CN113061190B - HIV N-peptide Fusion Peptide Inhibitor MTQ-N36 and Its Application - Google Patents

Abstract

The invention discloses an HIV N peptide fusion inhibitor MTQ-N36 and application thereof, wherein the amino acid sequence of the N peptide fusion inhibitor is shown as SEQ ID NO. 1. The N peptide fusion inhibitor of the invention selects 20 amino acids (12-31) in MTQ to be connected with N36 (HXB 2, env, 546-581) of HIV-1, and ensures that a complete alpha spiral structure can be formed. In addition, the invention also detects the inhibition effect of the N-peptide fusion inhibitor on different subtypes of HIV-1 pseudoviruses and cell membrane fusion, the alpha helix proportion and stability of a 6-strand helix bundle (6 HB) formed by CHR and the interaction capacity with CHR. The results show that the N-peptide fusion inhibitor of the invention is more stable and has a broad spectrum and enhanced HIV-1 inhibiting ability compared with the known IZN36 inhibitor. Therefore, the invention provides more choices with inhibition potential for the development of anti-HIV drugs.

Description

HIV N-peptide Fusion Peptide Inhibitor MTQ-N36 and Its Application

technical field

The invention relates to an N-peptide fusion inhibitor, and also relates to the application of the inhibitor in inhibiting HIV-1, and belongs to the technical field of polypeptide inhibitors (drugs).

Background technique

HIV (human immunodeficiency virus, HIV) is an enveloped spherical virus particle, and the envelope protein is composed of surface glycoprotein gp120 and transmembrane glycoprotein gp41. gp41 contains an extracellular region, a transmembrane region and a cytoplasmic region, among which the extracellular region is the key to promoting membrane fusion. The extracellular region contains five functional regions: fusion peptide (FP), fusion peptide proximal (FPPR), N-terminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal region (MPER). FP contains multiple hydrophobic amino acids, which can be inserted into the cell membrane to promote the formation of 6HB; NHR and CHR contain multiple heptahydrophobic repeat sequences, and the amino acid arrangement of each heptad repeat sequence is divided into a, b, c, d, e, f, g bit. Among them, NHR can spontaneously form a helix to form a trimer coiled-coil core. The process of HIV invading target cells is divided into three steps: (1) gp120 binds to CD4 through the CD4 binding site (CD4bs), resulting in changes in the variable regions V1 and V2, exposing the binding sites of the co-receptors CCR5 and CXCR4, After the V3 region binds to the co-receptor, the conformation of gp120 is changed, which in turn changes the conformation of gp41, and the hydrophobic FP is exposed, inserted and fixed on the surface of the host cell membrane; (2) 3 CHRs spontaneously form a trimer with the host cell in an antiparallel manner The NHR of the coiled-coil combines to form 6HB; (3) The viral envelope and the host cell membrane are gradually drawn closer to form a fusion pore, and the viral genome enters the cell through the fusion pore to complete the fusion process. The fusion process of viruses provides precise targets for the development of antiviral drugs. Fusion peptide inhibitors are artificially synthesized peptides derived from gp41 NHR or CHR sequences, which act on viruses or fusion intermediates in the natural state, combine with the corresponding part of gp41 to form heterologous 6HB, and inhibit CHR and NHR from forming the virus's own 6HB, thereby Inhibits membrane fusion and viral entry into target cells. The inhibition mechanism of N-peptide inhibitors is divided into two aspects. On the one hand, it interacts with the NHR of gp41 in the form of monomer or dimer to form a heterotrimeric coiled-coil, thereby interfering with the formation of viral 6HB. On the other hand, the trimeric coiled-coil is formed spontaneously, targeting the exposed gp41 CHR to form heterologous 6HB, thereby preventing the formation of the virus's own 6HB, and the inhibitor in the form of a trimer has good solubility and significantly increased inhibitory potential.

In 2002, the International Intellectual Property Organization (World Intellectual Property Organization, WIPO) disclosed a group of trimerized polypeptides (WO/2002/024735), including IZN17, IZN23, and IZN36, and introduced trimers on the basis of monomeric inhibitors Modified isoleucine zipper (IZ). IZ contains 4 sets of seven repeat sequences similar to Ile-Glu-Lys-Lys-Ile-Glu-Ala (d-e-f-g-a-b-c), these charged amino acid residues form a good electrostatic interaction with the amino acid residues of the adjacent helix, located in The glutamic acid Glu at the b position forms a salt bridge with the lysine Lys at the f position in the same helix, which improves the stability of the coiled coil. These characteristics make the structure of the inhibitor introduced into IZ a stable trimeric coiled coil, and the inhibitory effect reaches nanomolar level (Eckert DM, Kim PS. Design of potent inhibitors of HIV-1entry from the gp41 N-peptide region. Proc NatlAcadSci U SA. 2001;98(20):11187-11192.).

In 2009, WIPO disclosed the trimerization motif MTQ, the core of its amino acid sequence is four seven repeat sequences: Glu-Ile-Ala-Lys-Ile-Lys-Glu-Glu-Gln-Ala-Lys-Ile- Lys-Glu-Lys-Ile-Ala-Glu-Ile-Glu-Lys-Arg-Ile-Ala-Glu-Ile-Glu-Lys, the seven amino acid residues of the above-mentioned seven repeated sequences are represented by g-a-b-c-d-e-f in turn, and the iso Leucine (Ile) is introduced into the a and d positions to form the hydrophobic core of the coiled-coil; glutamine (Gln) is introduced into the a-position of the second seven-repeat sequence, which is beneficial to the polymerization number and direction of the coiled-coil, forming a parallel coiled-coil Structure; the combination of Glu-Lys-Glu at the g-c-f position forms a helical salt bridge, which improves the stability of the trimerization motif. The G-position arginine (Arg) of the fourth hepta-repeat sequence plays an important role in trimer formation, and improves the trimerization ability of the motif. Ile-Lys-Glu before the N-terminal g position, and Arg-Ile-Ala after the C-terminal f position are "protective amino acids", which make the internal amino acids form a coiled-coil structure of a seven-repeat sequence. The Gly-Gly-Ser-Gly-Gly sequence upstream of the N-terminal Ile-Lys-Glu of the motif is a flexible linker sequence (linker) between the motif and the target protein, which can keep the trimerization motif relative to the structure of the target protein Independently, it can also be used as a "protective amino acid" at the N-terminal to increase the helical components in the coiled-coil and make the helical structure more stable. Therefore, the present invention selects an appropriate length of MTQ to connect with N36 to ensure that the synthetic N-peptide inhibitor can form a complete α-helical structure.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the shortcomings of the existing N-peptide inhibitor monomers such as poor solubility, easy aggregation, low bioavailability, and only micromolar inhibitory effects. The N-peptide fusion inhibitor that is more stable and more effective in inhibiting HIV-1 is named MTQ-N36.

The technical problem to be solved by the present invention is achieved through the following technical solutions:

An HIV N-peptide fusion inhibitor of the present invention is named MTQ-N36, and the amino acid sequence of the N-peptide fusion inhibitor is shown in SEQ ID NO:1.

The N-peptide fusion inhibitor of the present invention selects 20 amino acids (12-31) in MTQ to be connected with N36 (HXB2, Env, 546-581) of HIV-1 to ensure that a complete α-helical structure can be formed. At the same time, it also detects The inhibitory effect of fusion peptide inhibitor MTQ-N36 on different subtypes of pseudoviruses and cell membrane fusion, the ratio and stability of α-helix of 6-helix bundle (6HB) formed with CHR, and the ability to interact with CHR were investigated. The results show that, compared with the known IZN36 inhibitor, the N-peptide fusion inhibitor of the present invention is more stable and has a broad spectrum, and the inhibitory ability to HIV-1 is also enhanced.

The nucleotide sequence encoding the HIV N-peptide fusion inhibitor is also within the protection scope of the present invention.

At the same time, the present invention also proposes the use of the HIV N-peptide fusion inhibitor in the preparation of HIV-inhibiting drugs.

Wherein, preferably, the HIV virus is HIV-1.

Compared with the prior art, the beneficial effects of the present invention are:

The present invention proposes a novel HIV N-peptide fusion inhibitor named MTQ-N36. The N-peptide fusion inhibitor selects 20 amino acids (12-31) in MTQ and HIV-N36 from the N-peptide fusion inhibitor. The N36 (HXB2, Env, 546-581) of 1 is connected, and the present invention predicts the structure of the N-peptide fusion inhibitor through molecular conformational simulation to ensure that it can form a complete α-helix. Pseudovirus infection inhibition experiments and cell fusion inhibition experiments were used to detect the inhibitory effect of N-peptide inhibitors on different subtypes of pseudoviruses and cell membrane fusion. Circular dichroism (CD) was used to detect the ratio and stability of the coiled-coil formed by N-peptide inhibitor and the α-helix of 6HB formed by CHR (C34). Non-denaturing gel electrophoresis (Native-PAGE) was used to detect the amount of 6HB formed by the interaction between the N-peptide inhibitor and CHR. The results show that, compared with the known IZN36 inhibitor, the N-peptide fusion inhibitor of the present invention is more stable, has a broad spectrum, and has stronger inhibitory ability to HIV-1.

Description of drawings

Fig. 1 is a schematic diagram of molecular conformational simulation of N-peptide fusion inhibitor MTQ-N36.

Fig. 2 is a graph showing the inhibitory effect of N-peptide fusion inhibitor MTQ-N36 on the infection of different subtypes of HIV-1 pseudoviruses. IZN36 is a trimer control.

Fig. 3 is a result diagram of the coiled-coil formed by the N-peptide fusion inhibitor and the ratio and stability of the 6HB helix formed with C34.

Circular dichroism (CD) analysis of the α-helix structure (A) formed by the N-peptide fusion inhibitor and the stability of the inhibitor itself (C); the α-helix structure of 6HB formed by the N-peptide fusion inhibitor and C34 (B) and 6HB stability (D).

Fig. 4 is a graph showing the result of N-peptide fusion inhibitor and C34 forming 6HB by Native-PAGE.

The first swimming lane is N36, because it carries a positive charge, it moves out of the gel plate during electrophoresis, so there is no band; the second swimming lane is C34, because it has a negative charge, a band appears. The third, fourth and fifth lanes are 6HB formed by N-peptide fusion inhibitor and C34.

detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1

The amino acid sequence of N-peptide fusion inhibitor MTQ-N36 is:

Lys-Ile-Lys-Glu-Glu-Gln-Ala-Lys-Ile-Lys-Glu-Lys-Ile-Ala-Glu-Ile-Glu-Lys-Arg-Ile-Ser-Gly-Ile-Val-Gln- Gln-Gln-Asn-Asn-Leu-Leu-Arg-Ala-Ile-Glu-Ala-Gln-Gln-His-Leu-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Gln- Leu-Gln-Ala-Arg-Ile-Leu (SEQ ID NO: 1).

1. Conformational simulation of N-peptide fusion inhibitors

SWISS Model was used to predict the spatial conformation of N-peptide inhibitors, and pymol software was used to analyze the molecular conformation of N-peptide inhibitors. The model diagram of the molecular conformational simulation of the N-peptide fusion inhibitor MTQ-N36 is shown in Figure 1. The structure of the N-peptide fusion inhibitor is predicted by molecular conformational simulation to ensure that a complete α-helix can be formed.

2. Inhibitory effect of N-peptide fusion inhibitors on different subtypes of HIV pseudovirus infection

1. Construction of Env pseudovirus: First, extract Env eukaryotic expression plasmid and backbone plasmid psG3Δenv. Then the eukaryotic cell transfection experiment was carried out: human embryonic kidney 293T cells (purchased from ATCC, USA) were spread in a 6-well plate at a density of 5×10 ⁵ cells/well, and placed in a cell culture at 37°C and 5% CO ₂ Cultivated in a box. When the cell growth area is 85%-90% of the bottom area of the well plate, transfect 1 μg of the Env eukaryotic expression plasmid into 293T cells, the transfection reagent used is Lipofectamine LTX (purchased from Invitrogen, USA), and the transfection operation refers to Reagent instructions. The HIV-1 strains and subtypes selected in the experiment are shown in Table 1, and HIV pseudoviruses of different subtypes were constructed according to the above methods.

HIV-1 strains and subtypes selected in the experiment in Table 1

2. Pseudovirus semi-tissue cell infection dose (TCID50) detection: Dilute the pseudovirus with a culture solution containing DEAE, and the final concentration of DEAE is 7.5 μg/mL. Add 12.5 μL of the pseudovirus constructed above into the wells for 5-fold serial dilution, spread the TZM-bl cells in a 96-well plate at a density of ¹ ×104/well, and place them in an atmosphere at 37°C and 5% CO ₂ cultured in a cell culture incubator. After culturing for 48 hours, an equal amount of Bright-GloTM Luciferase assay substrate was added, 100 μL of the mixture was added to a 96-well black chemiluminescence detection plate, and the luminescence value was read by a ModulusTMII detector.

3. Pseudovirus infection inhibition experiment: Pseudovirus was diluted to a final concentration of 100 times TCID50/well. The N-peptide inhibitors IZN36 and MTQ-N36 were diluted with culture medium containing DEAE, and the final concentration of DEAE was 7.5 μg/mL. Two-fold serial dilution was performed, and the diluted N-peptide inhibitors were sequentially added to a 96-well deep-well plate, and mixed with the virus at a volume ratio of 1:1. Aspirate the culture medium in the 96-well cell culture plate, add 100 μL of the incubated virus-N-peptide inhibitor mixture into the corresponding wells, and spread TZM-bl cells in 96 wells at a density of ¹ ×104/well In the plate, culture was continued for 48 hours at 37°C. After culturing for 48 hours, an equal amount of Bright-Glo ^TM Luciferase assay substrate was added, 100 μL of the mixture was added to a 96-well black chemiluminescence detection plate, and the luminescence value was read by a Modulus ^TM II detector. The results are shown in Table 2 and Figure 2 .

The inhibitory effect of table 2 N peptide inhibitors on HIV different subtype pseudovirus infection

4. Cell fusion inhibition experiment: firstly extract the Env eukaryotic expression plasmid and pSCTZα, pRev and pSCTZω (Carol D. Weiss). 1) 293T cell transfection: 293T cells (purchased from ATCC, USA) were plated in a 6-well plate at a density of 5×10 ⁵ cells/well, and cultured in a cell culture incubator at 37° C. and 5% CO ₂ . When the cell growth area was 85%-90% of the bottom area of the well plate, 0.1 μg Env expression plasmid, 1 μg pSCTZα and 0.6 μg pRev were transfected into 293T cells. The transfection reagent used was Lipofectamine LTX (purchased from Invitrogen, USA), Refer to the instructions of the reagents for the transfection operation. 2) RC4 cell transfection: RC4 cells (from Oregon Health and Science University) were spread in a 6-well plate at a density of 5×10 ⁵ cells/well, and cultured in a cell culture incubator at 37°C and 5% CO ₂ . When the cell growth area was 85%-90% of the bottom area of the well plate, 1.6 μg pSCTZω was transfected into RC4 cells. The transfection reagent used was Lipofectamine 2000 (purchased from Invitrogen, USA). For the transfection operation, refer to the reagent manual. 3) Fusion of 293T cells and RC4 cells: the transfected 293T cells were plated in a 96-well plate at a density of 1×10 ⁴ cells/well. Put into the incubator and incubate for 6h. 4) After 6 hours, spread the transfected RC4 cells into the same 96-well plate at a density of 1×10 ⁴ cells/well. 5) The N-peptide inhibitors IZN36 and MTQ-N36 were serially diluted in a 96-well deep-well plate, then added to a 96-well cell culture plate, and incubated overnight. 6) Configure and operate according to the requirements of the instructions of the GalactoStar kit, and finally put it into the Modulus ^TM II detector to read the fluorescence value. The results are shown in Table 3.

The inhibitory effect of table 3N peptide inhibitor on cell-cell fusion experiment

3. α-helix formation and stability of N-peptide inhibitors

Circular dichroism experiment (CD): N peptide was dissolved in ultrapure water, C peptide was dissolved in PBS buffer solution with pH 7, centrifuged at 12000r/min for 10min, no insoluble matter was found. Take 9 μL of 6M guanidine hydrochloride and add 1 μL of storage solution to make it 10-fold diluted and denatured. Use NanoDrop 2000 to read the value of protein A280, and calculate and quantify the N-peptide and C-peptide. Mix the peptide inhibitor buffer with the stock solutions of IZN36, MTQ-N36 and C34 to prepare a mixture with a concentration of 10 μM and a final volume of 200 μL, and incubate at 37°C for 30 min. The chirascan circular dichroism spectrometer was used for detection at 20°C, wherein the scanning range was 300-190 nm, the band width was 1.0 nm, the reaction time was 4.0 s, and the scanning speed was 5 nm/min. The background value of the buffer is removed to obtain the graph of the alpha helix. The α-helical conformation is characterized by negative peaks often at 208nm and 222nm. Alpha helix formation was detected by negative values at 208 and 222 nm. At the selected wavelength of 222nm, the temperature was raised from 4°C to 95°C at a rate of 1°C/min to detect the thermal stability of the peptide, smooth the obtained curve, and calculate the midpoint temperature, which is the Tm value, and The Tm value is directly proportional to the stability of the polypeptide. Reverse denaturation at 95°C to 4°C is detected in the same way. The result is shown in Figure 3.

4. The formation of 6HB by N-peptide inhibitors and C34

Non-denaturing gel electrophoresis experiment (Native-PAGE): The stock solution of N-peptide inhibitor IZN36 or MTQ-N36 and C34 was prepared with a peptide inhibitor buffer solution containing 20mM sodium phosphate salt and 0.2M sodium chloride to a concentration of 40μM , Samples with a final volume of 25 μL, incubated in a water bath at 37°C for 30 minutes. Take 4 μL of 6×Non-reduced Loading Buffer (0.35M Tris-HClpH6.8, 30% glycerol, 10% SDS, 0.012% bromophenol blue) and add it to 20 μL of the above-mentioned processed sample, 125V, 2h electrophoresis. After the electrophoresis was completed, the gel was taken out and placed in Coomassie Brilliant Blue R-250, and placed in an air shaker for staining for 2 hours. After the staining is completed, put it into the decolorization solution, put it in an air shaker at room temperature to shake the decolorization until the decolorization is complete. Photographs were taken with the GIS-2010 gel imaging analysis system. The result is shown in Figure 4.

sequence listing

<110> Harbin Medical University

<120> HIV N-peptide fusion peptide inhibitor MTQ-N36 and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 56

<212> PRT

<213> artificial sequence

<400> 1

Lys Ile Lys Glu Glu Gln Ala Lys Ile Lys Glu Lys Ile Ala Glu Ile

1 5 10 15

Glu Lys Arg Ile Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg

20 25 30

Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile

35 40 45

Lys Gln Leu Gln Ala Arg Ile Leu

50 55

Claims (3)

1. An HIV N peptide fusion inhibitor is named as MTQ-N36, and is characterized in that the amino acid sequence of the N peptide fusion inhibitor is shown as SEQ ID NO. 1.

2. A nucleic acid encoding the hiv N-peptide fusion inhibitor of claim 1.

3. The use of the HIV-N peptide fusion inhibitor of claim 1 in the preparation of a medicament for inhibiting HIV-1.

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