Application of PHLPP1 inhibitor in resisting mycobacterial infection
By regulating the host immune system through inhibitors targeting PHLPP1, the problems of long tuberculosis treatment time and drug resistance are solved, and effective control of mycobacterial infection and reduction of inflammation are achieved.
Patent Information
- Application Number
- CN202510708217.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-29
- Publication Date
- 2025-10-03
AI Technical Summary
Existing tuberculosis treatment regimens have problems such as long treatment time, many adverse reactions, and poor effectiveness for multidrug-resistant tuberculosis patients, and the host-directed therapy (HDT) target is unclear.
Develop inhibitors targeting PHLPP1, including small interfering RNA, shRNA or antisense oligonucleotides, to block the expression or activity of PHLPP1, regulate the host immune system, and enhance the killing ability against mycobacteria.
By inhibiting PHLPP1, the proliferation of mycobacteria in host cells is significantly reduced, infection and inflammatory damage are reduced, and a new anti-tuberculosis treatment strategy is provided.
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Figure CN120733033A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology and relates to the role and application of PHLPP1 inhibitors in combating mycobacterial infections. Specifically, the invention provides the use of PHLPP1 inhibitors in the preparation of drugs for treating mycobacterial infectious diseases and / or drugs for alleviating symptoms. Background Art
[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) complex, primarily a respiratory infection. Pulmonary tuberculosis is the most common form, causing varying degrees of lung damage and potentially leading to disseminated tuberculosis with multi-organ lesions, seriously endangering human health. TB is an ancient disease that was once effectively controlled thanks to the advent of anti-TB drugs and Bacillus Calmette-Guérin (BCG) vaccination. However, since the 1990s, TB has experienced a resurgence, driven by factors such as globalization-induced population mobility, weakened awareness of TB prevention, the limited protective effect of BCG, the emergence of drug-resistant TB, and the spread of HIV / AIDS. According to the World Health Organization's Global Tuberculosis Report, the global TB epidemic remains severe, with statistics showing that the number of new TB cases worldwide has remained stable at 9-11 million per year in recent years. Although the use of anti-tuberculosis drugs such as rifampicin and isoniazid has achieved good clinical treatment effects, the subsequent emergence of multidrug resistant tuberculosis (MDR-TB) and even extensively drug-resistant tuberculosis (XDR-TB) has kept the cure rate of tuberculosis at a low level (globally averaging no more than 60%).
[0003] The current mainstream treatment for tuberculosis (TB) is chemotherapy, which targets Mycobacterium tuberculosis and aims to overcome its highly evolved survival and pathogenic mechanisms. However, this treatment approach has drawbacks such as prolonged treatment duration, numerous adverse reactions, and tissue damage, and is not effective in patients with multidrug-resistant / rifampicin-resistant TB. The human immune system plays a crucial role in the infection, pathogenesis, treatment, and prognosis of TB. Therefore, host responses may represent a new and useful target for anti-TB therapy. Host-directed therapy (HDT), a new adjunctive therapy for TB, targets the host immune system. Compared with mainstream chemotherapy, HDT targets the host immune system by modulating host immune pathways against M. tuberculosis, enhancing the host's protective immune response and killing capacity against M. tuberculosis. It also reduces inflammatory responses, thereby reducing inflammation and pathological damage caused by bacterial infection, thereby synergizing with anti-TB drugs to achieve better therapeutic efficacy. Furthermore, HDT does not directly target M. tuberculosis itself and is generally not believed to contribute to the development of drug resistance.
[0004] Currently, the HDT target for tuberculosis is still unclear, but the potential mechanisms of action of HDT candidate drugs are generally believed to include the following four aspects: ① triggering the host's infected cell autophagy pathway to enhance innate immunity against Mycobacterium tuberculosis; ② enhancing acquired immunity by enhancing antigen-specific T cell responses; ③ blocking angiogenesis and affecting granuloma structure, making it easier for drugs to reach the lesions and increasing drug utilization; ④ inhibiting the pro-inflammatory response of infected cells to reduce tissue damage.
[0005] PHLPP1 (pleckstrin homology domain leucine-rich repeat protein phosphatase 1), also known as SCOP, PPM3A, or PLEKHE1, is a member of the protein serine / threonine phosphatase family. PHLPP1 has been shown to directly dephosphorylate conserved sites on proteins such as AKT, protein kinase C (PKC), and S6 kinase (S6K), thereby regulating various cellular biological functions. Current research on PHLPP1 focuses on its target molecules, AKT and STAT1. For example, PHLPP1 dephosphorylates AKT, which can reduce regulatory T cell receptor signaling activation and affect T cell development. Dephosphorylating STAT1 regulates macrophage phagocytosis, thereby participating in inflammatory immune responses. However, whether PHLPP1 functions in mycobacterial infection remains unclear, and no relevant reports have been reported. Summary of the Invention
[0006] In response to the above problems and based on the actual treatment needs of tuberculosis, the present invention has developed an active substance that can effectively block the invasion of mycobacteria, effectively resist mycobacterial infection and control tissue inflammatory damage caused by mycobacterial infection, so as to control mycobacterial infectious diseases and their related symptoms and / or other complications.
[0007] The research process of the present invention is as follows: Analysis of the transcriptome data of blood immune cells of tuberculosis patients found that the transcription level of PHLPP1 in the immune cells of active tuberculosis patients was significantly increased. This result was also verified in the macrophage cell line after mycobacterium infection, suggesting that PHLPP1 plays a role in the interaction between Mtb and the host.
[0008] The Mtb model bacterium Mycobacterium marinum (Mm) was further used as the experimental bacterium to detect the proliferation of Mm in peritoneal macrophages and bone marrow-derived macrophages of PHLPP1 knockout mice. It was found that the proliferation of Mm in macrophages was significantly reduced after PHLPP1 knockout, thereby confirming that PHLPP1 inhibitory substances play an important role in resisting mycobacterial invasion and infection.
[0009] Based on the above research, the technical solution of the present invention is as follows:
[0010] One of the main objectives of the present invention is to provide the use of PHLPP1 inhibitors in resisting mycobacterial infection and further provide their use in treating mycobacterial infectious diseases and related diseases or symptoms. The drugs or pharmaceutical compositions disclosed herein can be used to effectively resist mycobacterial infection and control the occurrence of infectious diseases.
[0011] In a first aspect, the present invention provides use of an inhibitor targeting PHLPP1 in the preparation of a drug for treating mycobacterial infectious diseases and / or infection-related diseases and / or a drug for alleviating symptoms.
[0012] Preferably, the mycobacterium infection is an infectious disease caused by one or more mycobacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium marinum, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium gordonii, Mycobacterium fortuitum, Mycobacterium ulcerans, Mycobacterium hemophilus, and Mycobacterium terrestris.
[0013] Mycobacterium tuberculosis is the primary pathogen of human tuberculosis; the remaining mycobacteria are nontuberculous mycobacteria (NTM). Although they do not primarily cause tuberculosis, their pathogenesis and treatment are still closely linked to the immune system. This study demonstrates that PHLPP1 is abnormally highly expressed in immune cells of hosts infected with these NTM. Knocking out or inhibiting its activity can significantly reduce its proliferation.
[0014] Furthermore, diseases related to Mycobacterium tuberculosis complex (MTBC) mainly include pulmonary tuberculosis (most common) and extrapulmonary tuberculosis, such as lymphadenopathy: cervical lymphadenopathy (common in children); tuberculous pleurisy / peritonitis: serous effusion; bone and joint tuberculosis: such as spinal tuberculosis (Pott's disease); genitourinary tuberculosis: renal tuberculosis, epididymal tuberculosis; tuberculous meningitis; miliary tuberculosis: hematogenous dissemination leading to miliary nodules in multiple organs, etc.
[0015] Nontuberculous mycobacteria (NTM) infections are more common in immunocompromised individuals. Common pathogens include Mycobacterium avium complex (MAC) and Mycobacterium kansasii. Symptoms are often seen in lung disease (chronic cough, cavitary or nodular lesions (similar to tuberculosis), often seen in patients with chronic lung diseases such as COPD and bronchiectasis), lymphadenitis (cervical lymphadenitis is common in children), and skin and soft tissue infections (such as skin ulcers caused by Mycobacterium marinum infection and Buruli ulcers caused by Mycobacterium ulcerans).
[0016] Preferably, the inhibitor is selected from one or more of the following: small interfering RNA (siRNA), shRNA or antisense oligonucleotide targeting the PHLPP1 coding sequence; a compound that inhibits the expression or activity function of the PHLPP1 protein;
[0017] Furthermore, the sequence of the interfering RNA, shRNA or antisense oligonucleotide targeting the PHLPP1 coding sequence is shown in any one of the following SEQ ID NOs. 1 to 5:
[0018] 01: CCUUACUCAUCUCAACUUA (SEQ ID NO.1);
[0019] 02:GGAGGAUCCUUUACCCUGA (SEQ ID NO.2);
[0020] 03:GUAAAUAACUUCCGUGACA (SEQ ID NO.3);
[0021] 04: UAAAGGCACUCUAUGCUUC (SEQ ID NO.4);
[0022] 05: GAAGUCAAGUGUAGAU (SEQ ID NO. 5).
[0023] In a second aspect, the present invention provides a recombinant vector targeting PHLPP1, characterized in that it comprises an interfering RNA or antisense oligonucleotide targeting the PHLPP1 coding sequence inserted into the expression vector, the sequence of which is shown in any one of SEQ ID NOs. 1 to 5 above.
[0024] Preferably, the expression vector can be selected from existing technologies, such as a plasmid vector, a phage vector or a viral vector, wherein the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, etc.
[0025] In a third aspect, the present invention provides use of the aforementioned recombinant vector targeting PHLPP1 in the preparation of a drug for treating mycobacterial infectious diseases or related diseases.
[0026] In a fourth aspect, the present invention provides a pharmaceutical composition selected from liposomes or nanomaterials encapsulating siRNA, shRNA or antisense oligonucleotide targeting the PHLPP1 coding sequence, recombinant expression vectors targeting PHLPP1, or compounds that inhibit the expression or activity of PHLPP1 protein.
[0027] Furthermore, the pharmaceutical composition is preferably used as an anti-mycobacterial infection drug, and is preferably used in combination with current anti-mycobacterial infection drugs.
[0028] The experimental results confirmed that the proliferation of mycobacteria in the immune cells of mice with PHLPP1 knockout or inhibition was significantly reduced, suggesting that PHLPP1 inhibitors can be used as HDT drugs to target the host immune system, regulate the host's immune pathways against mycobacteria, enhance the host's protective immune response, and improve its ability to kill mycobacteria.
[0029] Beneficial guarantees and effects of the present invention: The present invention provides the use of PHLPP1 inhibitors in products for treating mycobacterial infectious diseases and / or diseases and / or symptoms associated with mycobacterial infection. Experiments have confirmed that PHLPP1 knockout or reduced expression inhibits mycobacterial invasion and infection of cells, suggesting that PHLPP1 inhibitors have the effect of inhibiting mycobacterial invasion and infection, providing useful strategies and new explorations for the treatment of mycobacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The present disclosure will be further described below in conjunction with the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present disclosure and are not intended to limit the scope of the present disclosure.
[0031] Figure 1 The results showed that the transcriptional expression level of PHLPP1 in the immune cells of patients with active tuberculosis was significantly increased. The transcriptome data of peripheral blood immune cells of patients with pulmonary tuberculosis were analyzed to evaluate the transcription level of PHLPP1 gene. The results showed that the transcriptional level of PHLPP1 in the immune cells of patients with active tuberculosis was significantly increased ( Figure 1 A); Compared with uninfected cells, the transcription level of PHLPP1 in Mtb-infected THP-1 cells increased significantly at different time points ( Figure 1B), (***, P<0.001; **, P<0.01; UI: uninfected).
[0032] Figure 2 The results showed that Mycobacterium marinum significantly reduced the proliferation of macrophages in PHLPP1 knockout mice. Bone marrow-derived macrophages and peritoneal macrophages from PHLPP1 knockout mice and control mice were infected with Mycobacterium marinum at an MOI of 1. The results showed that the proliferation of Mycobacterium marinum in PHLPP1 knockout macrophages was significantly reduced (***, P < 0.001; **, P < 0.01; ns, no significance). Figure 2 A shows the proliferation of Mycobacterium marinum in bone marrow macrophages after infection. Figure 2 B shows the proliferation of Mycobacterium marinum in peritoneal macrophages after infection.
[0033] Figure 3 It was shown that using siRNA interference to reduce the expression of PHLPP1 can significantly reduce the proliferation of Mycobacterium marinum in peritoneal macrophages. Figure 3 A is the efficiency verification of PHLPP1 small interfering RNA; Figure 3 B and 3C show the intracellular proliferation of mouse peritoneal macrophages after infection with Mycobacterium marinum at MOI 1 and 3, respectively (***, P<0.001). DETAILED DESCRIPTION
[0034] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods, such as PCR. Such methods are well known to those skilled in the art and are described in numerous publications.
[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.
[0036] Example 1: The transcription level of PHLPP1 is significantly reduced in immune cells of patients with active tuberculosis
[0037] Using published transcriptome data, we applied bioinformatics tools to normalize the data and perform differential expression analysis, focusing on evaluating the transcriptional differences of the PHLPP1 gene between active tuberculosis patients and healthy controls. The results showed that the transcriptional level of PHLPP1 was significantly increased in the immune cells of active tuberculosis patients ( Figure 1A); Compared with uninfected cells, the transcription level of PHLPP1 in Mtb-infected THP-1 cells increased significantly at different time points ( Figure 1 B).
[0038] Example 2: Mm proliferation in bone marrow macrophages of PHLPP1 knockout mice was significantly reduced.
[0039] (1) Construction of mouse model: PHLPP1 knockout mice (genotype: PHLPP1- / -, constructed by Suzhou Saiye Biotechnology Co., Ltd.) and corresponding control mice (wild type, genotype: PHLPP1+ / +) were used. The mice were kept under the same environmental conditions and adapted for at least one week before the experiment.
[0040] (2) Isolation and culture of bone marrow-derived macrophages: Cells were extracted from the bone marrow of PHLPP1 knockout mice and control mice; bone marrow cells were inoculated in a culture medium containing M-CSF (macrophage colony-stimulating factor) and cultured for 7 days to induce macrophage differentiation.
[0041] (3) Isolation of peritoneal macrophages: Peritoneal macrophages were collected from PHLPP1 knockout mice and control mice by intraperitoneal injection of broth culture medium. Three days later, peritoneal fluid was collected, centrifuged, and the cells were resuspended in culture medium. After counting, the cells were plated in 24-well cell culture plates.
[0042] (4) Bacterial infection: Infection was performed with Mm at an MOI of 1. Isolated bone marrow-derived macrophages and peritoneal macrophages were seeded into 24-well plates and cultured at 32°C, 5% CO2 after infection. Cell samples were collected at different time points (6 h, 48 h, and 72 h) after infection to assess Mm proliferation.
[0043] (5) Assessment of bacterial proliferation: The plate count method was used to assess Mm proliferation: At an appropriate time point after infection, cells were harvested and lysed to release bacteria. The bacterial suspension was diluted and inoculated onto a suitable culture medium and cultured for 10 days to allow colonies to form. The number of colonies on each plate was counted to calculate the Mm proliferation level in each sample.
[0044] (6) Data analysis: Statistical software (e.g., GraphPad Prism) was used to analyze the data and compare the proliferation of Mm in PHLPP1 knockout mice and control mice. The student's T test was used to assess the significance of the differences between the groups, and P < 0.05 was considered statistically significant.
[0045] Example 3: Interference with PHLPP1 expression inhibits the proliferation of mycobacteria in mouse macrophages
[0046] (1) Isolation of peritoneal macrophages: Peritoneal macrophages from PHLPP1 knockout mice and control mice were collected by intraperitoneal injection of broth culture medium. Three days later, peritoneal fluid was collected and primary peritoneal macrophages from wild-type mice were obtained. The cells were centrifuged and resuspended in culture medium, counted and plated in 24-well cell culture plates. A mixture of five siRNAs targeting mouse PHLPP1 (sequences are shown in SEQ ID NO. 1 to 5) and a control small RNA (all synthesized by Shanghai JiKai Gene Medical Technology Co., Ltd.) were transfected. 72 hours after the peritoneal macrophages were transfected with the above siRNAs, Mm (MOI = 1 or MOI = 3) was added and cultured at 32°C and 5% CO2 after infection. Cell samples were collected at different time points (6h, 48h and 72h) after infection to evaluate the proliferation of Mm.
[0047] (2) Assessment of bacterial proliferation: The plate count method was used to assess Mm proliferation: At an appropriate time point after infection, cells were harvested and lysed to release bacteria. The bacterial suspension was diluted and inoculated onto a suitable culture medium and cultured for 10 days to allow colonies to form. The number of colonies on each plate was counted to calculate the Mm proliferation level in each sample.
[0048] (3) Data analysis: Statistical software (e.g., GraphPad Prism) was used to analyze the data and compare the proliferation of Mm in PHLPP1 knockout mice and control mice. The t-test was used to assess the significance of the differences between the groups, and P < 0.05 was considered statistically significant.
[0049] The specific sequence of siRNA that specifically interferes with PHLPP1 expression (PHLPP1-siRNA) is as follows:
[0050] siRNA-01:CCUUACUCAUCUCAACUUA (SEQ ID NO.1);
[0051] siRNA-02:GGAGGAUCCUUUACCCUGA (SEQ ID NO.2);
[0052] siRNA-03: GUAAUAACUUCCGUGACA (SEQ ID NO.3);
[0053] siRNA-04: UAAAGGCACUCUAUGCUUC (SEQ ID NO.4);
[0054] siRNA-05:GAAGUCAAGUGUGUAGAU (SEQ ID NO. 5).
[0055] The results showed that compared with the control transfection group ( Figure 3Compared with A), the expression level of PHLPP1 mRNA in macrophages transfected with the above-mentioned mixture of 5 siRNAs targeting PHLPP1 decreased significantly, indicating that these siRNAs can effectively interfere with the expression of PHLPP1 in macrophages. Mm proliferation in macrophages after PHLPP1 interference was significantly reduced ( Figure 3 B. Figure 3 C).
[0056] The results indicate that interfering with PHLPP1 expression using siRNA can inhibit the proliferation of mycobacteria in macrophages, thereby limiting mycobacterial infection.
[0057] The undescribed parts of the present invention are the same as the prior art or are implemented using the prior art. The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned specific implementation methods, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.
Claims
1. Use of a PHLPP1 inhibitor in the preparation of a drug for treating mycobacterial infectious diseases and / or associated diseases or a drug for improving symptoms.
2. The use according to claim 1, characterized in that The PHLPP1 inhibitor is selected from one or more of the following: siRNA, shRNA or antisense oligonucleotide targeting the PHLPP1 coding sequence; and a compound that inhibits the expression or activity of the PHLPP1 protein.
3. The use according to claim 2, characterized in that The sequence of the siRNA, shRNA or antisense oligonucleotide targeting the PHLPP1 coding sequence is shown in any one of SEQ ID NOs. 1 to 5.
4. The use according to claim 1, characterized in that The mycobacterial infectious disease is an infectious disease caused by one or more mycobacteria selected from the following group: Mycobacterium tuberculosis, Mycobacterium marinum, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium gordonii, Mycobacterium fortuitum, Mycobacterium ulcerans, Mycobacterium hemophilus, and Mycobacterium terrestris.
5. A recombinant expression vector targeting PHLPP1, characterized in that: The invention comprises an interfering RNA or an antisense oligonucleotide inserted into the expression vector and targeting the PHLPP1 coding sequence, wherein the sequence is shown in any one of SEQ ID NOs. 1 to 5.
6. Use of the recombinant expression vector targeting PHLPP1 according to claim 5 in the preparation of a drug for treating mycobacterial infectious diseases and / or associated diseases or a drug for improving symptoms.
7. A pharmaceutical composition, characterized in that The drug is selected from liposomes or nanomaterials encapsulating siRNA, shRNA or antisense oligonucleotide targeting PHLPP1 coding sequence, recombinant expression vectors targeting PHLPP1, or compounds that inhibit PHLPP1 protein expression or activity function.
8. Use of the pharmaceutical composition according to claim 7 as an adjuvant therapeutic agent against mycobacterial infection.
9. The use according to claim 8, characterized in that The pharmaceutical composition is used in combination with other anti-mycobacterial infection drugs.