BACKGROUND OF THE INVENTION

Field of the Invention

The invention generally relates NK-lysin peptides as antimicrobial or antiviral agents alone or in combination with other agents, including β-lactam and/or other antibiotics, and with interleukin-2 or silver nanoparticles. The invention further relates to methods for treating microbial or viral infection with a pharmaceutical composition comprising NK-lysin peptides.

Background

Antimicrobial peptides (AMPs) are immune response molecules that have been found in various organisms. AMPs play a fundamental role in first line of defense against invading pathogens to inhibit properties that mediate fitness to cause infection. Moreover, AMPs also perform immune-modulatory functions. NK-lysin is a cationic AMP with antibacterial activity that was first identified from pig gastrointestinal tract. It was also found to be secreted from the granules of natural killer and cytotoxic T cells, and thus acquired the name of NK-lysin. NK-lysin is referred to as granulysin in humans and is a saposin-like protein family member. The peptide possesses a strong positive charge conferred by its conserved cysteines residues, which are also responsible for disulfide bonds formation inside the amino acids chain. The 3-D structural characteristics have been shown to increase its antimicrobial potentialities. The ability to move through bacterial membrane is critically due to the number of non-polar amino acids at the side chain of the NK-lysin peptide. Accordingly, these non-polar amino acids are highly conserved and account for 27-51% of the amino acid residues in NK-lysin homologues within all animal species.

Peptide binding and disruption of cell membranes is facilitated by non-polar amino acids such as Ile and Leu. NK-lysin and granulysin peptides comprise a saposin domain with minor changes in domain length. The domain is folded in 4-5 helical bundles, depending upon the species, having three disulfide bonds between six cysteine residues that stabilize the peptide conformational structure.

The topic of antibiotic resistance is dynamic, with new data and concepts shaping and reshaping the field on a continuous basis. The topics of greatest interest in this field include β-lactam antibiotic resistance and specifically β-lactamases, the enzymes that are capable of hydrolysing β-lactam antibiotics. Since the 1980s, the number of β-lactamases identified has increased significantly; however, alternative ways were taken into consideration by healthcare and pharmaceutical Industry to improve and invent new routes to combat and prevent multi-drug resistance pathogens by using biocompatible non-toxic agents. For example, uptake of silver nanoparticles has been used to prevent infection with both gram-positive and negative bacteria. Recently, silver nanoparticles combined with antimicrobial peptides and/or antibiotic have been investigated as a means to eliminate bacterial resistance and improve antimicrobial molecules.

Several biomolecule classes, including cytokines and chemokines, have exhibited antibacterial activities in addition to their immune-regulatory properties. The binding affinity between positively charged chemokines and negatively charged phospholipids in the cell membrane bilayer may underly the mode of action that causes membrane disruption. Interleukin-2 (IL-2) is an example of a cytokine that has multiple roles in defences against disease. IL-2 plays an important role in managing the immune system, and it is also used as a medication to treat different oncological diseases. A combination of interleukin and lysozyme shows highest activity in the micromolar range of antibiotics. It was recently reported that IL-2 has bacteriolytic activity, but the physiologically significant for this has not yet been demonstrated.

Various studies have demonstrated that innate immune response molecules may have antiviral effects on enveloped and non-enveloped viruses and teach that membrane disruption is a mode of action induced against viral infection. For example, Levy (J Leuk Bio76:909, November 2004) teaches that lymphocytes such as NK-cells express granulysin or the non-human homologue NK-lysin and that granulysin is a membrane-active peptide with activity against a range of bacteria, includingM. tuberculosisand that the IL-2 inducible NK-lysin and granulysin also has antimycobacterial activity. Levy teaches that KN-lysin binds LPS and has an inhibitory effect. Jacobs et al. (Antimicrob Agents Chemo 47(2):607) teach that porcine NK-lysin and a shortened analog NK-2 have antimicrobial activity againstT. cruzi. Jacobs teaches that NK-2 is less cytotoxic to mammalian cells than NK-lysin, while retaining antimicrobial activity against bacteria, yeast and protozoa by disruption of microbial membranes. U.S. Pat. No. 8,703,134 teaches a fusion protein to target various microorganisms, comprising an immunoglobulin and a biocide comprising IL-2 or granulysin. Synthetic NK-lysin peptides, which correspond with functional region helices 2 and 3, showed similar hydrophobicity ranging from 40 to 43% and net positive charge of 5.0-7.9. The peptides comprise 20-30% basic residues, which contribute to high effectiveness againstS. aureusandE. coli. The high alphahelicity of the bovine peptide did not have much effect onM. bovisand M haemolytic isolates (Chen et al.,PLoS One;2016; 11(7):e0158882), but in contrast bovine NK lysin peptide was highly effective towardsH. somniisolates (Dassanayake et al.PLoS One.2017; 12(8):e0183610; and Dassanayake et al. 2018; PLoS One13(5): e0197677). Studies which were done on chicken NK lysin peptide show their antimicrobial activity is similar to other NK lysin peptides (Harwig et al.FEBS Lett.1994; 342:281-5; Lehrer and Ganz.Curr Opin Immunol.2002; 14:96-102; Goitsuka et alProc Natl Acad Sci USA.2007; 104:15063-8; Lee et al.Proc Natl Acad Sci USA.2012; 109:12087-92; and Lee et al.Poult Sci.2014; 93:864-70). Research by (Jacobs et al., 2003, PNAS Nov. 25, 2003 100 (24) 14339-14344) shows that the mammalian NK-lysin peptide rapidly permeabilized the plasma membrane of the protozoan parasiteTrypanosoma cruzi, resulting in the release of cytosolic enzymes within minutes after exposure. They also found that the NK-lysin and NK-2 killed trypanosomes residing inside the human glioblastoma cell line 86HG39 but only NK-2 left the host cells apparently unharmed, whereas NK-lysin was not safe. Pheasant cathelicidin-1 (Pc-CATH1) showed similar data, where the growth ofE. coliwas restricted after 1 hour of exposure, and even after 6 hours of being exposed the bacteria did not start growing (Wang et al., 2011, Curr. Microbiol.62, 703-709. doi: 10.1007/s00284-010-9767-2). Melimine peptide and its derivative exhibit a powerful antimicrobial activity againstP. aeruginosabecause it is smaller in size (17-29 aa), which is possibly why it is capable of covering the cytoplasmic membrane ofP. aeruginosa. The smaller length of the MeI4 peptide is thought to take more time to move across the outer membrane or to interact with the inner membrane ofP. aeruginosato destroy the bacteria, or that it needs to align itself inside the membrane more effectively so as to start its activity.

Levashov et al. (Acta Naturae.2017; 8:1(33):81-87) demonstrated that IL-2 has a bacteriologic activity in the micromolar range, and that a combination of IL-2 and lysozyme has bacteriolytic activity in the micromolar range. The physiological significancy of bacteriolytic activity for this cytokine remains unclear.

Though studies on NK-lysin peptides have been carried out in terms of their antibacterial activity against some microbial pathogens, the potential of bovine, human and chicken NK-lysin peptides as antimicrobial and/or antiviral agents, alone or in combination with other agents, has not been fully established. Furthermore, the potential for sensitizing antibiotic-resistant bacterial pathogens to beta-lactam antibiotics is an ongoing and urgent need.

SUMMARY OF THE INVENTION

NK-lysins from chicken, bovine and human are used as antiviral and antibacterial agents. Gram-negative and gram-positive microorganisms, includingStreptococcus pyogenes, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Shigella sonnei, Klebsiella pneumoniaeandSalmonella typhimurium, are susceptible to NK-lysin treatment. The NK-lysin peptides and compositions comprising these peptides are also effective as a treatment against Rotavirus species. Combinations of NK-lysin with IL-2 or silver nanoparticles are also effective againstS. typhimurium. Expression of β-lactamase genes, including CTX-M-1, M-8 and M-9 is inhibited in response to treatment with NK-lysins. Bacterial resistance to β-lactam antibiotics is at least partially overcome by inhibiting β-lactamase activity. NK-lysin peptides are also used to test for inhibition of infectivity, which ranged from 50-90% depending on NK-lysin species.

In one embodiment, the invention is a method for treating an infection in a patient in need thereof, comprising the step of administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising an NK-lysin peptide, wherein the infection is caused by a pathogen selected from the group consisting ofStreptococcus pyogenes, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Klebsiella pneumoniae, Shigella sonneiandSalmonella typhimurium. The NK-lysin peptide is derived from a chicken, bovine or human protein or peptide. The chicken NK-lysin peptide has the amino acid identity of SEQ ID NO:1, the bovine NK-lysin peptide has the amino acid identity of SEQ ID NO:2 and the human NK-lysin peptide, also known as granulysin, has the amino acid identity of SEQ ID NO:3.

In another embodiment, the invention is a method for treating a rotaviral infection in a subject in need thereof, comprising the step of administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising an NK-lysin peptide, wherein the NK-lysin peptide has an amino acid identity selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. In one embodiment of the invention the antiviral agent is a bovine NK-lysin peptide having the amino acid identity of SEQ ID NO:2.

In yet another embodiment, the invention is a method of treating a bacterial infection wherein the bacterial infection is caused by a pathogen that is or is suspected to be resistant to β-lactam antibiotics, comprising the step of co-administering at least one β-lactam antibiotic and a therapeutically effective amount of NK-lysin peptide, wherein the therapeutically effective amount of NK-lysin peptide is sufficient to inhibit expression of at least one bacterial β-lactamase gene selected from the group consisting of CTX-M-1, M-8 and M-9. The NK-lysin peptide has an amino acid identity selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

