conjugated C20- and C60- Fullerene nanostructures.
Another embodiment of the present invention relates to the results of Drug likeliness, absorption, distribution, metabolism, excretion and toxicity (ADMET) studies of Nipah virus (NiV) phosphoprotein and nucleoprotein inhibitors.
4. Drug likeliness, absorption, distribution, metabolism, excretion and toxicity (ADMET) studies of Nipah (NiV) phosphoprotein and nucleoprotein inhibitors:
The assessment of docking efficiency, target specificity, drug-likeliness and basic absorption and dissolution in the human gastro intestinal tract (GI) and plasma through in silico approaches, Quantitative structure-activity relationship (QSAR) based Absorption, distribution, metabolism, excretion and toxicity (ADMET) studies are undertaken to gain insights into the pharmacokinetics of andrographolide and stigmasterol and are presented in tabular format as shown in Table 1 as presented below. The experiments are crucial for assessing the benefit versus risk ratio of utilizing these in silico identified anti-chaperone molecules and the resulting nanostructures.
Table 1: in silico absorption, distribution, metabolism, and toxicity (ADMET) study data on andrographolide and stigmasterol using a) admet SAR 3.0 and b) virtual models for property evaluation of chemicals within a global architecture
The results indicated the potential for both andrographolide and stigmasterol to traverse the GI tract and blood-brain barrier (BBB), a significant advantage against Nipah (NiV) induced encephalitis, wherein the virus migrates through endothelial tight junctions toward brain tissues, causing severe oxidative stress and inflammation. Caco-2 permeability analysis suggests better paracellular movement in the GI tract for stigmasterol possibly due to its high lipophilicity, although oral availability relies on various factors. Both compounds have been found to be substrates and non-inhibitors of P-glycoprotein implying enhanced effectiveness at higher doses to overcome P-mediated drug efflux. With the exception of CYP3A4, the ligands showed no substrate or inhibitory activity toward major hepatic drug-metabolizing enzymes. Toxicity simulations revealed no mutagenic or carcinogenic or teratogenic potential for andrographolide and stigmasterol consistent with their natural origin and safety profile as constituents of herbs, edible materials and nutraceuticals.
Another embodiment of the present invention relates to the results of homology modeling, ligand functionalization and molecular docking of andrographolide- and stigmasterol-conjugated C20 Fullerene and C60 Fullerene conjugates.
5. Homology modeling, ligand functionalization and molecular docking of functionalized andrographolide and stigmasterol C20 Fullerene conjugates
The potential of Andrographolide and Stigmasterol in inhibiting the Nipah virus (NiV) RNA dependent RNA polymerase studied in silico. RNA polymerase for RNA synthesis (RdRp) being pivotal for Nipah (NiV) replication with conserved residues and lack of homology with human proteins targeting RdRp along with chaperone-governing residues to enhance antiviral therapy effectiveness without causing adverse effects. Since the structure of RdRp is not experimentally resolved, its three-dimensional structure (residues: 1-971; template: Protein data bank (PDB 6v85A)) has been homology modeled (Fig. 6, A) and verified through various analyses including Ramachandran plot (99.4% residues lying within allowed regions) and distortion analysis. Structural alignment between models generated in normal (green; single template; 6v85A) and intensive modes (blue; two-template system; PDB: 6v85A and 7youA) (Fig. 6, B) showed consistency in quality and conservation. The modeled loop portion (in magenta colour) showed that lacked homologous template structures generated using ab initio approach have not been included in active sites for further experiments. The potential drug-binding sites in NiV RdRp study is presented in (Fig. 6, C). Active sites also included additional residues known to bind with RdRp. Interestingly both andrographolide and stigmasterol inhibited NiV-RdRp with appreciable binding affinities (B.E.: -7.5 and -9.1 kcal/mol, respectively) suggest their capability to target viral RNA synthesis and recruitment for effective virion budding and replication. To further enhance binding affinity and potency, the ligands are strategically functionalized with phosphate (for andrographolide) or phosphate and carboxylic groups (for stigmasterol) at designated positions covalently conjugated to C20-fullerene quantum dots of size ~ 0.4 nm. The yielded functionalized andrographolide-conjugated C20- fullerene conjugates (ACFN) shown in (Fig. 6, D) and functionalized stigmasterol-conjugated C20 Fullerene conjugates (SCFN) shown in (Fig. 6, G). Since nucleotide triphosphates, the natural substrates of RdRp recognize the viral enzyme primarily through phosphate groups via hydrogen bonds, electrostatic interactions and other intermolecular interactions such that phosphate functionalization enhance the docking efficiency toward RdRp. Fullerenes are selected for their potential antiviral, antioxidant and free radical scavenging activity. Moreover, fullerenes demonstrated successful crossing of the Blood-brain barrier (BBB) suggesting that conjugating Andrographolide and Stigmasterol to Fullerene could aid their facile paracellular transport toward the tight junctions of BBB potentially combating Nipah virus (NiV) in brain (antiviral) and treating NiV-induced encephalitis (antioxidant, anti-inflammatory effects). C20 fullerene quantum dots chosen for their small size (~0.4 nm), low molecular weight (240.2) and spherical shape (icosahedral) are relatively less recognized by the reticuloendothelial system. As anticipated, both andrographolide and stigmasterol displayed significant binding affinity towards NiV RdRp (B.E.: -10.50 and -9.55 kcal/mol, respectively) in the functionalized conjugated form (Fig. 6, E, H). The binding affinities with Nipah virus (NiV) chaperone-governing residues of NiV phosphoprotein and nucleoprotein also have substantially improved binding energies (B.E.: -6.47 and -13.50 kcal/mol, respectively). Further, (Fig. 6, F, I) suggest the andrographolide and stigmasterol to simultaneously inhibit NiV-RNA synthesis, recruitment, packaging and new virion budding more effectively when delivered in the form of functionalized C20 Fullerene conjugates. Further, Fig. 4 table presents the docking results of unconjugated and C20 Fullerene conjugates of andrographolide and stigmasterol with NiV phosphoprotein, nucleoprotein and RdRp along with their closely interacting amino acid residues (<4 Å distance between the ligand atoms and NiV target residues).
The detailed analysis of the interacting residues elucidated the binding mechanism underlying the inhibition of the target enzyme (NiV RdRp) by the ligand (ACFN). The interactions between ACFN and RdRp are predominantly stabilized by Vander Waals forces, hydrophobic interactions and by the formation of four crucial hydrogen bonds (Fig. 7, A). Specifically, phosphate group in ACFN acts as an oxygen donor forming hydrogen bonds with the hydrogen atoms in the amino groups of Lys 547 and Lys 555 within the enzyme. Furthermore, while dihydrofuran ring of ACFN established a hydrogen bond with Lys 25, amide linkage in ACFN formed another hydrogen bond with hydroxyl group of Tyr 487. Hydrophobic interactions by Tyr 32, Val 491, and Leu 492 of RdRp with fullerene moiety of ACFN enhanced the binding affinity. Moreover, the intermolecular and Van der Waals interactions involve residues Lys 59, Ser 60, Val 486, and Pro 488 which contribute towards the overall stability of the complex leading to a binding affinity of -10.50 kcal/mol indicative of effective enzyme inhibition. In the case of ACFN-NiV phosphoprotein complex, residues Ile 13, Phe 16, Ile 17, and Ile 24 interact hydrophobically with C20 Fullerene moiety, while Gln 21, Gln 25, Tyr 28, Ser 32, and Ile 33 contribute to binding affinity through Van der Waals interactions ensuring their overall stability. Additionally, phosphate group of functionalized ACFN formed a hydrogen bond with Arg 30 and two hydrogen bonds are formed between Ser 31 and ACFN (Fig. 7, B). Interestingly, in the case of SCFN, the binding with NiV RdRp or nucleoprotein in addition to hydrophobic interactions, Vander Waals and hydrogen bonds salt bridges are found to significantly contribute to the stability of ligand protein complexes by providing strong electrostatic interactions between charged groups. Specifically, the positively charged amines in Arg 361, Lys 893, Lys 907, and Arg 911 of RdRp electrostatically attracted the negatively charged diphosphate groups of SCFN (Fig. 7, C). Similarly, for SCFN-NiV nucleoprotein complex, anionic phosphate moiety is specifically recognized by positively charged amines in Glu 190, Arg 193, and Lys 178 of nucleoprotein (Fig. 7, D). Some scarce hydrogen bonds formed between SCFN and RdRp (Leu 552), and SCFN and NiV nucleoprotein (Leu 348). Interestingly, ACFN and SCFN also interacted covalently with their Nipah virus (NiV) targets. For ACFN-NiV RdRp complex, a covalent bond formed between carbonyl oxygen atom of ACFN with a hydroxyl group of Tyr 487 (Fig. 7, E). For ACFN-phosphoprotein docked complex, two covalent bonds found between hydroxyl groups of ACFN and Ser 31 of phosphoprotein (Fig. 7, F). For SCFN-NiV RdRp complex, diphosphate (oxygen) group covalently interacted with amine group (nitrogen atom) of Lys 907 of RdRp (Fig. 7, G) and for SCFN- nucleoprotein complex, two covalent bonds found as: (a) diphosphate group (oxygen) of SCFN with hydroxyl group (oxygen) of Thr 185 and (b) diphosphate group (oxygen) of SCFN with amine group (nitrogen) of Glu 190 (Fig. 7, H). Thus, phosphate functionalization of the ligands followed by covalent conjugation of C20- fullerene aided to achieve better molecular recognition and binding stability of andrographolide and stigmasterol toward NiV RdRp and their respective targets, phosphoprotein and nucleoprotein.
6. In silico gene expression, drug-likeliness, toxicity, and QSAR studies of ACFN and SCFN
Following docking efficiency assessment, analysis of gene expression modulation by C20-ACFN and C20-SCFN in silico is presented in Table 2. The ACFN showed potential upregulation of VDR protein whose increased expression is associated with combating hypertension, inflammation and cognitive disorders, the hallmark symptoms of Nipah virus (NiV)-induced encephalitis. Further, potential downregulation of TMSB15A gene associated with hepatomegaly and neurotoxicity (symptoms often seen in NiV-infected patients) is also observed. Moreover, ACFN indicated upregulation of genes SQLE, SFRP1, CACNG4, CHRNE and FOXO4 linked to combating similar disorders. SCFN was found to potentially inhibit MDM2 protein, whose increased expression is associated with chromosome breakage, hepatomegaly and inflammation along with potential upregulation of VDR, CACNG4, and CHRNE genes. Shared upregulation of VDR, CACNG4, and CHRNE genes between ACFN and SCFN which suggests influence by the unique molecular architecture of C20- fullerenes such as sp2-hybridized carbons and delocalized π-electrons, possibly offering antioxidant properties to mitigate oxidative stress related damage.
Drug-likeness, toxicity and Quantitative structure-activity relationship (QSAR) studies of ACFN and SCFN quantum dots is conducted in silico and the results are presented in tabular format in (Table 2 A, B, C). Both ACFN and SCFN demonstrated favorable GI absorption and potential Blood-brain barrier (BBB) penetration crucial for systemic antiviral defense and treating NiV-induced encephalitis. Neither ACFN nor SCFN exhibited hepatoxicity, carcinogenicity, mutagenicity or mitochondrial membrane potential modulation aligning with the biocompatibility and generally regarded as safe (GRAS) category of andrographolide, stigmasterol and fullerenes. QSAR studies suggested favorable interaction of ACFN with biological targets due to increased rotatable bonds, hydrogen bond donors and acceptors facilitating interactions in physiological medium. Molar Refractivity (MR), Topological polar surface area (TPSA) and octanol-water coefficient values suggested efficient interaction of ACFN through polar bonds and hydrophobic interactions with potential for high penetration in cell membranes. SCFN displayed similar QSAR profiles with increased hydrophobicity offset by diphosphate and monocarboxylic groups, promoting stable interactions with biological targets, supported by their binding affinity with NiV targets (-9.5 kcal/mol for RdRp and -13.5 kcal/mol for nucleoprotein).
Table 2: (a) in silico gene expression, (b) drug-likeliness and toxicity data, and (c) Quantitative structure-activity relationship (QSAR) studies on functionalized Andrographolide-conjugated C20 Fullerene nanostructures (C20-ACFN) and functionalized Stigmasterol-conjugated C20 Fullerene (C20-SCFN) quantum dots.

7. Homology modeling, ligand functionalization and molecular docking of andrographolide and stigmasterol C60 Fullerene conjugates
After evaluating the docking efficiency, the nature of chemical interactions, QSAR and toxicity of C20-based ACFN and SCFN systems, the in silico studies were extended to C60-based ACFN and SCFN for targeting the viral RdRp and the chaperone proteins. These studies were crucial for understanding the specificity of molecular recognition and binding affinity towards NiV protein targets, considering variations in the nanoparticle size (~0.4 for C20 Vs ~0.7 nm for C60-fullerenes), structure (pentagonal Vs Pentagonal plus hexagonal faces) and symmetry (C60 has more symmetry) between C20- versus C60- fullerenes. Modified C60 fullerenes (~0.7 nm) were modeled and covalently conjugated with phosphate/carboxyl-functionalized andrographolide or stigmasterol to obtain C60-ACFN or C60-SCFN. The structures have been geometrically optimized, energy minimized and utilized for the molecular docking studies. Fig. 8, A1-A4 and B1-B4 illustrates the docking results of C60-ACFN with NiV phosphoprotein and RdRp, showing significant binding affinities of -7.6 kcal/mol and -10.4 kcal/mol, respectively. C60-SCFN also showed potent inhibition of NiV nucleoprotein (B.E.: -11.0 kcal/mol, Figure 1, C1-C4) and RdRp (B.E.: -12.1 kcal/mol, Figure 1, D1-D4) as indicated by the docking simulations. Similar to the C20-based ACFN and SCFN (C20-ACFN, C20-SCFN), the functionalized andrographolide and stigmasterol exhibited strong and specific binding affinities towards the NiV target proteins in the C60 Fullerene-based conjugated forms (C60-ACFN and C60-SCFN), potentially inhibiting the NiV RNA synthesis, recruitment, packaging, and assembly.
Detailed analysis of interacting residues revealed that C60-ACFN-NiV phosphoprotein interactions are stabilized by the combination of hydrophobic interactions, hydrogen bonds and pi-stacking interactions (Fig. 8, A). Specifically, residues ILE 13, ILE 17, GLN 25, and ILE 33 contributed to hydrophobic stabilization, while oxygen atoms in C60-ACFN formed hydrogen bonds with ILE 13 and ILE 17. Pi-stacking interactions were observed between several atoms in C60-ACFN and TYR 28 of the NiV phosphoprotein, contributing to the overall stability.
For C60-ACFN - NiV RdRp docked complex (Fig. 8, B), residues PRO 13, GLU 14, TYR 62, ILE 184, ASN 213, PRO 232 and GLU 233 interacted hydrophobically. Seven hydrogen bonds formed between C60-ACFN oxygen atoms and residues LYS 25, SER 60, SER 187, PRO 212, ASN 213, and TYR 62 (two H-bonds) of NiV RdRp led to a binding affinity of -10.4 kcal/mol.
The interactions of stigmasterol with NiV nucleoprotein were also found to be significant when delivered as C60- fullerene conjugates (C60-SCFN, Fig. 8, C). Residues ALA 182, TYR 258, GLU 261, THR 262, ALA 265, ALA 323, PRO 324, TRP 331, ALA 347, LEU 348, ARG 352, TYR 354 interacted hydrophobically with the alkyl groups of C60-SCFN. Interestingly, two hydrogen bonds formed: one between an oxygen atom of ARG 352 (H-bond donor) with an oxygen atom of C60-SCFN (acceptor) and another between an alpha carbon of GLY 263 (H-bond donor) and C60-SCFN (acceptor), the net effect leading to a binding affinity of -11.0 kcal/mol. Moreover, C60-SCFN conjugates indicated effective inhibition of NiV RdRp, in silico (Fig. 8, D). Specifically, residues PRO 13, GLU 14, LYS 25, TYR 32, ILE 184, ASN 213, PRO 232, GLU 233, PRO 488 stabilized the binding of the nanomolecules/nanostructures with the viral protein hydrophobically while a nitrogen atom from the amine group in Val 491 of NiV RdRp (H-bond donor) was found to make H-bonds with an oxygen atom (acceptor) from the phosphate group of C60-SCFN. Similarly, a nitrogen atom from the amine group in His 55 of NiV RdRp (H-bond donor) was found to make H-bonds with another oxygen atom (acceptor) from the phosphate group of C60-SCFN.
Further, pi-cation interactions between the aromatic group of C60-SCFN with LYS 25 of RdRp and salt bridges formed between the carboxylic group of C60-SCFN with Lys 25, and the phosphate group of C60-SCFN with His 55, further stabilized the interactions, resulting in a significant binding affinity of -12.1 kcal/mol, indicative of an effective enzyme inhibition. Thus, C60-based ACFN and SCFN showed high potential for inhibiting NiV nucleoprotein, phosphoprotein, and RdRp similar to C20-based counterparts (ACFN and SCFN).
8. Drug-likeliness, toxicity and QSAR studies of C60-ACFN and SCFN
Analysis of the potential inhibitory properties of C60-ACFN and C60-SCFN in Nipah virus (NiV) phosphoprotein, nucleoprotein and RdRp studies were conducted on the drug-likeness, toxicity, and quantitative structure-activity relationship (QSAR) of modified C60 conjugated functionalized andrographolide (C60-ACFN) and modified C60 conjugated functionalized stigmasterol (C60-SCFN), in silico and the results have been presented in Table 3 as shown below.
Table 3: the in silico drug-likeliness, toxicity and quantitative structure-activity relationship (QSAR) studies on functionalized andrographolide-conjugated C60 fullerene (C60-ACFN) and functionalized stigmasterol-conjugated C60 Fullerene molecules (C60-SCFN ).
Similar to their C20 based counterparts, ACFN and SCFN, both C60 ACFN and C60 SCFN showed favorable gastrointestinal (GI) absorption and potential blood-brain barrier (BBB) penetration. These characteristics are crucial for systemic antiviral defense and for treating the virus inflammation in the brain and NiV induced encephalitis. Importantly, in silico studies have also revealed that neither C60 ACFN nor C60 SCFN exhibit potential hepatotoxicity, carcinogenicity, mutagenicity, or mitochondrial membrane potential modulation, which align with the biocompatibility and Generally Recognized as Safe (GRAS) status of andrographolide, stigmasterol, and fullerenes. QSAR studies indicated that C60 ACFN has favorable interactions with biological targets, attributed to increased numbers of rotatable bonds and hydrogen bond donors and acceptors, which facilitate interactions in physiological environments. The molecular refractivity (MR), topological polar surface area (TPSA), and octanol-water partition coefficient values suggested that C60 ACFN can efficiently interact with cell membranes through polar bonds and hydrophobic interactions. Similarly, C60 SCFN demonstrated comparable QSAR profiles, with increased hydrophobicity relatively less balanced despite the presence of diphosphate and monocarboxylic groups, owing to the hydrophobicity contributed by C60 fullerenes. Nevertheless, the apparent small size (~0.7 nm) together with its large surface area (~6.33 nm2) aids to enhance stable interactions with biological targets. This stability was supported by their binding affinities to NiV targets, with C60 ACFN showing a binding affinity of -11.0 kcal/mol for nucleoprotein and -12.1 kcal/mol for RdRp.

ADVANTAGES OF THE PRESENT INVENTION

1. An advantage of the present invention discloses Andrographolide-conjugated C20 and C60 fullerene molecules and Stigmasterol-conjugated C20 and C60 fullerene molecules that have the potential to target at least three key proteins of Nipah virus (NiV) that are RNA dependent RNA polymerase (RdRp) and chaperone-governing sites in phosphoprotein and in nucleoprotein which are structurally conserved and critical for the virus for its activation, replication, propagation and formation of the new virions. These molecules can also form nanostructures through self-assembly.
2. Another advantage of the present invention is 'Synergistic Antiviral Activity' which have the potential to simultaneously target multiple stages of the viral replication cycle due to combined inhibitory effect of RNA polymerase RNA synthesis (RdRp) and viral chaperone to show synergistic effects and enhance antiviral activity by targeting each component individually.
3. Another advantage of the present invention is 'Disruption of Viral RNA Synthesis and Packaging' which inhibits RNA polymerase for RNA synthesis (RdRp) and impairs viral ribonucleic acid (RNA) synthesis by inhibition of viral chaperones which disrupts RNA recruitment, packaging and assembly. The combined inhibitions disrupt multiple critical steps in the viral replication cycle for more profound inhibition of viral replication and assembly.
4. An advantage of the present invention is 'Reduced Risk of Resistance Development' Since two distinct viral components (RdRp enzyme activity and chaperone activity) are simultaneously targeted and the likelihood of the Nipah virus (NiV) developing drug resistance to both inhibitors simultaneously gets reduced as one inhibitor compensate for the activity of the other thereby limiting the emergence of drug resistant strains.

5. Another advantage of the present invention is 'Blockade of Viral Spread and Propagation' by impairing viral RNA synthesis, recruitment, packaging and assembly which results in combinatorial inhibition which limits the production of infectious viral particles and reduces viral spread and propagation within the host.

6. Another advantage of the present invention is that such working mechanism provides possibility for broad spectrum antiviral activity of the developed Andrographolide and Stigmasterol-conjugated C20 Fullerene and C60 Fullerene molecules and the resulting nanostrcutures, as the targeting of conserved proteins across multiple strains of Nipah virus (NiV) and other related viruses within the Paramyxoviridae family reduces the off-target effects on the host cellular processes to result in improved safety profiles.

7. Yet another advantage of the present invention is 'Enhanced Inhibition Efficacy and Viral Control' which provides a comprehensive strategy for controlling Nipah virus (NiV) infection to improve the inhibition efficacy and viral control by disrupting the formation of viral nucleocapsids and maturation and assembly of new virions. , Claims:We Claim:

1. Functionalized fullerene conjugated nanomolecules for the inhibition of Nipah virus infections having formula (I)




(I)
and formula (II)






(II)
wherein formula (I) comprises a C20 Fullerene nanostructure and formula (II) comprises a C60 Fullerene nanostructure, wherein 'R1' group is a functionalized diterpene lactone or a functionalized phytosterol that is covalently conjugated
2. The functionalized fullerene conjugated nanomolecules as claimed in claim 1, wherein the nanomolecule is Andrographolide Conjugated Fullerene nanomolecule (ACFN) when the diterpene lactone is Andrographolide functionalized with two phosphate groups.

3. The functionalized fullerene conjugated nanomolecules as claimed in claim 1, wherein the nanomolecule is Stigmasterol Conjugated Fullerene nanomolecule (SCFN) when the phytosterol is Stigmasterol functionalized with a diphosphate and a monocarboxylate group.
4. The functionalized fullerene conjugated nanomolecules as claimed in claim 1, wherein the nanomolecules work by combinatorial inhibition of RNA-dependent RNA polymerase and viral activity-governing residues in the phosphoprotein or nucleoprotein of the Nipah virus.

5. A method of preparation of the functionalized Andrographolide Conjugated Fullerene nanomolecule (ACFN) as claimed in claim 2, comprising the steps of:
(a) carboxylating the C20 Fullerene and the C60 Fullerene nanostructures;
(b) purifying the obtained C20 Fullerene and the C60 Fullerene using column chromatography;
(c) analysing the purified carboxylated C20 Fullerene and the C60 Fullerene obtained in step (b) using organic spectroscopic techniques;
(d) reacting methyl iodide, phosphorous oxychloride/phosphorus iodide in the sodium hydroxide base and under inert gas conditions in a separate reaction;
(e) stirring the reaction mixture of step (d) at 400 rpm to 600 rpm at temperature of 4°C for 12 hours;
(f) increasing the temperature to 25°C gradually;
(g) incubated the reaction mixture at 800 rpm to 1000 rpm for additional 12 hours;
(h) quenching the excess of the reagents;
(i) extracting and purifying the product obtained after step (h) using column chromatography or high performance liquid chromatography;
(j) analyzing the purified product obtained in step (i) with organic spectroscopic technique;
(k) aminating the product obtained in step (j) using a suitable aminating agent, organic acid and a solvent mixture;
(l) centrifuging, washing and purified the aminated product obtained in step (k) using chromatography;
(m) characterising the purified aminated product obtained in step (l) using high performance liquid chromatography and mass spectroscopy;
(n) conjugating the obtained aminated product with the purified carboxylated C20 Fullerene and C60 Fullerene obtained in step (b); and
(o) washing and purifying the conjugated product obtained in step (n).

6. A method of preparation of the functionalized Stigmasterol Conjugated Fullerene nanomolecule (SCFN) as claimed in claim 3, comprising the steps of:
(a) carboxylating the C20 Fullerene and the C60 Fullerene nanostructures;
(b) purifying the obtained C20 Fullerene and the C60 Fullerene using column chromatography;
(c) analysing the purified carboxylated C20 Fullerene and the C60 Fullerene obtained in step (b) using organic spectroscopic techniques;
(d) methylating Stigmasterol using known concentrations of methyl iodide, potassium carbonate or sodium hydroxide slowly in a suitable amount of organic solvent and co-solvent mixture;
(e) neutralising the reaction mixture obtained in step (d) using an acid;
(f) purifying the obtained product in step (e) using column chromatography;
(g) charaterizing the purified product obtained in step (f) with organic spectroscopic technique;
(h) dispersing the methylated product after characterization in step (g) in an mixture of organic solvent and phosphorus oxychloride to form an intermediate phosphate ester product;
(i) adding a suitable nucleophile donor such as ethylene glycol;
(j) extracting and purifying the obtained product in step (i) using column chromatography or high performance liquid chromatography;
(k) analysing the resultant product using using mass spectroscopy and nuclear magnetic resonance spectroscopy;
(l) aminating the product obtained in step (k) using an aminating agent;
(m) centrifuging, washing and purified the aminated product obtained in step (l) using chromatography;
(n) characterising the purified aminated product obtained in step (l) using high performance liquid chromatograpy and mass spectroscopy technique;
(o) conjugating the obtained aminated product after characterisation in step (m) with the purified carboxylated C20 Fullerene and C60 Fullerene obtained in step (b); and
(p) washing and purifying the conjugated product obtained in step (o).

7. The method of preparation of the functionalized Andrographolide and Stigmasterol Conjugated Fullerene nanomolecules as claimed in claim 5 and claim 6, wherein the aminating agent comprises of mixture of primary or secondary amines together with sodium cyanoborohydride, acetic acid, methylene chloride, methanol or dichloromethane.

8. The method of preparation of the functionalized Andrographolide and Stigmasterol Conjugated Fullerene nanomolecules as claimed in claim 5 and claim 6, wherein the organic spectroscopic techniques for characterisation and analysis of products are mass spectroscopy and nuclear magnetic resonance spectroscopy.

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