# US 5,703,044 A · Synergistic antifungal protein and compositions containing same

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- Number: US 5,703,044 A
- Document ID: US5703044A
- Status: Granted
- Kind: A
- Application number: 08/456,430
- Filed: 1995-06-01
- Granted: 1997-12-30
- Earliest claimed: 1987-10-02
- Assignees: Novartis Finance Corporation
- Inventors: Walden K. Roberts; Claude P. Selitrennikoff; Bridget E. Laue; Sharon L. Potter
- Web page: https://patentgrep.com/patent/US5703044A

## CPC

- A61K 36/899: Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
- A01N 65/44: Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
- A61K 38/168: {from plants}
- C07K 14/415: from plants
- C12N 15/8282: {for fungal resistance}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Novel plant proteins (SAFPs) which synergize the activity of antifungal antibiotics are identified. SAFPs are demonstrated to synergize antifungal antibiotics, such as nikkomycins, polyoxins and amphotericins. SAFPs alone also display antifungal activity against several species of fungi, including strains of Candida, Trichoderma, Neurospora and strains of the plant pathogens Fusarium, Rhizoctonia and Chaetomium. Synergistic antifungal compositions containing SAFP and antifungal antibiotics are provided. In particular, synergistic compositions of corn-SAFP (zeamatin), sorghum-SAFP (sormatin) or oat-SAFP (avematin) and nikkomycin are found to be effective as antifungal compositions, especially against the opportunistic human pathogen Candida albicans. Method for employing SAFPs and synergistic compositions containing them for the inhibition of fungi are provided. In addition, a method for purifying SAFP from grain meal is provided.

## Claims

### Claim 1

A method of inhibiting the growth of a fungus which comprises applying to said fungus or a habitat of said fungus zeamatin in substantially pure form in an antifungally effective amount or an amount sufficient to enhance the antifungal activity of an antifungal antibiotic selected from the group of antifungal antibiotics which comprises nikkomycins, polyoxins and amphotericins, wherein said zeamatin is a protein isolatable from corn, having a molecular weight of about 22 kD under reducing conditions upon SDS-PAGE, and having the N-terminal amino acid sequence A V F V V N Q C P F T V W A A S V P V G G G R Q L R G E.

### Claim 2 (depends on 1)

The method of claim 1 wherein said fungus is a strain selected from the group consisting of strains of Candida, Tricholerma Neurospora, Rhizoctonia, Fusarium and Chaetomium.

### Claim 3 (depends on 1)

The method of claim 1 wherein said fungus is a plant pathogen.

### Claim 4 (depends on 1)

The method of claim 1 wherein said zeamatin protein is obtainable from corn steepwater or a protein concentrate thereof.

### Claim 5

A method of inhibiting the growth of a fungus which comprises applying to said fungus or a habitat of said fungus sormatin in substantially pure form in an antifungally effective amount or an amount sufficient to enhance the antifungal activity of an antifungal antibiotic selected from the group of antifungal antibiotics which comprises nikkomycins, polyoxins and amphotericins, wherein said sormatin is a protein isolatable from sorghum, having a molecular weight of about 25 kD under reducing conditions upon SDS-PAGE, and having the N-terminal amino acid sequence A V F T V V N R C P Y T V W A A S V P V G G.

### Claim 6 (depends on 5)

The method of claim 5 wherein said fungus is a strain selected from the group consisting of strains of Candida, Trichoderma Neurospora, Rhizoctonia, Fusarium and Chaetomium.

### Claim 7 (depends on 5)

The method of claim 5 wherein said fungus is a plant pathogen.

### Claim 8 (depends on 5)

The method of claim 5 wherein said sormatin protein is obtainable from a partially purified protein extract of sorghum.

### Claim 9

A method of inhibiting the growth of a fungus which comprises applying to said fungus or a habitat of said fungus avematin in partially purified form in an antifungally effective amount or an amount sufficient to enhance the antifungal activity of an antifungal antibiotic selected from the group of antifungal antibiotics which comprises nikkomycins, polyoxins and amphotericins, wherein said avematin is a protein isolatable from oat and having a molecular weight of about 22 kD under reducing conditions upon SDS-PAGE.

### Claim 10 (depends on 9)

The method of claim 9 wherein said fungus is a strain selected from the group consisting of strains of Candida, Trichoderma Neurospora, Rhizoctonia, Fusarium and Chaetomium.

### Claim 11 (depends on 9)

The method of claim 9 wherein said fungus is a plant pathogen.

### Claim 12

A method of inhibiting the growth of a fungus which comprises applying to said fungus or a habitat of said fungus an antifungal composition which comprises an antifungal antibiotic and an antifungal protein, said antifungal antibiotic and said antifungal protein being present in concentrations sufficient to inhibit the growth of said fungus, said antifungal protein being a protein selected from
(i) zeamatin isolatable from corn, having a molecular weight of about 22 kD under reducing conditions upon SDS-PAGE, and having the N-terminal amino acid sequence A V F T V V N Q C P F T V W A A S V P V G G G R Q L N R G E;
(ii) sormatin isolatable from sorghum, having a molecular weight of about 25 kD under reducing conditions upon SDS-PAGE, and having the N-terminal amino acid sequence A V F T V V N R C P Y T V W A A S V P V G G; and
(iii) avematin isolatable from oat and having a molecular weight of about 22 kD under reducing conditions upon SDS-PAGE;
and said antifungal antibiotic being selected from the group of antifungal antibiotics which comprise nikkomycins, polyoxins and amphotercins.

### Claim 13 (depends on 12)

The method of claim 12 in which said fungus is selected from the group of fundi consisting of Candida and Rhodotorula.

### Claim 14 (depends on 13)

The method of claim 13 in which said fungus is a strain of Candida.

### Claim 15 (depends on 14)

The method of claim 14 in which said fungus is a strain of Candida albicans.

### Claim 16 (depends on 12)

The method of claim 12 wherein said antifungal antibiotic of said composition is a nikkomycin.

### Claim 17 (depends on 12)

The method of claim 12 wherein said nikkomycin is present at a concentration greater than or equal to about 0.03 times the MIC of said nikkomycin alone.

### Claim 18 (depends on 12)

The method of claim 12 wherein said nikkomycin is present at a concentration greater than or equal to about 0.01 times the MIC of said nikkomycin alone.

### Claim 19 (depends on 12)

A method according to claim 12 wherein the fungus is a strain of Candida albicans, the antifungal antibiotic is a nikkomycin that has anti-Candida activity, and the antifungal protein is zeamatin.

### Claim 20 (depends on 19)

The method of claim 19 wherein said nikkomycin is nikkomycin X.

### Claim 21 (depends on 19)

The method of claim 19 wherein said nikkomycin is nikkomycin Z.

### Claim 22 (depends on 19)

The method of claim 19 wherein said nikkomycin is present at a concentration greater than or equal to about 0.03 times the MIC of said nikkomycin alone.

### Claim 23 (depends on 19)

The method of claim 19 wherein said nikkomycin is present at a concentration greater than or equal to about 0.01 times the MIC of said nikkomycin alone.

### Claim 24 (depends on 19)

The method of claim 19 wherein the zeamatin is present in amounts sufficient to lower the MIC of nikkomycin by about 33-100 fold relative to the MIC of nikkomycin alone for said strain of Candida albicans.

### Claim 25 (depends on 12)

A method according to claim 12 wherein the fungus is a strain of Candida albicans, the antifungal antibiotic is a nikkomycin having anti-Candida activity, and the antifungal protein is sormatin.

### Claim 26 (depends on 12)

A method according to claim 12 wherein the fungus is a strain of Candida albicans, the antifungal antibiotic is a nikkomycin that has anti-Candida activity, and the antifungal protein is avematin.