WO2018085954A1

WO2018085954A1 - Antimicrobial peptides

Info

Publication number: WO2018085954A1
Application number: PCT/CL2017/050063
Authority: WO
Inventors: Jorge ARAYA ROJAS; Hernan SEGUA FRANCO; Marco RAMIREZ GALLARDO; Jorge GONZALEZ CORTES; Alejandro CATALAN RODRIGUEZ; Iván NEIRA CORTES; Patricio ORREGO LILLO; Kely ORDENES GONZALEZ; Daniel MIERES CASTRO; William CORTES ARAYA; Camilo URBINA ALONSO
Original assignee: Universidad De Antofagasta
Priority date: 2016-11-14
Filing date: 2017-11-14
Publication date: 2018-05-17
Also published as: CL2016002890A1

Abstract

The invention is intended primarily for use in the development of products used to inhibit and kill bacteria, and especially for use in the treatment of infectious diseases caused by bacteria, in particular in the treatment of bacteria that do not respond to treatment with antibiotics or that have developed resistance to existing antibiotics.

Description

PEPTIDES WITH ANTIMICROBIAL ACTIVITY

Scope

The invention described herein has its main field of application in the development of products for inhibiting and destroying bacteria, with special applicability to the treatment of infectious and contagious diseases caused by bacteria, in particular in the treatment of those bacteria that do not respond to antibiotic treatment or that They have developed resistance to existing antibiotics.

Background

Antimicrobial Peptides (PAMs)

Since Csordás et al. (1968) made their first description, these peptides have been recorded both in vertebrates (Nicolás and Mor, 1995: Romeo et al., 1988) and in invertebrates (Bulet et al., 1999: Steiner and cois., 1981), with a wide phylogenetic distribution, including humans (Selsted and cois., 1983: Lazarovici and cois., 1986: Frank and cois., 1990: Shiff, 1994: Ganz and Lehrer, 1994: Hubert et al., 1996: Hoffmann et al., 1999) In vertebrates they have been found in the skin, on the epithelial surfaces (mucous membranes) and in the blood. As well as, in the hemolymph of insects and in the poisonous secretions of bees, wasps and in arachnids, within invertebrates. They are relatively small molecules, encoded by genes, have low molecular weight, approximately less than 10 kDa, (15 to 100 amino acids), amphipathic, cationic, sequence length and variable structure.

In 1981 it was characterized, from the hemolymph of the pupa of a giant moth (Hyalophora cecropia), a peptide that produced lysis of bacteria in culture (Steiner et al., 1981). At first these antibacterial peptides were considered as exclusive to insects, but nowadays they are known to be widely distributed in the animal kingdom. Two years later in 1983, defensins were identified in rabbit alveolar macrophages (Selsted et al., 1983). In 1985 the PGLa peptide was isolated from the mucous secretion of Xenopus skin, although its bactericidal activity was not fully tested (Andreu et al., 1985b). Zasloff (1987), found a group of antlbacterial peptides, magainins, in frog skin secretions. A short time later, bactenecin was found in bovine neutrophils (Romeo et al., 1988). Peptides with a certain similarity to mammalian defensins were found in two genera of insects, in Sarcophaga and in Phormia (Matsuyama and Natori, 1988; Lambed et al., 1989); and Cecropin Pl isolated from the intestine of the insect-like Cecropin-like pig (Lee et al., 1989). At the same time, the first proline-rich antibacterial peptides (Casteeis et al., 1989; Casteeis et al., 1990) were described in bees, peptides of this type have subsequently been isolated in bovine neutrophils (Frank et al., 1990) and in the small intestine of the pig (Agerberth et al., 1991). Antibacterial peptides are distributed throughout the evolutionary scale, having also been isolated in mollusks (Hubert et al., 1996), crustaceans (Shiff, 1994) and fish (Lazarovici et al., 1986).

These PAMs are broad spectrum, non-specific, with activity against a wide range of microorganisms, including enveloped viruses, Gram negative and Gram positive bacteria, protozoa, yeasts and filamentous fungi (Bulet et al., 2004; Yount et al., 2006; Guaní-Guerra ef a /., 2010).

It has recently been shown that in addition to their antimícrobial activity, PAMs can encompass a number of other diverse biological functions and are, in fact, multifunctional molecules. Thus, it has been proven that these peptides have antitumoral effects, mitogenic activity and, above all, participate in signal transduction mechanisms as immuno modulators in the release and / or production of cytokines (Kamysz et al., 2003; Bowdish and cois., 2005 ;. Brown and Hancock, 2006; Yount and cois., 2006; Easton and cois., 2009; Lai and Gallo, 2009; Guani-Guerra and cois., 2010).

PAMs constitute a defense mechanism widely distributed in plants, insects and vertebrates These molecules that are produced by different tissues and cell types, are classified into different families, according to their secondary structure (Ganz, 2003). The most important structure is that of the amphipathic peptide with 2-4 beta structures, amphipathic alpha-helix, loop structure and extended structure. The amino acid composition, its net charge (generally cationic), as well as its amphipathic and size characteristics, favor its interaction with lipid bilayers, mainly those that form the cytoplasmic membranes of pathogens (bacteria, fungi, enveloped viruses and parasites), in those that form pores.

More recently it has been proven that its range of action has extended to the bacterial cytoplasm, where they alter the formation of the septum of the cytoplasmic membrane, inhibit the synthesis of the cell wall, that of nucleic acids, that of proteins, as well as some activities enzymatic (Boman et al., 1993; Subbalakshmi and Sitaram, 1998; Patrzykat et al., 2002; Brodgen, 2005). Their mechanisms of action have been intensively studied, they would form channels or pores in cell membranes, causing rupture and loss of cellular physiological integrity (Hwang et al., 1998).

This action is basically determined by the structural conformation of the peptides, which acts by disrupting the integrity of the white cell membrane. The cationic part of the peptide is nicely attracted to the negatively charged cell walls of bacteria and fungi and / or the cytoplasmic membranes of other cells, after this first electrostatic interaction, the insertions of the peptides permeabilize the microbial cell membranes through of its hydrophobic portion. Thus, microorganisms and other cells are destroyed through membrane destabilization and / or pore formation (Brogden, 2005; Yount and cois., 2006). The detailed mechanism of pore formation has been described in detail by Brogden (2005) and Salditt et al., (2006). Beyond this direct interaction with microbial membranes, a growing number of evidences suggest that PAMs may have other mechanisms to inactivate the various pathogenic agents. Recent studies have shown that peptides can be transferred to the cytoplasm of microorganisms to act on specific intracellular targets. Thus, once inside, the peptides would interfere with some essential metabolic functions, with various protein targets, nucleic acids and in cell wall synthesis, leading to bacterial cell death (Kamysz et al., 2003; Brogden, 2005 ; Yount and cois., 2006; Hale and Hancock, 2007; Nicolás, 2009). Moreover, it seems that many peptides can be multifunctional microblids, since they would act simultaneously on the cell membrane and / or interfering in the metabolic pathways of pathogenic microorganisms (Yount et al., 2006).

On the other hand, more recently, other groups of antimlcrobial molecules have been identified, which do not fit the classic definition of PAMs. These are anionic peptides, of more than 10 kDa, multlfunctional proteins that contain antlmicrobial sub-domains that break under certain conditions and generate fragments that resemble the classic antimlcrobial peptides (Brogden, 2005; Yount and cois., 2006).

PAMs were initially classified into two main categories depending on whether or not they contain cysteine residues to form disulfide bridges. At present, they are divided into four large families based on their secondary structures: 1). Linear helix peptides, lack cysteine residues, sometimes have a hinge in the center (Gennaro and Zanettl, 2000; Tossi and cois., 2002) represented by cecropins (Boman and cois., 1981: Akuffo and cois. , 1998), magaininas (Moore et al., 1991) and melitin (de Grado et al., 1982). In aqueous solution many of these peptides have no defined structure, which adopt a random structure and can form a structure of α helix or double α helix in organic solvents, triphenylethanol, SDS, micelles, phospholipid vesicles, llposomes or lipid A or al joining the entire molecule or a part of the cltoplasmátl membranes adopts helix structure (Gennaro and Zanettl, 2000). Other examples of this group are cathelicidin LL-37, which in Aqueous solution exhibits a spectrum of circular dlcroism, consistent with a state of structural disorder. However, in 15 mM solution of bicarbonate or sulphites, the peptide adopts a helical structure. In the case of buforin II and cathelicldin, the extension of the helix correlates directly with the bactericidal activity against Gram positive and Gram negative; as the content of α-helix increases, antimlcrobial activity increases (Park et al., 2000). 2). Peptides rich in proline and arginlna or another amino acid, formed by cationic peptides, rich in certain amino acids, which adopt an extended helical structure, (Otvos, 2002). This group includes bactericins and PR-39, which are rich in proline residues (33 to 49%) and arglnine (13 to 33%), profenin that is rich in proline (57%) and phenylalanine (19%) and Indolicidin rich in tryptophan. These peptides lack cysteine residues and are linear, although some may form extended spin structures (Brogden, 2005). 3). Peptide with β-fold, is formed by cathonic and anlonic peptides, which contain cysteine residues, form disulfide bonds and stable β-folded structures, which comprise alpha and betadefensins (Harder and cois., 1997; Schróder and Harder, 1999 ), and the tachyplesln / protegrin group (Nakamura et al., 1988: Iwanaga et al., 1994; Kokryakov et al., 1993), which contains several disulfide bridges, and adopt either alpha or beta folds or beta fork -, it is sometimes associated with a propeller-cutter as in scorpion defenses (Coclancich and cois., 1993) This group includes porcine leukocyte protegrin, consisting of 16 amino acid residues, including 4 clsteins that form two intramolecular disulfide bridges, and a diverse family of defensins (Brogden, 2005). In mammals, 55 alpha-defensins have been identified, including HNP 1-4 peptides (Human Neutrophll Peptlde) (Ganz and Lehrer, 1994; Lehrer and Ganz, 1999) and cryptidines, which contain 29 to 35 amino acids including 6 cysteines that form 3 bridges. 4). Neutral or anonymous antimicrobial peptides, only partly form a helix, such as bacteriocins, some peptides are rich in glycine, with a size between 7 to 8 kDa. In mammals, these are found in surfactant extracts, bronchuloalveolar fluids and respiratory tract epithelial cells (Brogden, 1998; Brogden, 1999), are produced in millimolar concentrations (mM), require zinc as a cofactor for antimicrobial activity and are active against Gram positive and Gram negative bacteria ; they are similar to the propeptide peptide neutralizing zymogens of larger size, have antimicrobial activity when they are alone (Brogden, 1997).

The production of these peptides, which are encoded in the genome, is highly regulated, since in addition to their antimicrobial effect, they participate directly regulating other tissue processes, such as inflammatory processes through chemotaxis functions, angiogenesis promotion and tissue repair. damaged and, in the modulation of the immune response (Kozuella et al., 2007; Schauber and Gallo, 2007). Consequently, the name of antimicrobial peptides that were initially assigned does not involve all the multiple functions performed by these important bioactive molecules, it is for this reason - being involved in various defensive mechanisms - is that today they are called host defense peptides ( PDH). Host defense peptides

The fact that microbial agents lack cholesterol in their cytoplasmic membranes probably allows the specific action of PDHs by increasing their selectivity and on the contrary the existence of cholesterol in the membranes of the host cell can serve as a protector of the deleterious effects of peptides On the other hand, in many cases, cells with a high transmembrane potential are more susceptible to the lethal effects of the peptides. Said potential, being negative inside the cell, can provide an electroendosomal force such that it helps the integration of PDH with positive charge in the white membrane (Iwasaki et al., 2009). There is ample evidence that the activity of most PDHs is not receptor-mediated, demonstrated by the fact that synthetic PDHs composed only of D-amino acids (Bessalle et al., 1990; Wade et al., 1990; Hetru et al., 2000), or by D- and Lamlnoaclds in random sequences (Shai and Oren, 1996; Papo, and cois., 2002) are biologically active as are their counterparts consisting of L-amino acids. This observation highlights the uniqueness of the mechanism of action of PDH compared to most chemopharmaceuticals, which are direct inhibitors of enzymes with chirality of active sites.

Today, there will be 1,400 to 1,500 different PDHs, 77% of which correspond to those with antibacterial activity, 28% antifungal, 6% antiviral and 6% anticancer (Ladokhín and White, 2001). Globally, several research groups are working in this area, it has grown so much that today there is a database of antimicrobial peptides, called APD2 (http://aps.unmc.edu/AP/main.php), in She can look for peptides from different families (bacteriocins, cyclotides or defensins), from different animal sources such as: fish, amphibians, birds or mammals. For peptides from plants, there is another page, exclusive to them, the database is called PhytAMP (http: //phytamp.pfba- lab-tun.org/). (Oren and Shai, 1998).

Peptides in Arachnid poisons

Spider venoms contain a large amount and diversity of toxins that target a wide range of receptors, channels and enzymes in a wide range of vertebrate and invertebrate species. Each poison, in its composition, would have its own peculiarities (Foelix, 1996; Saez and cois., 2010). Spider species described range from 30 thousand to 50 thousand species reported in the world (Rosenthal et al., 1989), while scorpionidae would be represented by about 1500 species in the world (Possani, 2004). Chemically, the venom of these species has been defined as a "multicomponent system," arachnid venoms contain salts, small organic molecules, proteins and among other components of low molecular weight peptides ranging from 3 to 10 KDa (Escoubas and cols., 2000; Rash and Hodgson, 2002; Tedford et al., 2004; Estrada et al., 2007; Vassilevsk and cois., 2009), each of them can be separated by high pressure liquid chromatography (HPLC) and those components with antimicrobial, anti-cancer, anti-inflammatory or analgesic activity can be accurately determined.

At the University of Mexico (UNAM), it has been shown that Mexican tarantulas, far from being a danger to humans, represent an alternative to combat some health problems that are in force. The toxins mainly target ionic membrane channels because, by joining them, they prevent the flow of ions in neurons causing an imbalance in the action potentials, which produces among other cellular effects, a muscular paralysis in insects (Escoubas and cois. , 2000; Escoubas and Rash, 2004).

Although only a small number of spider venom peptides have been pharmacologically characterized, the variety of biological activities described are impressive (Vassilevski et al., 2009). In addition to the well-known neurotoxic effects of spider venom, which contain peptides with antiarrhythmics, antibiotics, analgesic activity, antiparasitic inhibitor, cytolytic, hemolytic, and enzymatic activity. On the other hand, the raw poison of Macrothele raveni has antitumor activity on human hepatocarcinoma cells (BEL-7402), but the responsible component has not yet been identified (Gao et al., 2005; Gao et al., 2007). Other toxins, such as the latrotoxins of Latrodectus mactans and other related species, induce the release of neurotransmitters and have played an important role in the dissection of the exocytosis process of Ushkaryov and cois synaptic vesicles, 2004).

Peptides of the ICK type, U1-TRTX-Pc1 a (Psalmopeotoxin I) and U2-TRTX-Pc1 a (Psalmopeotoxin II), isolated from the venom of the Psalmopoeus cambridgei tarantula are effective against intra-erythrocyte forms of Plasmodium falciparum (Staines and cois. , 2005). Interestingly, this is the same spider from which Psalmotoxin p-TRTX-Pd a was isolated, the most potent blocker known from ASICI a (Escoubas et al., 2003). Psalmotoxin is a peptide of 40 acidic amino acids, which has 6 cysteines linked by three disulfide bridges. The three-dimensional structure consists of a compact core joined by disulfide bridges from which, three turns and the N and C ends arise. The main element of the structure is an antiparallel with three chains, β sheets (Escoubas et al., 2000). Yan and Adams (1998) describe Licotoxins an antimicrobial peptide obtained from the wolf spider, Lycosa carolinensis, two Licotoxins I and Licotoxins II peptides strongly inhibit the development of Escherichia coli Candida glabatra (Adáo et al., 2008). Similarly, Kuhn-Nentwig and cois. (2002), describe a new family of highly basic antimicrobial peptides, isolated from the Cupiennius salei spider (Ctenidae). Cupiennin 1 a would interact with phospholipid bait, being able to alter the cytoplasmic membranes of prokaryotes and eukaryotes (Pukala et al., 2007). A conservative prediction, based on proteomic and transcriptomic studies, suggests that spider venoms contain more than 10 million bioactive peptides, so it is certainly a valuable resource for the discovery of new drugs (Saez et al., 2010) .

On the other hand, scorpion venom is a complex mixture, composed of a wide range of substances. It contains mucopolysaccharides, hyaluronidase, phospholipase, peptides of relatively low molecular weight, molecules such as serotonin, histamine, protease inhibitors and histamine releasers. It is also a rich source of toxic peptides that affect the function of ion channels, excitable and non-excitable cells (Simard and Watt, 1990; Possani et al., 2000). It has been proposed that 100,000 different peptides would exist among the 1500 different species existing worldwide, of which only 0.02% are known to interact with ion channels (Possani et al., 2000), 191 with Na channels + (Rodríguez de la Vega and Possani, 2005), 121 with K + channels (Rodríguez de la Vega and Possani, 2004), 5 with Cl - channels (Debin et al., 1993), 2 with Ca2 + channels ( Possani et al., 2000) and 2 to selective channels to RYR peptides (Valdivia and Possani, 1998). Cysteine-free antimicrobial peptides were also identified and characterized from the venom of six scorpion species. These peptides have the ability to integrate into bacterial membranes or those of mammals and transmembrane pores form (Verdonck et al., 2000; Moerman et al., 2002). Non-antlmlcroblane-free Clstein peptides have also been isolated from scorpion venom. These peptides have activity either potentiating bradykinin (Ferreira et al., 1993; Meki et al., 1995.), or the activity of the peptides is simply unknown at present (Ali et al., 1998). Most of the efforts in the discovery of new peptides in the scorpion venom have focused on toxic ion channels. Characteristics of these peptides include pore formation and / or antimicrobial activity. These peptides may have the ability to fight cancer, a variety of bacterial skin lesions or fungal infections (Elgar et al., 2006).

Recently, numerous studies show the effect of poisonous substances with anticancer properties, whose mechanism observed and described, has been the inhibitory capacity on cell adhesion and proliferation. In the case of the scorpion venom Odontobuthus doriae, an inducing effect of apoptosis and inhibition of DNA synthesis in neuroblastoma cells (Zargan et al., 2010) has been demonstrated, in the scorpion venom Tityus discrepans two new peptides have been described (neopladine 1 and 2) that were active against human breast carcinoma cells (D'Suze et al., 2010). On the other hand, there are drugs based on the mechanism of action of metabolites of poisons and are used as therapeutic agents in a variety of ailments, including Captopril that would act as an antihypertensivist and comes from bradykinin-enhancing peptides existing in the Bothrops jararaca venom. (Ferreira et al., 1965; Hayashi and Camargo, 2005). Cui and cois., (2010), cloned a peptide with antitumor and analgesic activity from genomic DNA of the Chinese scorpion Buthus martensii Karsch (BmKAGAP).

Blue scorpion venom, Rhopalurus junceus initially marketed under the name of Escozul, is used in the treatment of different pathologies of tumor origin, the venom of Blue scorpion has a molecular composition similar to that described for other species with a molecular mixture of less than 14 kDa (Betancourt et al., 2008). The therapeutic use of blue scorpion toxin (Rhopalurus junceus) was discovered after a decade of work by Cuban biologist Misael Bordier, who initiated the scorpion hatchery and its research at the Guantanamo School of Medical Sciences at the end of the 80s of the last century. The toxin, once formulated and sterilized, constitutes the natural product Escozul. It is attributed to antimicrobial and antitumor properties demonstrated through some preclinical studies (Rodríguez et al., 2004). Today, the Escozul, is being produced on a large scale by the LabioFam Laboratory and sold worldwide with a new trade name of Vidatox 30 CH, is a homeopathic medicine, whose active ingredient is the scorpion venom "Rhopalurus junceus ", an endemic arthropod from Cuba. It is a product with analgesic, anti-inflammatory and antitumor properties successfully applied in humans for three years, after having experimented in various biological models.

Scorpions as a group of arthropods have a wide range of peptides in their poisons, of which a large number have antimicrobial activity, which have received increasing attention based on the growing need for alternatives to combat pathogenic bacteria. (Harrison and cois, 2014).

Scorpion venom of the genus Caraboctonus sp.

For the scorpion genus Caraboctonus sp. There are no publications describing the poison or its components.

Tarantula venom of the genus Aphonopelma sp.

Poison of two species of the genus Aphonopelma sp. (A. seemani found in Costa Rica, and A. spp. Found on the Island of Saint Kitts) was collected and characterized and its hyaluronidase, protease and phospholipase activities were compared with other previously known poisons. The finding of peptides as such is not reported (Hooper and cois, 1986).

Savel-Niemann (1989) describes a series of peptides called ESTX isolated from the Eurypelma californicum venom that have a variation in amino acid at position 26. It is reported that they were found to be homologous with venom toxins of various scorpions and with a toxin of the Spider venom Segestria florentina. Later Pan and Yu (2010) cite Savel-Niemann describing that Eurypelma californicum is actually Aphonopelma californicum, and that the ESTxl toxin found by Savel-Niemann in this spider has a high degree of homologamlento with the HnTxll toxin reported by these authors found in the poison of the Chinese bird spider (Haplopelma hainanum).

Poison of two species of the genus Aphonopelma sp. has been reported with a clotoxic effect in various lines of

The insecticidal activity of peptides of the venom of the tarantula Brachypelma albiceps has been described, and it has been established that its amlnoacidic sequence is highly similar to the insecticidal peptides of the genus Aphonopelma sp. reported as coming from the USA. (Corzo and cois, 2009).

There are no applications or patents granted that specifically protect any peptide of any type obtained from scorpions of the genus Caraboctonus sp. or tarantulas of the genus Aphonophelma sp. That said, if there are publications of patent applications or granted patents in which reference is made to peptides with identified sequences that can be defined as homologous to those of the genus Aphonopelma sp.

Of particular interest in this regard is the granted patent US8399026 B2 (Peptides solated from splder venom, and uses thereof: Peptides isolated from spider venom and its uses), which, in its description of the invention, establishes that said Invention in one of its embodiments, the peptide purified or isolated from spider venom may comprise or consist of an amino acid sequence having 2 to 50 consecutive amino acids, of 10 at 50 consecutive amino acids, from 20 to 50 consecutive amino acids, 25-45 consecutive amino acids, from 29 to 40 consecutive amino acids, 29-35 consecutive amino acids, at least 29, and in most of the 35 consecutive amino acids. In a further embodiment, the purified or isolated peptide has a molecular weight in the range of 3000 to 5000 daltons, 3,250 to 4,750 daltons, 3,500-4,500 daltons, 3550 to 4,300 daltons, or 3,600 to 4,250 daltons. Said patent, in its detailed description of the invention, states that the term "spider" refers to spiders of the family Theraphosidae, which includes the genus Aphonopelma sp.

Description of the Invention

Low molecular weight peptides (between 5 and 35 kDa) have been obtained from the venom of the endemic poisonous arthropod species of Chile Caraboctonus keyserlingi and

Aphononopelma aberrans, purified by HPLC and these purified peptides have been evaluated in their ability to be used as an antibacterial active substance.

The invention disclosed herein, in its manifestation or main embodiment, is described sequentially below:

Scorpion:

• Scorpion specimens of the endemic species of Chile Caraboctonus keyserlingi are captured. The collected scorpions are identified by a morpho-taxonomic study to ensure that they work with specimens of the indicated species. Additionally, the geographical location of the collection helps to ensure that the species is effectively described as its natural habitat.

• The poison is extracted by electrostimulation.

• The extracted venom is preferably subjected to a process (CLAR, or HPLC) of separation of chromatographic peaks corresponding to the peptide fractions of interest by means of the high resolution liquid chromatography technique. Preferably a Phenomenex Luna® 5 μιη C18 100 Á, LC column will be used Column 250 x 10 mm. Conditions, linear gradient from 0 to 60% acetonitrile with (TFA) at 0.1% for 50 minutes with a flow rate of 2ml / min, 40 ° C. Alternatively, as a way to focus the separation and reduce the HPLC process time, it is to perform an electrophoretic separation of the venom by Tricine SDS-PAGE at 16% / 6%. This preprocessing methodology is used to separate proteins in the 1-100 kDa mass range, and is an electrophoretic approximation of choice for the resolution of proteins smaller than 30 kDa, which are of interest for their antimicrobial action. Once the peptide bands smaller than 35 kDa are obtained, this band is cut from the gel, the peptides are eluted with appropriate solvents, and the peptides contained in the band are purified by HPLC.

• HPLC purified peptides are obtained in the solution of Acetonitrile / trifluoroacetic acid in which they pass through the chromatographic separation column. This solvent mixture is relatively volatile, so after a short period of drying, the pure dry peptide is finally obtained. This dry peptide is resuspended in 50 μΙ ultra pure water, to achieve a concentration of the peptide between 5 and 20 pgr / l, preferably 10 pgr / l, this being the final product obtained.

• The peptide fractions identified with antimícrobiana activity are called PO and P1.

Tarantula:

· Tarantula specimens of the endemic species of Chile are captured

Aphononopelma aberrans The collected tarantulas are identified by a morpho-taxonomic study to ensure that they work with specimens of the indicated species. Additionally, the geographical location of the collection helps to ensure that the species is effectively described as its natural habitat.

· The poison is extracted by electrostimulation. • The extracted venom is preferably subjected to a process (CLAR, or HPLC) of separation of chromatographic peaks corresponding to the peptide fractions of interest by means of the high resolution liquid chromatography technique. Preferably, a Phenomenex Luna® 5 μιη C18 100 Á, LC Column 250 x 10 mm column will be used. Conditions, linear gradient from 0 to 60% acetonitrile with 0.1% acidotrifluoroacetic acid (TFA) for 50 minutes with a flow rate of 2ml / min, 40 ° C. Alternatively, as a way to focus the separation and reduce the HPLC process time, it is to perform an electrophoretic separation of the venom by Tricine SDS-PAGE at 16% / 6%. This preprocessing methodology is used to separate proteins in the 1-100 kDa mass range, and is an electrophoretic approximation of choice for the resolution of proteins smaller than 30 kDa, which are of interest for their antimicrobial action. Once the peptide bands smaller than 35 kDa are obtained, this band is cut from the gel, the peptides are eluted with appropriate solvents, and the peptides contained in the band are purified by HPLC.

· HPLC purified peptides are obtained in the solution of

Acetonitrile / trifluoroacetic acid in which they pass through the chromatographic separation column. This solvent mixture is relatively volatile, so after a short period of drying, the pure dry peptide is finally obtained. This dry peptide is resuspended in 50 μΙ of ultrapure water, to achieve a concentration of the peptide between 5 and 20 pgr / l, preferably 10 pgr / l, this being the final product obtained.

• The peptide fractions identified with antimícrobiana activity are called P5 and P6, the latter being subsequently subjected to a second chromatographic separation step that allows two peptide fractions called P6.1 and P6.2 to be separated.

· Peptide fractions isolated and purified from the poison of A. aberrans were subjected to analysis using the MALDI-TOF Autoflex Speed (Bruker) equipment using the internal sample preparation protocol, using alpha-cyanohydroxycinmic acid matrix. The protocol consists of a mixture of 1uL of a saturated solution of the respective matrix (TA 30-30/70 ACN: 0.1% TFA) with 1 uL of sample solution. The purified fraction P5 consists of 9 peptide molecules whose molecular weight in Daltons is respectively: 3,480.7, 3,649.0, 3,771, 9, 3,788.9, 3,821, 4, 3,883.1, 4,265.7, 4,630.0, and 4,763 , 5, the largest component in terms of mass of this P5 fraction being the 3,883.1 Daltons peptide (greater than 60%). The purified fraction P6 consists of 5 peptide molecules whose molecular weight in Daltons is respectively: 2403.7, 3,887.7, 4,166.5, 4,186.4 and 4,264.9, the largest component in terms of mass of this fraction P6 being the 4,264.9 Daltons peptide (greater than 60%).

Definitions

As used herein, the term "peptide" means a compound that is composed of two or more amino acids linked by covalent bonds that are formed by the removal of an H2O molecule from the amino group of an amino acid and the carboxyl group of the following amino acid

The term "isolated" for the purposes of the present invention designates a biological material (nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a peptide present in the natural state in an animal is not isolated, however the same peptide separated from the adjacent amino acids in which it is naturally present is considered "isolated."

The term "purified" does not require that the material be present in a form with absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition. A peptide is in the "purified" state after purification of the starting material or natural material by at least an order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

As used herein, the term "substantially pure" describes a peptide or other material that has been separated from its native contaminants. Typically, a monomeric peptide is substantially pure when at least about 60 to 75% of a sample exhibits a single peptide backbone. Minor variants or chemical modifications usually share the same peptide sequence. Generally, a substantially pure peptide will comprise more than about 85 to 90% of a peptide sample, and in particular will be more than 95% pure, more than 97% pure, or will be more than about 99% of purity. Normally, purity is measured on a polyacrylamide gel, with homogeneity determined by staining. Alternatively, for certain purposes, a high resolution will be necessary and HPLC or a similar means will be used for purification. For most purposes, a simple polyacrylamide gel chromatography column will be used to determine purity.

Examples

The peptides that constitute the invention described herein have their main use as antibacterial agents, a series of examples of their application having been demonstrated that demonstrate this practical utility, described below:

Antibacterial activity of PO and P1 Peptides obtained from Caraboctonus keyserlingí venom applied on bacterial cultures growing in gelled medium:

The example corresponds to the description of Figures 1A and 1 B. Bacterial suspensions of 100 μΙ with 0.2 OD were inoculated to 9.9 ml of LB medium with 0.8% agar, then plated and cultured at 37 ° C for 7 h after the addition on the gelled raw venom agar and dilutions thereof. Positive control corresponds to 5 μΙ of antibiotic (ATB) (carbenicillin 30 μg) dissolved in water, while the negative control 5 μΙ of water, mobile phase (FM) was also evaluated. Total venom (16.2 μς / μΙ) P0 (0.026 μς / μΙ) P1 (0.0128 μς / μΙ) P2 (0.59 μς / μΙ) P3 (1, 416 Mg / μΙ) P4 (0.94 μς / μΙ) P5 (0.88 μς / μΙ) P6 (0.114 μς / μΙ) P7 (0.234 μς / μΙ). (A) Activity of raw venom and PO to P5 fractions on S. aureus (ATCC®29213), (B) Activity of raw venom and PO to P5 fractions on E. coli (ATCC®25922). Antibacterial activity of the purified PO and P1 fractions on gram positive and gram negative bacteria is clearly observed.

Antibacterial activity of peptides P5 and P6 obtained from the venom of Aphonopelma aberrans applied on bacterial cultures growing in gelled medium:

The example corresponds to the description of Figure 2. Bacterial suspensions of 100 μΙ with 0.2 OD were inoculated to 9.9 ml of LB medium with 0.8% agar, then plated and cultured at 37 ° C by 7 h after the addition on the gelled agar of the fractions P0 to P7 obtained by fractionation in HPLC. In order to concentrate each fraction, 6 fractionations of the poison were performed and 100 μΙ of sample was injected for each collection. Positive control corresponds to 5 μΙ of antibiotic (ATB) (carbenicillin 30 μg) dissolved in water, while the negative control 5 μΙ of water, mobil phase (FM) was also evaluated. Activity of the fractions on E. coli (ATCC® 25922), P0 (3.12 μς / μΙ), P1 (0.034 μς / μΙ), P2 (0.344 pg / pl), P3 (1.028 Mg / μΙ), P4 (4.01 μς / μΙ), P5 (4.46 μς / μΙ), P6 (4.55 μς / μΙ) and P7 (1.55 μς / μΙ). Antibacterial activity of the purified fractions P5 and P6 is observed.

Antibacterial activity of Peptides P0 and P1 obtained from the venom of Caraboctonus keyserlingi applied on bacterial culture growing in liquid medium:

The example refers to Figure 3. 10 μΙ (10 gr / ml) of the purified peptides P0 and P1 are added to 100 μΙ of bacterial culture in liquid medium logarithmic growth phase (adjusted to <0.1 OD595 nm) . Negative controls were also included using 10 μΙ of ultrapure water (type I). Microbial growth (S. aureus) was measured as the increase in OD595 nm in a Bio-Rad microplate reader, model 550 after incubation at 37 ° C for 5 hours under agitation with some modifications as described by Bulet and cois., (1995), Ehret-Sabatler and cois., (1996) and Haeberll (2000). It can be seen that both peptides P0 and P1 have a growth inhibitory action of S. aureus.

Antibacterial activity of peptides P5 and P6.1 obtained from the venom of Aphonopelma aberrans applied on bacterial culture growing in liquid medium:

The example refers to Figure 4. 10 μΙ (10 pgr / ml) of the purified peptides P5 and P6.1 are added to 100 μΙ of bacterial culture in liquid medium in logarithmic growth phase (adjusted to <0.1) OD595 nm). Negative controls were also included using 10 μΙ of ultrapure water (type I). Microbial growth (S. aureus) was measured as the increase in OD595 nm in a Blo-Rad mlcroplate reader, model 550 after incubation at 37 ° C for 5 hours under agitation with some modifications as described by Bulet and cois., (1995), Ehret-Sabatier and cois., (1996) and Haeberli (2000). It can be seen that both peptides P5 and P6.1 have a growth inhibitory action of S. aureus.

Antibacterial activity of Peptides P0 and P1 obtained from the venom of Caraboctonus keyserlingi in bactericidal antibacterial assay in 96-well microplates:

This example refers to Figures 5 and 6. The microorganisms were cultured in liquid nutrient medium LB for E. coi After incubation for 16-18 h at 37 ° C under stirring (200 rpm) the bacteria were collected by centrifugation (4300 rpm for 10 min at 4 ° C), and resuspended in phosphate buffer pH 7.4 (PBS) and adjusted to 0.2 OD595 nm. Briefly, in order to measure the antibacterial activity of the P0 and P1 fractions obtained by analytical HPLC, different concentrations in a volume of 10 μΙ were added in 96-well microplates with pH 7.4 saline phosphate buffer (PBS) that previously contained a volume of 100 μΙ of bacterial culture. At the same time, gentamiclna (128 μg / mL) was used as a positive control for Gram negative microorganisms, the negative controls were also included using ultrapure water (type I). After incubation of the microplate at 25 ° C for 12 hours as described by Haerberll et al., (2000), the optical density of the suspension was measured bacterial For this, a Bio-Rad model 550 microplate reader was used, in which, after stirring, the wavelength was measured at 595 nm, in order to detect if there was an increase or decrease in the absorbance of E. coli. In Figures 5 and 6, it can be seen that the PO and P1 peptides from C. keyserlingi purified on an analytical column have an evident bactericidal activity on E. coli, said activity in both cases is concentration dependent.

Antibacterial activity of peptides P5 and P6.1 obtained from the venom of Aphonopelma aberrans in bactericidal antibacterial assay in 96-well microplates: This example refers to Figures 7 and 8. The microorganisms were cultured in liquid nutrient medium LB for E coli After incubation for 16-18 h at 37 ° C under stirring (200 rpm) the bacteria were collected by centrifugation (4300 rpm for 10 min at 4 ° C), and resuspended in phosphate buffered buffer pH 7.4 (PBS) and adjusted to 0.2 OD595 nm. Briefly, in order to measure the antibacterial activity of the P0 and P1 fractions obtained by analytical HPLC, different concentrations in a volume of 10 μΙ were added in 96-well microplates with pH 7.4 saline phosphate buffer (PBS) that previously contained a volume of 100 μΙ of bacterial culture. At the same time, gentamicin (128 pg / mL) was used as a positive control for Gram negative microorganisms, negative controls were also included using ultrapure water (type I). After incubation of the microplate at 25 ° C for 12 hours as described by Haerberli et al., (2000), the optical density of the bacterial suspension was measured. For this, a Bio-Rad model 550 microplate reader was used, in which, after stirring, the wavelength was measured at 595 nm, in order to detect if there was an increase or decrease in the absorbance of E. coli. In Figures 7 and 8, it can be seen that peptides P5 and P6.1 from A. aberrans and purified on an analytical column show an evident bactericidal activity on E. coli, said activity in both cases is concentration dependent. The examples described herein are only part of the possibilities of application of the present invention and do not constitute in any way an exhaustive description thereof, it being possible for anyone skilled in the art to find multiple other applications of the invention in the scope of application. stated, considering all of them tacitly described as part of the present invention.

Claims

1. A peptide fraction obtained from the Caraboctonus keyserlingi venom called PO, characterized in that said PO peptide fraction has a molecular weight between 5 and 35 Kilodaltons, preferably less than 10 Kllodaltons.

2. A peptide fraction obtained from the venom of Caraboctonus keyserlingi called P1, characterized in that said peptide fraction P1 has a molecular weight between 5 and 35 Kilodaltons, preferably less than 10 Kllodaltons.

3. A peptide fraction obtained from the Aphanopelma aberrans venom called P5, characterized in that said peptide fraction P5 has a molecular weight between 3,480.7 and 4,763.5 Daltons, the peptide being the largest component in terms of mass of this fraction of 3,883.1 Daltons.

4. A peptide fraction obtained from the Aphanopelma aberrans venom called P6, characterized in that said peptide fraction P6 has a molecular weight between 2403.7 and 4,264.9 Daltons, the peptide being the largest component in terms of mass of this fraction of 4,264.9 Daltons.

5. The peptide fraction of claim 1, characterized by presenting antimlcrobial activity against Staphylococcus aureus (ATCC®29213) and on Escherichia coli (ATCC®25922). 6. The peptide fraction of claim 2, characterized by presenting activity antimicrobial against Staphylococcus aureus (ATCC®29213) and on Escherichia coli (ATCC®25922).

The peptide fraction of claim 3, characterized by presenting antimicrobial activity against Staphylococcus aureus (ATCC®29213) and on Escherichia coli (ATCC®25922).

The peptide fraction of claim 4, characterized by presenting antimicrobial activity against Staphylococcus aureus (ATCC®29213) and on Escherichia coli (ATCC®25922).

A method of producing the peptide fractions of claims 1, 2, 5 and 6, characterized in that said production method consists in extracting the venom from Caraboctonus keyserlingi by electrostimulation, then by which the extracted poison is preferably subjected by means of the High performance liquid chromatography technique to a process of separation of chromatographic peaks that will correspond to the peptide fraction of interest. Preferably, a Phenomenex Luna® 5 μιη C18 100 Á, LC Column 250 x 10 mm column will be used. Conditions, linear gradient from 0 to 60% acetonitrile with 0.1% acidotrifluoroacetic acid (TFA) for 50 minutes with a flow rate of 2ml / min, 40 ° C. Alternatively, as a way to focus the separation and reduce the HPLC process time, it is to perform an electrophoretic separation of the venom by Tricine SDS-PAGE at 16% / 6%. This preprocessing methodology is used to separate proteins in the 1-100 kDa mass range, and is an electrophoretic approximation of choice for the resolution of proteins smaller than 30 kDa, which are of interest for their antimicrobial action. Once the peptide bands smaller than 35 are obtained kDa, this band is cut from the gel, the peptides are eluted with appropriate solvents, and the peptides contained in the band are purified by HPLC. Peptides purified by HPLC are obtained in the solution of Acetonitrile / trifluoroacetic acid in which they pass through the chromatographic separation column. This solvent mixture is relatively volatile, so after a short period of drying, the pure dry peptide is finally obtained. This dry peptide is resuspended in 50 μΙ ultra pure water, to achieve a concentration of the peptide between 5 and 20 pgr / l, preferably 10 pgr / l, this being the final product obtained. 10. A method of producing the peptide fractions of claims 3, 4, 7 and 8, characterized in that said production method consists in extracting the Aphonopelma aberrans venom by electrostimulation, then by which the extracted venom is preferably subjected by means from the technique of high performance liquid chromatography to a process of separation of chromatographic peaks that will correspond to the peptide fraction of interest. Preferably, a Phenomenex Luna® 5 μιη C18 100 Á, LC Column 250 x 10 mm column will be used. Conditions, linear gradient from 0 to 60% acetonitrile with 0.1% acidotrifluoroacetic acid (TFA) for 50 minutes with a flow rate of 2ml / min, 40 ° C. Alternatively, as a way to focus the separation and reduce the HPLC process time, it is to perform an electrophoretic separation of the venom by Tricine SDS-PAGE at 16% / 6%. This preprocessing methodology is used to separate proteins in the 1-100 kDa mass range, and is an electrophoretic approximation of choice for the resolution of proteins smaller than 30 kDa, which are of interest for their antimicrobial action. Once the peptide bands smaller than 35 kDa are obtained, this band is cut from the gel, the peptides are eluted with appropriate solvents, and the peptides contained in the band are purified by HPLC. Peptides purified by HPLC are obtained in the solution of Acetonitrile / trifluoroacetic acid in which they pass through the chromatographic separation column. This solvent mixture is relatively volatile, so after a short period of drying, the pure dry peptide is finally obtained. This dry peptide is resuspended in 50 μΙ ultra pure water, to achieve a concentration of the peptide between 5 and 20 pgr / l, preferably 10 pgr / l, this being the final product obtained.

11. The peptide fraction of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, characterized in that they are used as broad-spectrum antibacterial agents.