Anti-HIV peptide and modified anti-HIV peptide

A peptide having anti-HIV activity consists of an amino acid chain represented by Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cy s-Glu-Val-Glu-Asp-Gln-Lys-Glu. The Cys underlined in the above amino acid chain may be replaced by Phe, Ser, Cys.sup.Acm or Cys.sup.Bzl.

BACKGROUND OF THE INVENTION 
This invention relates to an anti-HIV (human immunodeficiency virus) 
peptide and a modified anti-HIV peptide. 
The anti-HIV peptide of the present invention functions to inhibit 
infection of HIV (human immunodeficiency virus) and may be applied to 
treatment or prevention of manifestation of acquired human 
immunodeficiency syndromes (AIDS). 
As a drug or medicament for treatment or prevention of manifestation of 
AIDS by HIV, inhibitors for reverse transcriptase owned intrinsically by 
HIV and necessitated for replication of the virus particles, are 
practically used. However, these inhibitors are not desirable because of 
the powerful toxicity thereof against the normal cells. Although proper 
vaccination may be thought of as a measure against the manifestation of 
the disease, difficulties are met in the evolution of a proper vaccine 
because the antigenicity of the protein covering the outer surface of the 
HIV may be changed easily by mutation and no reports have been made on the 
examples of success in the evolution of suitable vaccines. 
Thus, the evolution of drugs or medicaments which are more powerful and 
lower in toxicity is progessing briskly. For example, various medicaments 
have been proposed for preventing the infection by inhibiting the binding 
of HIV to the cells. 
A first one of such medicaments is an antibody capable of being bound to gp 
120 or gp 41 which is the protein covering the outer surface of HIV. Such 
antibody may be prepared in the form of an antiserum or as a monoclonal 
antibody by inoculating a suitable animal with the protein. However, since 
the antigenicity of the above mentioned protein is not necessarily 
constant, it is necessary to find an antibody against the amino acid 
sequence which is not subject to variation. Moreover, since the usually 
available antibody is derived from animals, the antibody itself exhibits 
immunogenicity with respect to a human so that the antibody cannot be used 
repeatedly. 
In the second place, it has been attempted to administer an antibody 
against the CD4 molecule, which is the HIV receptor on the cell, to 
thereby sheath the cell for exempting the cell from infection by HIV. 
Although the HIV may be prevented from being bound to the cell, the 
function of the normal cells is affected simultaneously. 
In the third place, it has been attempted to apply the CD4 molecule itself, 
which is the HIV receptor, to the treatment for obviating the problem in 
employing the antibody. It is generally recognized that the CD4 molecule, 
which is soluble, is effective to prevent propagation of infection by 
binding the gp 120 of HIV, while not interfering with the function of 
normal cells, such as that of macrophages, or the class II specific 
interaction among the T cells. This soluble CD4 molecule has already been 
prepared by application of a genetic engineering technique (Hussey, R.E. 
et al., Nature, 331, 78, 1988). 
However, the soluble CD4 molecule, when used actually as the anti-HIV drug, 
presents problems in connection with the shortness of the time period 
during which the efficacy of the drug in the blood is reduced to half, 
non-sustained efficacy and larger dosage. Thus, it has been felt necessary 
to improve the soluble CD4 molecule (Capon, D.J.et al., Nature, 337, 
525-531, 1989). 
In the fourth place, a method has been proposed which takes advantage of a 
region of the peptide of the CD4 molecule capable of being specifically 
bound with the gp 120 of HIV. This peptide is comprised only of a portion 
taking part in binding with gp 120 and may be bound with gp 120 in the 
same manner as is the soluble CD4 molecule so that the peptide is highly 
unlikely to be involved in any other unnecessary reactions and hence 
exhibits high specificity. Also, this peptide may be prepared easily as 
drugs in various ways, since it is prepared by chemical synthetic methods. 
As such chemically synthesized peptide, a peptide having 63 amino acid 
residues has been proposed, as in Hayashi, Y. et al., Archives of 
Virology, 105, 129-135, 1989. This peptide, however, is inconvenient since 
it cannot be mass-produced without difficulties by the peptide synthesis 
technique because of its longer amino acid chain length. Another peptide 
having 19 amino acid residues has also been proposed by Lisfson, J.D. et 
al., Science, 241, 712-716, 1988. Although shorter in amino acid chain 
length, this peptide is not satisfactory in anti-HIV activity. Thus, there 
is a strong demand for a peptide which has a shorter amino acid chain 
length and yet is superior in anti-HIV activity. 
SUMMARY OF THE INVENTION 
An object of this invention is to provide a shorter amino acid chain 
section of the amino acid chain of the CD4 molecule capable of inhibiting 
infection or propagation of HIV, that is an anti-HIV peptide and a 
modified anti-HIV peptide. 
In accordance with the present invention, there is provided a peptide 
having anti-HIV activity, the peptide consisting of an amino acid chain 
represented by 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cys 
-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu. 
In accordance with the present invention, there is also provided a peptide 
having anti-HIV activity, the peptide consisting of an amino acid chain 
represented by 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Phe 
-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu. 
In accordance with the present invention, there is also provided a peptide 
having anti-HIV activity, the peptide consisting of an amino acid chain 
represented by 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Ser 
-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu. 
In accordance with the present invention, there is also provided a modified 
peptide having anti-HIV activity, the peptide consisting of an amino acid 
chain represented by 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cys 
Acm-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu 
wherein Acm represents an acetoamidomethyl group. 
In accordance with the present invention, there is also provided a modified 
peptide having anti-HIV activity, the peptide consisting of an amino acid 
chain represented by 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cys 
.sup.Bzl -Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu 
wherein Bzl represents a benzyl group. 
PREFERRED EMBODIMENTS OF THE INVENTION 
The present invention is explained in more detail hereinbelow. 
The present inventors have prepared, on the basis of the amino acid 
sequence determined by the DNA base sequence coding the CD4 molecule 
(Maddon, P.J.et. al., Cell, 47, 333 to 348, 1986), various partial 
peptides of the CD4 molecule and added the partial peptides to an HIV 
receptive cell strain MT-4 and to the HIV infected system to investigate 
into the anti-HIV activity, that is the activity inhibiting cell 
extinction or denaturation caused by HIV infection. As a result thereof, 
the present inventors have found that the following partial peptides 
represented by the sequences (1) to (5) below, which are the partial 
peptide covering 68th to 94th amino acids as counted from the N-terminal 
end and partial peptides obtained by partially modifying or changing the 
firstly mentioned partial peptides, exhibit a highly superior anti-HIV 
activity. Incidentally, each amino acid residue is shown by three letter 
abbreviation. 
(1) 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cy 
s-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu. 
(2) 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Ph 
e-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu. 
(3) 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Se 
r-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu. 
(4) 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cy 
s.sup.Acm -Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu (Acm: acetoamidomethyl group). 
(5) 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cy 
s.sup.Bzl -Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu (Bzl:benzyl group) 
In accordance with the present invention, the anti-HIV peptide or the 
modified anti-HIV peptides (1) to (5) above may be prepared by, for 
example, a first method, i.e. a solid-phase method employing an Fmoc amino 
acid (Sheppard, R.C. et. al., J. Chem. Soc., Chem. Comm., 165-166, 1985). 
Specifically, an Fmoc amino acid ester of pentafluorophenyl Pfp), 
corresponding to the C-terminal end of the peptide represented by any of 
the sequences (1) to (5) above, is bound to a high molecular carrier or 
substrate of, for example, p-alkoxybenzyl alcohol resin, in , for example, 
dimethylformamide, in the presence of 4-dimethylaminopyridine. Then, 
another Fmoc amino acid easter of Pfp to be bound next is then bound by 
condensation reaction to both the high molecular carrier and to the 
previously bound Fmoc amino acid Pfp ester. By the similar sequence of 
operations, the corresponding Fmoc amino acid Pfp esters are formed on the 
high molecular carrier. It is noted that threonine (Thr) and serine (Ser) 
are preferably introduced with the use of 
1-oxo-2-hydroxy-dihydro-benzotriazine (DHBT) ester. After termination of 
the coupling reaction of each amino acid, the high polymer carrier having 
the peptide bound thereto is treated with a 20 wt.% solution of piperidine 
in dimethylformamide for eliminating the Fmoc groups at the N-terminal 
ends. It is then acted upon by, for example, TFA-tioanisole at room 
temperature in the presence of n-cresole for eliminating the whole 
protective groups, while at the same time the desired partial peptides are 
recovered from the high polymer carrier and purified as an ultimate 
product. 
The second method for synthesizing the anti-HIV peptide and the modified 
anti-HIV peptide of the present invention is a solid phase method 
employing the Boc-amino acid according to Merrifield, J.Am. Chem. Soc., 
85, 2149 (1963). In this second method, when an amino acid side chain is 
modified as represented by the chains (4) and (5) above, a stable modified 
amino acid is introduced into the peptide under the condition of 
eliminating the protective groups. 
Besides the above mentioned methods of producing the partial peptides and 
modified partial peptides, the peptides may also be produced by other 
conventional chemical synthetic methods, methods of producing a DNA 
corresponding to the desired peptide and introducing the DNA into a 
suitable vector for production in animal cells or microorganisms to 
produce the desired partial peptide, or methods of chemically modifying 
the produced peptide in a suitable manner. 
The anti-HIV peptide or modified anti-HIV peptide according to the present 
invention may be produced more advantageously because the length of the 
amino acid chain thereof is less than half that of the conventional 
peptide as proposed in Hayashi, Y. et. al., Archives of Virology, 105, 
129-135, 1989, and hence the time and labor involved in the synthesis may 
be reduced to one fourth or less. With the amino acid chain length thus 
reduced, the antigenicity or toxicity such as inhibition of the function 
of the in vivo CD4 molecule may be suppressed to a minimum, while the 
excellent anti-HIV activity is demonstrated. On the other hand, the 
anti-HIV effect may also be augmented by modifying the side chain of a 
suitable amino acid residue or by replacing a specific amino acid residue 
by another amino acid.

EXAMPLES OF THE INVENTION 
The present invention will be explained in more detail with reference to 
several Examples. 
EXAMPLE 1 
For synthesizing the peptide consisting of the following amino acid chain : 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cys 
.sup.Acm -Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu (Acm: acetoamidomethyl group), 
the glutamic acid at the C terminal end of the peptide was introduced by 
the ester linkage on a p-alkoxy benzyl alcohol Resin (0.2 mmol) (0.35 meq 
OH/g, Polystyrene 1% Divinyl benzene Copolymer, manufactured by Kokusan 
Chemical Works, Ltd.) in dimethylformamide (DMF), using 1 mmol of its 
derivative Fmoc-Glu(Acm)-OPfp active ester and 0.2 mmol of DMAP 
(4-dimethyl aminopyridine) as a catalyst. 
The protective groups for the side chains of the Fmoc-amino acid 
derivatives (manufactured by Milligen Division of Millipore Ltd.) were 
Asp(OB.sub.u t), Glu(OBu.sup.t), Thr(Bu.sup.t), Ser(Bu.sup.t), 
Tyr(Bu.sup.t), Lys(Boc), His(Boc), Arg(Mtr) and Cys(Acn). Each 
condensation reaction for production of the peptide chain was performed, 
with the exception of that for threonine and serine, with the use of 2.5 
eq. of Pfp(pentafluorophenyl) active ester in DMF in the presence of 0.2 
mmol of HOBT(1-hydroxybenzotriazole). On the other hand, threonine and 
serine were introduced using DHBT(3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 
3-benzotriazine)ester. 
Each Fmoc group was removed by using a 20 wt.% piperidine solution in 
dimethylformamide, while each coupling reaction was monitored with 
ninhydrine. 
After the termination of all of the coupling reactions, the Fmoc groups 
remaining at the N-terminal end was eliminated with a 20 wt.% of 
piperidine in DMF to set the NH.sub.2 -group free. Then, for eliminating 
all of the protective groups and separating the peptide from the resin, 
the resin with the peptide affixed thereto was treated with 
TFA-thioanisole for three hours at room temperature in the presence of 
m-cresole and filtered through a glass filter to remove the resin. The 
filtrate was condensed at room temperature and ether was added thereto to 
produce powders, which were then recovered and dissolved in a formic acid 
ammonium buffer so as to be then eluted in 0.5N AcOH by "Sephadex G-25" 
(manufactured by Pharmacia). The eluted product was then desalted and 
purified so as to be then purified by HPLC. 
For fractionation by HPLC, the eluted product was caused to flow at a flow 
velocity of 1 ml per minute through a column of "Nucleosil 100 5c18" 
(4.0.times.150 mm)column at a concentration gradient of 10 to 60 % of 
acetonitrile in 0.1 wt.% TFA and monitored at 210 and 260 nm. The 
objective anti-HIV peptide of the present invention (partial peptide 68 to 
94) was eluted at a holding time of 16.8 minutes. A portion of the 
produced fraction was analysed by using "835S amino assist analyzer" 
manufactured by Hitachi Seisakusho KK and identified as the objective 
partial peptide. 
EXAMPLE 2 
For preparing a peptide consisting of the following amino acid chain : 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cys 
.sup.Bzl -Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu 
(Bzl:benzyl group), 0.05 mmol scal. of Boc - Glu(OBzl) -Pam resin, 
manufactured by Applied Biosystems Inc., was used as the starting material 
of glutamic acid which is the C-terminal end of the peptide. The peptide 
chain was subjected to sequential extension, using an automatic peptide 
synthesizer "model 430A" manufactured by Applied Biosystem Inc. (Program 
version 1.40). Side chain protection of the Boc amino acid derivative 
employed in the peptide chain extension (manufactured by Peptide 
Laboratories) was by Asp(cHex), Cys(Bzl), Glu(cHex), Lys(CIZ), Ser(Bzl), 
Thr(Bzl), Thr(Bzl) and Tyr(BrZ). Each condensation reaction for extending 
the peptide chain was performed in DMF by synthesizing 2.0 eq. of acid 
anhydrides of Boc-amino acids corresponding to the sequence in a CH.sub.2 
Cl.sub.2 (dichloromethane)-DMF liquid mixture using DCC 
(dicyclohexycarbodiimide) except aspartic acid and glutamic acid. The 
condensation reaction for aspartic acid and glutamic acid was performed by 
using an HOBt(1-hydroxybenzotriazole)ester for each of Boc-Asn and 
Boc-Gln. 
Each Boc group was removed by using a 50 wt.% TFA (trifluoroacetic acid) 
solution in CH.sub.2 Cl.sub.2. 
After the termination of all of the amino acid condensation reactions, the 
Boc group at the N-terminal end as well as the whole protective groups 
were removed by treatment in a 10 wt.% anisole/anhydrous HF (hydrogen 
fluoride) under ice cooling at -5.degree. C. for 30 minutes. 
Simultaneously, the peptide was separated from the resin and HF was 
distilled off in vacuum. The residues were washed with ether and the 
peptide was dissolved in TFA. After the resin was removed by a glass 
filter, the filtrate was concentrated in vacuum and dried ether was added 
to the residues to produce powders. 
The produced powders, which were the crude peptide product, were purified 
by reversed phase HPLC for fractionation in the same way as in Example 2. 
EXAMPLES 3 to 5 
The peptides consisting of the following amino acid chains were synthesized 
in the same way as in Example 1. 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cys 
-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu.(Example 3) 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Phe 
-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu.(Example 4) 
Asn-Phe-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Ser 
-Glu-Val-Glu-Asp-Gln-Lys-Glu-Glu.(Example 5) 
EXAMPLE 6 
A cultured solution of the MOLT-4 cells, which were already sustainedly 
infected by HIV-1(HTLV-IIIB), was used as the HIV solution and diluted in 
ten stages. Into these diluted cultured solutions was introduced at a rate 
of 1 mg/ml. each of the peptide produced in Example 3 and, as controls, a 
peptide consisting of 74th to 94th amino acids (abbreviated to control 
74-94), a peptide consisting of 86th to 104th amino acids (abbreviated to 
control 86-104), a peptide consisting of 86th to 107th amino acids 
(abbreviated to control 86-107) and a peptide consisting of 105th to 120th 
amino acids (abbreviated to control 105-120), which were synthesized in 
the same method as described in Example 1. Mixtures of the diluted 
cultured solutions and these peptides were maintained at room temperature 
for 30 minutes. These mixtures each were mixed with HIV-sensitive MT - 4 
cell at a rate of 1.times.10.sup.6 cells/ml and a vol/vol mixture ratio of 
1 : 1 so that the total volume was 0.2 ml. Each of the resulting mixtures 
was introduced into an RPMI - 1640-10% FCS culture liquid and maintained 
in a carbonic gas incubator at 37.degree. C. for four days. For measuring 
the anti-HIV activity of each partial peptide, the infectious index or 
HIV-1 titer was measured by an immunofluorescent method employing a 
monoclonal antibody against the gag protein p18 of HIV, and the HIV 
activity inhibit rate, that is the rate of lowering the HIV-1 titer by the 
above mentioned partial peptide containing diluted HIV solutions, with the 
partial peptide acting as the inhibitor, was measured in accordance with 
the following formula : 
100-(HIV-1 titer of inhibitor/ 
HIV-1 titer of control).times.100 
The results are shown in Table 1. 
TABLE 1 
______________________________________ 
Partial Peptides 
in Inhibitor HIV-1 Titer 
Inhibit Rate (%) 
______________________________________ 
(control) 10.sup.3.5 0 
68-94(Ex. 3) 10.sup.1.5 99 
74-94 10.sup.2.5 90 
74-104 10.sup.2.5 90 
86-104 10.sup.2.5 90 
86-107 10.sup.2.5 90 
105-120 10.sup.3.5 0 
______________________________________ 
It is noted that the HIV-1 titer represents the limit dilution ratio at 
which the diluted virus solution demonstrates the infectious potential. 
Thus, the higher the infectious potential, the higher becomes the 
infectious index. In other words, stronger anti-HIV activity is 
demonstrated when the infection is not caused with a virus solution 
thicker in concentration than the control. In the above table, the control 
is the virus solution which is free from various partial peptides acting 
as the above mentioned inhibitor. 
It is seen from Table 1 that the anti-HIV higher than the inhibit rate of 
99% was noticed with the partial peptide prepared in Example 3, that is 
the peptide consisting of the 68th to 94th amino acids counted from the 
N-terminal end of the CD4 molecule, and that only weak anti-HIV activity 
was noticed with the partial peptides further away from the N-terminal end 
than the partial peptide of Example 3. 
EXAMPLE 7 
The HIV-titers were measured in the same way as in Example 6 except that 
the partial peptides prepared in Examples 1 to 5 were used as the partial 
peptides constituting the inhibitors, and that the HIV-1 titer of the 
control was set to 10.sup.6.5, to find the inhibit rates. The results are 
shown in Table 2. 
TABLE 2 
______________________________________ 
Partial peptides 
in Inhibitor HIV-1 Titer 
Inhibit Rates (%) 
______________________________________ 
(control) 10.sup.6.5 0 
Peptide of Ex. 3 
10.sup.3.5 99.9 
Peptide of Ex. 1 
10.sup.3.5 99.9 
Peptide of Ex. 2 
10.sup.1.5 99.999 
Peptide of Ex. 4 
10.sup.3.5 99.9 
Peptide of Ex. 5 
10.sup.4.5 99 
______________________________________ 
It is seen from the results of Table 2 that both the anti-HIV peptides and 
the modified anti-HIV peptides of the present invention exhibit superior 
inhibitive effects with respect to the HIV activity. 
Although the present invention has been described with reference to the 
specific examples, it should be understood that various modifications and 
variations can be easily made by those skilled in the art without 
departing from the spirit of the invention. Accordingly, the foregoing 
disclosure should be interpreted as illustrative only and is not to be 
interpreted in a limiting sense. The present invention is limited only by 
the scope of the following claims.