ANTIVIRAL PEPTIDES

Abstract: Peptides of a sequence which is similar or identical to the amino terminal sequence of the F1 chain of mumps virus. A variety of peptides are disclosed ranging from oligopeptides to dodecapeptides.

Description: DESCRIPTION ANTIVIRAL PEPTIDES Technical Field The present invention relates to synthetic oligo- and polypeptides, compositions containing same, and methods of use, including the treatment of viral infections. Background Art Generally speaking, antiviral therapy has proceed¬ ed along two separate but interrelated pathways. The first is the development of compounds that act by directly influencing the virus/host cell interaction as a means of aborting the infection. The second is more broadly directed at altering -the host's immune functions to prevent viral entry or enhance viral clearance at the level of the host organism. Both approaches have required identification of the specific viral agent in question and detailed understanding of the events of viral replication, in terms of molecular interactions at the cellular level and pathogenic mechanisms at the level of the entire host (see generally Server, A. C. and olinsky, J. S., Human Motor Neuron Diseases, edited by Lewis P. ■ Rowland (Raven Press, New York, 1982), the chapter entitled "Approaches to Antiviral Therapy, " at pages 519-546.) Among specific antiviral chemotherapeutic agents are drugs that interfere with virus adsorption to, penetration into, and uncoating in the host cell. Inhibition of these early steps in virus replication, which occur prior to utilization of a significant number of host processes, would be associated with limited host toxicity. Few drugs, however, have been shown to act at these steps in virus infection. Among these are included heparin, amantadine, and a number of oligopeptides. It has been suggested that a number of carboben- zoxyoligopeptides, which inhibit infection by some members of the herpes, orthomyxovirus, and para- myxovirus families may act by preventing virion penetration. Activity of these agents in culture was independently reported by several groups in 1968. See, for example. Miller, F. A. e_t al., Applied Micro¬ biology 16: 1489-1496 (1968); or Nicolaides, E.. et al.. Journal of Medicinal Chemistry 11; 74-79 (1968). Carbobenzoxy D-phenylalanine-L-phenylalanyl-nitro-L- arginine (SV-4814) is among the most effective of these agents, with the greatest activity shown against measles virus (Miller, F. " A. et al. , supra) . The site of action of this agent in inhibiting measles virus infection has been demonstrated (Norrby, E., Virology 4_4: 559-608 (1971)). SV-4814 has no effect on virus adsorption or virus-induced hemagglutination, yet it markedly prolongs the period during which adsorbed virions are susceptible to neutralization by extra¬ cellular antibody. It has been concluded therefore that the drug inhibits virion penetration, which, in the case of measles, is thought to occur by fusion of virion envelope with the host cell membrane. The activity of the drug is generally thought to derive from its sequence homology with the amino-terminus of the viral protein that mediates fusion (fusion, or F, protein). For example, Richardson, et al. (Virology 105; 205-222 (1980)), has demonstrated that the inhibition of paramyxovirus activity by SV-4814 and closely related oligopeptides was amino acid sequence-specific. For example, in the case of Sendai virus, the most effective inhibitor was the' oligo- peptide with the closest sequence homology to the amino-terminus'of the viral fusion protein. Although the ultimate value of these peptides 'as clinically useful antiviral agents still remains to be determined, preliminary studies indicate that these inhibitors of the prior art have limited toxicity in vitro and can be safely administered orally to labora¬ tory animals. Richardson, e_t al., supra, have also demonstrated that oligopeptides with activity against measles a-nd other ' paramyxoviruses and at least one orthomyxovirus (influenza A) can be synthesized, pointing- to a great potential for these agents. Among oligopeptides of relevance to those claimed in the present invention can be mentioned the fol¬ lowing: Cbz-D-Phe-L-Phe-Gly (where Cbz is the carbobenzoxy group), Cbz-L-Phe-L-Phe, Cbz-D-Phe-L- Phe-L-(N0 2 )Arg, Cbz-Gly-L-Phe-L-Phe, and Cbz-Gly-L- Phe-L-Phe-Gly (see Server and Wolinsky, supra, page 523). These oligopeptides are inhibitors of Sendai virus activity. Cbz-Phe-D-Ala has also been tested against measles and herpes viruses (see Nicolaides, Ξ. et al., supra) . It would be of great interest to extend the anti¬ viral peptide findings of the prior art to other viral species, including mumps virus. The fusion (F) protein of mumps virus is composed of two disulfide-linked glycopeptides, F, and F«, that are generated by proteolytic cleavage of a precursor (F ) glycopeptide chain (see, for example, Ri a, et al., Journal of Genetic " Virology 46; 501-505 (1980), or Merz, et aL , Ibid 64; 1457-1467 (1983)). Studies of the F, N-terminus of mumps virus have been hampered by the difficulty in obtaining sufficient quantities of purified protein. It appeared worthwhile, however, to initiate and carry out an investigation of mumps virus in order to provide further potentially clinically useful anti¬ viral peptides. DISCLOSURE, OF THE. INVENTION The invention relates to a peptide comprising the following sequence: X-NH-Ala-Gly-Y wherein X is D- or ; where Z is hydrogen, C-i-Cg acyl, aroyl or an amino-protecting group, such as benzyloxycarbonyl, t-butyloxycarbonyl, tosyl, trityl, phthaloyl, or trifluoroacetyl; Y is selected from the group consisting of Ile, lie-Ala, Ile-Ala-Ile, Ile-Ala-Ile-Gly, Ile-Ala-Ile-Gly-Ile, Ile-Ala-Ile-Gly-Ile-Ala, Ile-Ala-Ile-Gly-Ile-Ala-Ala, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr-Ala, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr-Ala- Ala, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr-Ala- Ala-Glu, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr-Ala- Ala-Glu-Val, and Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr-Ala- Ala-Glu-Val-Thr. The amino acid residue corresponding to the car¬ boxyl- terminus of any of the shown peptides can either be the true carboxyl terminus, or its salts, esters or amides, or it may be linked to a further amino acid residue or residues. In other words, the shown pep¬ tides may be part of longer oligo or polypeptides. The same is true of the amino end of the shown peptides. If either the shown N-terminus or the shown C-termi- nus, or both, are flanked by additional peptide sequences, then the overall polypeptide should, pref¬ erably, not be identical with the naturally occurring F. or F n mumps virus proteins. Preferably the C-terminus residue has the formula Y-CO-R 1 wherein Y is as indicated above, R 1 is OM or NR 2 R 3 , where M is hydrogen, a pharmaceutically acceptable salt, or a C,-C fi branched or unbranched alkyl ester; and R 2 and R3 are the same or different and selected from the group consisting of hydrogen, C-,-Cg branched or unbranched alkyl, or both R 2 and R 3 taken together with the nitrogen atom form a five or six membered heterocyclic ring. These peptides are useful as antiviral agents, by themselves or in combination, alone or in appropriate compositions. They are also useful as immunogens for the generation of antisera, for example in vaccination procedures in which case they may be mixed with or conjugated to appropriate carrier molecules or adjuvants. Another use for the peptides of the inven¬ tion is as antigen analogues in labelled form for competitive immunoassay procedures in the detection of a virus such as mumps virus. BRIEF DESCRIPTION OF DRAWINGS Figure 1 shows the immunoaffinity purification of the mumps virus F protein. Figure 2 shows a fluorogram for an SDS-PAGE under reducing conditions. The proteins of mumps virus are shown in lane V. The designations to the left of lane V used to label the polypeptide bands are as follows: HN: hemagglutinin neuraminidase; NP: nucleocapsid pro¬ tein; F,: F, polypeptide chain of the F protein; P: polymerase; M: membrane protein; F 2 : F 2 polypeptide chain of the F protein. Electrophoretic mobilities of protein standards of known molecular weights are indicated to the right of lane II. Standards used were myosin H chain (200.0 kDA); phosphorylase B (92.5 kDA); bovine serum albumin (68.0 kDA); ovalbumin (43.0 kDA); alpha-chymotrypsinogen (25.7 kDA); betalactoglo- bin (18.4 kDA); and cytochrome C (12.3 kDA). Figure 3 shows the isolation of the F, and F 2 polypeptide chains of the F protein of mumps virus by high performance size exclusion chromatograph . Figure 4 shows a fluorogram for an SDS-PAGE per¬ formed under reducing conditions for the fractions of Figure 3. Lane 0 shows the F protein of mumps virus prior to the separation of the F, and F 2 polypeptide chains. Aliquots from pools I and II are shown in lanes I and II, respectively. The electrophoretic mo¬ bilities of protein standards of known molecular weight (See Figure 2) are indicated to the right of lane II. Figure 5 shows a comparison of the N-terminal sequence of the F, polypeptide of the mumps virus with those of three other paramyxoviruses. The dashed lines indicate identity with the mumps virus sequence. Sequence data for SV5, Sendai virus and NDV are from Richardson, e_t al., (supra, 1980). BEST MODE FOR CARRYING OUT INVENTION The peptide sequences of the invention comprise a first amino acid residue (position 1) which is phenyl- alanine. This residue may be either in the D- or L-configuration, the D-configuration being preferred. The remaining amino acid residues are preferably in the L-configuration. The phenylalanine residue at position 1, furthermore, can be " either in the free amine form (that is, Z is hydrogen), or in acylated or protected amine form. c ι~ C 6 acyl groups such as acetyl, trifluoroacetyl, propionyl, butyryl or benzoyl are useful. The amino group of the phenylalanine res¬ idue may, however, also be protected by an appropriate amino-protecting group. Among the preferred protect¬ ing groups are benzyloxycarbonyl (Cbz), t-butyloxycar- bonyl, tosyl, trityl, phthalolyl, or trifluoroacetyl. The carboxy terminus residue of the sequence, which may be any of several as indicated above, is either in free acid form (wherein R is OH), or in ester, amide or pharmaceutically acceptable salt form. C,-C fi branched or unbranched alkyl esters are pre¬ ferred and C,-C 6 branched or unbranched alkyl amides, either primary or secondary, are also preferred. Alternatively, cyclic amides, such as piperidine and pyrrolidine can also be utilized. The esters or amides can also be utilized as intermediates or stable forms of the peptides of the invention. Readily known hydrolysis, either chemical or enzymatic, can then be utilized to generate the free acids or salts. Among the preferred pharmaceutically acceptable salts are alkaline and alkaline-earth metal salts such as lithium, sodium, potassium, rubidium, cesium, cal¬ cium, magnesium, manganese, barium, or other metals such as zinc, copper, and so forth. Other pharmaceu¬ tically acceptable salts include amine cations derived from primary, secondary or tertiary amines. Examples of primary amines are methylamine, dimethylamine, tri- methylamine, ethylamine, dibutylamine, triisopropyla- mine, N-methylhexylamine, decyla ine, allylamine, cyclopentylamine, dicyclohexylamine, benzylamine, dibenzylamine, alpha-phenylethylamine, beta-phenyl- ethylamine, ethylene diamine, diethylene triamine and the like. Aliphatic, cycloaliphatic and aryl-aliphatic amines containing up to and including about 18 carbon atoms as well as heterocyclic amines, such as piperi- dine, morpholine, pyrrolidine, piperazine, and lower alkyl derivatives thereof, such as 1-methylpiperidine, 4-ethylmorpholine, 1-isopropylpyrrolidine, 2-methyl- pyrrolidine, 1,4-dimethylpiperazine, as well as amines containing water solubilizing or hydrophilic groups such as mono-, di- and tri-ethanolamine, epinephrine, procaine, and the like, can also be used. Suitable pharmacologically acceptable quaternary ammonium cations are tetrametylammonium, tetraethylam- monium, benzyltriethylammoniurn, phenyltriethylam- monium, and the like. The peptides can be by themselves or as part of acid addition salts of such acids as hydrohalic acids, carboxylic- acids, and the like. Suitable hydrohalic acids include HC1, HBr, HF. Suitable carboxylic acids include acetic -acid, trifluoroacetic acid, benzoic acid, and the like. The peptides can be by themselves or be a part of longer sequences. In other words, the sequence cor¬ responding to these peptides, when part of longer pep¬ tides, are still part of the present invention, and should be so construed, especially if performing the same functions as- herein described. Among the preferred peptides of the invention are Cbz-D-Phe-L-Ala-L-Gly; Cbz-D-Phe-L-Ala-L-Gly-L-Ile; and Cbz-D-Phe-L-Ala-L-Gly-L-Ile-L-Ala. The peptides of the invention can be utilized as antiviral compounds. The antiviral activity is utilizable not only for mumps virus, but also for other kinds of viruses such as Sendai, SV-5, measles and the like. The latter viruses have substantial sequence homology in the N-terminus of their F, pro¬ teins with that of the mumps virus described in this invention. Another use for the peptides of the present inven¬ tion is as immunogens, either by themselves or prefer¬ ably in association, either covalent or non-covalent, with appropriate carriers and/or adjuvants. Standard carriers such as bovine serum albumin, human serum albumin, keyhole limpet hemocyanin, tetanus toxoid, diptheria toxoid, thyroglobulin, ovalbumin, and puri¬ fied protein derivative can be used and the peptides associated therewith. Upon inoculation into an appro¬ priate host, under appropriate protocols, the host develops antibodies thereto. The antibodies can then be utilized in various processes such as passive immunization, immunoaffinity purification, immunoassay techniques, and the like. Lymphocytes derived from -li¬ the host can be processed by known fusion techniques to produce appropriate hybridomas. The immunization using the peptides and/or carriers or adjuvants can also be used as a primary vaccination step, in which case, the peptides of the invention can form part of vaccine compositions. Yet another use for the peptides of the invention is as antigen analogues in immunoassay procedures. A peptide of the invention can be labelled as, for example, by covalent linking to an enzyme (for exam¬ ple, alkaline phosphatase, or horseradish peroxidase), a radiolabel (or example, 14C or 3H), or a fluorescent dye (fluoroscein) , and used in a competitive immuno¬ assay mode. Competition between the labelled peptide of the invention and mumps virus (or any other virus having a close homology relationship to the peptide) for the active site of a given antibody and eventual determination of the label gives a quantitative measure of the amount of virus present within a given sample. Immunoassays techniques are well known to those of skill in the art. The peptides can also be insolubilized by attach¬ ment thereof to appropriate insoluble solid phases such as latex particles or beads, and used in agglu¬ tination techniques for the determination of antiviral antibodies in a sample. When used as antiviral agents, the peptides are formulated in appropriate carriers. They may be employed in dosage form such as tablets, capsules, powder packets or liquid solutions, suspensions or elixirs. Sterile liquid formulations such as solu- tions or suspensions can be prepared for parenteral use. In such compositions, the active ingredient will ordinarily always be present in an amount of 0.5% by weight based on the total weight of the composition and up to 99.9% by weight thereof. Administration can be by any means that promptly ensures their antiviral effect. The compounds, can be administered parenterally, subcutaneously, intraven¬ ously, intramuscularly, intraperitoneally, by trans¬ fusion or topically. The dosage administered will depend upon the age, health and weight of the recipient, kind of concurrent treatment if any, frequency of treatment, and the na'ture of the effect desired. Generally,- daily oral dosage of the active ingredient compounds will be from about 50 mg to 150 mg per kg of body weight. The peptides can be prepared by methods well known in the art of polypeptide synthesis (see, for example, Bodanszky, M., et al., "Peptide Synthesis (Second Edition)," G. A. Olah, Editor (John Wiley & Sons, New York, 1976) or Considine, "Chemical and Process Tech¬ nology Encyclopedia," European Patent Application 22-824. When synthesis is carried out in homogeneous phase, the peptide is obtained through the condensa¬ tion of an amino acid or peptide containing a free carboxyl group and an amino acid or peptide containing a. free amino group. A definite sequence can be con¬ structed by repeating condensation with individual amino acid residues following the sequence by stepwise elongation or in some case, by the condensation between two preformed peptide fragments. In such condensations, the amino and carboxy groups which are not to participate in the reaction must be blocked. Blocking groups should be readily introduced, stable to the condensation reaction and removed selectively from the accomplished peptide. Amino groups can be protected with a wide range of reagents. N-alkoxycarbonylation with benzyl or t-butyl chlorocarbonate is most widely used since the blocking group is readily decomposed by heating .with HBr in glacial acetic acid. Other examples for amino block¬ ing are propylation, tritylation, and phthaloylation or trifluoroacetylation. Carboxyl groups are pro¬ tected usually through formation of the ester. Once the peptide bond is formed, the ester linkage is hydrolyzed selectively in dilute alkaline solution at room temperature. Hydroxyl groups should also be adequately blocked during peptide condensation reac¬ tions. The blocking must be stable to unmasking of the alpha-amino or alpha-carboxy group for stepwise condensation and must be readily removed at .the final stage. Formation of the peptide bonds from amino and carboxyl components is accomplished by activation of the carboxyl components as acid halides, anhydrides, or esters. Peptide synthesis has been automated by use of solid phase supports (Merrifield) synthesis. Thus, both homogeneous and heterogeneous synthetic procedures can be used to prepare the peptides of the present invention. The final products can be purified by various chromatographic procedures such as silica gel, alumina gel, gel permeation, ion exchange, high performance liquid chromatography, hydrophobic chromatography, and the like. Another method of synthesis is by expression of a genetic sequence coding for any desired peptide of the invention. The genetic sequence may be present in an appropriate host such as a microorganism, i.e., a bac¬ terium or yeast, or in a tissue culture host. ' Genetic sequences coding for any desired peptide can be con¬ structed, integrated into an appropriate vehicle such as a plasmid or other vector, and transformed into the desired hosts. Having now generally described the invention, the same will become better understood by reference to certain specific examples, which are included for pur¬ poses of explanation only and are not intended to be limiting unless otherwise specified. I. DETERMINATION OF THE SEQUENCE OF THE AMINO TERMINUS OF THE MUMPS VIRUS Fl PROTEI A. MATERIALS AND METHODS Virus: The Kilham strain of mumps virus (Kilham, L., Journal of the American Medical Association 146: 1231-1232 (1951) ) was grown in CV-1 cells in the 3 presence of [ H]-leucine and was isolated as described in Server, et al. (Infection and Immunity 35: 179-186 (1982)). Immunoaffinity Chromatography: Monoclonal antibodies against the glycoproteins of mumps virus were gener¬ ated and characterized as described in Server, et al . , supra. The antibodies were precipitated from mouse ascites fluids with ammonium sulfate and coupled to an activated affinity support, Affigel-10 (Bio-Rad), according to the procedure of Staehelin, et al. , Journal of Biological Chemistry 256: 9750-9754 (1981). Immunoaffinity chromatography was performed using a modification of the method of Staehelin, et al. , supra. Purified mumps virus was disrupted in 0.025 M Tris HC1, pH 7.5 containing 0.15 M NaCl, 0.1% SDS, 1.0% (w/v) sodium deoxycholate, 1.0% (w/v) Triton X-100, 0.002 M phenylmethyl-sulfonyl fluoride and 100 Kallikrein Inhibitor Units/ml Aprotinin (Sigma) (Buffer A). The virus lysate was centrifuged at 95,000 x g for one hour, and the supernatant was applied to the immunoaffinity column (3 ml bed volume) at a flow rate of 10 ml/hr. Following application of the virus lysate, the column was washed with Buffer A (13 bed volumes), Buffer B (0.025M Tris HC1, pH 7.5 containing 0.5 M NaCl and 0.2% Triton X-100M26 bed.volumes) and Buffer C (0.15 M NaCl with 0.1% Triton X-100H10 bed volumes) at a flow rate of 30 ml/hr. Specifically bound virus glyσoprotein was eluted from the column with 0.2 N acetic acid, pH 2.5 containing 0.15 M NaCl and 0.1% Triton X-100 (Buffer D) at a flow rate of 10 ml/hr and precipitated from Buffer D: one volume of protein solution was mixed with 4 volumes of methanol: acetone (1:1 v/v), heated at 37°C for 2 min, and the protein precipitate pelleted by centrifugation at 10,000 x g for 30 min. Alkylation and Reduction; The mumps fusion protein was reduced with 4.7 mM dithiotreitol in 7 M guani- dine-HCl/0.5 M Tris-HCl, pH 8.2 containing 0.005 M EDTA at 37°C for 90 minutes. Excess iodoacetic acid was added, and the reaction was incubated for 1 hr at 37°C and stopped by addition of glacial acetic acid. High Performance Size Exclusion Chromatography; The mixture of the reduced and alkylated chains was lyophilized and redissolved in 7.5% trifluoroacetic acid/92.5% (v/v) elution buffer. The chains were separated on a Spherogel TSK-SW 3000 analytical column (Beckman) (0.75 x 30 cm) with an SW pre-column (Beckman) (0.75 x 10 cm) using a buffer of glacial acetic acid/propan-2-ol/water (20/15/65, v/v/v) (Brown, et al.. Analytical Biochemistry 112; 128-134 (1981)) at a flow rate of 0.5 ml/min with detection at 280 nm. Amino Acid Analysis; Amino acid compositions were determined using a Beckman 6300 analyser with Hewlett- Packard 3390A integrators after acid hydrolysis for samples in sealed, evacuated tubes (Pyrex, 6 x 50 mm) at 100°C for .24 hr in constant boiling HC1 (0.025 ml) (Pierce, Sequanal grade) (Moore, S., Chemistry and Biology of Peptides, Meienhofer, J., ed. (Ann Arbor Science, Ann Arbor, MI, 1972), pp. 629-653). Ninhy- drin and two channel (440 and 570 nm) integration pro¬ vided analysis of all amino acids, except tryptophan, cysteine, asparagine, glutamine, with 1-7% confidence values at 100 pmol/amino acid and with lower limit of detection of 25 pmol. Protein Sequence Analysis; Protein sequence analysis of the mumps fusion protein (F, ~) and the isolated F, and F 2 chains were determined with Applied Biosystems 470A protein sequencer. The sequence program (Hewick, R. M., e_t al., Journal of Biological Chemistry 256; 7990-7997 (1981)) contains one coupling step (44°C, 26 min), a single cleavage (44°C, 6.7 min), and automated conversion of the 2-anilino-5-thiazoliήone derivatives (Pth-amino acids) using 25% trifluoroacetic acid (50°C, 33 min). Polybrene (Tarr, G. E., et al. , Analytical Biochemistry 84; 622-627; Klapper, D. G., et al. , Analytical Biochemistry 85: 126-131 (1978)) was added to the glass filter disc, and five sequence cycles were run to reduce Polybrene-related contami¬ nants. The Pth-amino acids were identified by high performance liquid chromatography on IBM cyano columns, using a 15 mM sodium acetate buffer (pH 5.8) and a complex gradient of acetonitrile/methanol (92.5:7.5, v/v) (Hunkapiller, M.W. and Hood, L.E., Methods of Enzymology 91; 486-493) . SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE): SDS-PAGE was performed under reducing conditions, according to the procedure of Laemmli, U. K. , Nature (London) 227: 680-685 (1970). After electrophoresis, the gels were processed for fluorography by the method of Bonner, W.M. and Laskey, R.A., European Journal of Biochemistry 46: 83-88 (1974). Prestained protein standards (Bio-Rad) were used for molecular weight determinations. B. RESULTS Immunoaffinity Purification of Mumps Virus F Protein: Radiolabelled mumps virus was disrupted with detergent in Buffer A and used for the serial extraction of the virus glycoproteins (Figure 1). The HN glycoprotein was removed quantitatively by immunoaffinity chroma¬ tography using an anti-HN monoclonal antibody ' (Figure 2, lanes V,0). The effluent from the first column with the remaining mumps virus proteins was applied to a second immunoaffinity column containing an anti-F monoclonal antibody (Figures 1, 2). As shown in Figure 2, removal of the F protein from the virus pro-- tein mixture was incomplete (lanes 0, I), although residual F protein could be removed by reapplication of the effluent to the column. The F protein, elated from the column at acid pH (Figure 1, peak II), con¬ tained no detectable contamination by other radio- labeled virus proteins (Figure 2, lane II). Moreover, amino acid analysis of the eluent from the column prior to the application of virus proteins indicated that no appreciable antibody was lost from the immuno- affinity matrix during acid elution. The purified F protein was precipitated from the detergent-containing eluent with methanol/acetone prior to reduction and alkylation or sequence analysis. Purification of the F. and F 2 Chains: Purified F protein was reduced and alkylated under denaturing conditions. The sample was applied to a high performance size exclusion chromatography column equilibrated with acetic acid/propan-2-ol/water (20/15/65, v/v/v). Three peaks were detected by UV absorbance at 280 nm (Figure 3). The broad peak eluting in the inclusion volume (12-19 ml) was shown to contain guanidine-HCl by an analysis of the reduction and alkylation buffer in the absence of protein. Fractions from the other two peaks were pooled, and an aliquot from each pool was analyzed by SDS-PAGE. As illustrated in Figure 4, the F-, and F 2 chains were separated from one another: pool I contained F, (lane I) and pool II contained F 2 (lane II). Protein Sequence Analysis of the F-, and F 2 chains: The purified F, and F 2 peptide chains were sequenced by automated Edman degradation with a gas phase sequenator (Table I): T_J-le I Sequence Data for the F- ^ and F, Peptide Chains and for the F Protein a Cycle F l F 2 F protein Cyde 1 Phe 81 Val 797 Phe 180 Val 307 1 2 Ala 55 Asn 675 Ala 230 Asn 255 2 3 Gly 71 He 270 Gly 190 lie 257 3 4 lie 64 Asn 574 lie 226 Asn 191 4 ' 5 Ala 55 He 301 Ala 193 lie 301 5 6 lie 80 Leu 274 lie 272 L≤u 195 6 7 Gly 35 Gin 321 Gly 164 Gin 142 7 8 lie 48 Gin 380 lie 193 Gin 208 8 9 Ala 51 lie 428 Ala 168 He . 212 9 10 Ala 60 Gly 377 Ala 190 Gly 196 10 11 Leu 36 Tyr 395 Leu 163 Tyr 171 U 12 Gly 134 lie 203 Gly 140" He 57 12 13 Val 86 Lys ■ 75 Val 138 Lys 53 13 14 Ala 31 Gin 117 Ala 129 Gin 67 14 15 Thr 15 Gin 183 Thr 54 Gin 107 15 16 Ala 22 Val 187 Ala 96 Val 56 16 17 Ala 15 Arg 277 Ala 118 Arg 63 17 18 Gin 16 Gin 74 Gin 109 18 19 Val 28 Leu 128 Val 92 Leu 78 19 20 Thr 13 Ser 67 Thr 35 Ser 18 20 21 Ala 13 Tyr 91 Ala 71 Tyr 78 21 22 Ala 23 Tyr 134 Ala 91 Tyr 107 22 23 Val 9 Ser 25 Val 60 Ser 9 23 AL* 213 858 •453 RY C 95.3% 9 .3% 95. .3% 93 .2% Yields in piccmoles of 3-pr__rryl-2-thiohydantoin(Pth)-ai_ino acids plus phenylthiocar- ___τyl(Ptc)-e_nino acids, when present, minus background of corresponding amino acids. AL - amounts loaded based on amino acid analyses (this application) and molecular weight estimates (Harrier and Cααpans, Virology 47: 354-362 (1983). RY - repetitive yields calculated fran"yields of representative, stable, Pth-amino acids. Twenty three amino acid residues were identified for each chain in the following sequences: F.. NH 2 -Phe-Ala-Gly-Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu- 1 5 10 Gly-Val-Ala-Thr-Ala-Ala-Gln-Val-Thr-Ala-Ala-Val- 15 20 F 2 NH 2 -Val-Asn-Ile-Asn-Ile-Leu-Gln-Gln-Ile-Gly-Tyr- 1 5 10 Ile-Lys-Gln-Gln-Val-Arg-Gln-Leu-Ser-Tyr-Tyr-Ser- 15 20 An analysis of the intact F protein revealed a mixture sequence with two residues per degradative cycle,.cor¬ responding to the residues of the F, and F 2 chains (Table I) . II. SYNTHESIS OF PEPTIDES CORRESPONDING TO THE AMINO TERMINUS OF THE MUMPS VIRUS Fl PROTEIN Peptides of five and fourteen amino acids corres¬ ponding to the amino terminal sequence of the mumps virus Fl protein were synthesized with either a L-Phe or D-Phe at the amino terminus. Using standard solid phase methods (Barany, G. and Merrifield, R.B. (1980) In: "The Peptides" (Gross, E. and Meienhofer, J., Editors) Volume 2, Academic, New York; Rosenblatt, M. , Goltzman, D., eutmann, H.T., Tregear, G.W. , and Potts, J.T., Jr. (1976) Journal of Biological Chemis¬ try 251:159-164), the peptides were synthesized on solid supports of hydroxymethyl resin (Peninsula derivatized initially with Boc-Ala (0.15 to 0.4 meq alanine/g by coupling in the presence of dicyclohexyl- carbodii ide and dimethylaminopyridine in dimethyl- fprmamide (DMF). Deprotection of the amino terminal t-butyloxycarbonyl (Boc) protecting group was with 40% trifluoroacetic acid in dichloromethane (10 ml/g) for 30 min at ambient temperature. After washing with dichloromethane (3 x 10 ml/g resin), the neutraliza¬ tion was with 25% diisopropylethylamine in dichloro¬ methane (10 ml/g resin). After additional washing with dichloromethane (3 x 10 ml/g resin), the subse¬ quent Boc-amino acid (3 molar excess) was. coupled in DMF (10 ml/g resin) using dicyclohexylcarbodiimide (-3 ' .3. molar excess) for 2 hours at room temperature. The resin was washed first with DMF and then with dichloromethane (3 x 10 ml/g resin) to- remove the unreacted Boc-amino acid. The procedure was repeated for the addition of the next Boc-amino acid. After the N-terminal most amino acid was added, and the Boc protecting group was removed, the resin was washed with ethanol and dried in vacuo. The peptide was cleaved from the resin by HF (10 ml/g resin) with anisole (1 ml/g resin) at 0 C for 60 minutes (Sakakibara, S. (1971) in "Chemistry and Biology of Amino Acids, Peptides, and Proteins" (B. Weinstein, Editor), Volume 1, pages 51-85, Dekker, New York). After drying the resin _in vacuo, the residual anisole was extracted by diethyl ether, and the peptide was extracted from the resin by glacial acetic acid. The peptides were recovered by lyophilization of the glacial acetic acid diluted with distilled water (1:10, v/v). The N-terminus of the pentapeptides was blocked with benzyloxycarbonyl (Cbz) (Bergmann, M. and Zervas, L. (1932) Ber. Dtsch. Che . Gr. 65:1192-1201). Peptides were characterized by amino acid analysis and analytical reverse phase high performance ' chroma¬ tography. The sequence of selected peptides was confirmed by protein sequence analysis (Hewick, R.M., Hunkapiller, M.W., Hood, L.E., and Dryer, W.J. (1981) Journal of Biological Chemistry 256:7990-7997) . Sub-nanomole quantities of the F protein of mumps virus were obtained by immunoaffinity chromatography using an anti-F monoclonal antibody. The high degree of specificity afforded by the use of a monoclonal antibody allowed the isolation of purified F protein with no detectable contamination. This material was subjected to amino acid sequence analysis both before and after reduction and alkylation, and twenty-three amino acids from the N-terminus of each of the peptide chains of the F protein were identified. The N-term¬ inus of the F, chain of mumps virus shows a high degree of sequence homology with the corresponding regions of the F proteins of SV-5, Sendai virus, and NDV (Figure 5). Fourteen of the first twenty amino acid residues at the N-terminus of the F-, chains are conserved among the four viruses; amino acid substitu¬ tions, when they occur, are invariably at positions 2, 4, 5, 8, 9, and 19 in the sequence. Having now fully described the invention it will be appreciated that the same can be performed within a broad and equivalent range of parameters, structures and conditions without affecting the spirit or scope thereof of or any embodiment thereof.

Claims: CLAIMS: 1. A peptide comprising the following sequence: X-NH-Ala-Gly-Y wherein X is D- or L-Z-N CO- where Z is hydrogen, Cl, -C c o acyl, aroyl or an amino-protecting group; and Y is selected from the group consisting of He, He-Ala, Ile-Ala-Ile, Ile-Ala-Ile-Gly, Ile-Ala-Ile-Gly-Ile, Ile-Ala-Ile-Gly-Ile-Ala, Ile-Ala-Ile-Gly-Ile-Ala-Ala, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr-Ala, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr-Ala- Ala, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr-Ala- Ala-Glu, Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr-Ala- Ala-lu-Val, and Ile-Ala-Ile-Gly-Ile-Ala-Ala-Leu-Gly-Val-Ala-Thr-Ala- Ala-Glu-Val-Thr. 2. The peptide of Claim 1 wherein X is Z-D-NH-CH-CO- 3. The peptide of Claim 1'wherein X is Cbz-D-NH-CH-CO- 4. The peptide of any of Claims 1, 2 or 3 where¬ in Y is He. 5. The peptide of any of Claims 1, 2 or 3 where¬ in Y is He-Ala. 6. The peptide of any of Claims 1, 2 or 3 where¬ in Y is He-Ala-He. 7. The peptide of any of Claims 1, 2 or 3 where¬ in Y is He-Ala-He-Gly. 8. The peptide of Claim 1 wherein the C- termin¬ us thereof has the formula Y-CO-R 1 wherein Y is as indicated; R 1 is OM or NR 2 R 3 ; where M is hydrogen, a pharmaceutically acceptable salt, or a C,-C 8 branched or unbranched alkyl group; and R 2 and R3 are the same or different and selected from the group consisting of hydrogen, C-,-Cg branched or unbranched alkyl, or both R 2 and R-. taken together with the nitrogen atom form a five or six membered heterocyclic ring. 9. The peptide of Claim 8 wherein R is OM. 10. The peptide of Claim 9 wherein M is H or a pharmaceutically acceptable salt. 11. A compound of the formula Cbz-D-HN-CH-CO-NH-Ala-Gly-Ile-Ala-COOM I where M is hydrogen or a pharmaceutically accept¬ able salt. 12. A compound of the formula Cbz-D-HN-CH-CO-NH-Ala-Gly-Ile-Ala-He-COOM where M is hydrogen or a pharmaceutically accept¬ able salt. 13. A composition useful for antiviral treatment comprising an effective amount of a peptide according to Claim 1 together with a pharmacologically inert carrier. 14. A method of treatment for a viral infection in a subject, which comprises administering to said subject an antivirally effective amount of a peptide according to Claim 1.

Inventors: SERVER ALFRED, SERVER, ALFRED
Applicants: GEN HOSPITAL CORP, THE GENERAL HOSPITAL CORPORATION
Application year: 1985
Main classification: A61K37
IPC codes: A61K38, C07K5, C07K7, C07K14