Phialomustin b (1) for treatment of parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS)

Abstract

Phialomustin B (1) for treatment of Parkinson's disease (PD) and Amyotrophic lateral sclerosis (ALS) The present invention provides Phialomustin B (1) a natural product isolated from the endophytic fungus Phialophora Mustea from the Corms of Crocus sativus. The present invention describes therapeutic potential of Phialomustin B (1) as Keap1 inhibitor for Parkinson's disease (PD) and SOD-1 modulator for Amyotrophic lateral sclerosis (ALS). The present invention also provides a pharmaceutical composition comprising an effective amount of Phialomustin B (1) of Formula I.

Description

Phialomustin B (1) for treatment of Parkinson’s disease (PD) and Amyotrophic lateral sclerosis (ALS)FIELD OF THE INVENTION

[0001] The present invention relates to the discovery of a novel therapeutic potential of Phialomustin B (1) a natural product molecule which is isolated and reported first time from the endophytic fungus Phialophora Mustea from the Corms of Crocus sativus. The invention relates to the discovery of new therapeutic potential of 1 depicting Keapl inhibitor for Parkinson’s disease (PD) and SOD-1 modulator for Amyotrophic lateral sclerosis (ALS).BACKGROUND OF THE INVENTION

[0002] Endophytic fungi are one of the richest sources of novel bioactive secondary metabolites which display a broad spectrum of biological activities viz., antimicrobial, immunosuppressive, anticancer, antioxidant, and neurological disorder etc.[Ali et. al. 2015, RSC Adv.5, 95307-95312, Strobel, et. al, J. Nat. Prod. 2004, 67, 257-268]. The proficiency of endophytes to produce diverse secondary metabolites inspired us to explore the isolation of the bioactive metabolite from an endophytic fungus, Phialophora mustea isolated from Crocus sativus. Recent literature indicated that only 12 molecules were reported from the genus Phialophora, however, no such study has been carried out on Phialophora mustea. Our previous report for isolation of four distinct azaphilone derived molecules PhialomustinA-D exhibit Antimicrobial and cytotoxic potential [Ali et. al. 2015, RSC Adv.5, 95307-95312]. The present invention reveals the discovery of novel therapeutic potential of Phialomustin B (1). Keapl is a cytosolic repressor protein of the transcription factor, Nrf2. Nrf2 is the protein responsible to activate the transcription of the antioxidant responsive element (ARE) pathway. Direct inhibition of the protein-protein interaction between Keapl -Nrf2 serves as a therapeutic strategy to overcome cellular oxidative stress generated in neurodegenerative diseases.

[0003] Superoxide Dismutase 1 (SOD1) is an antioxidant enzyme that serves in conversion of superoxide radicals to molecular oxygen and hydrogen peroxide. Clinically significant mutations have been described in this enzyme which causes structural aberrations and is known to cause a toxic gain of function. This leads to loss of motor neurons in brain stem and spinal cord causing a terminal neurodegenerative disease called, amyotrophic lateral sclerosis (ALS).The Keapl-Nrf2 defence system and Parkinson’s disease (PD)

[0004] The transcription factor Nuclear erythroid 2-related Factor (Nrf2) is pledged to influence many ARE driven cytoprotective genes. The Nrf2 factor expression inside the cell is compactly controlled by the kelch-like ECH-associated protein 1 (Keapl) (Ganan-Gomez et al., 2013, Free Rad. Biol. & Med. 65, 750-764). The Keapl (624 aa) protein is homologous to the Drosophila actin binding protein, kelch. The Keapl protein is mainly localized in the cytoplasm of cell. The Keapl - Nrf2 pathway is the major cell protection mechanism and it will curb the oxidative stress as well as xenobiotic stress. The Keapl protein acts as a negative regulator of Nrf2 transcription factor and hence modulates the cellular level of Nrf2. The Nrf2 transcription factor is dynamic and expressed continuously at normal physiological conditions. Under homeostasis condition, Nrf2 interacts with Keapl and undergoes ubiquitination followed by 26S proteasomal degradation thus maintaining optimal level of Nrf2 inside the cells (Deshmukh et al., 2017, Biophys. Rev. 9(1), 41-56). However, under the oxidative / xenobiotic stress conditions, the Keapl protein undergoes phosphorylation / redox modifications which in-turn halt the ubiquitination and degradation of Nrf2 by negating the Keapl -Cul3-Rbxl E3 ubiquitin ligase complex. Hence, cytosolic level of Nrf2 decreases and helps in the translocation and accumulation of Nrf2 in the nucleus. Inside the nucleus, Nrf2 with help of other transcription factors, it activates the ARE / EpRE promoter region. The activation of ARE promoter leads to the transcription of genes encoding detoxifying enzymes and antioxidant proteins. Upon availing the basal cellular homeostatic conditions, Nrf2 translocate back to the cytoplasm and it undergoes proteasomal degradation with the help of Keapl and Cul3-Rbxl core ubiquitin complex(Villeneuve et al., 2010, Antioxidant & redox signaling, 13(11), 1699- 1712). The activation of Nrf2 is crucial for the regulation of antioxidant enzymes and proteins for all living cells, especially neurons. These antioxidant enzymes and proteins helps in curbing the oxidative stress, leading to cell protection as well as cell survival. The Keapl-Nrf2-ARE pathway is a potential drug target in various neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and Parkinson’s disease (Calkins et al., 2009, Antioxidants & redox signalling, 77(3), 497-508). The protective role of Nrf2 in PD has also been studied in vivo and in vitro models. Dopamine analogue 6-hydroxyldopamine (6- HAD) which is highly neurotoxic is found to activate the Nrf2-ARE system which in turn, activates cellular defence mechanism to protect against oxidative stress (Jakel et al. 2007, Brain res. 1144, 192-201).Superoxide Dismutase 1 (SOD1) and Amyotrophic lateral sclerosis (ALS)

[0005] Superoxide Dismutase 1 (SOD1) is a relatively high studied member of the protein family, considering its critical role in the pathology of a non-curable neurodegenerative disease, Amyotrophic Lateral Sclerosis (ALS). It has metal ions Cu and Zn playing an essential role in the maintenance of the structural stability of the protein. It is a 153 amino acid containing monomer subunit with a molecular mass of 32 kDa and naturally exists as a homodimer. ALS causing SOD1 mutations that contribute to the development of familial amyotrophic lateral sclerosis have been intensively studied. The genetically inherited ALS (Lou Gehrig’s disease) is called familial amyotrophic lateral sclerosis (fALS), which forms about only the 10% of the total ALS cases. Studies reveal that around 20-25% of the ALS cases are caused due to mutations in the SOD1 protein. There has been around 180 mutations discovered in SOD1 protein sequence which gives rise to ALS symptoms. These mutations have been studied through in-vivo and in-vitro cellbased assays to extract the optimum folding mechanism and elucidate the diseased state for further research on its therapeutic target. Mutations in SOD1 cause the loss of metal ions which leads to the loss of structural stability and results in dimer dissociation followed by formation of insoluble protein aggregates (Milardi et al.2010, Mol. Bio. Sys., 6(6), 1032-1039). Studies reveal that the zinc deficient species form the majority of the intermediates in the protein misfolding mechanism of mutant SOD1 species, disclosing its contribution to the misfolding mechanism and gradually leading to the aggregate formation. Intensive research has been carried out on the change in structure and functionality of the SOD1 protein, causing formation of soluble or insoluble, mutated, monomer aggregates of the protein, leading to death of the motor neurons of the brain stem and spinal cord. The changes in the protein structure is believed to cause changes in the substrate affinity, impairment of binding of zinc, dimer dissociation and increased protein aggregation, all of which take place in a systematic unfolding mechanism (Rumfeldt et al. 2009, J. Mol. Boil., 3S5(1), 278-298).

[0006] The available commercial drugs for the treatment of Amyotrophic lateral sclerosis (ALS), Riluzole, helps prolong the survival for a small duration in ALS inflicted patients. Previously, crystal structures with modulators near the tryptophan oxidation site have been previously elucidated in the laboratory as well as other research groups (Manjula et al., 2018, FEBS let., 592(10), 1725-1737).

[0007] Therefore, there is a dire need in the art to develop a natural product based on long chain compounds as a Keapl inhibitor for Parkinson's disease (PD) and as a SOD-1 modulator for Amyotrophic lateral sclerosis (ALS).OBJECTIVE OF THE INVENTION

[0008] The main objective of the present invention is discovery of a novel therapeutic potential of Phialomustin B (1) a natural product molecule isolated from the endophytic fungus Phialophora Mustea of the Corms of Crocus sativus depicting Keapl inhibitor for Parkinson’s disease (PD) and Superoxide Dismutasel (SOD1) modulator for Amyotrophic lateral sclerosis (ALS).

[0009] The objectives of the present invention:1. Investigate the effect of 1 on the activation of transcription factor Nrf2 and its downstream antioxidant enzymes by the inhibition of Keapl protein using biochemical and cell-based assays in Parkinson’s disease cell model SHSY5Y cells.2. Investigate the effect of 1 on reduction of SOD1 aggregation (wild-type SOD1 and disease mutant SOD1) by biochemical and aggregation studies.SUMMARY OF THE INVENTION

[0010] Accordingly, the present invention provides an unsaturated fatty acid Phialomustin B (1) exhibiting Keapl inhibitor for Parkinson’s disease (PD) and Superoxide Dismutasel (SOD1) modulator for Amyotrophic lateral sclerosis (ALS).I.

[0011] In an embodiment of the present invention Phialomustin B (1) inhibits the Keapl-Nrf2 interaction by inducing cytoprotectivity in rotenone induced PD model of SHSY5Y cells.

[0012] In another embodiment of the present invention Phialomustin B (1) induces the translocation of Nrf2 and activation of downstream genes like HO-1, NQO1, GCLC etc. as observed by qRT-PCR and western blotting studies.

[0013] In yet another embodiment of the present invention Phialomustin B (1) binds to SOD1WTprotein at KD value of 5 pM through MST assay.

[0014] In an embodiment of the present invention Phialomustin B (1) inhibits aggregation in SOD1I113Tin a mutation specific manner under metal chelation conditions by EDTA.

[0015] In another embodiment of the present invention Phialomustin B (1) induces aggregation in another mutant SOD1A4Vunder metal chelation conditions by EDTA.

[0016] The present invention provides a method for inhibition of Keapl protein for treatment of Parkinson’s disease.

[0017] The present invention provides a method for modulating Superoxide Dismutasel (SOD1) for treatment of Amyotrophic lateral sclerosis (ALS).

[0018] The present invention provides a pharmaceutical composition for inhibition of Keapl protein for treatment of Parkinson’s disease.

[0019] The present invention provides a pharmaceutical composition for modulating Superoxide Dismutasel (SOD1) for treatment of Amyotrophic lateral sclerosis (ALS).

[0020] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0022] Figure 1 depicts the structure of compounds Phialomustin A-D (1- 4) , in accordance with an embodiment of the present disclosure.

[0023] Figure 2 depicts the Keapl -DC purification scheme (A) Ni-NTA affinity purification; (B) His-tag cleavage; (C) Re-affinity purification; (D) Anion exchange chromatography: (E) Gel filtration; (F) Gel filtration chromatogram, in accordance with various embodiments of the present disclosure.

[0024] Figure 3 depicts the quantitative binding studies of the Keapl -DC domain with 1, in accordance with an embodiment of the present disclosure.

[0025] Figure 4 depicts the effect of 1 on the cell survival in SH-SY5Y cells, in accordance with an embodiment of the present disclosure.

[0026] Figure 5 depicts the effect of Ion the protein expression level of cytosolic Nrf2. A. Representative Nrf2 immunoblot, B. Immunoblot analysis representing decrease in the Nrf2 protein expression level in cytosolic fraction after treatment with 1 (lOpM), in accordance with an embodiment of the present disclosure.

[0027] Figure 6 depicts the effect of Ion the protein expression level of nuclear Nrf2, (A) Representative Nrf2 immunoblot, (B) Immunoblot analysis representing significant increase in the Nrf2 protein expression level in nuclear fraction after treatment with 1 (lOpM), in accordance with an embodiment of the present disclosure.

[0028] Figure 7 depicts the effect of 1 on mRNA level of antioxidant enzymes, in accordance with an embodiment of the present disclosure.

[0029] Figure 8 depicts the effect of 1 on the protein expression level of GCLC, (A) Representative GCLC immunoblot, (B) Immunoblot analysis representing increase in the GCLC protein expression level after treatment with 1 (lOpM), in accordance with an embodiment of the present disclosure.

[0030] Figure 9 depicts the effect of 1 on the protein expression level of NQO1, (A) Representative NQO1 immunoblot, (B) Immunoblot analysis representing increase in the NQO1 protein expression level after treatment with 1 (lOpM), in accordance with an embodiment of the present disclosure.

[0031] Figure 10 depicts the SOD1 protein purification scheme, in accordance with an embodiment of the present disclosure.

[0032] Figure 11 depicts the quantitative binding studies of the SOD1 with 1, in accordance with an embodiment of the present disclosure.

[0033] Figure 12 depicts the analytical SEC chromatogram towards evaluation of anti- aggregation propensity of 1 on SOD1A4Vunder metal chelation conditions of EDTA at Ohrs (red line), 24hrs (yellow line) and 48hrs (green line), in accordance with an embodiment of the present disclosure.

[0034] Figure 13 depicts the analytical SEC chromatograms towards evaluation of anti- aggregation propensity of 1 on SOD1I113Tunder metal chelation conditions of EDTA at Ohrs (blue line), 24hrs (red line) and 48hrs (grey line) in a dose dependent manner of various proteins: ligand (P: L) ratios; (A) 1:5; (B) 1:10, and (C) 1:30, in accordance with an embodiment of the present disclosure.DESCRIPTION OF THE INVENTION

[0035] The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.Definitions:

[0036] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0037] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0038] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".

[0039] Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0040] The term “endophytic fungus” refers to an endophytic fungus endosymbioant, that lives within a plant for at least part of its life cycle without causing apparent disease.

[0041] The term “Keap 1” refers to a cytosolic repressor protein of the transcription factor.

[0042] The term “Nrf2” refers to the protein responsible to activate the transcription of the antioxidant responsive element (ARE) pathway. Keapl represses Nrf2 by directly sequestering it and directing the latter towards ubiquitination and proteosomal mediated degradation. Direct inhibition of the protein-protein interaction between Keapl-Nrf2 serves as a therapeutic strategy to overcome cellular oxidative stress generated in neurodegenerative diseases.

[0043] The terms “Superoxide Dismutase 1” and “SOD1” refer to an antioxidant enzyme that serves in conversion of superoxide radicals to molecular oxygen and hydrogen peroxide. Clinically significant mutations have been described in this enzyme which cause structural aberrations and is known to cause a toxic gain of function. This leads to loss of motor neurons in brain stem and spinal cord causing a terminal neurodegenerative disease called, amyotrophic lateral sclerosis (ALS).

[0044] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0045] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0046] Additionally reports suggest the possibility of long chain compounds to exhibit potentiality towards the stabilization of the dimer interface of SOD1, leading to reduction of aggregation of the protein which is pivotal in ALS syndrome.

[0047] The present invention describes the isolation of Phialomustin B (1) a new unsaturated lapidated natural product isolated from the endophytic fungus of the Corms of Crocus sativus exhibiting the novel therapeutic potential depicting Keapl inhibitor for Parkinson disease (PD) and SOD1 for Amyotrophic lateral sclerosis (ALS). In case of Parkinson’s disease, it was observed that inhibition of Keapl and Nrf2 interaction is mediated by 1 through direct structural interference derivatively through binding to Nrf2 interaction. This disruption in interaction induces cytoprotective activity in PD induced cells.

[0048] On the other hand, SOD1 is one of the most targeted protein in amyotrophic lateral sclerosis (ALS) due to the abundant cases of SOD1 mutations. The molecule,1, not only inhibits self-aggregation of SOD1 in a time- accelerated study but also forms a mutation specificity towards the ALS mutant SOD1I113T.

[0049] In an embodiment of the present disclosure, herein the four new Azaphilone derived molecules from 25 liters culture broth of Phialophora mustea were isolated (Figure 1). After extraction with Ethyl acetate (EtOAc) and concentrated under reduced pressure, afforded 6 g of crude extract, which was subjected to column chromatography over silica gel (230-400 mesh) using a gradient of hexane and ethyl acetate (100:0 to 0:100) to give fractions (Fr. 1-30). Phialomustin A (2 (27 mg) was isolated from fractions Fr. 10 which were eluted with hexane-EtOAC (7:3). Furthermore, fraction (14-30) were pooled on TEC profile (4g) which was rechromatographed over silica gel (230-400 mesh) with a step gradient of hexane and ethyl acetate (6:4) fr. 6-8 gave Phialomustin B (1) (500 mg) was obtained as a colourless viscous, furthermore column eluted with hexane-EtOAC (1:1). Phialomustin C (3) (2.3 g) as yellow solid and Phialomustin D (4) (20 mg) as a yellow solid. After screening all the four compounds Phialomustin B (1) were proven the most potent binding affinity with Keapl-DC. Treatment of PD induced rotenone model of SHSY5Y cells with Phialomustin B revealed increased cell viability due to Nrf2 translocation and activation of downstream ARE genes as revealed through western blotting studies. In case of SOD1, Phialomustin B revealed good binding to the SOD1 protein as seen through microscale thermophoresis. The positively anti-aggregation effects of Phialomustin B on a mutant SOD1 protein, SOD1I113Twas observed through the analytical size exclusion chromatography.

[0050] Furthermore, this invention discloses the

[0051] The present invention discloses the novel therapeutic potential of Phialomustin B (1) as a potential target for Keapl inhibitor for Parkinson’s disease(PD) and Superoxide Dismutasel (SOD1) modulator for Amyotrophic lateral sclerosis (ALS).

[0052] The objective of the present invention is to elaborate to understand the mechanism and utilize as pharmaceutical agents.

[0053] The Phialomustin B (1) embodiment is to provide a method for facilitating the transport of a unsaturated lapidated drug to the brain through intranasal, stereotactic, or intrathecal delivery, or delivery across the blood brain barrier with the present invention to a subject in need thereof.

[0054] The Phialomustin B(l) preferred embodiment, the pharmaceutical compositions are formulated for oral administration which can be formulated, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs. The pharmaceutical compositions can be prepared as per known standard methods and may be used anyone agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

[0055] For tablets formulation require the active ingredient in admixture with suitable non-toxic pharmaceutically acceptable excipients including, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch, or alginic acid; binding agents, such as starch, gelatin or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc.

[0056] The tablets can be uncoated, or they may be coated by known techniques in order to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed to further facilitate delivery of the drug compound to the desired location in the digestive tract.

[0057] Pharmaceutical compositions for oral use can also be prepared as hard gelatin capsules as active ingredient and mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

[0058] Pharmaceutical compositions can be formulated as oily suspensions by suspending the active compound(s) in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and / or flavouring agents may be added to provide palatable oral preparations.

[0059] These compositions can be preserved by the addition of an antioxidant such as ascorbic acid. The pharmaceutical compositions can be formulated as a dispersible powder or granules, which can subsequently be used to prepare an aqueous suspension by the addition of water. Such dispersible powders or granules provide the active ingredient in admixture with one or more dispersing or wetting agents, suspending agents and / or preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavouring and colouring agents, can also be included in these compositions.

[0060] Pharmaceutical compositions can be formulated as a syrup or elixir by combining the active ingredient(s) with one or more sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations can also optionally contain one or more demulcents, preservatives, flavouring agents and / or colouring agents.General Experimental Procedure:

[0061] Melting points of synthesised compounds were recorded on electrical melting point apparatus (Buchi, D-545) and are uncorrected.

[0062] High resolution mass spectra were obtained on Agilent 6540 (Q-TOF) high resolution mass spectrometer, in the electrospray (ESIMS) mode.

[0063] ’ H NMR spectra were recorded (Brucker Avance) DPX FT-NMR at 400 and 500 MHz and13C NMR at 125 MHz in CDCI3 and CD3OD, chemical shifts values are reported in 6 (ppm) units and coupling constants values in hertz. Tetramethyl silane (TMS) was used as internal standard.

[0064] Optical rotation was measured on a Perkin Elmer 341 polarimeter in a 1dm cell at 25°C. CD spectra were recorded using a JASCO J-810 CD spectrometer at a concentration of 1.0 x 10’4M in MeOH at 25 °C.

[0065] Both UV / vis and CD spectra were measured between 200 and 600 nm using 10 mm path-length quartz cuvettes. Infra-red spectra were recorded with a PerkinElmer spectrum 65 FT-IR spectrometer and wavelengths (n) are given in cm' i

[0066] Column chromatography was performed using silica gel (230-400 mesh; Merck) and Sephadex LH-20. Semipreparative HPLC was performed on an Agilent HPLC with a RP-18 column (250 x 4.6 mm, 5 pm; Agilent), a photodiode array detector and auto injector function (Agilent 1260 series).

[0067] Although the subject matter has been described in considerable detail with reference to certain embodiments thereof, other embodiments are possible. As such, the spirit and scope of the disclosure should not be limited to the description of the embodiments contained herein.Examples

[0068] Following are the examples given to further illustrate the invention and should not be construed to limit the scope of the present invention.Example 1

[0069] Fermentation and Isolation: The endophytic fungus was isolated as described in Ali et. al. 2015, RSC Adv.5, 95307-. Corms of Crocus sativus were thoroughly washed with running tap water, and surface sterilized with 1% sodium hypochlorite and 70% ethanol. Before the treatment with alcohol, traces of sodium hypochlorite were removed by washing in sterile distilled water. The outer tissues were removed and the internal tissues were cut into small pieces of 0.5 to 1 cm and plated on water agar (Himedia, India). The plates were incubated at 25°C for three weeks. Hyphal tips of the fungus, emerging out of the plant tissue, were picked and grown on potato dextrose agar (HiMedia, India) in pure culture. The culture was also submitted to the National Fungal Culture Collection of India under the Voucher No. NFCCI 3710. For the extraction of secondary metabolites, the culture wasgrown in shake flasks on PD broth at 25°C and 200 rpm for 15 days [Ali et. Al. 2015, RSC Adv.5, 95307-95312],

[0070] Extraction and Isolation: 20 litters culture broth of Phialophora mustea, extracted with Ethyl acetate (EtOAc) and concentrated under reduced pressure, afforded 6 g of crude extract, which was subjected to column chromatography over silica gel (230-400 mesh) using a gradient of hexane and ethyl acetate (100:0 to 0:100) to give fractions (Fr. 1-30). Furthermore, fraction (14-30) were pooled on TEC profile (4g) which was re-chromatographed over silica gel (230-400 mesh) with a step gradient of hexane and ethyl acetate (6:4) fr. 6-8 gave Phialomustin B (1) (800 mg) was obtained as a colourless viscous.Phialomustin B (2):Colourless viscous oil; [a] + 55.5 (c 1.5, CHCI3);XH NMR (400 MHz, CDCI3) 6 7.43 (d, J = 15.6 Hz, 1H), 5.81 (d, J = 15.6 Hz, 1H), 5.75 (d, J = 9.8 Hz, 1H), 2.54 (dd, J = 14.5, 7.0 Hz, 1H), 1.82 (s, 3H), 1.50 - 1.15 (m, 6H),I.01 (d, J = 6.5 Hz, 3H), 0.90 (t, J = 6.9 Hz, 3H);13C NMR (126 MHz, CDCI3) 6 173.01, 151.97, 149.75, 130.92, 114.31, 36.48, 32.91, 29.31, 22.40, 20.02, 13.65,I I.91; HRESIMS m / z 197.1540 [M+H]+(calcd for C12H20O2 197.1463).I. Activation of Nrf2 transcription factor and antioxidant enzymes by 1 i minhibition of Keapl-Nrf2 pathway1. Molecular cloning and recombinant protein production of Keapl-DC

[0071] The cDNA encoding mouse Keapl-DC domain (aa: 324-607) of Keapl was PCR amplified and digested with Ndel and Xhol restriction enzymes. The digested PCR product was inserted between Ndel and Xhol restriction sites into the expression vector pET28a with N-terminal His-tag, to get purified protein. The Keapl-DC protein expression conditions were standardized using 0.5 mM IPTG for 3hrs growth and overnight incubation at 15°C on shaking incubator. In-brief, Keapl-DC protein was affinity purified as a 6His-tagged protein and dialyzed against the dialysis buffer. The Keapl-DC protein was concentrated to 5 mg / ml using centricon filter (10 kDa) before loading on gel filtration column (Figure 2). The Keapl-DC protein molecular mass is 33 kDa. Purified protein was concentrated to 13.5 mg / ml using centricon, flash frozen and stored at -80°C forfurther usage. The overall yield of Keapl-DC protein was 80 mg / L of bacterial culture volume. Each purification step was analyzed using SDS-PAGE, to verify the protein purity.2. Microscale Thermophoresis assay

[0072] The Keapl-DC- 1 binding affinity was estimated by MST assay. The 25 nM concentration of fluorescently labelled Keapl-DC protein was kept constant and N16 stock solution was serially diluted in 16 steps. The highest concentration of ligand used in the assay was 100 pM. The samples were loaded using the capillaries and binding interaction was recorded. The samples were dissolved in the lx PBST and experiment was run in the same buffer. After data acquisition, data was analysed using MO Affinity Analysis software.

[0073] The MST response curve and traces were sharp and showed the binding affinity of WS15 towards the Keapl-DC protein (Figure 3). Concentrationdependent response behaviour of 1 with the Keapl-DC domain showed 61 nM binding affinity. The binding constant was determined at 61 nM for WS15 ligand, which showed WS15 significantly binding with the Keapl-DC protein.3. Cell viability assay (MTT assay)

[0074] The in-vitro cell based functional studies were performed using neuroblastoma derived cells SH-SY5Y to check the efficacy of the 1 in SH-SY5Y cell line was procured from National Centre for Cell Science (NCCS) Pune, India. SH-SY5Y cells were used as a Parkinson’s disease (PD) model to validate the 1 efficiency. The electron transfer chain inhibitor rotenone (1 pM) was used to mimic the Parkinson’s disease (PD) in SH-SY5Y cells.

[0075] To validate the cyto-protective effect of 1 on SH-SY5Y cell viability, cells were co-treated with 1 and 1 pM rotenone for 24 hours at various concentrations (0.1, 1, 10, and 100 pM) and the cell viability was determined by MTT assay. The 1 significantly increases the highest cell viability compared to rotenone-treated cells at 10 pMthe concentration (Figure 4). The SH-SY5Y cells were seeded for 12 hours and thereafter, treated with 1 compound in various concentrations (0.1, 1, 10, and 100 pM) for 24 hours. Three independent experiments performed in triplicate anderror bars indicate the standard deviation. Error bars represent the SD calculated over three independent experiments. *P < 0.05; The P-value was calculated using paired two-tailed t-test in comparison with control. The IC50 value was determined to be 10 pM for 1 and same used as an effective dose for further downstream studies. The IC50 values were calculated using GraphPad Prism software (Version 6.0).4. Effect of 1 on SH-SY5Y cells at translational level (Western Blot)

[0076] To investigate the effect of Ion protein expression levels of Nrf2, GCLC and NQO1 western blot assay was performed. The sample preparation and blotting experiments were carried out as mentioned in the methods section. The SH-SY5Y cells were exposed to 10 pM concentrations of 1 along with 1 pM rotenone for 24 hours. The Nrf2 protein expression was evaluated in the cytoplasmic (Figure 5A, B) and nuclear fraction (Figure 6A, B) samples. In Figure 5, *P < 0.05. The P-value was calculated using paired two-tailed t-test in comparison with control. Blot is representative of at least three independent experiments. In Figure 6, *P < 0.05. The P-value was calculated using paired two-tailed t-test in comparison with control. Blot is representative of at least three independent experiments.

[0077] The western blot analysis showed the Nrf2 translocation from cytosol to the nucleus (Figure 6A, B). Indicating 1 compound competitively binding to the Keapl protein and free the Nrf2 to activate the ARE driven antioxidant enzymes.5.Effect of 1 on SH-SY5Y cells at transcriptional level (qRT-PCR)

[0078] The qRT-PCR was performed to evaluate the effect of 1 on antioxidant enzymes at the transcriptional level, which were primarily regulated by Nrf2 activation. Here, the mRNA levels of HO-1, GCEC and NQO1 enzymes were checked. The SH-SY5Y cells were exposed to 10 pM concentrations of lalong with 1 pM rotenone for 24 hours. The mRNA level of GCEC and NQO1 enzymes were increased significantly compared to the rotenone treated cells (Figure 7). The mRNA level of antioxidant enzymes (HO-1, GCLC and NQO1) quantified with and without 1 (10 pM). Each experiment was performed in triplicate and data shownare the averages of three independent experiments. *P < 0.05; The P-value was calculated using paired two-tailed t-test in comparison with control. This result confirmed that Phialomustin B (1) significantly up-regulated the Nrf2 downstream GCLC and NQO1 antioxidant enzymes gene expression. Figure 8 depicts the effect of 1 on the protein expression level of GCLC, (A) Representative GCLC immunoblot, (B) Immunoblot analysis representing increase in the GCLC protein expression level after treatment with 1 (lOpM).6. Effect of 1 on GCLC and NQO1 antioxidant enzymes expression

[0079] Further, the protein expression levels of GCLC and NQO1 upon treatment of 1 were evaluated. The expression levels of GCLC (Figure8A, B) and NQO1 increased significantly as shown in (Figure 9A, B). *P < 0.05; The P-value was calculated using paired two-tailed t-test in comparison with control. Blot is representative of at least three independent experiments. From western blot analysis it was evident that 1 up regulated the expression levels of Nrf2, GCLC and NQO1, which revealed the cell protection by antioxidant enzymes.II. Anti-aggregation potential of 1 on ALS -causing SOD1 mutants1. Protein expression and production of SOI) 1w r. SOD1A4Vand SOD1I113T

[0080] The full length constructs of SODl-wild type (153 aa.) and its corresponding SOD1 mutants - A4V and I113T were used for protein production and biochemical studies. The plasmids (pETMl 1) containing the protein of interest was transformed into expression hosts E.coli BL21 DE3 cells (Manjula et al., 2018, FEBS let., 592(10), 1725-1737). The cells were grown at 37°C till the culture reaches OD600 = 0.6. The cultures were further subjected to IPTG induction. The protein expression was observed at 20°C and 0.5 mM IPTG induction for wild-type protein and 18 °C and 0.5 mM IPTG induction for the disease mutants, A4V and I113T. The cells were harvested by centrifugation of the culture at 7000 rpm. The cell pellet was dislodged and further process for cell lysis through sonication. The cells were suspended in a lysis buffer containing 30 mM KH2PO4, 300 mM NaCl,5 mM P-mercaptoethanol and 1 mM PMSF further aided by the presence of lysozyme, Dnase I and complete protease inhibitors. Sonication was performed in small batches of cell suspension at 3 sec ON and 5 sec OFF sonics cycle. The cell lysate was centrifuged at 12,000 rpm for 30 mins to separate the cell debris. The SOD1 proteins were designed with a hexa-histidine tag (6x His) at the N terminal. The supernatant was separated and subjected to Ni-NTA affinity purification to allow the separation of the His-tagged SOD1 from the host proteins. The bound SOD1 protein was gradient eluted using increasing concentration of imidazole. The SOD1 (wild-type and disease mutant) proteins eluted around 150-200 mM of imidazole. The eluted proteins were checked on SDS-PAGE to evaluate its homogeneity. The pure proteins were pooled together and dialyzed in the presence of TEV protease to cleave the His-tag of the proteins. The tag-digested proteins were passed through the Ni-NTA beads again to separate from undigested SOD1 and retain a homogeneous mixture of tag -cleaved SOD1 protein. The pure protein was pooled and concentrated before subjecting it to size exclusion chromatographic (Superdex 200pg 16 / 600, GE Healthcare). The dimer species of the wild-type and disease mutant SOD1 proteins eluted at near-around 75-85 ml (Figure 10). The homogeneity of the protein elutes were evaluated through SDS-PAGE. The homogenous elutes were pooled together and concentrated to be flash frozen in LN2 and frozen at -80°C.2. Estimation of binding affinity of 1 with SOD1WTusing microscale thermophoresis assay

[0081] The fluorescently labelled SOD1WTprotein and 16 different concentrations of the 1 from ImM to 30.5 nM were prepared to evaluate the binding interactions between SOD1WTprotein and WS15. MST traces showed smooth appearance and ligand concentration-dependent shift in magnitude. The binding affinity (Kd) was calculated based on the directional movement of molecules along a temperature gradient, and the protein-ligand interactions were measured using thermophoretic properties of the interacting molecules. The MST assay revealed that 1 had abinding affinity of 4.0 p M with SODlWT(Figure 11). Concentration-dependent response behaviour of 1 with the SOD1 domain showed 4.0 pM binding affinity.3. Size exclusion chromatograph (SEC) based aggregation assay

[0082] Evaluation of the anti-aggregation activity of 1 was performed on the disease mutants through size exclusion chromatography. The purified SOD1 mutants - A4V and I113T in their dimer conformation were tested on SEC analytical column (Superdex 200pg 10 / 300 GL, GE Healthcare). 50 p M of the protein was incubated with 250, 500 and 1500 pM of WS15 (5x, lOx, 30x) in a buffer system containing 20 mM Tris-HCl (pH = 7.4), 150 mM NaCl and 20mM DTT with the addition of 5 mM EDTA to induce aggregation. Negative (buffer only; buffer + EDTA; protein only; protein + WS 15) and positive (protein + EDTA) controls were setup for the experiment in parallel. Vehicle control (protein + DMSO) was also tested for the experimental setup. All the setup mixtures were incubated at 37°C with constant shaking for 48 hours. The samples were tested at 0, 24, 48hrs time -points. The chromatograms were analyzed to determine the extent of ligand-induced elevation or reduction of protein aggregation.4. Anti- Aggregation efficiency of 1 with SOD1 and its mutants under temperature and agitation based aggregation

[0083] Analytical size exclusion chromatograph studies of 1 was done with each, SOD1A4Vand SOD1I113T.SOD1A4V

[0084] Investigation of the anti-aggregation propensity of Phialomustin B (1) against SOD1A4Vshowed very insignificant difference in the elution pattern from the protein-only control (Figure 12). In comparison to the protein-control, treatment with 1 reduced the trimer population but there was no change in the rate of monomeric population. A slight development of higher aggregates were seen on the treatment of 1 on SOD1A4Vunder metal chelation conditions. Additionally, time dependent reduction in the monomer intensity and time-dependent increase in thedimer population was also visible at 48hrs, thus inferring the contribution of the monomeric population in the formation of the higher aggregates.SOD1I113T

[0085] Investigational studies to test the anti-aggregation propensity of 1 on II 13T under metal chelation conditions were performed. The dose dependent study of 1 reveals that the protein (P): ligand (L) ratios 1:5 and 1:10 does not show a significant change in reducing the aggregation of the protein in comparison to the protein-control. But an increase in the ratio to 1:30 reveals that 1 reduces aggregation through complete elimination of higher aggregates(Figure 13 (A), (B), and (C)). Also, the dimer population is able to sustain itself through the aggregating conditions in comparison to the negative control (in absence of 1) where the dimer population diminishes drastically to contribute to higher aggregate populations.ADVANTAGES OF THE INVENTION:

[0086] The present invention describes novel activity of a natural product molecule Phialomustin B (1) depicting Keapl inhibitor for Parkinson’s disease (PD) and SOD-1 modulator for Amyotrophic lateral sclerosis (ALS). Phialomustin B (1) inhibits the Keapl -Nrf2 interaction and induces cytoprotectivity in rotenone induced PD model of SHSY5Y cells. In addition, Phialomustin B (1) induces the translocation of Nrf2 and activation of downstream genes like HO-1, NQO1, GCLC etc. as observed by qRT-PCR and western blotting studies. At KD value of 5 pM, Phialomustin B (1) binds to SOD1WTprotein through MST assay. Phialomustin B (1) inhibits aggregation in SODlI113Tin both, mutation specific and dose-dependent manner under metal chelation conditions by EDTA. In the dimer condition, Phialomustin B (1) inhibits the formation of higher aggregate species and retains the protein, SOD1I113T. Phialomustin B (1) induces aggregation in another mutant SOD 1A4Vunder metal chelation conditions by EDTA. Lastly, Phialomustin B (1) induces the formation of monomeric species and gradually higher aggregate forms of the protein, SOD1A4V.

Claims

I / We claim:

1. Use of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, as Keapl inhibitor.Formula I2. Use of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, as Superoxide Dismutasel (SOD1) modulator.

3. Use of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, for the treatment of Parkinson’s disease (PD) or Amyotrophic lateral sclerosis (ALS).

4. Use of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, for the manufacture of a medicament in treating a disease or a condition.

5. The use as claimed in claim 4, wherein the disease or condition is Parkinson’s disease (PD) or Amyotrophic lateral sclerosis (ALS).

6. Use of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, as an inhibitor of Keapl-Nrf2 interaction or an inhibitor of aggregation in SOD1I113T7. The use as claimed in claim 6, wherein inhibition of Keap 1 -Nrf2 interaction is by inducing cytoprotectivity in rotenone induced PD model of SHSY5Y cells.

8. The use as claimed in claim 6, wherein inhibition of aggregation in SODlI113Tis in a mutation specific manner under metal chelation conditions by EDTA.

9. Use of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, as an inducer for the translocation of Nrf2, as an inducer ofaggregation in mutant SOD1A4Vand for activation of downstream genes such as HO-1, NQ01, and GCLC.

10. The use as claimed in claim 9, wherein Phialomustin B (1) of Formula I, acts an inducer of aggregation in mutant SOD1A4Vunder metal chelation conditions by EDTA.

11. A pharmaceutical composition comprising an effective amount of Phialomustin B (1) of Formula I, for use as claimed in any of the claims 1 to 10.

12. A method of treatment of a disease or a condition, the method comprising: administering an effective amount of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising Phialomustin B (1) of Formula I, to a subject in need thereof.

13. The method as claimed in claim 12, the disease or condition is Parkinson’s disease (PD) or Amyotrophic lateral sclerosis (ALS).

14. The method as claimed in claim 12, wherein administering is oral or intranasal.

15. A method of inhibition of Keapl, or Keapl-Nrf2 interaction or aggregation in SOD1I113T, the method comprising treating with an effective amount of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising Phialomustin B (1) of Formula I, to a subject in need thereof.

16. The method as claimed in claim 14, wherein the inhibition of Keapl-Nrf2 interaction is by inducing cytoprotectivity in rotenone induced PD model of SHSY5Y cells.

17. The method as claimed in claim 14, wherein the inhibition of aggregation in SOD1I113Tis in a mutation specific manner under metal chelation conditions by EDTA.

18. A method of modulation of Superoxide Dismutase 1 (SOD1), the method comprising treating with an effective amount of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, a pharmaceuticalcomposition comprising Phialomustin B (1) of Formula I, to a subject in need thereof. A method of inducing aggregation in a mutant SOD1A4Vor inducing translocation of Nrf2, the method comprising treating with an effective amount of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising Phialomustin B (1) of Formula I, under metal chelation conditions by EDTA, to a subject in need thereof. A method of activation of downstream genes, the method comprising treating with an effective amount of Phialomustin B (1) of Formula I, or its pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising Phialomustin B (1) of Formula I, to a subject in need thereof. The method as claimed in claim 20, wherein the method provides activation of downstream genes selected from HO-1, NQO1, or GCLC.

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