Use of PHT-427 in the preparation of a drug for inhibiting the activity of the core protease of monkeypox

Abstract

The present invention discloses the application of PHT-427 in the preparation of a drug for inhibiting the activity of the monkeypox core protease. By using high-throughput drug screening technology and through multiple screenings, a small molecule compound PHT-427 that can efficiently inhibit the core protease I7L was obtained. Through experiments, it was confirmed that the IC 50 value of PHT-427 for the monkeypox virus core protease I7L is 9 μM. In vitro enzyme activity experiments demonstrated that the compound PHT-427 can specifically bind to the active site of the monkeypox virus core protease, significantly inhibit the catalytic function of this protease, block the cleavage processing of viral polyproteins and the maturation and assembly of virus particles, and thereby exert an antiviral effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of PHT-427 in the preparation of drugs that inhibit the activity of monkeypox core protease. Background Technology

[0002] Monkeypox virus (MPXV) is an enveloped double-stranded DNA virus belonging to the genus Orthopoxvirus in the family Poxviridae. It is closely related to smallpox and cowpox viruses and shares similar antigenicity. It is divided into two branches: the West African branch, which is less pathogenic and has a mortality rate of approximately 3.6%, and the Central African (Congo Basin) branch, which is more pathogenic and has a mortality rate of up to 10% in unvaccinated smallpox populations. This virus can cause zoonotic monkeypox. It was first discovered in monkeys in 1958, and the first human case was reported in 1970. After infection with monkeypox virus, patients often present with symptoms such as fever, headache, swollen lymph nodes, and skin blisters. In severe cases, it can lead to encephalitis, sepsis, permanent vision loss, and even death.

[0003] Currently, no vaccines or drugs with proven efficacy have been approved for the prevention and treatment of monkeypox virus infection. Only two vaccines (ACAM2000 and Jynneos) and two antiviral drugs (Tecovirimat and Brincidofovir) have been approved by the U.S. Food and Drug Administration (FDA) for the prevention and treatment of smallpox viruses. Based on the safety of Tecovirimat in humans and its anti-monkeypox virus efficacy in animal models, the European Medicines Agency (EMA) approved it for the treatment of monkeypox virus infection. However, the latest clinical data show that Tecovirimat is not significantly more effective than placebo in the clinical treatment of monkeypox virus. Currently, monkeypox virus continues to cause intermittent outbreaks and circulate globally. Therefore, developing novel, low-toxicity, and highly effective anti-monkeypox virus drugs is not only crucial for addressing the current monkeypox virus epidemic but also has significant public health and social value in responding to potential future orthopox virus outbreaks. Summary of the Invention

[0004] The purpose of this invention is to overcome the lack of drugs for treating monkeypox virus in clinical practice and to provide an inhibitor that can inhibit the activity of monkeypox core protease.

[0005] To achieve the above objectives, the present invention provides the use of PHT-427 in the preparation of drugs that inhibit the activity of monkeypox core protease.

[0006] Optionally, the monkeypox core protease is the I7L protease.

[0007] Optionally, the PHT-427 inhibits monkeypox core protease activity by at least 70%.

[0008] Optionally, the drug may also include a pharmaceutically acceptable carrier.

[0009] Optionally, the pharmaceutically acceptable carrier includes any one or more of solvents, lubricants, antioxidants, preservatives, thickeners, pH buffers, and stabilizers.

[0010] Optionally, the drug can block viral replication by inhibiting the activity of the core protease I7L, thereby achieving the purpose of treating monkeypox virus infection.

[0011] Optionally, the dosage form of the drug is any one of injection, tablet, capsule, suspension, or emulsion.

[0012] Optionally, the route of administration of the drug includes any one of oral, transdermal, intravenous, or intramuscular injection.

[0013] This invention also provides the application of PHT-427 in the preparation of drugs against monkeypox virus infection.

[0014] Compared to the prior art, the beneficial effects of the present invention include at least the following: This invention demonstrates that compound PHT-427 can efficiently inhibit the activity of the monkeypox virus core protease I7L, achieving an inhibition rate of 70%, and possesses antiviral potential. PHT-427, by inhibiting the I7L protease, directly targets a key late-stage step in the viral life cycle—blocking the transformation of immature viral particles into infectious mature viral particles. This mechanism differs from existing drugs (such as Tecovirimat), providing a novel candidate drug molecule and a clear therapeutic target for monkeypox virus infections for which there are currently no effective treatments. Furthermore, due to the high conservation of the I7L protease within the orthopoxvirus genus, PHT-427, as a highly effective inhibitor, is not only effective against monkeypox virus but may also exhibit inhibitory activity against other orthopoxviruses such as smallpox virus and vaccinia virus, providing a broad-spectrum candidate drug reserve for addressing potential emerging or re-emerging infectious diseases of the orthopoxvirus genus in the future. Attached Figure Description

[0015] Figure 1 This is a preliminary fluorescence reading of the inhibition of the core protease by PHT-427 in the high-throughput screening of this invention.

[0016] Figure 2 The IC50 of PHT-427, obtained by screening in this invention, inhibits the core protease I7L. 50 Quantitative measurement chart. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Unless otherwise specified, the materials and reagents used in this invention are commercially available.

[0018] The chemical name of the compound PHT-427 of this invention is 4-dodecyl-N-1,3,4-thiadiazole-2-yl-benzenesulfonamide, and its chemical structural formula is shown in Formula I. Its CAS number is 1191951-57-1. Formula I The life cycle of ornithopox virus is highly conserved, involving five morphologies of viral particles during replication and infection: immature virion (IV), intracellular mature virion (IMV), intracellular enveloped virion (IEV), cell-associated enveloped virion (CEV), and extracellular enveloped virion (EEV). Monkeypox virus replication primarily occurs within the "viral factory" in the host cell cytoplasm, including viral DNA replication and transcription, accumulation of structural proteins, and viral particle assembly. The accumulation of viral structural proteins depends on the proteolytic action of two proteases: the core protease I7L and the G1L protease. The core protease I7L is a cysteine ​​protease that mainly acts in the late stages of viral assembly. Furthermore, the G7L protein, a crucial component of the intracellular mature viral core, also relies on the hydrolytic action of the core protease I7L to function.

[0019] Studies have shown that the core protease I7L plays a crucial role in the formation of infectious IMV particles of orthopoxvirus. This protease is highly conserved within the orthopoxvirus genus and has no homologous protein in humans, thus exhibiting extremely high selectivity. The core protease sequences of monkeypoxvirus, smallpoxvirus, and vaccinia virus share up to 99% homology, implying that inhibitors targeting the core protease may have broad-spectrum inhibitory effects against various orthopoxviruses. The core protease precisely recognizes the Ala-Gly-Xaa sequence and cleaves at the amide bond position of the glycine (Gly) residue, driving the viral particle from a non-infectious immature viral morphology (IV) to an infectious mature viral morphology (IMV). Mutations or deletions of the core protease result in loss of viral infectivity, suggesting that the monkeypoxvirus core protease is a potential target for antiviral drug development. Inhibiting the activity of the core protease can effectively block IMV formation, thereby inhibiting viral replication and proliferation. Proteases have been successfully used as antiviral drug targets in the treatment of HIV, HCV, and SARS-CoV-2. Therefore, elucidating the three-dimensional structure of the core protease and designing drugs based on the structure to develop highly efficient core protease inhibitors is not only of great scientific significance for the prevention and control of monkeypox virus, but also provides broad application prospects for addressing the potential threats of orthopoxvirus in the future.

[0020] To address the shortcomings of existing technologies, this invention provides the application of compound PHT-427 in the preparation of drugs that inhibit the activity of monkeypox core protease. In vitro enzyme activity experiments have demonstrated that compound PHT-427 can specifically bind to the active site of monkeypox virus core protease, significantly inhibit the catalytic function of the protease, block the cleavage processing of viral polyproteins and the maturation assembly of viral particles, thereby exerting an antiviral effect.

[0021] Based on a high-throughput inhibitor screening system for monkeypox virus core protease I7L, this invention screened approximately 17,000 compounds from FDA drug libraries, anti-infective drug libraries, natural product libraries, and lead compound libraries. The final concentration of small molecules added during screening was 20 μM. Proteins and small molecules were added using a Bravo automated liquid handling workstation and incubated for 5 min. Substrate was then added, and the inhibitory activity of the compounds was monitored using an EnVision microplate reader. Based on the initial reaction rate V0, the residual activity and the inhibition rate of the small molecules were calculated, initially identifying small molecule inhibitors with an inhibition rate greater than 70% against the monkeypox virus core protease. The inhibitors identified in the initial screening were then repeatedly screened using the compound library and additionally purchased small molecules to eliminate false positives and compounds exhibiting significant fluorescence signals under 340 nm excitation and 405 nm emission light, thereby reducing spectral interference and improving the authenticity and reliability of the screening results.

[0022] In vitro enzyme activity assay: In the experimental material preparation stage, the proteins and substrates were prepared first: the monkeypox virus core protease I7L was dissolved in enzyme activity buffer, and the final concentration was adjusted to 0.4 μM, ensuring that it was prepared and used immediately; the fluorescent substrate MCA-DDLQMVIAGAKSK (Dnp) was first dissolved with DMSO, and then diluted with enzyme activity buffer to a final concentration of 50 μM, aliquoted, and stored in the dark for later use. For compound library preparation, the 1 mM compound library stored at -80℃ was taken out, thawed naturally at room temperature, and centrifuged at 1000 rpm for 2 min to remove precipitates and impurities. After use, it was immediately sealed with sealing film and returned to the -80℃ freezer for storage to avoid repeated freeze-thaw cycles affecting activity.

[0023] The high-throughput screening operation was performed as follows: Using the Bravo automated liquid handling workstation, 40 μL of 0.4 μM monkeypox virus core protease was added to each well of a 384-well microplate. After the enzyme solution was dispensed, 1 μL of the treated compound (containing DMSO blank control group) was added to each well, and the plate was incubated at room temperature for 5 min. After incubation, 10 μL of 50 μM fluorescent substrate was added to each well, and the plate was immediately detected using an EnVision microplate reader. The detection parameters were set to excitation light 340 nm and emission light 405 nm. A total of 20 detections were performed, with each round of detection lasting approximately 43 s.

[0024] In the data processing and positive screening stage, the inhibition rate was first calculated: the fluorescence values ​​of the 3rd to 20th detection points were used to calculate the linear slope, which was compared with the slope of the DMSO blank control group to obtain the remaining enzyme activity of the sample. The inhibition rate was calculated according to the formula "inhibition rate (%) = (1 - sample group slope / blank group slope) × 100%". Small molecules with an inhibition rate ≥70% were defined as positive candidates, including PHT-427. Figure 1 The fluorescence readings show the inhibition of the core protease I7L by PHT-427 during high-throughput screening. After incubation with the core protease, PHT-427 resulted in low activity of the core protease, characterized by a flat and essentially unchanged fluorescence signal. In contrast, the core protease in the blank control group, with its normal activity, showed a continuously increasing fluorescence signal after reacting with the fluorescent substrate. This indicates that PHT-427 inhibits the activity of the core protease; the weak activity of the core protease and the lack of substrate reaction lead to a essentially unchanged fluorescence signal.

[0025] The positive small molecule rescreening was divided into two rounds. In the first round of rescreening, 1 μL of the solution corresponding to the positive candidate was taken and added to 40 μL of 0.4 μM protease. After incubation for 5 min, 10 μL of substrate was added. Three parallel samples and one blank control were set up for each small molecule. The candidate drugs obtained after repeated screening according to the initial detection method were defined as the second positive candidate drugs, including PHT-427.

[0026] The second round of screening requires purchasing positive small molecules that still show an inhibition rate ≥70% after the first round of screening. These molecules are then reconstituted in DMSO to the corresponding concentration, and the experiment is repeated using the initial screening method. Finally, a stable positive inhibitor with an inhibition rate ≥70% is confirmed. This invention, through one round of initial screening and two rounds of rescreening, confirms that PHT-427 exhibits a significant inhibitory effect on monkeypox virus core protease I7L.

[0027] After screening, a set of PHT-427 concentration gradients (four parallel experiments, including a DMSO blank control group) was set up using the Bravo automated liquid handling workstation. The inhibition rate at each concentration was measured following the same operating procedures as for high-throughput screening. Figure 2 As shown, the IC of PHT-427 50 The value is 9.00 ± 0.33 μM.

[0028] In some embodiments, the drug is used alone; in other embodiments, the drug is used in the form of a pharmaceutical composition.

[0029] In some embodiments, the drug also includes a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art. A thorough description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, phosphate buffer, Ringer's solution, physiological saline, balanced salt solution, glycerol, or sorbitol. Additionally, these carriers may contain auxiliary substances such as lubricants, flow aids, wetting agents or emulsifiers, pH buffers, and stabilizers such as albumin.

[0030] In some embodiments, the dosage form of the drug is any one of injection, tablet, capsule, suspension, or emulsion.

[0031] In summary, this invention provides the application of compound PHT-427 in the preparation of drugs that inhibit the activity of monkeypox core protease. In vitro enzyme activity experiments have demonstrated that compound PHT-427 can specifically bind to the active site of monkeypox virus core protease, significantly inhibit the catalytic function of the protease, block the cleavage processing of viral polyproteins and the maturation assembly of viral particles, thereby exerting an antiviral effect.

[0032] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Application of PHT-427 in the preparation of drugs that inhibit the activity of monkeypox core protease.

2. The application as described in claim 1, characterized in that, The core protease in the monkeypox is the I7L protease.

3. The application as described in claim 1, characterized in that, The PHT-427 inhibits monkeypox core protease activity by at least 70%.

4. The application as described in claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

5. The application as described in claim 4, characterized in that, The pharmaceutically acceptable carriers include any one or more of the following: solvents, lubricants, antioxidants, preservatives, thickeners, pH buffers, and stabilizers.

6. The application as described in claim 1, characterized in that, The drug inhibits the activity of the core protease I7L, thereby blocking viral replication and achieving the purpose of treating monkeypox virus infection.

7. The application as described in claim 1, characterized in that, The dosage form of the drug is any one of injection, tablet, capsule, suspension, or emulsion.

8. The application as described in claim 1, characterized in that, The drug can be administered via any of the following routes: oral, transdermal, intravenous, or intramuscular injection.

9. Application of PHT-427 in the preparation of drugs against monkeypox virus infection.

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