WO1994024150A2 - Pepstatin derivatives for treatment, prevention and detection of infections - Google Patents

Abstract

The subject invention provides novel proteinase inhibitors that find use in both the isolation of proteins and the treatment and prevention of animal and plant diseases, particularly diseases stemming from fungal infections. Novel proteinase inhibitors of the subject invention are the polypeptides according to the formula (I): A-B-G-C-Sta-Ala-Sta-E-F, wherein A is absent or an amino acid blocking group; C is either absent or peptide consisting of 1-3 amino acids; B is an amino acid, E is either absent or selected from the group consisting of glycine and β-alanine; F is either absent or a 1-5 amino acid peptides composed of amino acids that are either positively charged, negatively charged, or have hydroxyl groups on side chains; and G is an amino acid. Preferred embodiments of the compounds of formula (I) are: (II) Iva-Val-Val-Sta-Ala-Sta-Bal-D-Asp-D-Asp; (III) Iva-Val-Val-Sta-Ala-Sta-D-Ser-D-Ser; (IV) D-Asp-Bal-Val-Val-Val-Sta-Ala-Sta-Bal-D-Asp; and (V) Iva-Dab-Val-Sta-Ala-Sta. Also described are methods for assaying for the presence of fungal proteinases. The invention also provides kits for the performing assays for fungal proteinases.

Description

PEPSTATIN DERIVATIVES FOR TREATMENT, PREVENΗON AND DETECTION OF INFECTIONS

Background Applications

This application is a continuation-in-part of U.S. patent application 08/050,916 filed April 21, 1993.

Field of the Invention

The present invention is in the field of pepstatin analog proteinase inhibitors, more particularly proteinase inhibitors for use in the treatment and prevention of infections in plants and animals.

BACKGROUND Fungi and yeast cause numerous topical and systemic infections in both man and animals. The severity of fungal diseases ranges from mildly irritating to life threatening. Furthermore, fungi are known to be the etiological agents responsible for a variety of plant diseases. Pathogenic fungi produce a wide variety of extracellular proteinases. Many of these proteinases have been shown to play an important role in the development of disease in both plants and animals, i.e., extracellular proteinases are a virulence factor for fungal infections, for example see: MacDonald et_. al. , J. Med. Microbiol. J. Am. Med. Assoc. 13:423-35 (1980) , MacDonald et al . , J. Med. Microbiol. J. Am. Med. Assoc. 243:2409-11 (1980), Borg et al. , Infect. Immun. 56:626-31 (1988) , Rav et al ■ , Infect. Immun. 56:1942-9 (1988) ,

Kaminishi et al . , Infect. Immun. 58:2139-43 (1990) , MacDonald et al. , J. Gen. Microbiol. 129:431-8 (1983) , and Kwong-Chung et_. al. , Infect. Immun. 49:571-5 (1985). Proteinases have also been shown to play important roles in the pathogenesis of other infectious diseases, including diseases caused by viruses, bacteria, protozoan, and multicellular parasites.

Numerous inhibitors of proteinases, both intracellular and extracellular proteinases, are known to exist. Proteinase inhibitors may be highly specific for individual proteinases or may inhibit a wide variety of proteinases belonging to the same or different classes. Pepstatins are a well known class of proteinase inhibitor. Pepstatins are inhibitors of the activity of the enzyme pepsin and related proteinases. Several different, but structurally related, pepstatins can be found in nature. Pepstatins include certain pentapeptides that may be isolated from the broths of cultures of certain Streptomyces species. One pepstatin, pepstatin A, has the structure: Iva-Val-Val-Sta-Ala-Sta, (where Iva stands for isovaleric acid, Val stands for valine, Sta stands for statine, and Ala stands for alanine) . Some peptides containing the amino acid statine (4-amino-3-hydroxy-6-methylheptanoic acid) have been shown to be proteinase inhibitors.

Proteinase inhibitors have found a variety of uses in medicine. These medical uses typically involve the inhibition of proteinase activity of proteinases naturally produced within the human body by human cells. For example, inhibitors of renin have been used to treat hypertension. The use of proteinase inhibitors to treat viral diseases has been described, e.g., Current Communications in Molecular Biology: Viral Proteinases as Targets for Chemotherapy, Cold Spring Harbor Press (1989) .

Despite the use of proteinase inhibitors for therapeutic purposes, proteinase inhibitors have found at most minimal use with respect to treating or preventing infectious disease, including fungal infections. For example, see U.S. Patent 5,120,718; Ray et_. al . Infect. Imm. 58:508 (1990) ; and Tsuobi et al. J. Invest. Dermatol . 85:438 (1985) . Furthermore, proteinase inhibitors have never been used to treat or prevent plant infections. It is therefore of interest to provide proteinase inhibitors suitable for use in the treatment and prevention of diseases caused by fungi and other infectious organisms. Proteinase inhibitors also have various non- medical and non-agricultural uses. These non- medical/non-agricultural uses of proteinase inhibitors are at least partially based upon the proteinase inhibiting properties of the proteinase inhibitors. For example, proteinase inhibitors such as phenylmethylsulfonyl fluoride (PMSF) , benzamide, aprotinin (trasylol) , pepstatin, and the like are frequently used in the preparation of purified proteins obtained from natural sources, see for example Methods in Enzymology Vol. 182: Guide to

Protein Purification. Ed. Deutscher, Academic Press, San Diego, CA (1990) .

Summary of the Invention

The subject invention provides various methods for treating or preventing infections by administering proteinase inhibitors. The subject invention also provides for various novel proteinase inhibitors and compositions containing these novel proteinase inhibitors. The compositions may be formulated for pharmaceutical or agricultural use. Of particular interest are methods and compositions for the treatment or prevention of fungal infections in both plants and animals. One aspect of the subject invention is to provide pharmaceutical compositions comprising proteinase inhibitors, formulated for either topical, oral, or parenteral administration. Another aspect of the invention is to provide novel proteinase inhibitors that are analogs of the naturally occurring proteinase inhibitor pepstatin. The subject invention provides for novel polypeptide proteinase inhibitors having the formula (I) A-B-G-C-Sta-Ala-Sta-E-F.

Formula I represents a polypeptide where A represents either an amino acid blocking group, the absence of an amino acid blocking group of the D form of an amino acid with an aromatic side-chain, e.g. D- Trp, d-Phe, D-Nap (Napthylalamine) ; C represents either the absence of an amino acid or a 1-3 amino acid peptide; B represents an amino acid, preferably valine or an amino acid having one or more primary amine groups attached to a straight or branched aliphatic hydrocarbon side chain; E represents either jβ-alanine (Bal) , glycine, or the absence of an amino acid residue; and F represents either the absence of amino acids or a 1-5 amino acid polypeptide composed of amino acids selected from the group of amino acids consisting of positively charged amino acids, negatively charged amino acids, amino acids having hydroxyl groups on side chains. G represents an amino acid with an aliphatic side chain. Of particular interest for use as proteinase inhibitors in compositions for the treatment and/or prevention of infectious disease are compounds of formula I where A is an amino acid blocking group selected from the group consisting of isovaleric acid, D-Phe, D-trp, and t-Boc; B is selected from the group consisting of aminoglycine, ornithine, lysine, 2,4,-diamino butinoic acid, and 2,4, -diaminopropanoic acid; F is either absent or selected from the group consisting of serine, threonine, lysine, glutamic acid, cysteic acid, tyrosine, aspartic acid, histidine, and arginine, D-Asp, D-Asp-D-Asp (a dipeptide) , and D-Ser-D-Ser (a dipeptide) ; and G is valine. Particularly preferred embodiments of the subject novel proteinase inhibitors are the polypeptides:

(II) Iva-Val-Val-Sta-Ala-Sta-Bal-D-Asp-D-Asp,

(III) Iva-Val-Val-Sta-Ala-Sta-D-Ser-D-Ser (IV) D-Asp-Bal-Val-Val-Val-Sta-Ala-Sta-Bal-D-Asp and

(V) Tva-Dab-Val-Sta-Ala-Sta.

Another aspect of the subject invention is to provide methods for treating or preventing infections by administering compositions containing proteinase inhibitors, particularly compounds II, III, IV or V. Another aspect of the subject invention is to provide methods for increasing the yield of a desired protein in a protein purification scheme by adding at least one of the novel proteinase inhibitors of the subject invention to prevent proteolytic digestion of the protein to be isolated.

Another aspect of the invention is to provide assay for the declaration fungal aspartic proteinases, and consequently fungi producing the proteinase, in which the assays employ the step of binding the fungal proteinase to one or more of the proteinase inhibitors of the invention. Another aspect of the invention is to provide kits for performing the subject methods of detecting fungal aspartic proteinases.

Description of the Specific Embodiments Definitions The term "amino acid" as used with reference to the polypeptide formulae described herein refers to amino acid residues produced after the formation of a peptide bond. The term amino acid refers to amino acids that occur naturally in proteins and naturally occurring amino acids that are not naturally found in proteins (but may be found in small peptides and as individual amino acids) . An extensive list of non- protein amino acids and their properties can be found in Chemistry and Biochemistry of the Amino Acids, section on the non-protein amino acids, by Hunt, S., Barrett, G.C. ed. , Chapman and Hall, London, 1985. Although the term "amino acid" as used herein with reference to the subject compounds, refers to both amino acids found naturally in proteins and non- protein amino acids, the preferred embodiments of the subject invention use amino acids naturally occurring in proteins, unless specifically indicated otherwise. Amino acids are the L-stereoisomer form, unless indicated otherwise, e.g. preceded by a "D" . The amino acid (amino acid residue) abbreviations used in the formulae (II-V) are as follows: Val = L-valine; Ala = L-alanine; Iva = isovaleric acid; Bal = beta-alanine (β- alanine) ; Sta = statine; Dab = 2,4 diaminobutinoic acid; D-Asp = D-aspartic acid; and D-Ser = D-serine.

The term "amino acid blocking group" as used herein, refers to compounds that can be used to block, i.e., chemically combine with a functional group so as to prevent unwanted reactions with that functional group, the free terminal primary amine group on amino acids . Exemplary of amino acid blocking groups are protecting groups used to block the terminal primary amine groups on amino acids during in vitro synthesis of polypeptides, e.g., t- Boc (N^α-tert-butyloxycarbonyl) . Isovaleric acid (abbreviated Iva) is also exemplary of amino acid blocking groups; the structure of isovaleric acid is that of valine without the terminal amine group present on valine.

The Invention

The subject invention provides methods and compositions for treatment of infections caused by infectious organisms that produce an extracellular proteinase as a virulence factor, i.e., the extracellular proteinase plays a role in the pathogenesis of the disease caused by the infectious organism. Infectious organisms that produce proteinases as virulence factors may be prokaryotic, eukaryotic, or viral. Infectious organisms producing infections that may be treated and/or prevented by the subject methods and compositions include bacteria, fungi (the term "fungi" as used herein includes yeasts) , protozoans, and multicellular parasites. Of particular interest are diseases caused by fungi, including the fungi Candida albicans, Candida tropicalis, Saccharomyces cerevisiae, Histoplasma capsulatum, Aspergillus spp., Coccidioides spp. , Paracoccidioides spp. , Blastomvces spp. , Sporothrix spp. , Mucor spp. , Petriellidium spp. , and Trichosporon spp. Some of the animal diseases that may be treated/prevented by the methods and compositions of the subject invention can be found in Table I. Similarly., some of the plant diseases that may be treated/prevented by the methods and compositions of the subject invention can be found in Table II.

TABLE I

MOST MEDICALLY SIGNIFICANT FUNGI THAT CAUSE DISEASE IN HUMANS¹

FUNGUS DISEASE² ORGAN(S)⁵

Aspergillus species, Aspergiilosis: Respiratory tract and any other primarily fumigatus Respiratory tract

Allergic aspergiilosis

(asthma) Lung, other

Aspergilloma Lung, debilitated tissue, other;

Invasive aspergiilosis systemic

Blastomyces Blastomycosis: Primarily, respiratory tract dermatitidis

North American blastomycosis, Respiratory tract

Chicago disease,

Gilchrist's disease Systemic

Candida albicans Candidiasis (Candidosis): Any

Thrush, Mouth

Vulvo-vaginitis, Vagina yeast infection,

Candida balanitis Penis

Dermato-candidiasis, Skin diaper rash

Bladder infection Urinary tract

Endocarditis Heart

Endophthalmitis, Eye chorioretinitis

Meningitis Central nervous system (CNS) Debilitated tissue Systemic

Coccidioides immitis Coccidioidomycosis: Numerous, e.g.,

San Jaoquin Valley fever Respiratory tract

Posada-Wernicke's disease Subcutaneous tissue

Cryptococcus neoformans Cryptococosis: Numerous, e.g., Malade signal:

Cryptomeningitis CNS

Busse-Buschke's disease Subcutaneous tissue

European blastomycosis Respiratory tract, bones, joints, heart, kidney, liver, breast, prostate

Epidermophyton species Tinea cruris and pedis; Skin, subcutaneous tissue Ringworm, Athlete's Foot

Fusarium species Hyalohyphomycosis Primarily debilitated tissue, but also systemic Histoplasma capsulatum Histoplasmosis: Numerous, e.g., Darling's disease, Respiratory tract Ohio Valley disease GI tract Systemic

Microsporum species Tinea corpotis, favosa; Skin, subcutaneous tissue ringworm:

Mucor species Zygomycosis, mucormycosis: Primarily, skin, lung, GI tract Parracoccidioides brasiliensis Paracociidioidomycosis; Primarily, subcutaneous tissue South American blastomycosis: Respiratory tract

Pheumocystis carinii Pneumocystis carinii Primarily, respiratory tract pneumocia (PCP):

Phinosporidium seeberii Phinosporidosis: Primarily, nose, but also larynx, eye, ear, penis, vagina, anus

Sporothrix schenckii Sporotricosis: Primarily, subcutaneous tissue, but also respiratory tract, CNS and GI tracts, bones, joints

Torulopsis glabrata Torolopsosis: Primarily, vagina, uterus, fallopian tubes, heart, lung, CNS

Trichophyton species Tinea corpotis, cruris, favosa, Skin, subcutaneous tissue imbricata, pedis, and unguium; ringworm

1. This is a list of the more medically significant fungi that cause disease in humans. These species also cause disease in domestic and wild animals, including birds.

2. The first disease name is the preferred medical term for the overall disease, with common or historic names following the semi-colon; the names following the colon refer to organ-specific diseases and are related tot he organ listed on the same line in the column to the right.

In most cases this is a partial list of susceptible organs.

TABLE II

AGRICULTURALLY IMPORTANT PLANTS AND RELATED FUNGAL DISEASES

PLANT FUNGUS DISEASE

Bananas Mycosphaerella musicola Sigatoka leaf spot disease

Cocoa Phytophthora palmivora black pod

Marasmlus periclosus witches brown disease

Coconut and Oil Palm Phytophthora palmivora butt rot

Coffee Hemileia vastatrix leaf rust

Colletotrichum coffea-πum berry disease Mycena citricolor American blight Penicularia koleroga thread blight

Cotton Corticium solani sore-shin Pythirsm species sore-shin *Fusarium oxysporum and/or wilt, sore-shin and boll rot other species: Verticilllum albo-atmm wilt *Alternaria species: boll rot *Asperigillus species: boll rot •Rhizopus species: boll rot •Penicillium species: boll rot

Fruits (Citrus) Phytophthora species brown rot Diaporthe citri melanose Guignardia citricarpa black spot ^♦Penicillium digitatum common green mold •Penicillium italicum blue contact mold

Rubber Fomes lignosus and F. noxius white and red root disease Ganoderma pseudoferreum red root rot Oidlum heveae powdery mildew Gloeosporium alborubrum abnormal leaf fall Phytophthora palmivora leaf fall disease

Sorghum Sphacelotheca species covered and loose smut Trichometasperia turcica leaf blight Sugar Cane marasmius sacchari rot Phytium arrhenomanes root rot Sclerospora sacchari downy mildew

Sugar beet Phythium species black leg Aphanomyces cochlioides black leg Pleospora bjoerlingi (Phoma betae) black leg Cercospora beticola leaf spot Tea Exobusidium vexans Blister blight

Ustulina deusta root rot

Armillaria mellea root rot

Corticium theae black rot

Tobacco Phytophthora nicotianae Black shank

Thielaviopsis basicola Black root rot

Erysiphe cichoracearum powdery mildew

Fruits (Deciduous) Venturia inaequalis apple scab

V. pirina pear scab

Podosphaera leucotricha apple mildew

Nectria galligena apple canker

Pezicula albo bitter rot

P. nalicorticis perennial canker and fruit rot

Taphrine deformans leaf curl

Sphlerotinia fructicola brown rot

Sphaerotheca pannosa peach mildew

Grapes (Berries) Botrytis cinera botrythis' Maize (Corn) Puccinia species rust

Trichometasphaeria turcica leaf blight

Cochliobolus heterostrophus leaf spot

Diplodia zeae dry rot seed decay

Gibberella species pink ear rot and seedling blight

Ustilago species, primarily smut maydis

Olives Cycloconium oleaginum leaf spot Potatoes Phytophthora infestans blight

Synchytrium endobioticum wart disease

Rice Pyricularia oryzae blast

Cochliobolus miyabeanus brown spot

Gibberella fujikuroi foot rot Vines Plasmopara viticola downey mildew

Unicinula nector powdery mildew

Guignardia bibwelli black rot

Elsinoe ampelina anthrocnose

Wheat, barley, oats, and rye Puccinia species black stem, yellow, brown and crown rust

Ustilago species covered and loose smut

Phyrenophora graminea leaf stripe

Legend

This information on fungi that cause disease in plants is taken primarily from Stapley and Grayner (1969) and Rippon (1988) and represents only a partial list. The fiingi and the diseases they cause are grouped according to host, presented in alphabetical order. An asterisk to the left indicates documented incidences of the fungus causing disease in humans as well.

1. Not listed in Stapley and Gayner (1969) or Rippon (1988). Botrytitis cinerea also grows on household fruits (Movahedi and Heale, 1990a and 1990b). The methods of infection treatment and/or prevention of the subject invention comprise the step of administering an effective amount of a composition comprising a proteinase inhibitor capable of inhibiting the proteolytic activity of one or more extracellular proteinase produced by the organism causing the infection to be treated and/or prevented. Infections capable of being treated by the methods and composition of the subject invention may be caused by organisms producing one or more proteinases as virulence factors.

The subject invention provides for various compositions for the treatment or prevention of infections, where the compositions comprise either previously known proteinase inhibitors or the novel proteinase inhibitors of the subject invention. For examples of known proteinase inhibitors see Handbook of Enzyme Inhibitors. H. Zollner, VCH Publishers, Inc. NY, NY (1989) . The subject compositions may comprise more than one proteinase inhibitor.

The subject invention provides for novel polypeptide proteinase inhibitors having the formula

(I) A-B-G-C-Sta-Ala-Sta-E-F.

Formula I represents a polypeptide displayed in the amino (left) to carboxyl (right) terminus direction. The hyphens, "-", in formula I represent peptide bonds between the constituent amino acids (or amino acid analogs) ; however, the hyphen representing the bond between A and the rest of the molecule may be other than a peptide linkage when A is an amino acid blocking group that is not capable of being used to form a peptide bond. A represents either an amino acid blocking group or the absence of an amino acid blocking group. C represents either the absence of an amino acid residue or a 1-3 amino acid peptide. B represents an amino acid, preferably valine or an amino acid having one or more amine groups attached to a straight or branched aliphatic hydrocarbon side chain. E represents either 3-alanine (Bal) , glycine, or the absence of an amino acid residue. F represents either the absence of amino acids or a 1-5 amino acid polypeptide composed of amino acids selected from the group of amino acids consisting of positively charged amino acids, negatively charged amino acids, amino acids having hydroxyl groups on side chains. G represents an amino acid with an aliphatic side chain.

Of particular interest for use as proteinase inhibitors in compositions for the treatment and/or prevention of infectious disease are compound of formula I where A is selected from the group consisting of isovaleric acid, D-Phe, D-Trp, and t- Boc; B is selected from the group consisting of aminoglycine, ornithine, lysine, 2,4,-diamino butinoic acid, and 2,4, -diaminopropanoic acid; F is either absent or selected from the group consisting of serine, threonine, lysine, glutamic acid, cysteic acid, tyrosine, aspartic acid, histidine, and arginine, D-Asp, D-Asp-D-Asp (a dipeptide) , and D- Ser-D-Ser (a dipeptide) ; and G is valine.

Particularly preferred embodiments of the proteinase inhibitor compounds of formula I are compounds having the formulae:

(II) Iva-Val-Val-Sta-Ala-Sta-Bal-D-Asp-D-Asp

(III) Iva-Val-Val-Sta-Ala-Sta-D-Ser-D-Ser

(IV) D-Asp-Bal-Val-Val-Val-Sta-Ala-Sta-Bal-D-Asp and

(V) Iva-Dab-Val-Sta-Ala-Sta.

The order of the amino acid residues is given in the amino terminus to carboxyl terminus orientation. The novel compounds of the subject invention may be synthesized by conventional solid phase in vitro peptide synthesis techniques. Reviews of these synthesis techniques can be found in, among other places, Synthetic Peptides: A Users Guide, by Gregory Grant, W.H. Freeman Publisher (1992) , and U.S. Patent 4,617,149. By using the appropriate amino acid and amino acid analog synthesis precursors at the appropriate stages of m vitro peptide synthesis, peptides containing the amino acid sequence of compounds I may be produced, as well as the specific compounds of formula II, III, IV and V and various derivatives thereof, may be produced.

Whereas many proteinase inhibitors are known to the person of average skill in the art, some of the novel compounds of the subject invention are of particular interest because of the inhibitory specificity they possess for fungal aspartic proteinases and/or their increased aqueous solubility. The specificity (or lack thereof) of a proteinase has important therapeutic implications because of the multitude of important physiological roles play by proteinases in the mammalian body, e.g. blood clotting, inflammation, blood pressure regulation, etc. Thus, non-specific proteinase inhibitors may have adverse physiological effects.

The non-specific inhibitory activity of a proteinase inhibitor would be less significant when compositions containing the proteinase inhibitor are administered so as to avoid interacting with susceptible physiologically important proteinases, e.g., external application of the proteinase inhibitor containing composition.

Compounds according to formula I are novel proteinase inhibitors. The subject compounds are structural analogs of the proteinase inhibitor pepstatin. Some embodiments of the compound of formula I are designed so as to inhibit fungal aspartic proteinases at lower concentrations than concentrations of pepstatin required for equivalent inhibition of the same fungal aspartic proteinases, i.e., fungal aspartic proteinase specific proteinase inhibitors. Embodiments of formula I that are fungal aspartic proteinase specific are those in which C, i.e., the position immediately to the N-terminus side of the N-terminus side statine, is an amino acid that has one or more amine group on a straight or branched aliphatic side chain, e.g., aminoglycine, ornithine, lysine, Dab (2,4, -diamino butinoic acid) , Dap (2,4,- diaminopropanoic acid) . The compound according to formula V is an example of a fungal aspartic proteinase specific proteinase inhibitor. Compound V and the related fungal aspartic proteinase specific proteinase inhibitors of the subject invention are significantly poorer inhibitors of mammalian aspartic proteinases such as pepsin and renin.

Embodiments of the compounds of formula I in which F is hydrophilic are of particular interest because of the increased aqueous solubility (relative to pepstatin) conferred by the presence of the hydrophilic amino acids. The compounds of formulae II, III, and IV, are examples of the subject proteinase inhibitors that have increase aqueous solubility. As inhibitors of fungal aspartic proteinases, compounds II, III, IV, and V are preferred proteinase inhibitors for inclusion in compositions for the treatment and/or prevention of fungal infections. The specific proteinase inhibitors to be used in the subject compositions and methods may be selected on the basis of their ability to inhibit the proteolytic activity of one or more extracellular proteinases secreted by the infectious organism responsible for causing the disease to be prevented or treated. Methods of testing the effectiveness of specific proteinase inhibitors against the proteolytic activity of specific proteinases are well known to the person of average skill in the art. For example, the effectiveness of a proteinase inhibitor against proteinases secreted by a specific fungus may be tested by adding a chromogenic proteinase substrate, e.g., azocoll (Chavira, et al . Anal . Biochem. 136:446 (1984)) or a chromophoric peptide (Dunn et al, Biochem. J. 237:899-906 (1986)) to media from a culture of a fungal pathogen and subsequently testing if the proteinase inhibitor of interest is capable of inhibiting the proteolytic activity of the proteinase of interest as detected by the decreased proteolytic activity acting on substrate in the assay.

Numerous model systems exist for studying the effects of therapeutic compounds on fungal (and other) infections in animals and plants. These models for studying the effects of therapeutics on infection are known to the person average skill in the art of the field of infectious disease (including plant infectious disease) . The models may be used to demonstrate that the compounds of the subject invention may be used to treat (or prevent) infections and to determine the dosages required to treat (or prevent) infections. Descriptions of suitable model systems can be found, for example in Tsuobi et. al . J. Invest. Dermatol . 85:438 (1985) ,

Robinson et_. al . J. Allergy Clin. Immunol. 86:726-731 (1990), MacDonald et al. , J. Gen. Microbiol. 129:431- 8 (1983), and Bistoni e_t al . , Inf. and Immun. 51: 668-674 (1983) , and the like. The subject compositions comprising proteinase inhibitors may be formulated as pharmaceutical compositions so as to be adapted for certain types of administration, e.g., oral, parenteral, or topical. The proteinase inhibitor containing composition is preferably administered at the site of infection.

Consequently, the preferred form of formulation for a given proteinase inhibitor is dependent on the location of the infectious organism in the host animal or the location in a host where a given infectious organism would be expected to initially invade. For example topical infections are preferably treated or prevented by formulations designed for topical application, whereas systemic infections are preferably treated or prevented by administration of compositions formulated for parenteral administration.

Proteinase inhibitor compounds for use in the subject methods, including compounds according to formula I of the subject invention may be administered topically, orally, intranasally, by injection or by inhalation in the form of a pharmaceutical compositions comprising a proteinase inhibitor in the form of the original compound or optionally in the form of a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier which may be a solid, semi-solid or liquid diluent or an ingestible capsule, and such preparations comprise a further aspect of the invention. The proteinase inhibitors may also be used with carrier material . As examples of pharmaceutical preparations may be mentioned tablets, drops such as nasal drops, preparations for topical application such as ointments, jellies, creams and suspensions, aerosols for inhalation, nasal spray, liposomes, etc. Usually the active substance will comprise between 0.05 and 99%, or between 0.1 and 99% by weight of the preparation, for example between 0.5 and 20% for preparations intended for injection and between 0.1 and 50% for preparations intended for oral administration.

To produce pharmaceutical preparations in this form of dosage units for oral application containing a compound of the invention the active ingredient may be mixed with a solid, pulverulent carrier, for example lactose, saccharose, sorbitol, mannitol, a starch such as potato starch, corn starch, amylopectin, laminaria powder or citrus pulp powder, a cellulose derivative or gelatine and also may include lubricants such as magnesium or calcium stearate or a Carbowax^® or other polyethylene' glycol waxes nd compressed to form tablets or cores for dragees. If dragees are required, the cores may be coated for example with concentrated sugar solutions which may contain gum arabic, talc and/or titanium dioxide, or alternatively with a film forming agent dissolved in easily volatile organic solvents or mixtures of organic solvents. Dyestuffs can be added to these coatings, for example, to distinguish between different contents of active substance. For the preparation of soft gelatine capsules consisting of gelatine and, for example, glycerol as a plasticizer, or similar closed capsules, the active substance may be admixed with a Carbowax^® or a suitable oil as e.g. sesame oil, olive oil, or arachis oil. Hard gelatine capsules may contain granulates of the active substance with solid, pulverulent carriers such as lactose, saccharose, sorbitol, mannitol, starches (for example) potato starch, corn starch or amylopectin) , cellulose derivatives or gelatine, and may also include magnesium stearate or stearic acid as lubricants. By using several layers of the active drug, separated by slowly dissolving coatings sustained release tablets are obtained. Another way of preparing sustained release tablets is to divide the dose of the active drug into granules with coatings of different thicknesses and compress the granules into tablets together with the carrier substance. The active substance can also be incorporated in slowly dissolving tablets made for instance of fat and wax substances or evenly distributed in a tablet of an insoluble substance such as a physiologically inert plastic substance.

In order to obtain dosage units of oral preparations -- tablets, capsules, etc. -- which are designed so as to prevent release of and possible decomposition of the active substance in the gastric juice, the tablets, dragees etc. may be enteric- coated, that is provided with a layer of gastric juice-resistant enteric film or coating having such properties that it is not dissolved at the acidic pH in the gastric juice. Thus, the active substance will not be released until the preparation reaches the intestines. As example of such known enteric coatings may be mentioned cellulose acetate phthalate, hydroxypropyl-methylcellulose phthalates such as those sold under the trade names HP 55 and HP 50, and Edragit^®L and Eudragit^®S.

Effervescent powders are prepared by mixing the active ingredient with non-toxic carbonates or hydrogen carbonates of e.g. sodium, potassium or calcium, such as calcium carbonate, potassium carbonate and potassium hydrogen carbonate, solid, non-toxic acids such as tartaric acid, ascorbic acid, and citric acid, and for example aroma.

Liquid preparations for oral application may be in the form of elixirs, syrups or suspensions, for example solutions containing from about 0.1% to 20% by weight of active substance, sugar and a mixture or ethanol, water glycerol, propylene glycol and optionally aroma, saccharine and/or carboxymethylcellulose as a dispersing agent. For parenteral application by injection preparations may comprise an aqueous solution of a water soluble pharmaceutically acceptable salt of the active acids according to the invention, desirably in a concentration of 0.5 - 10%, and optionally also a stabilizing agent and/or buffer substances in aqueous solution. Dosage units of the solution may advantageously be enclosed in ampoules.

For topical application, especially for the treatment of herpes virus infections on skin, the preparations are suitably in the form of an ointment, gel, suspension, cream or the like. The amount of active substance may vary, for example between 0.05- 20% by weight of the active substance. Such preparations for topical application may be prepared in known manner by mixing the active substance with known carrier materials such as isopropanol, glycerol, paraffin, stearyl alcohol, polyethylene glycol, etc. The pharmaceutically acceptable carrier may also include a known chemical absorption promoter. Examples of absorption promoters are e.g. dimethylacetamide (U.S. Pat. No. 3,472,931) , trichloro-ethanol or trifluoroethanol (U.S. Pat. No. 3,891,757) , certain alcohols and mixtures thereof (British Pat. No. 1,001,949) . A carrier material for topical application to unbroken skin is also described in the British patent specification No. 1,464,975, which discloses a carrier material consisting of a solvent comprising 40-70% (v/v) isopropanol and 0-60% (v/v) glycerol, the balance, if any, being an inert constituent of a diluent not exceeding 40% of the total volume of solvent.

The dosage at which the proteinase inhibitors are administered may vary within a wide range and will depend on various factors such as for example the severity of the infection, the age of the patient, etc., and may have to be individually adjusted. As a possible range for the amount of proteinase inhibitor which may be administered per day be mentioned from about 0.1 mg to about 2000 mg or from about 1 mg to about 2000 mg.

The pharmaceutical compositions containing the proteinase inhibitors may suitably be formulated so that they provide doses within these ranges either as single dosage units or as multiple dosage units.

The subject invention also provides for compositions useful in agriculture containing proteinase inhibitors, including proteinase inhibitors according to the compound of formula (I) formulated for use in treating/preventing plant infections. The many forms of suitable formulation includes a powder or granule in a suitable carrier. Alternatively, the proteinase inhibitors may be applied as a suspension or dispersion, e.g. as an aqueous suspension with a suitable protectant such as methycellulose, dextran, dextrin, alginate, magnesium silicate. The composition may also be applied as a wettable powder.

Additionally, the composition may be formulated for application to seeds, may be applied as part of a seed coating composition, for instance mixed with xanthan gum, magnesium silicate, methylcellulose, gum arabic, polyvinyl pyrollidone, dextrins or dextrans . The subject invention also provides for numerous assays for the detection of fungal cells, either as contaminants or pathological agents, by means of the detection of fungal aspartic proteinase. The suject assay method find a variety of uses in diverse fields such as human medicine, veterinary medicine, agriculture, food preparation, and the like.

In the subject assay methods, proteinase inhibitors of the invention may be used to capture, i.e., immobilize, fungal aspartic proteinase in a composition for analysis. The subject proteinase inhibitors may be used to immobilize aspartic fungal proteinase because of the high affinity the proteinase inhibitors of the invention have for fungal aspartic proteinase. By providing for a method of immobilizing fungal aspartic proteinase, the invention provides a highly significant improvement over conventional immunoassays for the detection of fungal proteinases. Among the reasons that the immobilization of fungal aspartic proteinase through the subject proteinase inhibitors is an improvement over conventional immunoassay technique is that the proteinase inhibitors of the invention bind to the active site of aspartic fungal proteinases. Thus the proteinase inhibitors of the invention may be used to distinguish active fungal aspartic proteinases, which have a functional active site, from denatured proteinase, which lack a functional active site. Being able to distinguish active proteinases from inactive proteinases is a very important advantage because many compositions for analysis may be exposed to a variety of fungal proteinases, e.g., through the ingestion of food products, that would give rise to fungal proteinase fragments detectable by fungal proteinase specific fragments detectable by fungal proteinase specific antibodies, even though the composition lacks active fungal proteinases or fungal cells that produce the detected proteinases.

The subject proteinase inhibitors may be used in a variety of novel assays for the detection of fungal aspartic proteinases. The novel assays described herein are improvements of conventional assays for the detection of fungal proteinases, typically immunoassays such as ELISA, RIA, western blots, and the like. Detailed descriptions of conventional immunoassay that can be adapted for the detection of fungal proteinase can be found in numerous publications available to the person of ordinary skill in the art. Such publications include, J. Clarsen Immunochemical Techniques for the Identification and Estimation of Macromolecules: Laboratory Techniques in Biochemistry and Molecular Biology, 3rd. Edition, Elsevier, Amsterdam (1988) , Harlow and Lane Antibodies: A laboratory Manual, Cold Spring Harbor, Cold Spring Harbor, NY (1988) , and the like. The assay methods of the invnetion may be either qualitative or quantitative. The assay methods of the invention may be carried out by adding an immobilization step, i.e., immobilization of fungal aspartic proteinase step, to conventional immunoassays. The person of ordinary skill in the art could readily modify a conventional immunoassay so as to comprise an immobilization step employing one or more of the subject proteinase inhibitors to immobilize fungal aspartic proteinases. Typically, the proteinase inhibitor is attached to a solid substrate in manner that permits the proteinase inhibitor to bind to a fungal aspartic proteinase. For example, a composition for analysis may be contacted with microbeads containing an immobilized proteinase inhibitor of the invention. Aspartic fungal proteinase in the composition for analysis can then be exposed to the microbeads, whereby fungal aspartic proteinase are immobilized and consequently separated from the original composition for analysis. The microbeads suspected of containing the immobilized aspartic fungal proteinases may then be subjected to a conventional immunoassay, e.g., and ELISA, employing antibodies specific for the fungal proteinase of interest.

One example of an assay for the detection of fungal proteinases employs a solid substate such as a filter membrane, e.g., nitrocellulose, PVDF, and the like, that has been spotted with a proteinase inhibitor of the invention in such a way that the proteinase inhibitor is bound to the membrane. The membrane is then exposed to a blocking agent solution that contains a compound, typically an inexpensive protein, such as bovine serum albumin, rasein, and the like, a high enough concentration to block non- specific binding to the membrane. The membrane is then washed with a buffer, e.g., a phosphate buffered saline solution, to¹ remove excess blocking agent. The composition for analysis is then passed through the membrane so as to permit the binding of fungal aspartic proteinases to the proteinase inhibitors on the membrane. The membrane is then washed so as to remove the composition for analysis. One or more immunoreagents, . such as an antibody, specific for the fungal proteinase(s) of interest are then exposed to the membranes so as to bind to any immobilized fungal proteinases. The membrane is then washed again with buffer to remove unbound immunoreagent. The bound immunoreagent, e.g., antibody, can then be detected by any of a variety of methods, e.g., colloidal gold or enzymes conjugated to second antibody specific for the bound antibody. The assay protocol described above is only an example and should not be regarded as limitation on the claimed invention.

The assays of the invention may be. used to detect or measure the presence of fungal aspartic proteinase in a variety of compositions. These compositions for analysis may be performed on almost any composition suspected of containing a fungal aspartic proteinase. Examples of compositions for analysis include biological samples obtained from a human or animal, such as blood, lymph, stools, cerebral spinal fluid, synovial fluid, and the like. Other examples of compositions for analysis by the subject methods include food and pharmaceuticals. The invention also provides kits for performing the subject assays for fungal aspartic proteinases. Kits comprise a package unit having any or more containers containing one or more of the subject proteinase inhibitors. The kits may also include one or additional reagents for performing the assay methods of the invention. These additional reagents include antibodies specific for fungal aspartic proteinases, buffers, binding membranes, positive controls, negative controls, colormetric reagents, standards, and the like. Kits of the invention preferably include detailed instruction as to how to perform the subject methods. Reagent containers in the subject kits preferably contain reagents in unit quantities that obviate measuring steps when performing the subject methods.

EOUIVALENTS All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the fields of infectious disease, biochemistry, or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS What is claimed is:

1. A compound having the formula

(I) A-B-G-C-Sta-Ala-Sta-E-F

wherein A is absent or an amino acid blocking group, B is either absent or a peptide consisting of 1-3 amino acids, C is an amino acid, E is either absent or selected from the group consisting of glycine and _/β-alanine, F is either absent or a 1-5 amino acid peptides composed of amino acids that are either positively charged, negatively charged, or have hydroxyl groups on side chains, and G is an amino acid.

2. A compound according to Claim 1 wherein, A is an amino acid blocking group and G is valine.

3. A compound according to Claim 1 wherein,

C is selected from the group consisting of valine and amino acid residues having one or more amine groups attached to a straight or branched aliphatic hydrocarbon side chain.

4. A compound according to Claim 1, wherein F is a 1-5 amino acid peptide composed of amino acids that are either positively charged, negatively charged, or have hydroxyl groups on side chains.

5. A compound according to claim 1, wherein A is selected from the group consisting of isovaleric acid and t-Boc, B is absent, C is selected from the group consisting of aminoglycine, ornithine, lysine, 2,4,-diamino butinoic acid, and 2, 4, -diaminopropanoic acid, and F is selected from the group consisting of serine, threonine, lysine, glutamic acid, cysteic acid, tyrosine, aspartic acid, histidine, lysine, and arginine, D-Asp, D-Asp-D-Asp, and D-Ser-D-Ser.

6. A compound according to claim 1, wherein said compound is selected from the group consisting of

(II) Iva-Val-Val-Sta-Ala-Sta-Bal-D-Asp-D-Asp,

(III) Iva-Val-Val-Sta-Ala-Sta-D-Ser-D-Ser,

(IV) D-Asp-Bal-Val-Val-Val-Sta-Ala-Sta-Bal-D-Asp, and

(V) Iva-Val-Dab-Sta-Ala-Sta.

7. A method of treating or preventing an infection by an infectious organism, said method comprising the step, administering an effective amount of a composition comprising a proteinase inhibitor.

8. A method according to claim 7 wherein the proteinase inhibitor has the formula

(I) A-B-G-C-Sta-Ala-Sta-E-F

wherein A is absent or an amino acid blocking group, B is either absent or a peptide consisting of 1-3 amino acids, C is an amino acid, E is either absent or selected from the group consisting of glycine and β-alanine, F is either absent or a 1-5 amino acid peptides composed of amino acids that are either positively charged, negatively charged, or have hydroxyl groups on side chains, and G is an amino acid.

9. A compound according to Claim 8 wherein, A is an amino acid blocking group and G is valine.

10. A compound according to Claim 8 wherein,

C is selected from the group consisting of valine and amino acid residues having one or more amine groups attached to a straight or branched aliphatic hydrocarbon side chain.

11. A compound according to Claim 8, wherein F is a 1-5 amino acid peptide composed of amino acids that are either positively charged, negatively charged, or have hydroxyl groups on side chains.

12. A compound according to claim 8, wherein A is selected from the group consisting of isovaleric acid and t-Boc, B is absent, C is selected from the group consisting of aminoglycine, ornithine, lysine, 2,4,-diamino butinoic acid, and 2,4, -diaminopropanoic acid, and F is selected from the group consisting of serine, threonine, lysine, glutamic acid, cysteic acid, tyrosine, aspartic acid, histidine, lysine, and arginine, D-Asp, D-Asp-D-Asp, and D-Ser-D-Ser.

13. A compound according to claim 8, wherein said compound is selected from the group consisting of

(II) Iva-Val-Val-Sta-Ala-Sta-Bal-D-Asp-D-Asp,

(III) Iva-Val-Val-Sta-Ala-Sta-D-Ser-D-Ser, ( IV) D-Asp- Bal -Val-Val-Val-Sta-Ala-Sta-Bal-D-Asp, and

(V) Iva-Val -Dab- Sta-Ala- Sta .

14. A method for detecting a fungal proteinase, said method comprising the steps: incubating a composition for analysis with proteinase inhibitor having the formula

(I) A-B-G-C-Sta-Ala-Sta-E-F

wherein A is absent or an amino acid blocking group, B is either absent or a peptide consisting of 1-3 amino acids, C is an amino acid, E is either absent or selected from the group consisting of glycine and -alanine, F is either absent or a 1-5 amino acid peptides composed of amino acids that are either positively charged, negatively charged, or have hydroxyl groups on side chains, and G is an amino acid, wherein said proteinase inhibitor is immobilized on a solid substrate, whereby fungal aspartic proteinases present in said composition for analysis is bound to said proteinase inhibitor.

15. A method according to Claim 14 wherein, A is an amino acid blocking group and G is valine.

16. A method according to Claim 14 wherein,

C is selected from the group consisting of valine and amino acid residues having one or more amine groups attached to a straight or branched aliphatic hydrocarbon side chain.

17. A method according to Claim 14, wherein F is a 1-5 amino acid peptide composed of amino acids that are either positively charged, negatively charged, or have hydroxyl groups on side chains.

18. A method according to claim 14, wherein A is selected from the group consisting of isovaleric acid and t-Boc, B is absent, C is selected from the group consisting of aminoglycine, ornithine, lysine, 2,4,-diamino butinoic acid, and 2,4, -diaminopropanoic acid, and F is selected from the group consisting of serine, threonine, lysine, glutamic acid, cysteic acid, tyrosine, aspartic acid, histidine, lysine, and arginine, D-Asp, D-Asp-D-Asp, and D-Ser-D-Ser.

19. A method according to claim 14, wherein said compound is selected from the group consisting of

(II) Iva-Val-Val-Sta-Ala-Sta-Bal-D-Asp-D-Asp,

(III) Iva-Val-Val-Sta-Ala-Sta-D-Ser-D-Ser,

(IV) D-Asp-Bal-Val-Val-Val-Sta-Ala-Sta-Bal-D-Asp, and

(V) Iva-Val-Dab-Sta-Ala-Sta.

20. A method according to Claim 14, said method further comprising the step of contacting said immobilized fungal aspartic proteinase with an antibody capable of specifically binding said fungal aspartic acid proteinase.

21. A kit for detecting a fungal proteinase in a composition for analysis, said kit comprising a proteinase inhibitor having the formula

(I) A-B-G-C-Sta-Ala-Sta-E-F

wherein A is absent or an amino acid blocking group, B is either absent or a peptide consisting of 1-3 amino acids, C is an amino acid, E is either absent or selected from the group consisting of glycine and /3-alanine, F is either absent or a 1-5 amino acid peptides composed of amino acids that are either positively charged, negatively charged, or have hydroxyl groups on side chains, and G is an amino acid.

22. A kit according to Claim 21, said kit further comprising an antibody specific for a fungal aspartic proteinase.