WO2007068163A1

WO2007068163A1 - Hiv fusion inhibitor peptides and use thereof

Info

Publication number: WO2007068163A1
Application number: PCT/CN2006/001323
Authority: WO
Inventors: Qiuyun Dai; Jianwei Cheng; Yuxian He; Mingxin Dong; Junming He; Liankang Yu
Original assignee: Institute Of Biotechnology, The Academy Of Military Medical Sciences, Pla; Sichuan Kechuang Pharmaceutical Corporation Limited
Priority date: 2005-12-14
Filing date: 2006-06-14
Publication date: 2007-06-21
Also published as: CN1793170A

Abstract

Provided are a class of HIV virus fusion inhibitor peptides. Based on the mechanism of action of the HIV envelope with receptor molecules of cell membranes during fusion between viral and target cell membranes, active peptides which can bind the HIV envelope glycoprotein gp41 are designed. The action sites of the said peptides are located in the “long cavity” of the side of the trimeric NHR and two terminals of gp41, thereby efficiently inhibiting the copy of HIV. As compared to current drugs, the peptides have lower molecular weight, improved biological activity and better water-solubility. The HIV virus fusion inhibitor peptides can be used to produce compositions to inhibit infection of varied enveloped viruses.

Description

Polypeptide inhibiting HIV fusion and use thereof

TECHNICAL FIELD The present invention relates to a class of polypeptides that inhibit viral fusion, and more particularly to a class of polypeptides that inhibit HIV viral fusion. The invention also relates to the use of the polypeptide.

Background technique:

The life cycle of some enveloped viruses, such as Human Immunodeficiency Virus (HIV), Human Respiratory Syncytial Virus and Hepatitis B Virus, can be divided into four parts: virus and cell membrane fusion, genetic material entering the cell. Reverse transcription of viral DNA and integration into the chromosome of the host cell; transcription, translation, modification of viral proteins; assembly and budding of viral particles. What these viruses have in common is that there is a membrane fusion process in the life cycle, and the structures of the proteins involved in the fusion process are similar.

For example, in the treatment of HIV-infected drugs, the drugs currently used to treat HIV infection can be divided into three categories: reverse transcriptase inhibitors, protease inhibitors and entry inhibitors (including fusion inhibitors), respectively. Link (Jiang S., et al., Curr. Pharra. Des., 2002, 8 (8): 563-580).

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

HIV entry inhibitors inhibit the entry of HIV into cells, where fusion inhibitors act on viral and cell membrane fusion processes. Clinically, it has been found that the HIV virus fusion inhibitor alone can greatly reduce the viral load and has low side effects. HIV fusion inhibitors can also be combined with reverse transcriptase inhibitors and protease inhibitors to reduce the use of both drugs, thereby reducing side effects and preventing the emergence of new resistant strains.

The entry inhibitor acts on the stage before the virus enters the cell, ie from the beginning of the virus and cell contact to the membrane fusion. This phase consists of three main processes (Eckert DM, et al., Annu. Rev. Biochem. 2001, 70: 777-810): First, the surface glycoprotein gpl20 of the virus binds with high affinity to the cell surface receptor protein CD4 molecule. The conformation changes to allow the virus to adsorb to the host cell; then, gpl20 interacts with host cell surface co-receptors (eg, CCR5, CXCR4, etc.), the conformation is further altered, and is isolated from gp41, and the gp41 N-terminal fusion peptide is inserted into the host cell membrane. Finally, the CHR region of gp41 (C-terminal heptad repeats) folds back and is close to the α-helix trimer formed by its NHR region (N_terminal heptad reats), forming a hexamer α_helix, which brings the virus and The distance of the cells leads to the fusion of the viral envelope with the cell membrane.

The HIV entry inhibitors currently under study mainly target the viral surface glycoprotein gpl20/gp41 and the cell surface co-receptors CCR5/CXCR4 and CD4 receptors in the above process.

Entry inhibitors acting on gpl20 are (Thali M., et al., J. Virol. 1993, 67 (7): 3978-3988; Shaunak S., 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, PR0542, BMS-378806, 12P1. Entry inhibitors (fusion inhibitors) acting on gp41 (Wild CT, et al., Proc. Natl. Acad. Sci. USA. , 1994, 91 : 9770-9774 ; Jiang S., et al., Curr. Pharm. Des. , 2002, 8 (8) : 563-580 ; Root MJ , et al. , Science, 2001, 291: 884-888): DP107, T-20, T-1249, C34, IQN37, 5-Helix.

Entry inhibitors acting on the co-receptor CCR5/CXCR4 (Maeda K., et al., Curr. Opin. Pharmacol, 2004, 4(5): 447-452; Pierson TC, et al., Rev. Med. Virol 2004, 14: 255-270. ): AOP-RANTES, PR0140, UK- 427857, TAK-779, TAK-220.

Inhibitors of entry into the CD4 receptor are (Reimann K. A., et al., ADIS Res. Hum. Retroviruses., 1997, 12 (11): 933-943): TNX-355

Among the above-mentioned types of entry inhibitors, inhibitors acting on the co-receptor CCR5/CXCR4 are now considered to be inherently defective. It is well known that CCR5/CXCR4 is a molecule that exists on the surface of normal cells. In addition to being a co-receptor in the process of HIV virus entry, it is also a receptor for chemokines and inflammatory factors, so long-term competitive use of inhibitors can cause problems. Some small molecule antagonists have found side effects on the heart during clinical studies (Maeda Κ· , et al. , Curr. Opin. Pharmacol, 2004, 4 (5): 447-452).

Entry inhibitors that act on gpl20 are primarily antibodies that are screened or obtained from infected individuals with fewer small molecules. Currently, only 17b and 2G12 antibodies with broad neutralizing activity are obtained, and some of the other antibodies have neutralizing activity in vitro, but not in vivo. The current small molecule inhibitor is BMS-378806, which binds to a hydrophobic pocket on gpl20, and the small peptide (12P1) obtained by peptide library screening may have the same binding site. Inhibitors of entry into the CD4 receptor are currently the least studied because it is a very important molecule in vivo and its inhibition is likely to have serious side effects.

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

T-20 is derived from the CHR region of the HIV-1 LAI strain gp41 and consists of 36 amino acids (Wild, C. T., et al, Pro Natl. Acad. Sci. U. S. A 1994, 91: 9770-9774). The gp41 pre-fusion conformation can last for about 30 minutes, during which time T-20 can act on alpha-helix trimers formed by the NHR regions of three gp41 molecules (Melikyan GB, J. Cell. Biol., 2000, 151: 413-423). The competitive binding of T-20 prevents the CHR region of gp41 from binding to the NHR region, inhibiting the formation of helical hexamers, and thus the cell membranes of the virus and host are not close to each other, and the membrane fusion process is blocked.

Recent studies suggest that T-20 cannot form stable helical hexamers in solution with the NHR region polypeptide N36 derived from gp41, so it may interact with multiple sites of gp41 and gpl20 (Liu S., et Al., J. Biol. Chem, 2005, 280(12): 11259-11273), which is different from its design principle.

T-1249 is the second generation of T-20, consisting of 39 amino acids, derived from HIV-1 LAI, HIV-2 NIHZ, SIV mac251; ^CHR region (Schneider SE, et al, J. Pept. Sci. 2005, 11(11): 744-753). During clinical use, only one amino acid mutation in the CHR region of gp41 was found to cause resistance to T-20, but these strains are still sensitive to T-1249, which is thought to be possible to interact with the membrane. Capacity-related (Veiga AS, et al, J. Am. Chem. Soc., 2004, 126(45): 14758-14763).

Roche and its collaborators began researching TR-290999 and TR-291144 (13th CR0I Conference on Retroviruses and Opportunistic Infections Denver, Colorado, Feb 5-8, 2006) in 2004, each of which consisted of 38 The composition of 36 amino acids is said to be higher than T-20.

Although T-20, T-1249, TR-290999, and TR-291144 are currently the most effective peptide HIV fusion inhibitors, their common disadvantage is that molecular weight is larger than other anti-HIV drugs, making synthesis relatively difficult. .

Therefore, there is an urgent need for a polypeptide-inhibiting virus fusion agent having a shorter amino acid sequence for synthesis; in addition, the fusion agent needs to have a different action site than T-20 to fight against T-20-resistant virus; Solubility and stability to enhance efficacy and reduce drug use.

Summary of the invention:

It is an object of the present invention to provide a novel polypeptide which inhibits HIV viral fusion. The polypeptide has a short amino acid sequence, and the action site is different from the existing drug, and has good water solubility and high activity.

The design principle of the novel polypeptide provided by the invention

The surface glycoprotein of HIV-1 The NHR region of gp41 can be an ideal drug target because of its high degree of conservation, and the polypeptide derived from the CHR region can become a lead polypeptide of this drug because it can interact with the NHR region. The viral fusion inhibitors provided by the present invention are derived from a CHR sequence of gp41 of the RL42 strain of the B' subtype, which is the most widely spread and most infective in mainland China, and has the following amino acid sequence:

EIWNNMTWMEWEREIDNYTREIYTLIEESQNQ

Sequence analysis of the surface glycoprotein g _P 41 of HIV-1 revealed that there are two very conserved regions in the NHR region, one of which is "GIVQQQ" (Rimsky LT, et al, J. Virol., 1998, 72). : 986-993), any amino acid change in this region can cause T-20 resistance, and therefore is the main site of action of Τ-20; the other is Leu-565, Leu-566, Leu- 568, Thr -569, Val-570, Trp-571, Gly-572, lie- 573, Lys-574, Leu-576, Gin-577 and other 11 highly conserved amino acids consisting of a long hydrophobic domain "Cavity" (Eckert DM , et al, Cell, 1999, 99: 103).

In designing peptide inhibitors that bind to the NHR region, the inventors focused on sequences that bind to the "Cavity" domain on the NHR region of the trimer of gp41 and its ends. Previous inhibitors only considered CHR sequences. Tr _P 628, 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 weaker NHR binding ability to gp41, and introduced a hydrophilic amino acid to Increasing the water solubility of the inhibitor introduces an acidic or basic amino acid capable of generating an ionic bond at the "I" and "1+4" positions in the polypeptide sequence to increase the ability of itself to form a helix, which ultimately forms the present invention.

The present inventors have found that novel polypeptides of the general formula I have the effect of inhibiting viral fusion, and the primary structure of the polypeptide of the general formula I is as follows:

a -X _I -X ₂ WN-X ₃ -X ₄ -TWMEffER-X ₅ -IE-X ₆ -YTKLIY-X ₇ -IL-X ₈ -SQEX ₉ - β

Formula I

among them.

Selected from 'amino, acetyl, maleoyl, succinyl, tert-butoxycarbonyl, benzyloxycarbonyl or fatty acyl;

3⁄4 is selected from any one of amino acids or vacancies of V, L, I, M; X _{2 is} selected from any one of E, D, N amino acids or vacancies;

X ₃ is selected from E, D, N of any amino acid;

Any one of amino acids selected from K, M, Q or L;

X _{5 is} selected from hydrazine or hydrazine;

3⁄4 is selected from any one of Ν, Ε or D;

3⁄4 is selected from any one of D, E, K or R;

Any two identical or different amino acids selected from D, E, K or R;

Selected from Q or L;

β is selected from an amide group, a carboxyl group or a carboxyl group derivative;

The above other letters indicate the following amino acids -

D-aspartic acid; Ε-glutamic acid; I-isoleucine; Κ-lysine; L-leucine; Μ-methionine; Ν-asparagine; Q-glutamine; R- Arginine; S-serine; Τ-threonine; V-guanidine; W-tryptophan; Υ-tyrosine.

One or more amino acids of the polypeptide of the present invention may be replaced with an amino acid having a conformational D-form, a rare amino acid present in nature, or an artificially modified amino acid to increase bioavailability and enhance inhibitory activity. The amino acid of the D-form refers to an amino acid opposite to the amino acid constituting the L-form of the protein; rare amino acids present in nature include uncommon amino acids constituting the protein and amino acids not constituting the protein, such as: 5-hydroxylysine, methyl Histidine, Υ-aminobutyric acid, homoserine, etc.; Artificially modified amino acids refer to common L-type amino acids of constituent proteins modified by methylation, phosphorylation, and the like.

The polypeptides of the invention may be linked to macromolecule carriers or polypeptides including, but not limited to, proteins, polyethylene glycols, lipids and the like.

The polypeptide of the present invention includes a modification of its truncated portion and its truncated portion. The truncated portion of the above polypeptide is a peptide comprising 15-32 amino acids. The cut-off portion of the modifier refers to a modification using polyethylene glycol, a chemical small molecule such as maleimide, and a modification of the protein. A truncated portion of a modification refers to a derivative produced by the attachment of one or more amino acids of a polypeptide to polyethylene glycol, a chemical small molecule (e.g., maleimide), and a protein.

Polypeptides of the invention also include the above formula incorporating single and multiple amino acid inserts, substitutions and/or deletions.

The polypeptide of the present invention further includes the above polypeptide (Formula I) and its truncated polymer multimer, that is, several (1-4) identical polypeptides are passed through an amino acid such as lysine, cysteine, or the like. The molecules are joined together to form a multimer.

Among the polypeptides of the formula I, preferred are the following 18 polypeptides, the structures of which are as follows:

Peptide 1: NH ₂ - SEQ ID No : l-C0NH ₂

SEQ ID No : l= VEWNNKTWMEWERKIEEYTKLIYEILKKSQEQ

Polypeptide 2: N - SEQ ID No: 2- C0NH 2

SEQ ID No : 2=VEWNNMTWMEWERKIEEYTKLIYEILKKSQEQ

Peptide 3: NH ₂ - SEQ ID No : 3- C0NH ₂

SEQ ID No: 3 = VEWNNMTWMEWEREIENYTKLIYKILEESQEQ

Peptide 4: Ac- SEQ ID No : 4-C0NH ₂

SEQ ID No : 4=VEWNNMTWMEWEREIENYTKLIYKILEESQEQ

Peptide 5: Ac- SEQ ID No : 5-C0NH ₂

SEQ ID No: 5 = VEWNNKTWMEWEREIENYTKLIYKILEESQEQ

Peptide 6: NH ₂ -NEKDLLEWMEWEREIENYTKLIYKILEESQEQ-CONH ₂

Peptide 7 : NH ₂ -RINNIPWSEAMWMEWEREIENYTKLIYKILEESQEQ-CONH ₂

Peptide 8: NH ₂ - YDINYYT 丽 EWERKIEEYTKLIYEILKKSQEQ- C0NH ₂

Peptide 9: NH ₂ - ( CEKNEQELLWMEWEREIENYTKLIYKILEESQEQCONH ₂ ) ₂

Polypeptide 10: NH2- SEQ ID No: 6_C0 NH 2

SEQ ID No : 6= WNEMTWMEWEREIENYTKLIYKILEESQEQ

Peptide 11 : Ac- SEQ ID No : 7- CO NH ₂

SEQ ID No : 7=丽EMTWMEWEREIENYTKLIYKILEESQEQ Peptide 12: NH ₂ - SEQ ID No : 8 - CO NH ₂

SEQ ID No : 8= VEWNEMTWMEWEREIENYTKLIYKILEESQEQ

Peptide 13: NH ₂ - SEQ ID No : 9- CO N

SEQ ID No : 9= LEWNEMTWMEWEREIENYTKLIYKILEESQEQ

Peptide 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-C0 NH ₂

Peptide 18: NH ₂ -LEWNNMTWMEWEREIENYTKLIYKILEESQEL-C0 NH ₂

Among the above 18 polypeptides, polypeptides 1, 2, 3, 4, 5, 10, 11, 12, 13, 14 are particularly preferred. The polypeptide provided by the present invention is significantly different in sequence structure from T-20, C34, T-1249 and the like. The sequence of Τ-20 is: Ac- YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF- C0NH ₂ (Wild, CT, et al, Proc. Natl. Acad. Sci. US A 1994, 91 : 9770 - 9774); the sequence of C34 is Ac_WMEWDREINNYTSLHIS LIEESQNQQEKNEQELL-C0NH2 (Lu , M. , et al, J. Biomol. Struct. Dyn., 1997, 15: 465-471) ; The sequence of T-1249 is WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (Schneider SE et al, J. Pept. Sci. 2005, 11 (11) :744—753).

The present invention designs an active polypeptide which can bind to the viral surface glycoprotein gp41 according to the action mechanism of the HIV virus in the membrane fusion process and the receptor molecule on the cell membrane surface. The site of action of the present invention is located on the hydrophobic "Cavity" domain of the trimer NHR on the gp41 molecule and at both ends, and can completely inhibit viral replication down to nanomolar concentrations.

The advantages of the polypeptide provided by the present invention are -

1, easy to synthesize

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

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

The polypeptide sequences of the present invention differ from T-20, T-1249 and other reported fusion polypeptide sequences (Jiang S., et al., Curr. Pharm. Des., 2002, 8(8): 563-580), The site of action is also different. The reported polypeptides, such as T-20, C34, etc., mainly act on the hydrophobic "Cavity" of gp41 and its terminal binding. The peptides provided by the present invention and the hydrophobic "Cavity" domain of gp41 and two End combination.

3. Higher activity than natural peptides and existing drugs

We synthesized a 25-peptide of the C-terminus of the polypeptide of the present invention, which still failed to inhibit fusion of virus and cells in Ι μ ,, which is inferior to the inhibition of fusion activity by the native sequence peptide similar to the 25-peptide of the C-terminus reported in the literature. Consistent (EC ₅ . is 2. 8 ± 1. 4 μ Μ (Marc F, et al. Nature structural biology. 1999, 6 (10): 953-960). We also synthesized the front 12 amino acids of the present invention. Tetramer, which inhibits viral fusion, IC ₅ is 2. 6 ± 0.1 μμ. The sequence peptide (SLEQIWNNMTWMQWDK) similar to the terminal of the present invention reported in the literature has no activity detected (Hovanessian A G. , Et al. Immunity, 2004, 21: 617-627).

In the design of the peptide chain, the present invention mainly considers a sequence which binds to the sequence of the "Cavity" domain on the NHR region of the trimer of gp41 and the two ends thereof, and removes the sequence with weak binding ability. At the same time, a hydrophilic amino acid is introduced to increase the water solubility of the present invention, and an acidic or basic amino acid capable of generating an ionic bond is introduced at the "I" and "1+4" positions in the polypeptide sequence, so that the designed sequence has a strong The ability to form an alpha-helix to better bind to the above regions. The polypeptide of the present invention obtained by the above design has a much higher activity (about 500 times) than the activity of the natural peptide, wherein the activities of the polypeptides 3, 4, and 10 are higher than those of the existing polypeptide drug Τ-20 (see Example 4). Table 3), and the resistant strain of Τ-20 is still effective (see Example 5).

4, good water solubility

Since the hydrophilic amino acid is introduced into the polypeptide molecule, the polypeptide of the present invention is easily soluble in water, and the improvement of water solubility facilitates the improvement of the therapeutic effect of the drug and the development of the pharmaceutical dosage form.

The polypeptide of the present invention not only inhibits membrane fusion of HIV virus and cells, but also inhibits the entry of some enveloped viruses with similar membrane fusion processes into cells, such as: human respiratory syncytial virus (RSV), hepatitis Virus, etc. Method for preparing 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, engineering bacterial expression and the like. For example, similar to the synthetic route of T-20, the peptide is first synthesized on the resin, and then the polypeptide is linked in the liquid phase to form the polypeptide of the present invention; the DMA sequence which can express the polypeptide of the present invention is synthesized or extracted, and is linked to a certain The polypeptide of the present invention is obtained by transfecting into a cell of a eukaryotic or prokaryotic organism, expressing a protein or polypeptide containing the polypeptide sequence provided by the present invention, and extracting and purifying it.

Method of using the polypeptide of the present invention

The polypeptide of the present invention can be directly used for the treatment of HIV infection, or can be used in combination with one or more anti-HIV drugs for the purpose of improving the overall therapeutic effect. These anti-HIV drugs are derived from, but are not limited to, one or more of a reverse transcriptase inhibitor, a protease inhibitor, and an entry and fusion inhibitor. The above reverse transcriptase inhibitors include one or more of s AZT, 3TC, ddl, ddT, d4T, Abacavir, Nevirapine > Efavirenz 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. Polypeptides having a function of inhibiting viral entry.

The drug containing the polypeptide of the present invention or a truncated substance, derivative and composition thereof can be administered directly to a patient, or can be administered to a patient in combination with a suitable carrier or excipient to achieve the purpose of treating HIV infection. The carrier materials herein include: water-soluble carrier materials such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.; poorly soluble carrier materials such as ethyl cellulose, cholesterol stearate, etc.; enteric carrier materials, For example, cellulose acetate phthalate and carboxymethylethyl cellulose are preferred as water-soluble materials. Using these materials, the following dosage forms can be prepared: tablets, suppositories, solutions, capsules, aerosols, effervescent and drops, and the like, preferably solutions and aerosols.

Using the above dosage forms, the anti-HIV drugs and derivatives, truncates and analogs and compositions thereof provided by the present invention may be administered by injection, including intravenous injection, subcutaneous injection, intraluminal injection, etc.; Nasal administration; mucosal administration, such as nasal cavity, exerts systemic action on local or transmucosal absorption; intraluminal administration, such as transrectal and vaginal administration, local onset or absorption through the body. The above administration route is preferably administered by injection. BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 shows the conformation of the helical conformation of N36 and Peptide 3 and the interaction between the two. In the figure, A is a complex, Climbing is Peptide 3, and the country is 36;

Figure 2 is a schematic representation of the conformation of the helical conformation of N36 and polypeptide 6 and the interaction between the two, A is a complex, and the reference is a polypeptide 6, "is ^6;

Figure 3 is a diagram showing the conformation of the helical conformation of N36 and polypeptide 7 and the interaction between the two, A is a complex, and the reference is polypeptide 7, "" is ^6;

Figure 4 is a diagram showing the conformation of the helical conformation of N36 and polypeptide 9 and the interaction between the two; A is a complex, and the reference is polypeptide 9, which is ^6;

Figure 5 is a HPLC analysis diagram of polypeptide 3;

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

Figure 7 is a gel HPLC analysis of polypeptide 6 and its complex with N36, in which 1 is a complex, 2 is a polypeptide 6, and 3 is N36.

Figure 8 is a diagram showing the conformation of the helical conformation of N36 and polypeptide 10 and the interaction between the two. In the figure, A is a complex, ♦ is a polypeptide 10, and is a country;

Figure 9 is a diagram showing the conformation of the helical conformation of N36 and polypeptide 12 and the interaction between the two, A is a complex, and the reference is polypeptide 12, and the country is ^6;

Figure 10 is a diagram showing the conformation of the helical conformation of N36 and polypeptide 13 and the interaction between the two. A is a complex, and the reference is polypeptide 13, and || is ^6.

detailed description:

Example 1 Synthesis and purification of polypeptide

The polypeptide provided by the present invention can be synthesized on the ABI 433 solid phase synthesizer of Applied Biosystems, and the modification of the polypeptide is done manually. The amino acid used in the synthesis was protected by Fmoc (product of Advanced Chemtech, USA), and the resin used was Rink resin (product of Advanced Chemtech, USA). In the synthesis, 1-hydroxybenzotriazole (HoBt) (product of Advanced Chemtech, USA) was dissolved in N-methylpyrrolidone (匪P) (PE company) as an activator, using dicyclohexylcarbodiimide. (DCC) (Acros) as a coupling agent, using piperidine Piperidine (Shanghai Jill Biochemical) removes the protecting group. The amino acids all have an L-type chemical structure, which are sequentially coupled to the Rink resin.

The amount of Rink resin used and the amount of Fmc protected amino acid used are 1:5, and the protected amino acids are as follows: Fmoc-Ala-OH, Fraoc-Cys (Trt) -OH, Fmoc-As (OtBu) -OH, Fmoc-Glu ( 0tBu) _0H , Fraoc-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- Gin (Trt) -OH, Fmoc-Arg (Pbf) -OH , Fmoc-Ser (tBu) -OH , Fmoc- Thr (tBu) -OH , Fmoc-Val-OH , Fmoc-Trp (Boc) -OH, Fmoc- Tyr (tBu) - 0H. The coupling of the amino acids was carried out in accordance with the instrument operating procedures.

The N-terminus was acetylated by an artificial method. Peptide resin, N-methylpyrrolidone (NMP) (8-15 times the weight of the peptide resin), acetic anhydride (0.5 ml of lg resin), diisopropyl acetamide (diisopropylacetamide) was added to the reaction chamber. DIEA) (0.7 ml of lg resin), reacted at room temperature for 2 hours, filtered, and the resin was washed three times with dichloromethane, methanol and N-methylpyrrolidone (just P).

The above-mentioned synthesized lysate for resin (composition: bisthylthreitol (DTT) 5%, water (5%), trifluoroacetic acid (TFA) 88%, and triisopropylsilane (TIPS) 2%) ( The ratio of the amount of the resin to the lysate is: 0. 5- 1. 0 g of resin / 10 ml of lysate) cleavage 2. 5~3. 0 hours, filtered, and the filtrate is evaporated to remove most of the trifluoroacetic acid by a rotary evaporator. Precipitation was carried out with pre-cooled anhydrous diethyl ether, filtered to give a preliminary peptide, which was dissolved in dilute aqueous ammonia and the filtrate was lyophilized.

The above lyophilized crude peptide was purified by reverse-phase HPLC, and the purification column was a reverse phase C ₁₈ semi-preparative column (Zorbax, 300 SB-C18, 9. 4 awake x 25 cm), and the gradient eluent was acetonitrile with different gradients (including 0. 1% TFA) / water (containing 0.1% TFA), the target peak was collected, most of the acetonitrile was removed by rotary evaporation, and lyophilized to give a pure peptide. The molecular weight of each polypeptide was determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-T0F-MS Reflex III, Micromass), and the results were in agreement with theoretical calculations.

5摩尔ol, 1-hydroxybenzotriazole (HoBt), the amount of the Fmoc-protected amino acid is 0. 5ramol, 1-hydroxybenzotriazole (HoBt), the amount of the Fmoc-protected amino acid is 0. 5ramol, 1-hydroxybenzotriazole (HoBt) And dicyclohexylcarbodiimide (DCC) (Acros) as a condensing agent, piperidine (Shanghai Jill) deprotecting agent, according to the operating instructions of the American Applied Biosystems ABI 433 solid phase synthesizer The peptide-resin was synthesized by appropriately extending the coupling time (60-90 min) and the deprotecting reagent time (20-30 min). Peptide resin 0. 52 g.

5毫升三异。 Take the above-mentioned peptide-resin 0. 52g, into 10ml of lysate (composition: 0. 5g of decylthreitol (DTT), 0. 5ml of water, 8. 8ml of trifluoroacetic acid (TFA), 0. 2ml of three different Propyl silane (TIPS), cracked for 3.0 hours, filtered through a G3 glass core funnel, and most of the filtrate was evaporated to about 2 ml of residual liquid using a rotary evaporator, and then precipitated with 100 ml of pre-cooled anhydrous diethyl ether. After 1 hour, the G4 glass core funnel was filtered to obtain the initial peptide, and the solid was dissolved in 1% diluted aqueous ammonia, and the filtrate was lyophilized to obtain a crude peptide of 0.33 g, and the purity was about 60%.

The crude peptide was purified by HPLC, and the column was a reverse phase C ₁₈ semi-preparative column (Zorbax, 300 SB-C18, 9. 4 mm x 25 cra mobile phase: A, acetonitrile (containing 0.1% TFA); B, water (Including 0.1% TFA). The elution gradient is: 1- 5min 10-45% A; 5-30 min, 45-65% A, flow rate 3 ml / min, UV 214 nm detection, 5 mg per load The target fractions were collected, and most of the acetonitrile was removed by rotary evaporation, and lyophilized to give 20 mg of pure peptide. The molecular weight of the mass spectrometry was 4163. 20 Da (theoretical calculated value 4162. 67 Da).

The purity analysis of peptide 3 is shown in Figure 5, and the analytical conditions are as follows - column: Kromasil C- 18 column (Beijing Analytical Instrument Factory, 5 μ πι, 4. 6x250 匪). Mobile phase: hydrazine, acetonitrile (containing 0.1% TFA); B, water (containing 0.1% TFA). The elution gradient is: 1- 5min 10-35% A; 5-30 rain, 35-65% A, flow rate 1 ml I min, UV 214 nm detection

HPLC 1.

Note: More 6, 8, 9 more than 3, 5 (l-5min 10-25% A; 5-32 min, 25-80% A) and column (Kromasil C-18 column, 5 nm, 4 . 6x250 mm) Same outside.

Example 2 Determination of Secondary Structure of Polypeptide by CD Spectroscopy

Experimental purpose

By determining the change in the secondary structure (such as the helix content) of the polypeptide provided by the present invention and N36 (a sequence derived from the NHR region of HIV gp41, containing the polypeptide of the present invention and the site of action of T20, etc.) and their complexes To analyze the intermolecular interaction between the polypeptide of the present invention and N36. Strong interactions may also have higher activity, and vice versa. 2. Experimental instruments, reagents and methods

Instrument: JASCO J- 715- 150L

Parameter selection: Resolution 0. lnm, wave width 5. Onm, response time 4. 0s, wavelength 200-250 nm Lysis buffer: 50 mM Na3⁄4P0 ₄ (150 mM NaCl) pH=7. 2

Peptide concentration: 10 μ Μ

The determination of the secondary structure of the 18 polypeptides provided in the present invention in aqueous solution was carried out as follows:

Each of the polypeptides provided by Ν36 and the present invention was dissolved in water at a concentration of 2.5 mM, and then diluted to 10 μ3⁄41 in phosphate buffer to determine the secondary structure under the above parameters. Then, an equal amount of hydrazine 36 and the present invention were mixed, and then diluted to a respective concentration of 10 μΜ in a phosphate buffer solution, and incubated in a 37 ° C water bath for 30 minutes, and the mixture after the incubation was separately measured under the above parameters. Level structure.

3. Experimental results

Figure 1- ί shows the change in the helical conformation of N36 and the interaction of polypeptides 3, 6, 7, 9, 10, 12, and 13, respectively, and the HPLC analysis of the above polypeptide.

A typical α-helix shows a double negative peak at 208 nm and 222 nm on the CD map. Figures 1 to 4 and Figures 8 to 10 show changes in the conformation of N36 and the helical conformation of the polypeptide of the present invention and the interaction between the two. As can be seen from the figure, the polypeptide of the present invention interacts with N36 derived from the HIV gp41 NHR region, and the complex of the two forms a distinct negative peak at 220 nm. Molar optical rotation of the composite [ ^ Calculated as follows: lO x n x C x L

Where: Θ is the ellipticity value (in mdeg) measured on a circular dichroic chromatograph; n is the number of residues in the polypeptide, (the number of residues in the complex is 1/2 of the sum of the number of two peptide residues) Calculate); C is the molar concentration of the polypeptide (the concentration of the complex is calculated as ΙΟ μ Μ); L is the optical path of the quartz cup, in centimeters.

4 Conclusion

Most of the polypeptides provided by the present invention increase the amount of helix after interaction with hydrazine 36 in solution, indicating that these polypeptides interact with hydrazine 36 and form a complex, and the spiral formation tendency of the complex increases.

Example 3 Gel HPLC detection of the binding of the polypeptide of the present invention to Ν36

1. Purpose and principle of the experiment

The aim is to demonstrate whether there is a strong interaction between the two by detecting whether the polypeptide of the invention binds to Ν36. In the gel column, the substance is separated by the molecular weight, and the substance having a large molecular weight is first eluted. In this experiment, if the polypeptide forms a complex with Ν36, it will elute first when the molecular weight is increased. If the complex is not formed, the mixture will form two equal-high peaks corresponding to the polypeptide and oxime 36 provided by the present invention.

2. Experimental instruments, reagents and methods.

Gel column: TSK- G3000SWxl, 5 μ ηι, 7. 8 Wake up X 300mm (Japan T0S0H company) Instrument: Bio_Rad 13507 pump, Bio-DimensionTM UV/VIS detector

Mobile phase: 50 mM Na P0 ₄ (containing 150 mM NaCl) pH=7.22

Gradient: 0. 8 ml/min gradient elution

Detection wavelength: UV 214nm

5 raM。 The N, and the polypeptide was dissolved in deionized water, a concentration of 2. 5 raM. Then, diluted with 50 mM NaH ₂ P0 ₄ (containing 150 mM NaCl, pH = 7.2) to 0. 208 mM, and the diluted solution was each subjected to 30 μl injection analysis, and eluted according to the above conditions. Take 30 μl of each diluted solution, mix and incubate in a 37 ° C water bath for 30 min, and take all the mixture for loading. The analytical conditions are the same as above.

3. Experimental results

In this test, the polypeptide of the present invention and N36 form a complex, and the molecular weight is increased, first eluted, and then eluted as a monomer molecule. Figure 6 shows the first peak (t = 12. 2 min) which is the elution peak of polypeptide 4 and its complex with N36; the first peak shown in Figure 7 is peptide 6 and its complex with N36 De-peaking (t = 12.50 min).

Retention time of each polypeptide and complex formed with N36

4 Conclusion

The polypeptide provided by the invention can form a multimeric complex with N36 in phosphate buffer, indicating a strong interaction between the two. Example 4 Inhibition of HIV by the polypeptide of the present invention

The multifunctional HIV entry inhibitor provided by the present invention has an effect of inhibiting fusion of a virus and a cell membrane, and is determined by the following experiment. The cells and viruses used are from the US NIH AIDS Research and Reference Reagent Program.

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

HIV-1 prion-infected human lymphocytes H9 were stained with the fluorescent dye Calcein-AM (Molecular Probes, Inc., Eugene, OR) and then mixed with human lymphocyte MT-2 in a certain ratio and then applied to a 96-well plate. The 9 polypeptides of the present invention (see Table 3) were separately added and cultured at 37 ° C for 2 hours, using cells to which no polypeptide of the present invention was added as a control. Observation unfused and fused cells in the scale with a fluorescence inverted microscope (Zeiss, Germany) and counted using GraphPad Prism software (GraphPad Software Inc., San Diego, CA) and half the amount calculated percentage fusion inhibitor (IC _5. ), 90% inhibitor amount (IC ₉ .). (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

Human lymphocytes MT-2 were suspended in RPMI-1640 medium (Gibco Laboratories, Grand Island, NY) containing 10% fetal bovine serum (Gibco Laboratories) at a cell density of 5 x 104 cells/ml, and then plated at a volume of each well. 200 μ 1 . Take 100 times TCID ₅ . (half the infectious dose) Concentration of HIV-1 prions was infected. The polypeptide well of the present invention was added to the above-mentioned infected virus medium by gradient dilution, and the well without the polypeptide of the present invention was used as a control, followed by culturing overnight. The next day was changed to fresh medium. After 37 days of culture, the culture supernatant was collected 100 μl, an equal volume of water containing 5% Triton X-100 was added, 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. C F and VN (p24) are the experimental results of Experiment 1 and Experiment 2, respectively.

Inhibitory activity of 18 peptides against HIV virus

Description of Table 3:

1, noun explanation

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

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

3. Each of the above values is measured four times and averaged.

Example 5 Inhibition of fusion of a polypeptide of the present invention against a T-20 resistant HIV cell line

The procedure was the same as in Experiment 1 of Example 4, except that the clinically isolated T-20 resistant strain (92US657) was used to infect the cells, and the others were identical.

The polypeptide 3 provided by the present invention can completely inhibit the replication of the virus in cells at 1.0 μM, while the T-20 is

3. 2 uM still can not inhibit the replication of the virus.

Conclusion: The replication activity of peptide 3 inhibited the virus was higher than that of the control drug T-20.

Claims

Rights request

1. Formula I polypeptide:

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

Formula I

among them:

^ selected from amino, acetyl, maleoyl, succinyl, t-butoxycarbonyl, benzyloxycarbonyl or fatty acyl;

X, selected from any one of V, L, I, M amino acids or vacancies;

Any one of amino acids or vacancies selected from E, D, and N;

Any one of amino acids selected from E, D, and N;

Any one of amino acids selected from K, M, Q or L;

Selected from K or E;

3⁄4 is selected from any one of N, E or D;

X _{7 is} selected from any one of D, E, K or R;

Any two identical or different amino acids selected from D, E, K or R;

Selected from Q or L;

The other letters above represent the following amino acids:

D-aspartic acid; Ε-glutamic acid; I-isoleucine; Κ-lysine; L-leucine; Μ-methionine; Ν-asparagine; Q-glutamine; Arginine; S-serine; Τ-threonine; V-valine; W-tryptophan; Υ-tyrosine.

2. The polypeptide of formula I according to claim 1, wherein a is selected from the group consisting of an amino group or an acetyl group; and hydrazine is selected from the group consisting of an amide group.

3. The polypeptide of formula I according to claim 1, wherein is, L or a vacancy; 3⁄4 is E or a vacancy; is E or N; X _{4 is} selected from K or M; _{3⁄4 is} selected from K or E; X _{8 is} selected from E or N; X _{7 is} selected from E or K; two identical or different amino acids selected from E and K.

4. The formula I according to claim 1 is the following polypeptide: - polypeptide 1: NH ₂ - SEQ ID No: l-C0NH ₂

Peptide 2: NH ₂ - SEQ ID No : 2-C0NH ₂

Peptide 3: Leg SEQ ID No : 3-C0NH ₂

Peptide 4: Ac- SEQ ID No : 4-C0NH ₂

Peptide 5: Ac- SEQ ID No : 5-C0NH ₂

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

Peptide 11: Ac- SEQ ID No : 7-C0 NH ₂

Peptide 12: NH ₂ - SEQ ID No : 8-C0 NH ₂

Peptide 13: NH ₂ - SEQ ID No : 9-C0 NH ₂

Peptide 14: NH ₂ - SEQ ID No : 10- CO N3⁄4

5. Use of a polypeptide according to any one of claims 1, 2, 3 or 4 for the preparation of a medicament for inhibiting viral envelope fusion.

6. Use of a polypeptide of the general formula I for the preparation of a medicament for inhibiting viral envelope fusion according to claim 5, which is an HIV virus, a human respiratory syncytial virus or a hepatitis virus.

7. Use of a polypeptide according to any one of claims 1, 2, 3 or 4 for the manufacture of a medicament for the treatment of HIV infection.

A pharmaceutical composition for inhibiting viral envelope fusion, which comprises the polypeptide of any one of claims 1, 2, 3 or 4.

The pharmaceutical composition for inhibiting viral envelope fusion according to claim 8, wherein the virus is HIV virus, human respiratory syncytial virus or hepatitis virus.