Patent Application
20240000786
The present disclosure relates to the field of immunology. The present disclosure relates to a pharmaceutical composition comprising small molecules for enhancing the intracellular nitric oxide in vitro in human and mouse leukocytes and endogenous induction of nitric oxide in mice and their use for induction of anti-viral and anti-inflammatory host response.
Nitric oxide (NO) is a highly potent signaling molecule involved in a variety of physiological processes. NO is produced by neuronal nitric oxide synthase (nNOS or NOS1), inducible nitric oxide synthase (iNOS or NOS2) and endothelial nitric oxide synthase (eNOS or NOS3) which regulate nervous, immune and endothelial system functions respectively (Förstermann, U., and Sessa, W. C. (2012) European heart journal 33, 829-837Nitric oxide synthases: regulation and function).
A variety of biological functions such as inflammation, vasodilation, platelet aggregation/adhesion, leucocyte adhesion, vascular smooth muscle proliferation, angiogenesis and other physiological processes are mediated by Nitric Oxide (Niedbala, W., Besnard, A.-G., Jiang, H. R., Alves-Filho, J. C., Fukada, S. Y., Nascimento, D., Mitani, A., Pushparaj, P., Alqahtani, M. H., and Liew, F. Y. (2013) The Journal of Immunology 191, 164-170 Nitric Oxide-Induced Regulatory T Cells Inhibit Th17 but Not Th1 Cell Differentiation and Function; Niedbala, W., Alves-Filho, J. C., Fukada, S. Y., Vieira, S. M., Mitani, A., Sonego, F., Mirchandani, A., Nascimento, D. C., Cunha, F. Q., and Liew, F. Y. (2011) Proceedings of the National Academy of Sciences 108, 9220-9225. Regulation of type 17 helper T-cell function by nitric oxide during inflammation; Lundberg, J. O., Gladwin, M. T., and Weitzberg, E. (2015) Nature Reviews Drug Discovery 14, 623-641. Strategies to increase nitric oxide signalling in cardiovascular diseaseBogdan, C. (2015) Trends in immunology 36, 161-178. Nitric oxide synthase in innate and adaptive immunity: an update; Bogdan, C. (2001) Nature immunology 2, 907-916 Nitric oxide and the immune response 21.; Bauer, H., Jung, T., Tsikas, D., Stichtenoth, D., FRÖLICH, C., and Neumann, C. (1997) Immunology 90, 205-211 Nitric oxide inhibits the secretion of T-helper 1- and T-helper 2-associated cytokines in activated human Tcells′pa; Jon O. Lundberg, Mark T. Gladwin and Eddie Weitzberg, Strategies to increase nitric oxide signalling in cardiovascular disease,doi:10.1038/nrd4623, 7 Aug. 2015, Nature Reviews; Almudena Garcia-Ortiz and Juan M. Serrador, Nitric oxide Signaling in T Cell-Mediated Immunity, https://doi.org/10.1016/j.molmed.2018.02.002.; Wanda Niedbala, Beilei Cai, Haiying Liu, Nick Pitman, Lynda Chang and Foo Y. Liew, Nitric Oxide Induces CD4+CD25+ Foxp3− regulatory T cells from CD4+CD25− T cells via p53, IL-2, and OX40, www.pnas.org/cgi/doi/10.1073/pnas.0703725104; Wanda Niedbala, Jose C. Alves-Filho, Sandra Y. Fukada, Silvio Manfredo Vieira, Akio Mitani, Fabiane Sonego, regulation of type 17 helper T-cell function by nitric oxide during inflammation, www.pnas.org/cgi/doi/10.1073/pnas.1100667108.).
More importantly, NO plays an integral role in defense against bacterial, viral and parasitic pathogens in immune cell signaling and biochemical pathways and in regulating pathways of host inflammation (Hassan Fahmi, Daniel Charon, Michelle mondange and Richard Chaby, Endotoxin-Induced Desensitization of mouse Macrophages is Mediated in part by Nitric Oxide Production, http://iai.asm.org/, Dec. 29, 2019.; Mirela B., Maria C. Almeida, Evelin C. Carnio and Luiz G. S. Branco, Role of nitric oxide in tolerance to lipopolysaccharide in mice, doi:10.1152/japplphysiol.01243.2004.; Devangi R. Mehta, Ali A. Ashkar, Karen L. Mossman, The nitric oxide pathway provides innate Antiviral protection in Conjunction with the Type 1 Interferon Pathway in Fibroblasts, doi:10.1371/journal.pone.0031688; Fabio Lisi, Alexander N. Zelikin and Rona Chandrawati, Nitric Oxide to Fight viral Infections, DOI: 10.1002/advs.202003895,2021.; Howlin, R. P., Cathie, K., Hall-Stoodley, L., Cornelius, V., Duignan, C., Allan, R. N., Fernandez, B. O., Barraud, N., Bruce, K. D., and Jefferies, J. (2017) Molecular Therapy 25, 2104-2116. Low-Dose Nitric Oxide as Targeted Anti-biofilin Adjunctive Therapy to Treat Chronic Pseudononas aeruginosa Infection in Cystic Fibrosis.; Jeney, V., Ramos, S., Bergman, M.-L., Bechmann, L, Tischer, J., Ferreira, A., Oliveira-Marques, V., Janse, C. J., Rebelo, S., and Cardoso, S. (2014) Cell Reports 8, 126-136 Control of disease tolerance to malaria by nitric oxide and carbon monoxide).
NO is produced at various concentrations (nm-μm) in cells by enzymatic action of NOS using L-arginine as substrate. Among the three isoforms, eNOS and nNOS are constitutively expressed while iNOS is inducible in immune cells during their activation. Synthesis and release of NO is tightly regulated by molecules involved in several regulatory pathways. Fine-tuning of NOS activity is mediated post-transnationally by protein-protein interactions.
Nitric oxide synthase interacting protein (NOSIP) has emerged as a central modulator of biological activity of NOS. NOSIP interacts with all three isoforms of NOS with high affinity and is co-expressed in cells (Dedio, J., König, P., Wohlfart, P., SCHROEDER, C., KUMMER, W., and MÜLLER-ESTERL, W. (2001) The FASEB Journal 15, 79-89 NOSIP, a novel modulator of endothelial nitric oxide synthase activity.; Dreyer, J., Schleicher, M., Tappe, A., Schilling, K., Kuner, T., Kusumawidijaja, G., Müller-Esterl, W., Oess, S., and Kuner, R. (2004) Journal of Neuroscience 24, 10454-10465 Nitric oxide synthase (NOS)-interacting protein interacts with neuronal NOS and regulates its distribution and activity). It inhibits synthesis of NO by relocating NOS from the plasma membrane to intracellular compartments thereby uncoupling NOS from plasma membrane caveolae (Dedio, J., König, P., Wohlfart, P., SCHROEDER, C., KUMMER, W., and MÜLLER-ESTERL, W. (2001) The FASEB Journal 15, 79-89 NOSIP, a novel modulator of endothelial nitric oxide synthase activity).
Expression, kinetics and regulation of NOSIP in mammalian cells and organs are largely unexplored. Therefore, NOSIP is a major NOS interacting protein that down-regulates biological activity of all three isoforms of nitric oxide synthases viz., nNOS, iNOS and eNOS. NOSIP indirectly regulates the level of NO produced by different cells/organs.
NO plays a central role in a wide range of pathological conditions such as infectious diseases, cardiovascular diseases, hypertensive pregnancies, rheumatoid arthritis, systemic lupus erythematous, multiple sclerosis, septic shock etc., (Förstermann, U., et al. (2016); Howlin, R. P., et al. (2017)). Most of these diseases are associated with impaired NO production and/or activity. Decreased availability of NO has been attributed to imbalance between its synthesis and degradation (Förstermann, U., et al. (2016)). Consequently, many chemical NO donors have been designed for potential use for management of several pathologies (Schairer, D. O., Chouake, J. S., Nosanchuk, J. D., and Friedman, A. J. (2012) Virulence 3, 271-279 The potential of nitric oxide releasing therapies as antimicrobial agents). However, such NO donors have very limited clinical application since NO is unstable with very short half-life (Dreyer, J., Schleicher, M., Tappe, A., Schilling, K., Kuner, T., Kusumawidijaja, G., Müller-Esterl, W., Oess, S., and Kuner, R. (2004) Journal of Neuroscience 24, 10454-10465 Nitric oxide synthase (NOS)-interacting protein interacts with neuronal NOS and regulates its distribution and activity; Schairer, D. O., Chouake, J. S., Nosanchuk, J. D., and Friedman, A. J. (2012) Virulence 3, 271-279 The potential of nitric oxide releasing therapies as antimicrobial agents; Levine, A. B., Punihaole, D., and Levine, T. B. (2012) Cardiology 122, 55-68 Characterization of the Role of Nitric Oxide and its Clinical Applications; Reger, N. A., Meng, W. S., and Gawalt, E. S. (2017) Journal of functional biomaterials 8, 20 Antimicrobial Activity of Nitric Oxide-Releasing Ti-6Al-4V Metal Oxide; Fitzhugh, A. L., and Keefer, L. K. (2000) Free Radical Biology and Medicine 28, 1463-1469. Diazeniumdiolates:: Pro- and antioxidant applications of the “NONOates”; Li, B., Ming, Y., Liu, Y., Xing, H., Fu, R., Li, Z., Ni, R., Li, L., Duan, D., and Xu, J. (2020) Frontiers in Pharmacology 11, 923 Recent Developments in Pharmacological Effect, Mechanism and Application Prospect of Diazeniumdiolates; Arlene Bradley Levine, David Punihaole, T. Barry Levine, Characterization of the role of nitric oxide and its clinical Applications, DOI:10.1159/000338150,19 june, 2012; Maurizio Forte, Valeria Conti, Antonio Damato, Mariateresa Ambrosio, Annibale A. Puca, Targeting Nitric Oxide with Natural Derived Compounds as a Therapeutic Strategy in Vascular Diseases, http://dx.doi.org/10.1155/2016/7364138, Hindawi Publishing Corporation; David O. Schairer, Jason S. Chouake, Joshua D. Nosanchuk and Adam J. Friedmam, The Potential of nitric oxide releasing therapies as antimicrobial agents, http://dx.doi.org/10.4161/viru.20328.).
Nitric Oxide (NO) displays a variety of biological activity such as a) vasodilation b) anti-inflammation c) anti-thrombotic d) anti-proliferative e) neuroprotective f) anti-bacterial g) anti-viral effect etc., Thus induction of Nitric Oxide in vivo has been perceived to have several pharmacological benefits. Search for molecules that induce NO has been a major activity in the area of drug development. Currently the following approaches are being pursued—a) consumption or administration of Nitrate/Nitrite salts and their conversion into NO by bacterial nitrate reductase in the gut, b) Dietary supplementation of L-arginine to increase substrate levels for Nitric Oxide Synthase enzyme to generate more NO by cells, c) administration of chemical NO donors which have very short half-life. Such approaches are disclosed in WO1996016645A1 and US20210290663A1.
Therefore, there exists a need in the art for molecules that can induce Nitric Oxide production in the cells.
It is therefore an object of the present disclosure to propose a composition for increasing intracellular nitric oxide concentration.
It is an object of the present invention to demonstrate ability of NOSIP to interact with iNOS and inhibit its enzyme activity in a dose dependent manner to generate Nitric Oxide.
Another object of the invention is to demonstrate affinity of small molecules of the composition to interact with NOSIP for potential use as antagonists.
Another object of the invention is to induce human and mice cells to generate Nitric Oxide in vitro by treatment with Small Molecule ‘NOSIP antagonists’.
Yet another object of the invention is to generate endogenous Nitric Oxide in mice by administration of Small Molecule ‘NOSIP antagonists’.
Yet another object of the invention is to demonstrate up-regulation of type 1 Interferons viz., IFN-α and IFN-b genes and Inflammatory stimulatory genes (ISGs) in vitro by small molecule “NOSIP antagonists”.
Still another object of the invention is to therapeutically use Small Molecule ‘NOSIP antagonists’ to inhibit generation of inflammatory cytokines in mice induced by administration of bacterial endotoxin, Lipopolysaccharide (LPS).
These and other objects and advantages of the invention will be apparent from the ensuing description.
According to this invention is provided a composition comprising five small molecules wherein the molecules inhibit nitric oxide synthase interacting proteins and inducible Nitric oxide synthase interaction.
In one embodiment, the present invention discloses a pharmaceutical composition for enhancing intracellular nitric oxide in a subject, the said composition comprising 1-{3-[9-methoxy-7-(2-methoxyphenyl)-2,3,4,5-tetrahydro-1,4-benzoxazepine-4-carbonyl]phenyl}imidazolidin-2-one; N-tert-butyl-2-{N-[(furan-2-yl)methyl]-2-[5-(4-methylphenyl)-2H-1,2,3,4-tetrazol-2-yl]acetamido}-2-(4-hydroxyphenyl)acetamide; 3-[2-(2-{[(6-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)(4hydroxyphenyl) methyl]amino}-4-oxo-4,5-dihydro-1,3-thiazol-5-yl)acetamido]benzoic acid; 2-oxo-2-(1H-pyrrol-2-yl)ethyl 2-[(4-fluorophenyl)methoxy]benzoate, and 5-(3-{[(3-bromo-4-hydroxy-5 methoxyphenyl) methylidene]amino}-2 (butylimino)-2,3-dihydro-1,3-thiazol-4-yl)-2-hydroxybenzamide.
In another embodiment, the present invention discloses a method of preparing the composition.
It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. The detailed description is described with reference to the accompanying figures. Some embodiments of system or methods in accordance with embodiments of the present subject matter are now described, by way of example, and with reference to the accompanying figures, in which:
In the present document, the word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any embodiment or implementation of the present subject matter described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.
While the disclosure is susceptible to various modifications and alternative forms, specific embodiment thereof has been shown by way of example in the drawings and will be described in detail below. It should be understood, however that it is not intended to limit the disclosure to the forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure.
The terminology used herein is to describe particular embodiments only and is not intended to be limiting of example embodiments. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context indicates otherwise.
It will be further understood that the terms “comprise”, “comprising”, “includes” and/or “including” when used herein, specify the presence of stated features, steps, operations, elements and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components and/or groups thereof.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The term “equimolar concentration” implies having same molar concentration of solute in a solution. In the context of the present invention, the said term denotes the molar concentration of each of the 5 small molecule antagonists is same i.e., they are added in the ratio of 1:1:1:1:1 for preparing the stock solution and later suspending in phosphate buffer saline solution.
The term “MER” denotes the number of amino acid residues. 18 mer means a peptide (PP2) containing 18 amino acids.
According to the present disclosure, is provided a pharmaceutical composition comprising:
In an embodiment of the present disclosure, the components are small molecule antagonists that block the interaction between Nitric oxide synthase interacting proteins and inducible Nitric oxide synthase.
In one embodiment of the present disclosure, the components are present in an equimolar concentration in phosphate buffer saline.
In another embodiment of the present disclosure, the components either alone or in combination enhances Nitric Oxide level at 0.1 and 0.01 μM concentrations.
In yet another embodiment, the present disclosure provides a method for preparing the composition comprising selection of five different small molecules (viz; MCULE3765839647, MCULE8395438746, MCULE8094061955, MCULE8469877005, and MCULE4640800528) from chemical library, preparing 1M stock solution by dissolving 5 mg of the powder in 1 ml dimethyl sulfoxide, preparing cocktail of 1M solution by mixing 0.2 ml of each of the small molecule components as claimed in claim 1 making a total volume of 1 ml and storing at −20° C., preparing 1 ml cocktail of 100 uM stock solution in phosphate buffer saline by adding 0.1 ml of 1M stock in DMSO to 0.9 ml of phosphate buffer saline and storing for further use in −20 C in aliquots of 100 μl each, thawing 100 μM stock solution and addition of small molecule antagonists at different concentrations in phosphate buffer solution.
The small molecules are designated as ‘NOSIP antagonists’ and an equi-molar mixture of all 5 Small Molecule ‘NOSIP antagonists’ were used for biological experiments. Incubation of human peripheral blood mononuclear cells (PBMC) or murine Bone marrow derived Mononuclear cells (BMDM) in vitro with “NOSIP antagonists” induced production of Nitric Oxide by the cells. ‘NOSIP antagonists’ further potentiated TLR (Toll like receptors) agonists (LPS, PGN, CpG DNA, Flagellin) induced synthesis of Nitric Oxide by the above cells. A single dose of 100 ul-200 ul of 100 uM solution of “NOSIP antagonists” by intra-peritoneal route to mice resulted in production of Nitric Oxide in vivo as shown by elevated levels of Nitrate/Nitrite in plasma and peritoneal cavity in BALB/c mice. Mice administered with 100 ul of 100 uM “NOSIP antagonists” followed by injection of 5 mg/kg body weight of bacterial Endotoxin (Lipopolysaccharide, that induces inflammatory cytokines in circulation) resulted in decreased levels of inflammatory cytokines such as TNF-α and IL-1β. Macrophage cell line RAW incubated with “NOSIP antagonists” in vitro culture for 10 hrs at 37 C and analysed by Q-RT-PR were found to significantly activate for anti-viral genes viz., different subtypes of Interferon-a, Interferon-b, Interferon stimulated genes (ISGs).
The invention is now illustrated by way of non-limiting examples. The examples are intended to be purely exemplary of the invention, should therefore not be considered to limit the scope invention in any way. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.
Cloning, Expression and Purification of iNOS and NOSIP Proteins and their Inhibiting Interaction
The human iNOS oxygenase domain (SEQ ID No.: 24) containing 424 nucleotide sequence and NOSIP gene (SEQ ID No.: 23) containing 301 nucleotide sequences were synthesized by GenScript and cloned in pET22b expression vector. The recombinant pET22b: iNOS and pET22b: NOSIP plasmid was used to transform competent E. coli BL21 (DE3) cells. The proteins were over expressed using 0.2 mM IPTG at 25° C. The harvested cells were resuspended and sonicated in 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.05% Triton X-100 and protease inhibitor (Sigma). The expressed proteins were purified by nickel nitrilotriacetic acid column using 250 mM imidazole and gel filtration chromatography. The protein was quantified by Bradford assay (Sigma) and integrity of purified protein was checked on 12% SDS-PAGE.
In a calorimetric assay increasing concentrations of recombinant NOSIP were mixed with recombinant iNOS along with substrate L-Arginine and the resultant product, nitrate was quantified by using Griess reaction kit. The red bar in
Identification of iNOS Peptides that Block Interaction Between iNOS and NOSIP:
3-D structures of iNOS and NOSIP were analyzed by 7 different tools (PIPER, SWORM DOCK, PyDOCK, Z-DOCK, HADDOCK, ATTRACT, CLUSPRO) that resulted in prediction of four different iNOS peptides that can interfere with their interactions. The predictions were tested for validation by Specific Plasmon Resonance (SPR). One of the peptides (Peptide-2 abbreviated as PP-2) (SEQ ID No: 26) was found to block significantly interaction between recombinant iNOS and NOSIP as shown by SPR. The other 3 peptides (Peptide-1, SEQ ID No: 25), (Peptide3, SEQ ID No: 27), and (peptide-4, SEQ ID No: 28) were less efficient in inhibiting interaction between iNOS and NOSIP as shown in
Detailed methods used for the above analysis are the following:
Four different iNOS peptides were synthesized commercially by China peptides and iNOS peptides were used for molecular interaction studies with immobilized recombinant iNOS purified protein. The iNOS peptides mediated inhibition of rNOSIP and riNOS protein association in Bio-Rad XPR 36 surface plasmon resonance biosensor instrument. About 3 μg/ml of iNOS was immobilized on GLC chip by amine coupling method as suggested by manufacturer's instructions. 250 nM of iNOS peptides was used for inhibition assay with immobilized ligand(recombinant iNOS) in SPR analysis. Similar condition and concentration of small molecules were used for inhibition of recombinant protein association with immobilized ligand. It inhibited the association of rNOSIP and r iNOS purified protein significantly in this study. The rNOSIP protein was incubated with iNOS derived peptide/small molecules for 30 minutes in ice for inhibition assay before the SPR application in our study. The molecular interaction was carried out at 20° C. and kinetic parameters were determined, after fitting the association and dissociation curves to a 1:1 (Langmuir)-binding model. An activated channel without immobilized ligand was used to evaluate nonspecific binding. The response curves were also recorded on control surfaces. Results were calculated after subtraction of the control values using the ProteOn Manager software.
The collected sensorgram data were processed by reference subtraction using double-referencing with the in-line blank buffer. The integrated ProteOn Manager software was used to fit the data with the Langmuir model describing a 1:1 binding stoichiometry. Ka is the association rate constant, Kd is the dissociation rate constant, and KD is the equilibrium dissociation constant, defined by the Kd/Ka ratio for the analytes-ligand binding.
The 18 mer iNOS peptide (PP2) and small molecules were used for physical association of molecules in calorimetric analysis. The molecular association leads to thermodynamics changes such as Gibb's free energy change (ΔG), entropy change (ΔS), enthalpy change (ΔH) and number of binding sites (N) in interacting solution were investigated by isothermal calorimetry MICROCAL PEQ (Malvern) at 25° C. The sample cell were filled with peptides, small molecules and the reference cell was filled with 25 mM HEPES and 150 mM Sodium chloride, pH=7 and the syringe was filled with NOSIP solution (200 μM). The experiment consisted of multiple injections and each injection contained 1.5 μl of binding solution (recombinant NOSIP solution) with 120 second spacing time between subsequent injections. Sequential titrations were carried out and the sample solution was continuously stirred at 270 rpm by the rotating paddle attached to the syringe needle. Multiple injections of NOSIP made into the sample cell containing interacting solution of peptide and small molecules. The titration curves were analyzed using the analysis software provided with the calorimeter.
Identification of Small Molecules that Mimic iNOS Peptide 2 and Chemical Formulae and Structure 5 Small Molecules ‘NOSIP Antagonists’:
Using Peptide 2 of iNOS that significantly inhibited interaction between iNOS and NOSIP in SPR (as demonstrated in
Detailed Description of SPR and ITC for SM:
The 18 mer iNOS peptide (PP2) and five small molecules were used to analyze physical association in calorimetric analysis. The molecular association leads to thermodynamics changes such as Gibb's free energy change (ΔG), entropy change (ΔS), enthalpy change (ΔH) and number of binding sites (N) in interacting solution were investigated by isothermal calorimetry MICROCAL PEQ (Malvern) at 25° C. The sample cell were filled with peptides, small molecules and the reference cell was filled with 25 mM HEPES and 150 mM Sodium chloride, pH=7 and the syringe was filled with NOSIP solution (200 μM). The experiment consisted of multiple injections and each injection contained 1.5 μl of binding solution with 120 second spacing time between subsequent injections. Sequential titrations were carried out and the sample solution was continuously stirred at 270 rpm by the rotating paddle attached to the syringe needle. Multiple injections of NOSIP made into the sample cell containing interacting solution of peptide and small molecules. The titration curves were analyzed using the analysis software provided with the calorimeter. The SM binding affinity with NOSIP is described below in table below and it indicates the following trends in terms of hierarchy viz, CC4>CC11>CC6>CC5>CC13>Peptide 2 (iNOS). The biophysical parameters observed for the 5 small molecule designated as NOSIP Antagonists' are shown in Table 1 below:
The structures for 5 selected SM NOSIP Antagonists are shown below in Table 2:
Kinetic parameter of bi-molecular interaction of between iNOS and NOSIP in the presence and absence of 5 small molecules as shown by SPR.
The five small molecules bind with NOSIP and inhibit the association of recombinant NOSIP and recombinant iNOS. The rNOSIP protein was incubated with iNOS derived peptide or small molecules for 30 minutes in ice prior to SPR application. The molecular interaction was carried out at 20° C. and kinetic parameters were determined, after fitting the association and dissociation curves to a 1:1 (Langmuir)-binding model. An activated channel without immobilized ligand (recombinant iNOS) was used to evaluate nonspecific binding. The response curves were also recorded on control surfaces. Results were calculated after subtraction of the control values using the ProteOn Manager software.
The collected sensorgram data were processed by reference subtraction using double-referencing with the in-line blank buffer. The integrated ProteOn
Manager software was used to fit the data with the Langmuir model describing a 1:1 binding stoichiometry. Ka is the association rate constant, Kd is the dissociation rate constant, and KD is the equilibrium dissociation constant, defined by the Kd/Ka ratio for the analytes—ligand binding. The Ka, Kd and KD values of the inhibiting interaction between the small molecules and NOSIP and iNOS are shown in table 3 below.
The different Small Molecules interacted with NOSIP and resulted in inhibition of iNOS-NOSIP protein interaction kinetics in SPR analysis. The inhibition hierarchy of the small molecules were as follow: CC4>CC11>CC6>CC5>CC13>iNOS Peptide 2.
Murine Bone marrow derived mononuclear cells (BMDM—1 million cells per ml) were treated with or without NOSIP antagonists (equimolar concentration of a mixture of all 5 small molecule antagonists) at 0.1 and 0.01 μM concentrations for 30 min followed by addition of LPS (1 μg/ml), β-glucan (5 μg/ml), flagellin (200 ng/ml), Pam3CSK4 (100 ng/ml), poly I:C (50 μg/ml), and PGN (10 μg/ml) in triplicate wells and incubated for 24 h at 37° C. The culture supernatants were analysed by calorimetry for nitrite concentration using Griess reagent. The results (shown in
Bone marrow cells single cell suspension, small molecules treatment and DAF-FM (4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate) staining. The mouse femurs were aseptically isolated in Dubleco's phosphate buffer saline solution and kept immediately on ice. The muscles and residue tissues surrounding the femur were removed with sterile forceps and scissors. The femurs cut at both ends with sharp sterile scissors and marrow cells were flushed out using 26 gauge needle with ice cold DPBS. The collected sample was filtered through 70 m nylon cell strainer placed in a 50 ml falcon conical tube. The cells were washed and replaced with complete DMEM media by repeated centrifugation at 300 g for 10 minutes. The bone marrow cells were counted with a haemocytometer and 106 cells/ml were used for small molecules cocktail treatment in this studies. Further, titration of 2 and 4 uM concentration of cocktails for 1 h and 2 h was carried out. The treated cells were further incubated with 10 uM DAF-FM for 30 minutes at 5% CO2 at 370C. The cells were washed with DMEM incomplete media and further incubated with fresh complete DMEM media. The further incubation of cells for 30 minutes allowed complete de esterification. The cells were lysed/fixed with BD lysis/permeabilization buffer before the flow cytometer analysis at 495 nM/515 nM. The results are shown in
Normal human peripheral blood mononuclear cells (PBMC—1 million cells/ml) were treated with or without NOSIP antagonists (equimolar concentration of a mixture of all 5 small molecule antagonists) at 0.1 and 0.01 μM concentrations for 30 min followed by addition of LPS (1 μg/ml), β-glucan (5 μg/ml), flagellin (200 ng/ml), Pam3CSK4 (100 ng/ml), poly I:C (50 μg/ml), and PGN (10 μg/ml) in triplicate wells and incubated for 24 h at 37° C. The culture supernatants were analysed by calorimetry for nitrite concentration using Griess reagent. The results (shown in
A mixture of all 5 Small molecule ‘NOISP antagonists’ were injected by intra-peritoneal route (100 μl/mouse of 100 mM) and groups of mice were sacrificed at different time points and plasma and peritoneal lavage were harvested and nitrite levels were quantified using Griess reagent. The levels are shown in
Six to eight-week-old male C57BL/6 mice were intra-peritoneally injected with 200 μl of 100 μM SM ‘NOSIP Antagonists’, prepared by mixing equimolar concentration of five small molecules in PBS before each experiment. After 2 hrs, mice were administered 5 mg/Kg of LPS and after two hours the animals were euthanized, and blood was collected by cardiac puncture. Plasma levels of inflammatory cytokines TNFα, and IL-1β were measured using commercially available ELISA kits, as per the manufacturer's instruction (ebioscience, USA; TNFα #501128954; IL-1β #501129749). Pre-treatment of mice with ‘NOSIP Antagonists’ blocked induction of inflammatory cytokines by LPS. The results are shown in
PBMCs (1 million cells/ml) from healthy individuals' blood was isolated and were treated with or without NOSIP antagonists (either alone or mixture of equimolar concentration of all 5 small molecule antagonists at 0.1 and 0.01 μM) for 30 min in triplicate wells and incubated for 24 h at 37° C. The culture supernatants were analysed by calorimetry for nitrite concentration using Griess reagent, and the NO levels are shown as mean percent increase over untreated control. Results are shown in
Induction of NO Production by NOSIP Antagonist Cocktail is not Associated with Induction of Inflammatory Cytokines:
A mixture of all 5 Small molecule ‘NOISP antagonists’ were injected by intra-peritoneal route (100 μl/mouse of 100 μM) and groups of mice were sacrificed at different time points and plasma and peritoneal lavage were harvested and inflammatory cytokines (IL-1β, TNF-α and IL-6) levels were quantified using ELISA kits. Administration of NOSIP antagonist SM did not induce the inflammatory cytokines in plasma and in Peritoneal fluid. LPS (5 mg/kg) was used as a positive control. Higher dose of NOSIP antagonists SM cocktail (500 μl of 500 μM, i.p.) also did not influence inflammatory cytokine production in vivo. The results are shown in
The murine macrophage cell line, RAW 264.7 (ATCC® TIB-71™) was maintained in RPMI-1640 medium (PAN Biotech, Aidenbach Germany) supplemented with antibiotics, including penicillin (100 U/ml) and streptomycin (0.1 mg/ml), 2.0 mM L-glutamine (Himedia Laboratories Pvt. Ltd., Mumbai, India), and 10% heat-inactivated FBS (PAN Biotech, Aidenbach Germany) at 37° C. in a humidified incubator with 5% CO2. 8-12 week old male mice was sacrificed via cervical dislocation and legs were dissected. Bone marrow from tibia and femur bones was flushed out using a 25-gauge needle and a 10 ml syringe filled with DMEM (HiMedia Laboratory, Mumbai, India) containing 10% FBS (Gibco Laboratories, USA) under aseptic condition. The cell suspension was centrifuged at 2000 r.p.m for 5 min at room temperature to pellet cells. The supernatant was discarded and cells were resuspended in DMEM containing 10% FBS.
Quantitative Real-Time PCR (qRT-PCR) Analysis
For qRT-PCR analysis, RAW 264.7 cells were cultured in 6 well plate at 2×106 cells/2 ml in RPMI 1640 containing 10% FBS and incubated overnight at 37° C. and 5% CO2 in a humidified incubator. Next day, the cells were washed with RPMI 1640 media and either treated or untreated with different concentrations of SM (5 μM or 15 μM). The BMDM cell suspension was cultured in 24 well plate at 5×106 cells/1.5 ml in DMEM supplemented with 10% FBS, 1% antibiotic-antimycotic solution (Gibco Laboratories, USA). The cells were incubated overnight at 37° C. and 5% CO2 in a humidified incubator. Next day, the culture wells were gently washed 2 times with 1 ml with DMEM medium to remove non-adherent cells. The cells were either treated or untreated with LPS (1 μg/ml) or SM (5 μM or 15 μM). After 8 h, BMDM and RAW 264.7 cells were lysed in 1 ml TRIzol reagent (Thermo Fisher Scientific, IL, USA). Total RNA was extracted with molecular grade reagents according to the instructions provided by the manufacturer (Sigma; St. Louis, MO, USA). First-strand cDNA synthesis was carried out with 0.5 μg of total RNA using random primers and high capacity cDNA reverse transcription kit (Thermo Fisher Scientific, IL, USA). Real-time quantitative PCR was performed using SYBR select master mix (Thermo Fisher Scientific, IL, USA). At the end of PCR, dissociation curve analysis was performed to verify the amplification of a single product. Relative expression of inflammatory cytokines and interferon subtypes were calculated using 2−ΔΔCT method [Pfaffl, Michael W. “A new mathematical model for relative quantification in real-time RT-PCR.” Nucleic acids research 29, no. 9 (2001): e45-e45.].
Sequence of the Primers Used for qPCR
The forward and reverse primers of genes used were:
Significant induction of type 1 Interferons and absence of induction of inflammatory cytokines (TNF-α, IL-b and IL-6) were quantified by Q-RT-PCR and the results are shown in
SM NOSIP Antagonists Induce Several Interferon Stimulated Genes when Murine Macrophage Cell Lines RAW were Treated In Vitro for 8 Hrs:
RAW 264.7 were treated with different concentration of SM (5 μM or 15 μM). After 8 h of treatment, RNA was isolated, reverse transcribed to cDNA which was quantified by qRT-PCR. Values were normalized to R-actin mRNA content, and control cells were assigned a value of 1. Relative expression was calculated using the 2−ΔΔCT method. Values are expressed as Mean±SEM. Details of genes overexpressed by SM NOSIP antagonists are shown in
Q-RT PCR for Type 1 Interferon genes (14 a) and cytokine genes (14 b) in murine BMDM cells treated with SM NOSIP antagonists for 8 hrs: BMDM were treated with LPS (1 μg/ml) and different concentration of SM (5 μM or 15 μM). After 8 h of treatment, RNA was isolated, reverse transcribed to cDNA which was quantified by qRT-PCR. Values were normalized to R-actin mRNA content, and control cells were assigned a value of 1. Relative expression was calculated using the 2−ΔΔCT method. Values are expressed as Mean±SEM. Details of the results are shown in
Source and geographical origin of the biological materials and reagents used:
The animal and cells disclosed in the examples of the present invention were procured solely for the purpose of research and experimentation to generate additional data. The invention is based on a composition and do not claim or intend to commercialize any biological material.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202031050500 | Nov 2020 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2021/051070 | 11/15/2021 | WO |
