CN100455594C - Polypeptide for inhibiting HIV virus fusion and use thereof - Google Patents

Abstract

The invention provides a class of polypeptides for inhibiting HIV virus fusion. According to the action mechanism of HIV virus and the receptor molecule on the cell membrane surface during the membrane fusion process, the present invention designs an active polypeptide that can combine with the virus surface glycoprotein gp41. The action site of the polypeptide is located at the "long hole" on the side of the trimer NHR and both ends of the gp41 molecule, and can effectively inhibit the replication of HIV virus. Compared with the existing drugs, the polypeptide of the invention has small molecular weight, high activity and good water solubility. The medicament for inhibiting the infection of various enveloped viruses can be prepared by using the polypeptide of the invention.

Description

Polypeptide for inhibiting HIV virus fusion and use thereof

Technical field:

The invention relates to a class of polypeptides for inhibiting virus fusion, in particular to a class of polypeptides for inhibiting HIV virus fusion. The present invention also relates to the use of said polypeptide.

Background technique:

Some viruses with envelopes, such as human immunodeficiency virus (Human Immunodeficiency Virus, HIV), human respiratory syncytial virus and hepatitis B virus, the whole life cycle can be divided into four parts: virus and cell membrane fusion, genetic material enters the cell ; reverse transcription of viral DNA and integration into host cell chromosomes; transcription, translation, modification of viral proteins; assembly and budding of virus particles. What these viruses have in common is that there is a process of membrane fusion in the life cycle, and the structures of the proteins involved in the fusion process are similar.

Taking the drugs for the treatment of HIV infection as an example, the current clinical drugs for the treatment of HIV infection can be divided into three categories: reverse transcriptase inhibitors, protease inhibitors and entry inhibitors (including fusion inhibitors), which are respectively aimed at the different replication cycles of the above viruses. Links (Jiang S., et al., Curr. Pharm. Des., 2002, 8(8): 563-580).

Reverse transcriptase inhibitors and protease inhibitors are prone to drug resistance during use, which is an important reason for the current limited use of these two types of drugs. In addition, the long-term use of these two types of inhibitors has obvious toxic and side effects such as abnormal fat distribution and osteoporosis (Zhou Wei, et al. China AIDS and STD. 2005, 11(1): 73-75).

HIV entry inhibitors inhibit the entry of the HIV virus into cells, where fusion inhibitors act on the fusion of viral and cell membranes. During clinical use, it was found that the single application of HIV virus fusion inhibitors can greatly reduce the viral load, and the side effects are very low. HIV fusion inhibitors can also be used in combination with reverse transcriptase inhibitors and protease inhibitors, which can not only reduce the dosage of these two drugs, thereby reducing side effects, but also prevent the emergence of new drug-resistant strains.

Entry inhibitors act on the stage before the virus enters the cell, that is, from the contact between the virus and the cell to the membrane fusion. This stage mainly includes three processes (Eckert D.M., et al., Annu.Rev.Biochem.2001, 70:777-810): first, the surface glycoprotein gp120 of the virus binds with the cell surface receptor protein CD4 molecule with high affinity, The conformational change causes the virus to attach to the host cell; then, gp120 interacts with host cell surface co-receptors (such as: CCR5, CXCR4, etc.), the conformation changes further, and it is separated from gp41, and the fusion peptide at the N-terminal of gp41 is inserted into the host cell membrane ; Finally, the CHR region (C-terminal heptad repeats) of gp41 turns back and approaches the α-helix trimer formed by its NHR region (N-terminal heptad repeats), forming a hexameric α-helix, which brings the virus and the cell closer distance, resulting in the fusion of the viral envelope with the cell membrane.

The HIV entry inhibitors currently being studied mainly target the viral surface glycoprotein gp120/gp41 and the cell surface co-receptors CCR5/CXCR4 and CD4 receptors in the above process.

The entry inhibitor acting on gp120 has (Thali M., et al., J.Virol.1993, 67 (7): 3978-3988; ShaunakS., J.Pharmacol., 1994, 113: 151-158; Trkola A ., et al., J.Virol.1996,70:1100-1108; Wang T., et al., J.Med.Chem.2003,46:4236-4239; Ferrer M., et al., J. Virol.1999, 73(7):5795-5802): 17b, 2G12, PRO542, BMS-378806, 12P1.

The entry inhibitor (fusion inhibitor) acting on gp41 has (Wild C.T., et al., Proc.Natl.Acad.Sci.USA., 1994, 91:9770-9774; Jiang S., et al., Curr. Pharm.Des., 2002, 8(8):563-580; Root M.J., et al., Science, 2001, 291:884-888): DP107, T-20, T-1249, C34, IQN37, 5- Helix.

Entry inhibitor acting on co-receptor CCR5/CXCR4 (Maeda K., et al., Curr. Opin. Pharmacol, 2004, 4(5): 447-452; Pierson T.C., et al., Rev. Med. Virol .2004, 14:255-270.): AOP-RANTES, PRO140, UK-427857, TAK-779, TAK-220.

The entry inhibitor acting on the CD4 receptor has (Reimann K.A., et al., ADIS Res. Hum. Retroviruses., 1997, 12 (11): 933-943): TNX-355

Among the several classes of entry inhibitors mentioned above, inhibitors acting on the coreceptor CCR5/CXCR4 are now considered to have inherent defects. As we all know, CCR5/CXCR4 is a molecule that exists on the surface of normal cells. In addition to serving as a co-receptor in the HIV virus entry process, it is also a receptor for chemokines and inflammatory factors, so long-term use of inhibitors will cause problems. Competitive binding , Some small molecule antagonists have found side effects on the heart during clinical research (MaedaK., et al., Curr. Opin. Pharmacol, 2004, 4(5): 447-452).

The entry inhibitors acting on gp120 are mainly screened or antibodies obtained from infected persons, and there are fewer small molecules. At present, only two antibodies with broad neutralizing activity, 17b and 2G12, have been obtained. Some other antibodies have neutralizing activity in vitro, but not in vivo. The current small molecule inhibitors include BMS-378806, which binds to a hydrophobic pocket on gp120, and the small peptide (12P1) obtained through peptide library screening may have the same binding site.

Entry inhibitors acting on the CD4 receptor are the least studied because it is a very important molecule in the body, and its inhibition is likely to cause serious side effects.

Currently, only one peptide fusion inhibitor T-20 (trade name Fuzeon) is used clinically.

T-20 is derived from the CHR region of HIV-1 LAI strain gp41 and consists of 36 amino acids (Wild, C.T., et al, Proc. Natl. Acad. Sci. U.S.A 1994, 91: 9770-9774). The pre-fusion conformation of gp41 can last for about 30 minutes, during which time T-20 can act on the -helical trimer formed by the NHR regions of three gp41 molecules (Melikyan G.B., J.Cell.Biol., 2000, 151 : 413-423). The competitive combination of T-20 prevents the CHR region of gp41 from binding to the NHR region and inhibits the formation of hexamers. Therefore, the cell membranes of the virus and the host cannot approach each other, and the membrane fusion process is hindered.

Recent research results suggest that T-20 cannot form a stable helical hexamer in solution with NHR peptide N36 derived from gp41, so it may interact with multiple sites of gp41 and gp120 (Liu S., et al. al., J.Biol.Chem, 2005, 280(12): 11259-11273), which is different from its design principle.

T-1249 is the second-generation product of T-20, consisting of 39 amino acids, derived from the CHR regions of HIV-1 LAI, HIV-2 NIHZ, and SIV mac251 strains (Schneider S.E., et al, J.Pept . Sci. 2005, 11(11): 744-753). During clinical use, it was found that only one amino acid mutation in the CHR region of gp41 can cause strains resistant to T-20, but these strains are still sensitive to T-1249, which is considered to be likely to interact with the membrane. Ability-related (VeigaA.S., et al, J.Am.Chem.Soc., 2004, 126(45):14758-14763).

Roche and its collaborators have turned to study TR-290999 and TR-291144 since 2004 (13th CROIConference on Retroviruses and Opportunistic Infections Denver, Colorado, Feb 5-8, 2006). These two polypeptides consist of 38 and 36 peptides respectively. Amino acid composition, it is said that the activity is higher than T-20.

Although T-20, T-1249, TR-290999 and TR-291144 are currently the most effective polypeptide HIV fusion inhibitors, their common disadvantage is that the molecular weight is relatively large compared to other anti-HIV drugs, so the synthesis is relatively difficult .

Therefore there is an urgent need for polypeptides with shorter amino acid sequences to inhibit virus fusion agents so as to facilitate synthesis; in addition, fusion agents also need to have different sites of action from T-20 to combat T-20 drug-resistant viruses; Solubility and stability to enhance efficacy and reduce drug dosage.

Invention content:

The purpose of the present invention is to provide a novel polypeptide for inhibiting HIV virus fusion. The amino acid sequence of this type of polypeptide is short, the action site is different from that of existing drugs, and it has better water solubility and high activity.

The design principle of the novel polypeptide provided by the invention

The NHR region of the surface glycoprotein gp41 of HIV-1 can be used as an ideal drug target due to its high degree of conservation, and the polypeptide derived from the CHR region can become the lead polypeptide of this type of drug because it can interact with the NHR region. The virus fusion inhibitor provided by the present invention is derived from a CHR sequence of gp41 of the RL42 strain of the B' subtype, which is the most popular in the mainland of China and has the largest number of infected people. This sequence has the following amino acid sequence:

EIWNNMTWMEWEREIDNYTREIYTLIEESQNQ

The sequence analysis results of the surface glycoprotein gp41 of HIV-1 show that there are two very conserved regions in the NHR region, one of which is "GIVQQQ" (Rimsky L.T., et al, J.Virol., 1998, 72:986 -993), any amino acid change in this region can cause T-20 drug resistance, so it is the main action site of T-20; the other is Leu-565, Leu-566, Leu-568, Thr-569 , Val-570, Trp-571, Gly-572, Ile-573, Lys-574, Leu-576, Gln-577 and other 11 highly conserved long hydrophobic domain "Cavity" (Eckert D.M., et al. al, Cell, 1999, 99:103).

In designing a polypeptide inhibitor that binds to the NHR region, the inventors focused on the sequence that binds to the "Cavity" domain on the trimeric NHR region of gp41 and its two ends. Previous inhibitors only considered the sequence in the CHR sequence Trp628, Trp631, while ignoring the role of Trp623. In addition, the inventors designed a helical sequence that binds to the C-terminus of "Cavity" to increase the hydrophobic interaction with the gp41 NHR region; removed the sequence with weak binding ability to the trimeric NHR of gp41, and introduced hydrophilic amino acids to Increase the water solubility of the inhibitor, introduce acidic or basic amino acids that can generate ionic bonds in the "I" and "I+4" positions of the polypeptide sequence, so as to increase the ability to form a helix by itself, and finally form the present invention.

The present invention finds that the novel polypeptide having the general formula I has the effect of inhibiting virus fusion, and the primary structure of the polypeptide of the general formula I is as follows:

α-X ₁ -X ₂ WN-X ₃ -X ₄ -TWMEWER-X ₅ -IE-X ₆ -YTKLIY-X ₇ -IL-X ₈ -SQEX ₉ -β

Formula I

in:

α is selected from amino, acetyl, maleoyl, succinyl, tert-butoxycarbonyl, benzyloxycarbonyl or fatty acyl;

_X1 is selected from any amino acid or vacancy in V, L, I, M;

_X2 is selected from any amino acid or vacancy in E, D, N;

_X3 is selected from any amino acid in E, D, N;

_X4 is selected from any amino acid in K, M, Q or L;

_X5 is selected from K or E;

_X6 is selected from any amino acid in N, E or D;

_X7 is selected from any amino acid in D, E, K or R;

_X is selected from any two identical or different amino acids in D, E, K or R;

_X9 is selected from Q or L;

β is selected from amido, carboxyl or carboxyl derivatives;

The other letters above represent the following amino acids:

D-aspartic acid; E-glutamic acid; I-isoleucine; K-lysine; L-leucine; M-methionine; N-asparagine; Q-glutamine; R- Arginine; S-serine; T-threonine; V-valine; W-tryptophan; Y-tyrosine.

One or more amino acids of the polypeptide of the present invention can be replaced with D-type amino acids, rare amino acids in nature or artificially modified amino acids to increase bioavailability and inhibitory activity. Among them, D-type amino acids refer to amino acids that are opposite to L-type amino acids that make up proteins; rare amino acids that exist in nature include uncommon amino acids that make up proteins and amino acids that do not make up proteins, such as: 5-hydroxylysine, methyl Histidine, γ-aminobutyric acid, homoserine, etc.; artificially modified amino acids refer to the common L-type amino acids that make up proteins after modification such as methylation and phosphorylation.

The polypeptide of the present invention can be linked with macromolecular carriers or polypeptides, and these carriers or polypeptides include but not limited to: proteins, polyethylene glycol, lipids and the like.

The polypeptide of the present invention includes its truncated parts and modifications of the truncated parts. The truncated portion of the above-mentioned polypeptides includes peptides containing 15-32 amino acids. Modifications of truncated parts refer to modifications using polyethylene glycol, chemical small molecules (such as maleimide) and proteins. The modification of the truncated part refers to the derivatives produced after one or some amino acids of the polypeptide are linked to polyethylene glycol, small chemical molecules (such as maleimide) and proteins.

The polypeptides of the present invention also include single and multiple amino acid insertions, substitutions and/or deletions added to the general formula above.

The polypeptide of the present invention also includes the above-mentioned polypeptide (general formula I) and its truncated multimers, that is, several (1-4) identical polypeptides through amino acids, such as lysine, cysteine, or other Molecules link together to form polymers.

Among the polypeptides of general formula I, the following 18 polypeptides are preferred, and their structures are as follows:

Polypeptide 1: NH ₂ -SEQ ID No: 1-CONH ₂

SEQ ID No: 1 = VEWNNKTWMEWERKIEEYTKLIYEILKKSQEQ

Polypeptide 2: NH ₂ -SEQ ID No: 2-CONH ₂

SEQ ID No: 2 = VEWNNMTWMEWERKIEEYTKLIYEILKKSQEQ

Polypeptide 3: NH ₂ -SEQ ID No: 3-CONH ₂

SEQ ID No: 3 = VEWNNMTWMEWEREIENYTKLIYKILEESQEQ

Polypeptide 4: Ac-SEQ ID No: 4-CONH ₂

SEQ ID No: 4 = VEWNNMTWMEWEREIENYTKLIYKILEESQEQ

Polypeptide 5: Ac-SEQ ID No: 5-CONH ₂

SEQ ID No: 5＝VEWNNKTWMEWEREIENYTKLIYKILEESQEQ

Peptide 6: NH ₂ -NEKDLLEWMEWEREIENYTKLIYKILEESQEQ-CONH ₂

Peptide 7: NH ₂ -RINNIPWSEAMWMEWEREIENYTKLIYKILEESQEQ-CONH ₂

Peptide 8: NH ₂ -YDINYYTWMEWERKIEEYTKLIYEILKKSQEQ-CONH ₂

Peptide 9: NH ₂ -(CEKNEQELLWMEWEREIENYTKLIYKILEESQEQCONH ₂ ) ₂

Peptide 10: NH2-SEQ ID No: 6-CO NH ₂

SEQ ID No: 6 = WNEMTWMEWEREIENYTKLIYKILEESQEQ

Peptide 11: Ac-SEQ ID No: 7-CO NH ₂

SEQ ID No: 7 = WNEMTWMEWEREIENYTKLIYKILEESQEQ

Polypeptide 12: NH ₂ -SEQ ID No: 8-CO NH ₂

SEQ ID No: 8 = VEWNEMTWMEWEREIENYTKLIYKILEESQEQ

Polypeptide 13: NH ₂ -SEQ ID No: 9-CO NH ₂

SEQ ID No: 9 = LEWNEMTWMEWEREIENYTKLIYKILEESQEQ

Polypeptide 14: NH ₂ -SEQ ID No: 10-CO NH ₂

SEQ ID No: 10 = LEWNEMTWMEWEREIENYTKLIYKILEESQEL

Peptide 15: NH ₂ -WNNMTWMEWEREIENYTKLIYKILEESQEQ-CO NH ₂

Peptide 16: NH ₂ -WNNMTWMEWEREIENYTKLIYKILEESQEL-CO NH ₂

Peptide 17: NH ₂ -VEWNNMTWMEWEREIENYTKLIYKILEESQEL-CO NH ₂

Peptide 18: NH ₂ -LEWNNMTWMEWEREIENYTKLIYKILEESQEL-CO NH ₂

Among the above-mentioned 18 polypeptides, polypeptides 1, 2, 3, 4, 5, 10, 11, 12, 13, and 14 are particularly preferred.

The sequence structure of the polypeptide provided by the invention is significantly different from T-20, C34, T-1249 and the like. The sequence of T-20 is: Ac-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-CONH ₂ (Wild, CT, et al, Proc.Natl.Acad.Sci.USA 1994, 91:9770-9774); the sequence of C34 is Ac-WMEWDREINNYTSLHISLIEESQNQQEKNEQELL-CONH2 (Lu , M., et al, J.Biomol.Struct.Dyn., 1997, 15:465-471); The sequence of T-1249 is WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SchneiderS.E.et al, J.Pept.Sci.2005, 11( 11): 744-753).

According to the action mechanism of HIV virus and the receptor molecule on the surface of the cell membrane during the membrane fusion process, the invention designs an active polypeptide that can combine with the virus surface glycoprotein gp41. The action site of the present invention is located at the hydrophobic "Cavity" domain and both ends of the trimeric NHR on the gp41 molecule, and can completely inhibit virus replication at a concentration as low as nanomolar.

The advantage of polypeptide provided by the present invention is:

1. Easy to synthesize

T-20 and T-1249 consist of 36 and 39 amino acids respectively, and are synthesized by segmented synthesis and then ligation. The anti-HIV fusion polypeptide provided by the invention may only consist of 30-32 amino acids, but still has higher activity than T-20 and T-1249, and is easy to synthesize.

2. The peptide sequence and action site are different from existing drugs

The polypeptide sequence of the present invention is different from T-20, T-1249 and other reported fusion polypeptide sequences (Jiang S., et al., Curr.Pharm.Des., 2002, 8(8):563-580), The action sites are also different. The reported polypeptides, such as T-20, C34, etc., mainly act on the NHR hydrophobic "Cavity" of gp41 and its N-terminal binding. end-joint.

3. Higher activity compared with natural peptides and existing drugs

We have synthesized the 25 peptides at the C-terminus of the polypeptide of the present invention, which still cannot inhibit the fusion of viruses and cells at 1 μM, which is consistent with the results reported in the literature that the natural sequence peptides similar to the 25 peptides at the C-terminus of the present invention have low activity in inhibiting fusion ( EC ₅₀ is 2.8 ± 1.4 μ M (Marc F, et al.Nature structural biology.1999, 6 (10): 953-960). We also synthesized the tetramer of the front end 12 amino acids of the present invention, and it inhibits viral fusion The IC ₅₀ is 2.6±0.1 μ M. The sequence peptide (SLEQIWNNMTWMQWDK) similar to the N-terminus of the present invention reported in the literature has no activity (Hovanessian A G., etal. Immunity, 2004, 21: 617-627).

The present invention focuses on the sequence combined with the "Cavity" domain on the trimeric NHR region of gp41 and the sequence combined at both ends when designing the peptide chain, and the sequence with weaker binding ability is removed. At the same time, hydrophilic amino acids are introduced to increase the water solubility of the present invention, and acidic or basic amino acids that can generate ionic bonds are introduced into the "I" and "I+4" positions in the polypeptide sequence, so that the designed sequence has a strong The ability to form α-helices to better bind to the above regions. Compared with the natural peptide, the activity of the polypeptide of the present invention obtained through the above design has been greatly improved (about 500 times), and the activity of polypeptides 3, 4, 10, etc. is higher than that of the existing polypeptide drug T-20 (see Example 4). Table 3), and the drug-resistant strain of T-20 is still effective (see embodiment 5).

4. Good water solubility

Due to the introduction of hydrophilic amino acids into the polypeptide molecules, the polypeptides of the invention are all easily soluble in water, and the improvement of water solubility facilitates the improvement of drug efficacy and the development of drug dosage forms.

The polypeptide of the present invention can not only inhibit the membrane fusion of HIV virus and cells, in fact, it also has an inhibitory effect on the entry of some enveloped viruses with a similar membrane fusion process, such as: human respiratory syncytial virus (RSV), hepatitis virus etc.

The preparation method of polypeptide of the present invention

The polypeptide of the present invention can be produced by various methods: such as solid-phase synthesis, liquid-phase synthesis, and expression by engineering bacteria. For example, similar to the synthetic route of T-20, it is first synthesized in segments on the resin, and then connected to two polypeptides in the liquid phase to form the polypeptide of the present invention; a DNA sequence that can express the polypeptide of the present invention is synthesized or extracted, and connected to a vector, and transfected into eukaryotic or prokaryotic cells, express the protein or polypeptide containing the polypeptide sequence provided by the present invention, and obtain the polypeptide of the present invention through extraction and purification.

Methods of using the polypeptides of the invention

The polypeptide of the present invention can be directly used for the treatment of HIV infection alone, and can also be used in combination with one or more anti-HIV drugs to achieve the purpose of improving the overall therapeutic effect. These anti-HIV drugs come from (but not limited to) one or more of reverse transcriptase inhibitors, protease inhibitors and entry and fusion inhibitors. The aforementioned reverse transcriptase inhibitors include: one or more of AZT, 3TC, ddI, ddT, d4T, Abacavir, Nevirapine, Efavirenz and Delavirdine. The above protease inhibitors include: one or more of Saquinavir mesylate, Idinavir, Ritonavir, Amprenavir, Kaletra and Nelfinavir mesylate. The above-mentioned entry and fusion inhibitors include: T-20, T-1249, C34, IQN37, 5-Helix, TAK-779, SCH-C and naturally extracted proteins and other polypeptides that have the function of inhibiting virus entry.

The medicine containing the polypeptide of the present invention or its truncated products, derivatives and compositions can be directly administered to patients, or mixed with appropriate carriers or excipients and then administered to patients, so as to achieve the purpose of treating HIV infection. The carrier materials here include: water-soluble carrier materials, such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.; insoluble carrier materials, such as ethyl cellulose, cholesterol stearate, etc.; enteric carrier materials, such as acetic acid Cellulose phthalate, carboxymethyl ethyl cellulose, and the like are preferably water-soluble materials. Using these materials, the following dosage forms can be made: tablets, suppositories, solutions, capsules, aerosols, effervescents and drops, etc., preferably solutions and aerosols.

Using the above-mentioned dosage forms, the anti-HIV drugs and their derivatives, truncates and analogs and compositions provided by the present invention can be: administered by injection, including intravenous injection, subcutaneous injection, intracavitary injection, etc.; respiratory tract administration, such as Nasal administration; mucosal administration, such as nasal cavity, acts locally or acts systemically through mucosal absorption; oral administration, such as rectal and vaginal administration, acts locally or acts systemically through absorption. The above-mentioned route of administration is preferably administration by injection.

Description of drawings:

Figure 1 shows the helical conformation of N36 and polypeptide 3 and the conformational change after the interaction between the two. In the figure, ▲ is the complex, ● is polypeptide 3, and ■ is N36;

Figure 2 shows the helical conformation of N36 and polypeptide 6 and the conformational change after the interaction between the two, ▲ is the complex, ● is polypeptide 6, and ■ is N36;

Figure 3 shows the helical conformation of N36 and polypeptide 7 and the conformational change after the interaction between the two, ▲ is the complex, ● is polypeptide 7, and ■ is N36;

Figure 4 shows the helical conformation of N36 and polypeptide 9 and the conformation change after the interaction between the two; ▲ is the complex, ● is polypeptide 9, and ■ is N36;

Fig. 5 is the HPLC analysis figure of polypeptide 3;

Figure 6 is a gel HPLC analysis of polypeptide 4 and its complex with N36, in which 1 is the complex, 2 is polypeptide 4, and 3 is N36;

7 is a gel HPLC analysis of polypeptide 6 and its complex with N36. In the figure, 1 is the complex, 2 is polypeptide 6, and 3 is N36.

Figure 8 shows the helical conformation of N36 and polypeptide 10 and the conformational change after the interaction between the two. In the figure, ▲ is the complex, ● is polypeptide 10, and ■ is N36;

Figure 9 shows the helical conformation of N36 and polypeptide 12 and the conformational change after the interaction between the two, ▲ is the complex, ● is polypeptide 12, and ■ is N36;

Figure 10 shows the helical conformation of N36 and polypeptide 13 and the conformational change after the interaction between the two, ▲ is the complex, ● is polypeptide 13, and ■ is N36.

Detailed ways:

Synthesis and purification of embodiment 1 polypeptide

The polypeptide provided by the present invention can be synthesized on the ABI 433 solid phase synthesizer of Applied Systems Biosystems in the United States, and the modification of the polypeptide is done manually. The amino acid used in this synthesis is protected with Fmoc (U.S. Advanced Chemtech company product), and the resin used is Rink resin (U.S. Advanced Chemtech company product). During synthesis, 1-hydroxybenzotriazole (HoBt) (product of Advanced Chemtech, USA) was dissolved in N-methylpyrrolidone (NMP) (PE company) as an activator, and dicyclohexylcarbodiimide (DCC) was used (Acros Company) was used as a coupling agent, and piperidine (Piperidine) (Shanghai Jier Biochemical) was used to remove the protecting group. Amino acids all have an L-type chemical structure, and they are sequentially coupled to the Rink resin.

The usage amount of Rink resin and the usage amount of Fmc protected amino acid are carried out by 1:5, and protected amino acid is as follows: Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu( OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met -OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu )-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH. Coupling of amino acids was carried out according to the protocol of the instrument.

The N-terminus was acetylated manually. Add peptide resin, N-methylpyrrolidone (NMP) (consumption is 8-15 times of peptide resin weight), acetic anhydride (use 0.5ml on 1g resin), diisopropylacetamide (DIEA) ( Use 0.7 ml on 1 g of resin, react at room temperature for 2 hours, filter, and wash the resin three times with dichloromethane, methanol and N-methylpyrrolidone (NMP).

The resin after above-mentioned synthesis is used lysate (composition: dimercaptothreitol (DTT) 5%, water (5%), trifluoroacetic acid (TFA) 88%, and triisopropylsilane (TIPS) 2%) ( The ratio of the amount of resin to the lysate is: 0.5-1.0 g resin/10 ml lysate) cracked for 2.5-3.0 hours, filtered, and the filtrate was evaporated with a rotary evaporator to remove most of the trifluoroacetic acid, and then carried out with pre-cooled anhydrous ether Precipitate, filter to obtain the initial peptide, dissolve the solid with dilute ammonia water, and freeze-dry the filtrate.

The above-mentioned lyophilized crude peptide was purified by reverse-phase HPLC. The purification column was a reverse-phase _C18 semi-preparative column (Zorbax, 300SB-C18, 9.4mm×25cm), and the gradient elution solution was acetonitrile containing different gradients (containing 0.1% TFA )/water (containing 0.1% TFA), the target peak was collected, most of the acetonitrile was removed by rotary evaporation, and the pure peptide was obtained by lyophilization. The molecular weight of each polypeptide was determined by matrix-assisted laser desorption ionization-of-flight mass spectrometry (MALDI-TOF-MS Reflex III, Micromass Company), and the results were consistent with the theoretically calculated values.

The specific steps for the synthesis of polypeptide 3 are as follows: Weigh 0.17g Rink resin (0.1mmol, substitution rate 0.6g/mmol), the amount of each Fmoc protected amino acid is 0.5mmol, and 1-hydroxybenzotriazole (HoBt) and dicyclohexyl Carbodiimide (DCC) (Acros company) is the condensing agent, and piperidine (Piperidine) (Shanghai Jier company) deprotecting agent, according to the operating instructions of the ABI 433 type solid-phase synthesizer of Applied Systems Biosystems in the United States, appropriately prolong the coupling time (60-90min) and deprotection reagent time (20-30min), synthesize peptide-resin. 0.52 g of peptide resin was obtained.

Take 0.52g of the above-mentioned peptide-resin, put it into 10ml lysate (composition: 0.5g dimercaptothreitol (DTT), 0.5ml water, 8.8ml trifluoroacetic acid (TFA), 0.2ml triisopropylsilane (TIPS) ), cracked for 3.0 hours, filtered with a G3 glass sand core funnel, and evaporated most of the filtrate to a residual liquid of about 2ml with a rotary evaporator. The initial peptide was obtained by funnel filtration, the solid was dissolved with 1% dilute ammonia water, and the filtrate was lyophilized to obtain 0.33 g of crude peptide with a purity of about 60%.

0.1 g of the crude peptide was purified by HPLC on a reversed-phase C ₁₈ semi-preparative column (Zorbax, 300SB-C18, 9.4 mm×25 cm). Mobile phase: A, acetonitrile (containing 0.1% TFA); B, water (containing 0.1% TFA). The elution gradient is: 1-5min 10-45%A; 5-30min, 45-65%A, flow rate 3ml/min, UV 214nm detection, 5mg each time. The target fractions were collected, most of the acetonitrile was removed by rotary evaporation, and 20 mg of pure peptide was obtained by lyophilization. The molecular weight determined by mass spectrometry was 4163.20Da (theoretical calculation value 4162.67Da).

The purity analysis of polypeptide 3 is shown in Figure 5, and the analysis conditions are as follows:

Chromatographic column: Kromasil C-18 column (Beijing Analytical Instrument Factory, 5 μm, 4.6×250 mm). Mobile phase: A, acetonitrile (containing 0.1% TFA); B, water (containing 0.1% TFA). The elution gradient is: 1-5min 10-35%A; 5-30min, 35-65%A, flow rate 1ml/min, UV 214nm detection

The HPLC retention times of each polypeptide are shown in Table 1.

The peak time of the HPLC analysis of table 1 9 peptides

Peptide number 1 2 3 4 5 6 7 8 9 Retention time (min) 16.36 16.86 18.71 19.24 18.45 17.34 17.21 17.12 15.98

Note: The elution conditions of peptides 6, 8, and 9 are the same as those of peptide 3, and the analysis conditions of the remaining 5 peptides are except the elution gradient (1-5min, 10-25%A; 5-32min, 25-80%A) and chromatographic column (Kromasil C-18 column, 5μm, 4.6×250mm) are the same.

The CD spectrum determination of embodiment 2 polypeptide secondary structure

1. Purpose of the experiment

By measuring the changes in the secondary structure (such as helical content) of the polypeptide provided by the invention and N36 (a sequence from the NHR region of HIV gp41, containing the action site of the polypeptide of the invention and T20, etc.) and their complexes To analyze the intermolecular interaction between the polypeptide of the present invention and N36. The stronger the interaction, the higher the activity, and vice versa.

2. Experimental instruments, reagents and methods

Instrument: JASCO J-715-150L type

Parameter selection: resolution 0.1nm, wave width 5.0nm, response time 4.0s, wavelength 200-250nm

Dissolving buffer: 50mM NaH ₂ PO ₄ (containing 1 50mM NaCl) pH=7.2

Peptide concentration: 10μM

The secondary structure determination of 18 polypeptides provided by the invention in aqueous solution is carried out according to the following method:

N36 and each polypeptide provided by the present invention were respectively dissolved in water to a concentration of 2.5 mM, then diluted to 10 μM with phosphate buffer, and the secondary structure was respectively determined under the above parameters. Then equal amounts of N36 and the present invention were mixed and then diluted with phosphate buffer to a concentration of 10 μM, incubated in a water bath at 37°C for 30 min, and the incubated mixture was taken to determine the secondary structure of the mixture under the above parameters.

3. Experimental results

Figures 1-8 respectively show the changes in the helical conformation after the interaction between N36 and polypeptides 3, 6, 7, 9, 10, 12, and 13 and the HPLC analysis charts of the above polypeptides.

A typical α-helix shows double negative peaks at 208nm and 222nm on the CD diagram. Figures 1-4 and Figures 8-10 show the helical conformation of N36 and the polypeptide of the present invention and the conformational changes after the interaction between the two. It can be seen from the figure that the polypeptide of the present invention interacts with N36 derived from the NHR region of HIV gp41, and the complex of the two forms an obvious negative peak at 220nm. The molar optical rotation [θ] of the complex is calculated according to the following formula:

$<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; \&theta;</span>}<span\; class="notranslate">\; ]</span><span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; \&theta;</span>}}{\mathrm{<span\; class="notranslate">\; 10</span>}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; no</span>}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; C</span>}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; L</span>}}$

Wherein: θ is the ellipticity value (in mdeg) measured on the circular dichroism spectrometer; n is the number of residues of the polypeptide, (the number of residues of the complex is 1/2 of the sum of the number of residues of the two peptides Calculation); C is the molar concentration of the polypeptide (the concentration of the complex is calculated as 10 μM); L is the optical path of the quartz cup, in centimeters.

4 Conclusion

Most of the polypeptides provided by the present invention have increased helix content after interacting with N36 in solution, indicating that these polypeptides interact with N36 and form complexes, and the helix formation tendency of the complexes increases.

Example 3 Gel HPLC detection of the binding effect of the polypeptide of the present invention and N36

1. Experimental purpose and principle

The purpose is to prove whether there is a strong interaction between the two by detecting whether the polypeptide of the present invention binds to N36.

In a gel column, substances are separated according to their molecular weights, and substances with larger molecular weights are eluted first. In this experiment, if the polypeptide forms a complex with N36, it will be eluted first after the molecular weight increases. If the complex cannot be formed, the mixture will form two peaks of equal height, corresponding to the polypeptide and N36 provided by the present invention respectively.

2. Experimental instruments, reagents and methods

Gel column: TSK-G3000SWxl, 5μm, 7.8mm×300mm (product of Japan TOSOH company)

Instrument: Bio-Rad 13507 pump, Bio-Dimension ^TM UV/VIS detector

Mobile phase: 50mM NaH ₂ PO ₄ (containing 150mM NaCl) pH=7.2

Gradient: 0.8ml/min isocratic elution

Detection wavelength: UV 214nm

Step: Dissolve N36 and polypeptide 4 of the present invention in deionized water at a concentration of 2.5 mM. Then they were diluted to 0.208mM with 50mM NaH ₂ PO ₄ (containing 150mM NaCl, pH=7.2), and 30μl of the diluted solutions were injected and analyzed respectively, and eluted according to the above conditions. Take 30 μl of each diluted solution, mix and incubate in a water bath at 37° C. for 30 min, take the entire mixture and load the sample, and the analysis conditions are the same as above.

3. Experimental results

In this test, the molecular weight increases after the polypeptide of the present invention and N36 form a complex, which is eluted first, and the monomer molecule eluted later. The first peak (t=12.2min) shown in Figure 6 is the elution peak of polypeptide 4 and its complex with N36; the first peak shown in Figure 7 is the elution peak of polypeptide 6 and its complex with N36 (t=12.50 min).

Table 2 The retention time of each polypeptide and the complex formed with N36

4 Conclusion

The polypeptide provided by the invention can form a multimer complex with N36 in phosphate buffer solution, indicating that there is a strong interaction between the two.

Example 4 The polypeptide of the present invention has an inhibitory effect on HIV virus

The multifunctional HIV entry inhibitor provided by the present invention has the effect of inhibiting the fusion of virus and cell membrane, which is determined by the following experiments. The cells and viruses used are from the NIH AIDS Research and Reference Reagent Program in the United States.

Experiment 1: Inhibition of HIV-1-mediated cell-to-cell fusion

Human lymphocyte H9 infected with HIV-1 IIIB virus was stained with fluorescent dye Calcein-AM (Molecular Probes, Inc., Eugene, OR), and then mixed with human lymphocyte MT-2 in a certain ratio and added to a 96-well plate , were added with 9 polypeptides of the present invention (see Table 3), cultured at 37° C. for 2 hours, and the cells without the polypeptides of the present invention were used as a control. Fusion and non-fusion cells were observed and counted under a fluorescent inverted microscope (Zeiss, Germany) with a ruler, and the fusion percentage and half inhibitory dose (IC ₅₀ ) were calculated using GraphPad Prism software (GraphPad Software Inc., San Diego, CA) , 90% inhibitory dose (IC ₉₀ ). (Jiang S, et al. Procedings of SPIE. 2000, 3926, 212-219. and Jiang S, et al. J. Virol. Meth. 1999, 80, 85-96.) The results are shown in Table 3.

Experiment 2: Virus neutralization

Suspend human lymphocyte MT-2 in RPMI-1640 medium (Gibco Laboratories, Grand Island, NY) containing 10% fetal bovine serum (Gibco Laboratories) at a cell density of 5×104 cells/ml, then plate, volume per well for 200 μl. Infect with HIV-1 IIIB virus at a concentration 100 times _TCID50 (half the infectious dose). The wells of the polypeptide of the present invention are serially diluted and added to the above-mentioned infected virus culture medium, and the wells without the polypeptide of the present invention are used as a control, and then cultured overnight. Replace with fresh medium the next day. After four days of cultivation at 37°C, 100 μl of the culture supernatant was collected, added to an equal volume of water containing 5% Triton X-100, and the content of p24 was detected by ELISA (Jiang, S, et al. J. Exp. Med. 1991, 174, 1557-1563).

The results are shown in Table 3, and CF and VN (p24) are the experimental results of experiment one and experiment two respectively.

Table 3 The inhibitory activity of 18 polypeptides to HIV virus

Explanation to Table 3:

1. Explanation of terms

CF: cell fusion/cell fusion; VN: virus neutralization test/viral neutralization;

2. The purity of the polypeptides: the polypeptides used in the cell fusion and neutralization experiments were all purified, except for polypeptide 4 (purity 70%), the purity was greater than 90%. In the test, T-20 was used as a control, the purity of which was greater than 95%.

3. Each of the above values is measured four times, and the average value is taken.

Example 5 The fusion inhibitory effect of the polypeptide of the present invention on T-20 drug-resistant HIV cell lines

The method is the same as Experiment 1 of Example 4, except that the clinically isolated T-20 resistant strain (92US657) is used to infect the cells, and the rest are the same.

Polypeptide 3 provided by the present invention can completely inhibit virus replication in cells at 1.0uM, while T-20 cannot inhibit virus replication at 3.2uM.

Conclusion: The activity of polypeptide 3 in inhibiting virus replication is higher than that of the control drug T-20.

<110> Institute of Bioengineering, Academy of Military Medical Sciences, Chinese People's Liberation Army

<120> Polypeptides for Inhibiting HIV Virus Fusion and Uses thereof

<160>10

<170>PatentIn version 3.1

<210>1

<211>32

<212>PRT

<213> Artificial sequence

<400>1

Val Glu Trp Asn Asn Lys Thr Trp Met Glu Trp Glu Arg Lys Ile Glu

1 5 10 15

Glu Tyr Thr Lys Leu Ile Tyr Glu Ile Leu Lys Lys Ser Gln Glu Gln

20 25 30

<210>2

<211>32

<212>PRT

<213> Artificial sequence

<400>2

Val Glu Trp Asn Asn Met Thr Trp Met Glu Trp Glu Arg Lys Ile Glu

1 5 10 15

Glu Tyr Thr Lys Leu Ile Tyr Glu Ile Leu Lys Lys Ser Gln Glu Gln

20 25 30

<210>3

<211>32

<212>PRT

<213> Artificial sequence

<400>3

Val Glu Trp Asn Asn Met Thr Trp Met Glu Trp Glu Arg Glu Ile Glu

1 5 10 15

Asn Tyr Thr Lys Leu Ile Tyr Lys Ile Leu Glu Glu Ser Gln Glu Gln

20 25 30

<210>4

<211>32

<212>PRT

<213> Artificial sequence

<400>4

Val Glu Trp Asn Asn Met Thr Trp Met Glu Trp Glu Arg Glu Ile Glu

1 5 10 15

Asn Tyr Thr Lys Leu Ile Tyr Lys Ile Leu Glu Glu Ser Gln Glu Gln

20 25 30

<210>5

<211>32

<212>PRT

<213> Artificial sequence

<400>5

Val Glu Trp Asn Asn Lys Thr Trp Met Glu Trp Glu Arg Glu Ile Glu

1 5 10 15

Asn Tyr Thr Lys Leu Ile Tyr Lys Ile Leu Glu Glu Ser Gln Glu Gln

20 25 30

<210>6

<211>30

<212>PRT

<213> Artificial sequence

<400>6

Trp Asn Glu Met Thr Trp Met Glu Trp Glu Arg Glu Ile Glu Asn Tyr

1 5 10 15

Thr Lys Leu Ile Tyr Lys Ile Leu Glu Glu Ser Gln Glu Gln

20 25 30

<210>7

<211>30

<212>PRT

<213>Artificial sequence<

400>7

Trp Asn Glu Met Thr Trp Met Glu Trp Glu Arg Glu Ile Glu Asn Tyr

1 5 10 15

Thr Lys Leu Ile Tyr Lys Ile Leu Glu Glu Ser Gln Glu Gln

20 25 30

<210>8

<211>32

<212>PRT

<213> Artificial sequence

<400>8

Val Glu Trp Asn Glu Met Thr Trp Met Glu Trp Glu Arg Glu Ile Glu

1 5 10 15

Asn Tyr Thr Lys Leu Ile Tyr Lys Ile Leu Glu Glu Ser Gln Glu Gln

20 25 30

<210>9

<211>32

<212>PRT

<213> Artificial sequence

<400>9

Leu Glu Trp Asn Glu Met Thr Trp Met Glu Trp Glu Arg Glu Ile Glu

1 5 10 15

Asn Tyr Thr Lys Leu Ile Tyr Lys Ile Leu Glu Glu Ser Gln Glu Gln

20 25 30

<210>10

<211>32

<212>PRT

<213> Artificial sequence

<400>10

Leu Glu Trp Asn Glu Met Thr Trp Met Glu Trp Glu Arg Glu Ile Glu

1 5 10 15

Asn Tyr Thr Lys Leu Ile Tyr Lys Ile Leu Glu Glu Ser Gln Glu Leu

20 25 30

Claims (9)

1. general formula I polypeptide:

α-X ₁-X ₂WN-X ₃-X ₄-TWMEWER-X ₅-IE-X ₆-YTKLIY-X ₇-IL-X ₈-SQEX ₉-β

General formula I

Wherein:

α is selected from amino, ethanoyl, maleoyl, succinyl, tertbutyloxycarbonyl, carbobenzoxy-(Cbz) or fatty acyl group;

X ₁Be selected from any one amino acid or vacancy among V, L, I, the M;

X ₂Be selected from any one amino acid or vacancy among E, D, the N;

X ₃Be selected from any one amino acid among E, D, the N;

X ₄Be selected from any one amino acid among K, M, Q or the L;

X ₅Be selected from K or E;

X ₆Be selected from any one amino acid among N, E or the D;

X ₇Be selected from any one amino acid among D, E, K or the R;

X ₈Be selected from any two identical or different amino acid among D, E, K or the R;

X ₉Be selected from Q or L;

β is selected from amide group, carboxyl;

The following amino acid of above-mentioned other letter representation:

The D-aspartic acid; E-L-glutamic acid; The I-Isoleucine; K-Methionin; The L-leucine; The M-methionine(Met); The N-l-asparagine; The Q-glutamine; The R-arginine; The S-Serine; The T-Threonine; The V-Xie Ansuan; The W-tryptophane; Y-tyrosine.

2. the described general formula I polypeptide of claim 1, wherein α is selected from amino or ethanoyl; β is selected from amide group.

3. the described general formula I polypeptide of claim 1, wherein X ₁Be V, L or vacancy; X ₂Be E or vacancy; X ₃Be E or N; X ₄Be selected from K or M; X ₅Be selected from K or E; X ₆Be selected from E or N; X ₇Be selected from E or K; X ₈Be selected from two identical or different amino acid among E and the K.

4. the described general formula I of claim 1 is following polypeptide:

Polypeptide 1:NH ₂-SEQ ID No:1-CONH ₂

Polypeptide 2:NH ₂-SEQ ID No:2-CONH ₂

Polypeptide 3:NH ₂-SEQ ID No:3-CONH ₂

Polypeptide 4:Ac-SEQ ID No:4-CONH ₂

Polypeptide 5:Ac-SEQ ID No:5-CONH ₂

Polypeptide 10:NH2-SEQ ID No:6-CO NH ₂

Polypeptide 11:Ac-SEQ ID No:7-CO NH ₂

Polypeptide 12:NH ₂-SEQ ID No:8-CO NH ₂

Polypeptide 13:NH ₂-SEQ ID No:9-CO NH ₂

Polypeptide 14:NH ₂-SEQ ID No:10-CO NH ₂

5. arbitrary described polypeptide suppresses purposes in the medicine that peplos merges in preparation in the claim 1～4.

6. purposes according to claim 5, described virus are HIV virus, human respiratory syncytial virus or hepatitis virus.

7. arbitrary described polypeptide is preparing the purposes for the treatment of in the HIV infection medicine in the claim 1～4.

8. one kind is suppressed the pharmaceutical composition that peplos merges, and it is characterized in that containing arbitrary described polypeptide in the claim 1～4.

9. the pharmaceutical composition that merges of the described inhibition peplos of claim 8, described virus is HIV virus, human respiratory syncytial virus or hepatitis virus.

Images