Antibacterial application of pyrrolidin-piperdine derivatives containing composition to inhibit helicobacter pylori replication, preparation method thereof

Abstract

: Title: Antibacterial Application of Pyrrolidin-Piperdine Derivatives Containing Composition to Inhibit Helicobacter Pylori Replication, Preparation Method Thereof The present disclosure proposes a method for identification, optimizing, and preparing a therapeutic molecule (MA01027) for inhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori. The therapeutic molecule specifically targets and binds to a particular protein of interest, thereby enhancing the efficacy and specificity of the treatment. The therapeutic molecule is optimized for its binding affinity and selectivity for the target protein. The therapeutic molecule involves combining specific reactants under controlled conditions to achieve high yields and purity of the therapeutic molecule. The therapeutic molecule preparation includes purification steps to isolate the therapeutic molecule from the reaction mixture using standard techniques, thereby ensuring a high-quality final product. The therapeutic molecule binds to the CagA protein of H. pylori, inhibiting its replication by more than 90% and eliminating H. pylori-associated hemolysis.

Description

Antibacterial Application of Pyrrolidin-Piperdine Derivatives Containing Composition toInhibit Helicobacter Pylori Replication, Preparation Method ThereofDESCRIPTION:Field of the invention:

[0001] The present disclosure generally relates to the technical field of antimicrobial therapeutics, in specific, relates to an antibacterial composition containing pyrrolidin- piperdine derivatives of benzene tricarboxylic acid for inhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori (H. pylori), thereby preventing the replication and pathogenicity of H. pylori infections.Background of the invention:

[0002] Helicobacter pylori (H. pylori) is a gram-negative bacterium that colonizes in the human stomach, affecting more than half of the global population. It is well-established as a significant risk factor for various gastrointestinal diseases such as chronic gastritis, peptic ulcers, and gastric cancer. The ability of H. pylori to persist in the harsh acidic environment of the stomach and evade the host immune response makes it a particularly challenging pathogen to eradicate. Over the years, a growing body of research has been dedicated to understanding the molecular mechanisms underlying H. pylori infection and developing effective therapeutic strategies to combat its associated diseases.

[0003] The pathogenicity of H. pylori is largely attributed to its virulence factors, among which the cytotoxin-associated gene A (CagA) protein plays a crucial role. CagA is an oncogenic protein delivered into host gastric epithelial cells via a type IV secretion system. Once inside the host cells, CagA undergoes phosphorylation and interacts with various signaling molecules, leading to alterations in cell morphology, disruption of tight junctions, and increased cellular proliferation. These cellular changes are implicated in the development of gastric adenocarcinoma, making CagA a key target for therapeutic intervention.

[0004] Current treatment regimens for H. pylori infection typically involve a combination of antibiotics, such as clarithromycin, amoxicillin or metronidazole, along with a proton pump inhibitor (PPI). However, the effectiveness of these treatments has been declining due to the rising prevalence of antibiotic-resistant H. pylori strains. This antibiotic-resistance has become a major challenge in the management of H. py / or / -associated diseases, necessitating the development of novel therapeutic approaches that can overcome this obstacle.

[0005] In addition to antibiotic resistance, another challenge in the treatment of H. pylori infections is the bacterium's ability to evade the host immune system. H. pylori has evolved several mechanisms to avoid immune detection including the modulation of host immune responses and the alteration of its surface antigens. This immune evasion allows the bacterium to establish a chronic infection, which can persist for decades and increase the risk of developing severe gastrointestinal disorders. Targeting the molecular pathways involved in H. pylori's immune evasion strategies could offer a new avenue for therapeutic intervention.

[0006] Despite the challenges associated with treating H. pylori infections, significant progress has been made in understanding the bacteria's pathogenic mechanisms. Research has identified several key signaling pathways that are activated by CagA in host cells, such as the SHP-2, MARK, and Wnt / p-catenin pathways. These pathways are involved in cell proliferation, differentiation, and survival, and their dysregulation by CagA contributes to the oncogenic potential of H. pylori. Targeting these pathways could provide a targeted approach to prevent the progression of H. py / or / -associated diseases.

[0007] The identification of small molecules that can specifically inhibit the activity of CagA or its interaction with host signaling molecules has emerged as a promising strategy for combating H. pylori infections. Such inhibitors could potentially block the downstream effects of CagA, thereby preventing the pathological changes that lead to gastric cancer. However, the development of such inhibitors requires a deep understanding of the structural and functional aspects of CagA, as well as its interactions with host proteins.

[0008] Recent advances in structural biology have provided valuable insights into the three- dimensional structure of CagA and its interaction sites with host proteins. These structural studies have revealed potential binding pockets that could be targeted by small-molecule inhibitors. Additionally, high-throughput screening of chemical libraries has identified several candidate compounds that exhibit inhibitory activity against CagA. These developments have laid the foundation for the design and optimization of more potent and selective CagA inhibitors.

[0009] By addressing all the above-mentioned problems, there is a need for an antibacterial composition containing pyrrolidin-piperdine derivatives of benzene tricarboxylic acid for inhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori, thereby preventing the replication and pathogenicity of H. pylori infections. There is also a need for a method for preparing a therapeutic molecule that specifically targets and binds to a particular protein of interest, thereby enhancing the efficacy and specificity of the therapeutic treatment. There is also a need for a method for identifying a therapeutic molecule for binding affinity and selectivity for the target protein.

[0010] Additionally, there is also a need for a method for preparing a therapeutic molecule that includes purification steps to isolate the therapeutic molecule from the reaction mixture using standard techniques, thereby ensuring a high-quality final product. There is also a need for a method for identifying a therapeutic molecule that binds to the CagA protein of H. pylori, inhibiting its replication by more than 90% and eliminating H. py / or / -associated hemolysis. There is also a need for a method for screening lead therapeutic molecules that fit into the apoptosis-stimulating protein ASPP-2 binding pocket present in the CagA protein of H. pylori and effectively reduces the replication of bacteria. Further, there is also a need for a method for preparing a therapeutic molecule that can be produced in bulk in a pharmaceutical industry facility and can be distributed at economic prices.Objectives of the invention:

[0011] The primary objective of the present invention is to provide a method for screening, identification, synthesis and biological evaluation of a therapeutic molecule (MA01027) forinhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori (H. pylori), thereby preventing the replication and pathogenicity of H. pylori infections.

[0012] Another objective of the present invention is to provide a method for preparing a therapeutic molecule that specifically targets and binds to a particular protein of interest, thereby enhancing the efficacy and specificity of the therapeutic treatment.

[0013] Another objective of the present invention is to provide a method to screen and identify a therapeutic molecule that optimizes binding affinity and selectivity for the target protein.

[0014] Another objective of the present invention is to provide a method for preparing a therapeutic molecule that involves combining specific reactants (benzene tricarboxylic acid and pyrrolidin-piperdine) under controlled conditions to achieve high yields and purity of the therapeutic molecule.

[0015] Another objective of the present invention is to provide a method for preparing a therapeutic molecule that includes purification steps to isolate the therapeutic molecule from the reaction mixture using standard techniques, thereby ensuring a high-quality final product.

[0016] Another objective of the present invention is to provide a method for preparing a therapeutic molecule that binds to the CagA protein of H. pylori, inhibiting its replication by more than 90% and eliminating H. py / or / -associated hemolysis.

[0017] Yet another objective of the present invention is to provide a method for preparing a therapeutic molecule that fits into an apoptosis-stimulating protein ASPP-2 binding pocket present in the CagA protein of H. pylori and effectively reduces the replication of bacteria.

[0018] Further objective of the present invention is to provide a method for preparing a therapeutic molecule that can be produced in bulk in a pharmaceutical industry facility and can be distributed at economic prices.Summary of the invention:

[0019] The present disclosure proposes a method for preparing an antibacterial composition containing pyrrolidin-piperdine derivatives of benzene tricarboxylic acid for inhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key / critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0020] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method for the identification and preparing a therapeutic molecule (MA01027) for inhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori (H. pylori), thereby preventing the replication and pathogenicity of H. pylori infections.

[0021] According to one aspect, the invention provides a method for preparing a therapeutic molecule for inhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori. At one step, benzene tricarboxylic acid and 4-(pyrrolidi n-l-yl) piperidine are mixed in a reaction vessel to obtain a mixture. The benzene tricarboxylic acid is used in a molar excess relative to 4-(pyrrolidin-l-yl) piperidine.

[0022] At another step, l-(bis(dimethylamino)methylene)-lH-benzotriazole-l-oxide (TBTU), triethylamine (Et3N), and N,N-dimethylformamide (DMF) are added to the mixture, thereby obtaining a reaction mixture.

[0023] At another step, the reaction mixture is allowed to proceed at a temperature ranging from at least 20 °C to 80 °C for a time period sufficient to form a therapeutic molecule (MA01027). In one embodiment, the reaction mixture is carried out at a temperature of at least 40 °C to 60 °C. In one embodiment herein, the therapeutic molecule is used incombination with a broad-spectrum antibiotic to inhibit CagA-mediated pathogenicity and general H. pylori replication. The therapeutic molecule exhibits at least 90% inhibition of H. pylori replication in vitro.

[0024] At another step, the therapeutic molecule is isolated from the reaction mixture using standard purification techniques. Further, at another step, the therapeutic molecule is characterized using analytical methods, such as nuclear magnetic resonance (NMR), mass spectrometry (MS), and high-performance liquid chromatography (HPLC), to confirm structure and purity. The characterization of the therapeutic molecule confirms a purity level of at least 95%.

[0025] In one embodiment herein, the therapeutic molecule is characterized by a binding component (binding affinity) configured to interact with an apoptosis-stimulating protein of p53-2 (ASPP-2) binding pocket within the CagA protein. In one embodiment herein, the therapeutic molecule is characterized by a molecular structure that inhibits CagA from interacting with host cellular proteins, thereby preventing H. pylori from hijacking host cellular processes.

[0026] The molecular structure of the therapeutic molecule is computationally optimized for selective binding to the ASPP-2 binding pocket of the CagA protein, thereby minimizing off- target effects on other proteins within the host cells. In one embodiment herein, the therapeutic molecule is formulated with a slow-release delivery system to extend its activity within the host body, ensuring prolonged inhibition of CagA function and H. pylori pathogenicity.

[0027] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.Detailed description of drawings:

[0028] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0029] FIG. 1 illustrates a flowchart of a method for preparing a therapeutic molecule (MA01027) for inhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori, in accordance to an exemplary embodiment of the invention.

[0030] FIG. 2 illustrates a 2-dimensional structure diagram of the therapeutic molecule (MA01027), in accordance to an exemplary embodiment of the invention.

[0031] FIG. 3 illustrates a 3-dimensional model diagram of the therapeutic molecule bound in the pocket of the cytotoxin-associated gene A (CagA) protein, in accordance to an exemplary embodiment of the invention.

[0032] FIG. 4 illustrates a snapshot depicting the antibacterial assay that was performed using clinical patient samples of H. pylori to evaluate MA01027 along with standard antibiotics as controls, in accordance to an exemplary embodiment of the invention.

[0033] FIG. 5 illustrates a snapshot depicting the antibacterial activity of the therapeutic molecule using liquid brain-heart infusion (BHI) broth, in accordance to an exemplary embodiment of the invention.

[0034] FIG. 6 illustrates a graph representing a comparative analysis of the optical density (O.D.) of the liquid brain-heart infusion (BHI) broth in the presence and absence of the therapeutic molecule, in accordance to an exemplary embodiment of the invention.Detailed invention disclosure:

[0035] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0036] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a therapeutic molecule for inhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori (H. pylori), thereby preventing the replication and pathogenicity of H. pylori infections.

[0037] According to an exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of a method for preparing a therapeutic molecule (MA01027) for inhibiting cytotoxin- associated gene A (CagA) protein of Helicobacter pylori. The therapeutic molecule specifically targets and binds to a particular protein of interest, CagA protein from H. pylori thereby enhancing the efficacy and specificity of the treatment. The therapeutic molecule has optimized binding affinity and selectivity for the target protein. The preparation method of the therapeutic molecule involves combining specific reactants under controlled conditions to achieve high yields and purity of the therapeutic molecule. The preparation method of the therapeutic molecule includes purification steps to isolate the therapeutic molecule from the reaction mixture using standard techniques, thereby ensuring a high-quality final product.

[0038] At step 102, benzene tricarboxylic acid and 4-(pyrrolidin-l-yl) piperidine are mixed in a reaction vessel to obtain a mixture. The benzene tricarboxylic acid is used in a molar excess relative to 4-( pyrrolidin-l-y I) piperidine.

[0039] At step 104, l-(bis(dimethylamino)methylene)-lH-benzotriazole-l-oxide (TBTU), triethylamine (Et3N), and N,N-dimethylformamide (DMF) are added to the mixture, thereby obtaining a reaction mixture.

[0040] At step 106, the reaction mixture is allowed to proceed at a temperature ranging from at least 20°C to 80°C for a time period sufficient to form a therapeutic molecule (MA01027). This temperature range allows for optimal reaction conditions, ensuring the successful synthesis of the compound. The reaction time is adjusted based on empirical results to ensure that the therapeutic molecule (MA01027) is formed with high yield and purity.

[0041] At step 108, the therapeutic molecule is isolated from the reaction mixture using standard purification techniques. These techniques include filtration and solvent extraction, followed by chromatography methods such as silica gel chromatography. This purification process is essential to remove any impurities and by-products, ensuring that the final product is of high quality.

[0042] Further, at step 110, the therapeutic molecule is characterized using analytical methods, such as nuclear magnetic resonance (NMR), mass spectrometry (MS), and high- performance liquid chromatography (HPLC), to confirm structure and purity. The characterization of therapeutic molecule confirms a purity level of at least 95%.

[0043] In one embodiment herein, the therapeutic molecule is characterized by a binding component configured to interact with the apoptosis-stimulating protein of p53-2 (ASPP-2) binding pocket within the CagA protein. In one embodiment herein, the therapeutic molecule is characterized by a molecular structure that inhibits CagA from interacting with host cellular proteins, thereby preventing H. pylori from hijacking host cellular processes.

[0044] The molecular structure of the therapeutic molecule is computationally optimized for selective binding to the ASPP-2 binding pocket of the CagA protein, thereby minimizing off- target effects on other proteins within the host cells. In one embodiment herein, the therapeutic molecule is formulated with a slow-release delivery system to extend its activity within the host body, ensuring prolonged inhibition of CagA function and H. pylori pathogenicity.

[0045] According to another exemplary embodiment of the invention, FIG. 2 refers to a 2- dimensional structure diagram of the therapeutic molecule (MA01027). In one embodiment herein, the structure depicts a small, heterocyclic compound containing multiple nitrogen atoms (N), oxygen atoms (O), and aromatic rings. The arrangement of these atoms suggests that the therapeutic molecule is a complex molecule designed to interact with biological targets, emphasizing its structural specificity and functional capacity within the invention.

[0046] According to another exemplary embodiment of the invention, FIG. 3 refers to a 3- dimensional model diagram of the therapeutic molecule (MA01027) bound in the pocket of the cytotoxin-associated gene A (CagA) protein. In one embodiment herein, the three- dimensional model represents the therapeutic molecule as white stick model, fitted into a binding pocket on the surface of the protein. The surface representation of the protein is color-coded, with blue areas indicating positive electrostatic potential and red areas indicating negative potential. The snug fit of the therapeutic molecule in the pocket suggests strong binding affinity and effective interaction with the protein's binding pocket, which is a significant finding in terms of the compound's inhibitory potential.

[0047] In one embodiment herein, the docking results reveal key interactions between the MA01027 therapeutic molecule and the amino acid residues of the target protein. These interactions include hydrogen bonding and hydrophobic contacts, both of which are crucial for stabilizing the therapeutic molecule protein complex. The tight binding within the pocket implies a highly stable interaction, supporting the hypothesis that the therapeutic molecule (MA01027) can effectively interfere with the biological function of the protein.

[0048] According to another exemplary embodiment of the invention, FIG. 4 refers to a snapshot 400 depicting an antibacterial assay using clinical patient samples of H. pylori and standard antibiotics. In one embodiment herein, the snapshot 400 depicts various treatments including the therapeutic molecule (MA01027) and traditional antibiotics that are tested against bacterial cultures to observe their growth-inhibitory effects.

[0049] In one embodiment herein, the first plate labeled without inoculum serves as a negative control. It showcases the environment of the medium without the presence of any bacterial inoculum. This plate confirms that the medium itself does not promote bacterial growth in the absence of H. pylon, thereby validating the experimental conditions for subsequent tests.

[0050] In one embodiment herein, the second plate labeled control demonstrates the natural growth of H. pylori without any antimicrobial intervention. The bacterial colonies are densely populated across the medium, indicating that in the absence of therapeutic molecules or antibiotics, the bacterial growth remains unchecked, representing the baseline for further comparisons.

[0051] In one embodiment herein, the third plate labeled hemolysis highlights the virulence factor of H. pylori. The clear zones around the bacterial colonies are indicative of hemolysis, a process where red blood cells are lysed. This result shows how H. pylori might interact with host cells and emphasizes the need for therapeutic molecules like MA01027, which aim to inhibit the harmful effects of the CagA protein, thus preventing such destructive activities within the host.

[0052] In one embodiment herein, the fourth plate-labeled antibiotic serves as a comparative benchmark for the efficacy of conventional antibiotics against H. pylori. Antibiotics such as Ciprofloxacin (CIP), Clarithromycin (CLR)7and Tetracycline (TET) are represented by the clear inhibition zones around their respective discs, with measured areas of 8.299 cm2, 5.9419 cm2, and 2.40 cm2for Ciprofloxacin, Clarithromycin, and Tetracycline, respectively. These inhibition zones serve as reference points for evaluating the effectiveness of the new therapeutic molecule (MA01027).

[0053] In one embodiment herein, the fifth plate labeled amoxicillin depicts the specific effect of this commonly used antibiotic on H. pylori. The absence of reduction in bacterial colonies indicates loss of its efficacy and antibiotic-resistance of the H. pylori.

[0054] In one embodiment herein, the final plate labeled therapeutic molecule demonstrates the remarkable efficacy of the therapeutic molecule. The plate depicts a significant reduction in bacterial colony formation compared to the control and the Amoxicillin plate. This indicates that the therapeutic molecule is highly effective in inhibiting the growth of H. pylori, likely through its specific action on the CagA protein, which plays a crucial role in the bacterium's ability to interact with and hijack host cellular processes.

[0055] According to another exemplary embodiment of the invention, FIG. 5 refers to a snapshot 500 depicting the antibacterial activity of the therapeutic molecule using liquid brain-heart infusion (BHI) broth. In one embodiment herein, the snapshot 500 depicts the comparative antibacterial activity analysis using the liquid brain-heart infusion (BHI) broth to assess the efficacy of the therapeutic molecule against Helicobacter pylori. The experimental setup includes three distinct samples: a blank (no inoculum), a control (inoculated with H. pylori but untreated), and a sample treated with the therapeutic molecule.

[0056] The first sample, labeled "Blank," represents the baseline optical density (O.D.) at 600 nm wavelength (visible light range) measurement of the broth without bacterial inoculation, showing an O.D. value of 0.09. This low optical density indicates the absence of bacterial growth and serves as a control for the clarity of the broth. The second sample, labeled "Control," shows a significant increase in turbidity with an O.D. value of 1.11, confirming the presence of active bacterial growth in the broth. This sample is critical for demonstrating the growth potential of H. pylori under untreated conditions.

[0057] The third sample, treated with the therapeutic molecule and labeled accordingly, exhibits a drastically reduced O.D. value of 0.1, almost equivalent to the blank. This demonstrates the efficacy of the therapeutic molecule in inhibiting H. pylori growth. The reduction in turbidity suggests that the therapeutic molecule effectively prevents the replication of the bacteria, showcasing its potential as a targeted therapeutic agent against H. pylori infections.

[0058] Each of these samples is presented in standard laboratory bottles, clearly labeled to avoid any confusion during the experimental process. The results are pivotal in illustrating the specific inhibitory effects of the therapeutic molecule compared to the natural proliferation of the bacteria observed in the control. This setup not only provides a clear comparison of the effects of the treatment but also underscores the potential of the therapeutic molecule and effective solution to combat H. pylori- related infections, particularly in strains resistant to conventional antibiotics such as amoxicillin.

[0059] According to another exemplary embodiment of the invention, FIG. 6 refers to a graph 600 representing a comparative analysis of the optical density (O.D.) of the therapeutic molecule. In one embodiment herein, the graph 600 represents a comparative analysis of optical density (O.D.) at 600 nm, depicting the inhibitory effects of the MA01027 therapeutic molecule on the growth of Helicobacter pylori in liquid culture. The graph 600 includes three distinct bars corresponding to the blank, control, and the therapeutic molecule (MA01027)- treated samples.

[0060] The blank, represented by the blue bar on the left, shows an O.D. value of 0.09. This sample serves as a reference point for the baseline optical density without bacterial inoculation, ensuring that any change in absorbance observed in the other samples is due to bacterial growth. The control sample, represented by the red bar in the center, shows a significantly higher O.D. value of 1.11. This substantial increase in absorbance indicates the unchecked proliferation of H. pylori in the absence of any therapeutic intervention. The control sample serves as a critical comparison point, demonstrating the potential for bacterial growth under normal conditions.

[0061] The therapeutic molecule treated sample is represented by the red bar on the right, shows a dramatically reduced O.D. value of 0.1. This decrease in absorbance reflects the effective inhibition of H. pylori growth following treatment with the therapeutic molecule, showcasing its strong bacteriostatic or bactericidal properties. The near-identical O.D. values between the blank and the MA01027-treated sample (0.09 and 0.1, respectively) furtheremphasize the therapeutic efficacy of the therapeutic molecule in preventing bacterial growth.

[0062] The graph 600 clearly demonstrates the substantial difference in bacterial growth between the untreated control and the MA01027-treated sample, with the latter exhibiting minimal bacterial proliferation. The results highlight the potential of the therapeutic molecule (MA01027) as a promising therapeutic candidate against H. pylori, offering a novel approach to managing infections caused by this pathogen. The use of optical density at 600 nm as a quantitative measure of bacterial growth provides a reliable method for assessing the efficacy of antibacterial compounds such as the therapeutic molecule.

[0063] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, identification and a method for preparing a therapeutic molecule is disclosed. The proposed invention provides a method for preparing the therapeutic molecule (MA01027) for inhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori (H. pylori), thereby preventing the replication and pathogenicity of H. pylori infections.

[0064] The therapeutic molecule specifically targets and binds to a particular protein of interest, thereby enhancing the efficacy and specificity of the treatment. The therapeutic molecule optimizes binding affinity and selectivity for the target protein. The therapeutic molecule involves combining specific reactants under controlled conditions to achieve high yields and purity of the therapeutic molecule. The therapeutic molecule includes purification steps to isolate the therapeutic molecule from the reaction mixture using standard techniques, thereby ensuring a high-quality final product.

[0065] The therapeutic molecule (MA01027) binds to the CagA protein of H. pylori, inhibiting its replication by more than 90% and eliminating H. py / or / -associated hemolysis. The therapeutic molecule fits into the apoptosis-stimulating protein ASPP-2 binding pocket present in the CagA protein of H. pylori and effectively reduces the replication of bacteria.The therapeutic molecule can be produced in bulk in a pharmaceutical industry facility and can be distributed at economic prices.

[0066] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.

Claims

CLAIMS: l / We Claim:

1. A method for preparing a therapeutic molecule for inhibiting cytotoxin-associated gene A (CagA) protein of Helicobacter pylori, comprising: mixing benzene tricarboxylic acid and 4-(pyrrolidin-l-yl) piperidine in a reaction vessel to obtain a mixture; adding l-(bis(dimethylamino)methylene)-lH-benzotriazole-l-oxide (TBTU), triethylamine (Et3N), and N,N-dimethylformamide (DMF) to the mixture, thereby obtaining a reaction mixture; allowing the reaction mixture to proceed at a temperature various between 20°C and 80°C for a time period sufficient to form a therapeutic molecule;isolating the therapeutic molecule from the reaction mixture using standard purification techniques; and characterizing the therapeutic molecule using analytical methods include at least one nuclear magnetic resonance (NMR), mass spectrometry (MS), and high-performance liquid chromatography (HPLC), to confirm structure and purity.

2. The method as claimed in claim 1, wherein the therapeutic molecule is characterized by a binding component that is configured to interact with an apoptosis-stimulating protein of p53-2 (ASPP-2) binding pocket within the CagA protein.

3. The method as claimed in claim 1, wherein the therapeutic molecule is characterized by a molecular structure that inhibits the CagA protein from interacting with host cellular proteins, thereby preventing H. pylori from hijacking host cellular processes.

4. The method as claimed in claim 3, wherein the molecular structure of the therapeutic molecule is computationally optimized for selective binding to the ASPP-2 binding pocket of the CagA protein, thereby minimizing off-target effects on other proteins within the host cells.

5. The method as claimed in claim 1, wherein the therapeutic molecule is used both stand alone and in combination with a broad-spectrum antibiotic to inhibit CagA-mediated pathogenicity and general H. pylori replication.

6. The method as claimed in claim 1, wherein the reaction mixture is carried out at a temperature varies between 40 °C and 60 °C.

7. The method as claimed in claim 1, wherein the benzene tricarboxylic acid is used in a molar excess relative to 4-( pyrrol id i n-l-y I) piperidine.

8. The method as claimed in claim 1, wherein the characterization of the therapeutic molecule confirms a purity level of at least 95%.

9. The method as claimed in claim 1, wherein the therapeutic molecule exhibits at least 90% inhibition of the H. pylori replication in vitro.

10. The method as claimed in claim 1, wherein the therapeutic molecule is formulated with a slow-release delivery system to extend its activity within the host body, ensuring prolonged inhibition of CagA function and H. pylori pathogenicity.DATE AND SIGNATURE:Dated this 22ndday of December, 2024Patent Agent Name: Hima Bindu AttiINPA - 3925

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