WO2020065595A1 - Short leopeptides with antimicrobial activity against gram negative and gram positive bacteria - Google Patents

Abstract

The present invention relates to synthetic leopeptides of formula C_n-X₁-X₂-X₃-NH₂ , wherein: C_n is a fatty acid selected from the group consisting of C₁₂ to C16; X₁ is at least one glycine molecule; X₂ is at least two natural amino acids that have a positive net charge and/or are not proteinogenic; and X₃ can be present or absent, and when it is present it is at least one aliphatic amino acid. The lipopeptides have antimicrobial activity against pathogenic bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Serratia marcescens, Staphylococcus saprophyticus, Staphylococcus haemolyticus and Streptococcus salivarius), at minimum inhibitory concentrations (MIC) between 8 and 50 µM. In addition, they do not display toxicity in human red blood cells or in mosquito larvae in concentrations between 3.13 and 50.0 µM at an evaluated concentration of 100 µM.

Description

 SHORT LIPOPEPTIDES WITH ANTIMICROBIAL ACTIVITY AGAINST

NEGATIVE GRAM BACTERIA AND POSITIVE GRAM

FIELD OF THE INVENTION

The present invention belongs to the fields of organic chemistry, pharmaceutical chemistry and medicine, in particular, to short lipopeptides synthesized by Fmoc solid phase synthesis, with antimicrobial activity against Gram-negative and Gram-positive bacteria.

BACKGROUND OF THE INVENTION

Infectious diseases and antimicrobial resistance

Infectious diseases are caused by pathogenic microorganisms such as bacteria, viruses, fungi or parasites, which multiply in the host's tissues causing various symptoms (Brugueras MC and García MM. 1998. Antibacterials with systemic action. Part I. Beta-lactam antibiotics. Revista Cubana of Integral Medicine; 14: 347-361). Diseases such as tuberculosis, pneumonia, malaria, HIV, among others, have generated an increase in the mortality rate, causing social and economic consequences and threatening global public health (Sugden R, et al. 2016. Combatting antimicrobial resistance globally Nature Microbiology; l: l-2).

The World Health Organization (WHO - WHO, English World Health Organization) as the directing authority and coordinator of health action in the United Nations system, has reported high resistance to antibiotics that are used conventionally, of pathogenic bacteria such as Klebsiella pneumoniae, Escherichia cotí, Staphlylococcus aureus, Streptococcus pneumoniae, among others. Although antimicrobial resistance has been around since the first antibiotics came on the market in the 1930s, as several studies show (O'Neill J. 2016. Tackling drug-resistant infections globally: final report and recommendations. The Review on Antimicrobial Resistance: 1-80; Sugden et al. 2016. Op cit), the inadequate management given to these, such as excessive or empirical use, is one of the reasons that has led to the greater development of resistance on the part of of microorganisms (Singh S, et al. 2017. Understanding the mechanism of bacterial biofilms resistance to antimicrobial agents. The Open Microbiology Journal; 11: 53-62). Additionally, the excessive use of antibiotics in agriculture and in farm animals such as pigs and poultry contributes to increased resistance to antimicrobials, as has been reported for example in countries of the European Union (Carattoli A, et al. 2017 . Novel plasmid-mediated colistin resistance mcr-4 gene in Salmonella and Escherichia coli, Italy 2013, Spain and Belgium, 2015 to 2016. Euro Surveillance; 22: 30589; Kinross P, et al. 2017. Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) among human MRSA isolates, European Union / European Economic Area countries, 2013. Euro Surveillance; 22: l-l3).

This problem of antimicrobial resistance mainly affects patients with compromised immune system, with chronic underlying diseases and in routine or more complex surgical procedures, who receive treatments such as cancer chemotherapy, where the use of antibiotics is essential to avoid infections (Ma Q, et al. 2017. Antimicrobial resistance of Lactobacillus spp. from fermented foods and human gut. LWT - Food Science and Technology; 86: 20l-208; O'Neill J 2016. Op cit ).

Resistance manifests itself when a pathogenic microorganism is unaffected by exposure to a natural or synthetic antimicrobial drug that would usually inhibit its growth. The microorganism manages to avoid the action of the antimicrobial by means of acquired resistance mechanisms such as the production of enzymes that inactivate or destroy the drug, generating modifications at the molecular level, changing the objective of the antimicrobial compound or altering the permeability of the cell membrane (Holmes A , et al. 2016. Understanding the mechanisms and drivers of antimicrobial resistance. The Lancet; 387: l76-l87; Vignoli R and Seija V. 2000. Main mechanisms of antibiotic resistance. In: Topics of bacteriology and medical virology p. 649- 662).

BRIEF DESCRIPTION OF THE INVENTION

The present invention corresponds to synthetic lipopeptides of the formula

C „-XI-X ₂ -X3-NH ₂

where:

C _n is a fatty acid selected from the group consisting of Ci ₂ to C _IÓ ; Xi is at least one glycine molecule;

X ₂ is at least two net positively charged and / or non-proteinogenic natural amino acids; Y,

X ₃ can be present or absent and when present it is at least one aliphatic amino acid.

The length of the peptide sequence is from 3 to 5 amino acid residues, conjugated with a fatty acid between 12 and 16 carbon atoms, obtained by synthesis by the solid phase methodology Fmoc (9-Fluorenylmethoxycarbonyl), with a reaction yield of synthesis greater than 90% for all lipopeptides.

The new molecules called LIP 1, LIP 2, LIP 3, LIP 4, LIP 5, LIP 6, LIP 11 and LIP 12, are characterized by a purity greater than 90%, a secondary structure of random coil (random coil), determined by high efficiency liquid chromatography (HPLC), mass spectrometry (MS) and circular dichroism (CD) techniques, respectively. The molecular mass of lipopeptides determined by means of the protonated mass (M + H) is: LIP 1 = 485.4 Da, LIP 2 = 542.4 Da, LIP 3 = 613.5 Da, LIP 4 = 711, 5 Da, LIP 5 = 513.4 Da, LIP 6 = 570.5 Da, LIP 11 = 641.5 Da, LIP 12 = 739.6 Da.

Lipopeptides have antibacterial activity against pathogenic bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Serratia marcescens, Staphylococcus saprophyticus, Staphylococcus saprophycous, Staphylococcus saprophytic mM and verified by micrographs obtained by scanning electron microscopy (SEM). Additionally, they do not present toxicity in human erythrocytes at concentrations between 3.13 and 50.0 mM., Nor in the mosquito larvae of the species Aedes aegypti, at an evaluated concentration of 100 pM and they show stability against proteases present in serum human blood.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Structure of a synthetic lipopeptide. Lipopeptides have peptide sequences with a certain number of amino acids (n), where each contains a specific side chain (Ri or R ₂ ) and this sequence has a fatty acid conjugated to N-terminus that is given by a certain number of carbon atoms (x). The terminal carboxyl group of the peptide appears amidated, due to the synthesis process and the removal of the resin.

 FIG. 2 Solid phase lipopeptide synthesis scheme. The synthesis steps are indicated: deprotection of the resin, coupling and deprotection of amino acids for the formation of the peptide sequence, the conjugation of the fatty acid and the unpinning of the lipopeptide from the polymeric resin.

 FIG. 3 Molecular structures of the eight designed lipopeptides. Each lipopeptide presents its sequence and its net charge.

 FIG. 4 Chromatograms of lipopeptides synthesized by RP-HPLC. The chromatograms show a dead time of approximately 1.5 min and it is observed that the lipopeptides LIP 1 to LIP 4 have a retention time between 8.4 and 11.9 min and the lipopeptides LIP 5 to LIP 6 and LIP 11 to LIP 12 have a retention time between 10.6 and 14.2 min.

 FIG. 5 Mass spectra of lipopeptides synthesized by LC-MS. The spectra show the molecular ion of each of the lipopeptides and other signals corresponding to the formation of adducts.

 FIG. 6 CD spectra of lipopeptides synthesized at 5.0 mM in 30% TLE obtained in a spectropolarimeter. The spectra show a maximum and a minimum absorption, close to 200 nm and 220 nm, respectively.

 FIG. 7 Hemolytic activity of LIP 4 and LIP 12. The lipopeptides were evaluated in a concentration range of 3.13 to 50.0 pM in human erythrocytes that were incubated in 0.9% saline. Hemoglobin released by cell lysis was analyzed in a microplate reader at a wavelength of 545 nm.

 FIG. 8 SEM micrographs of P. aeruginosa cells treated with LIP 4. (A) Growth control for 20 h, (B) treatment for 30 min, (C) treatment for 2 h and (D) treatment for 20 h.

 FIG. 9 SEM micrographs of P. aeruginosa cells treated with LIP 12. (A) Growth control for 20 h, (B) treatment for 30 min, (C) treatment for 2 h and (D) treatment for 20 h.

FIG. 10 SEM micrographs of S. aureus cells treated with LIP 12. (A) Growth control for 20 h, (B) treatment for 30 min, (C) treatment for 2 h and (D) treatment for 20 h. FIG. 11 SEM micrographs of E. faecalis cells treated with LIP 12. (A) Growth control for 20 h, (B) treatment for 30 min, (C) treatment for 2 h and (D) treatment for 20 h.

 FIG. 12 SEM micrographs of E. coli cells treated with LIP 12. (A) Growth control for 20 h, (B) treatment for 30 min, (C) treatment for 2 h and (D) treatment for 20 h.

 FIG. 13 Stability of LIP 12 to blood serum proteases. The chromatograms obtained by RP-HPLC of each sample analyzed at different times are superimposed, in order to appreciate that there is no appearance of new species caused by proteolytic degradation.

DETAILED DESCRIPTION OF THE INVENTION Definitions

For purposes of interpreting this description, the following definitions will apply and when appropriate, terms used in the singular form will also include the plural form.

The terms used in the description have the following meanings unless the context clearly indicates otherwise:

Fatty acid: molecule of lipidic nature formed by a long linear hydrocarbon chain, of different number of carbon atoms with a carboxyl group at one end.

 ACN: Acetonitrile

 Therapeutic agent: chemical compound that is used to treat a disease.

 Amino Acid: An organic molecule with an amino and a carboxyl group attached to a central carbon and a variable side chain attached to the central carbon.

 Aliphatic Amino Acid: A hydrocarbon side chain amino acid such as alanine, valine, leucine, and isoleucine.

Non-proteinogenic amino acid: natural amino acids with biological function that are not part of proteins. Amphipathic: a molecule or compound that has the property of being both hydrophilic and hydrophobic.

 Anionic: compound that has a negative charge.

 Cationic: compound that has a positive charge.

 Minimum inhibitory concentration (MIC): minimum concentration of a compound that totally inhibits the growth of microorganisms.

 DCC: N, N’-dicyclohexylcarbodiimide

 DCM: Dichloromethane

 DMF: Dimethylformamide

 Fmoc: 9-Fluorenylmethoxycarbonyl

 Hydrophilic: compound that has an affinity for water and allows it to easily enter solution.

 Hydrophobic: compound that cannot be mixed with water.

 HOBT: l-Hydroxybenzotriazole

 Proteolytic inhibition: inhibition of the activity of a compound by its degradation by means of enzymes.

 IPA: Isopropyl alcohol

 Lipopeptide: molecule composed of a fatty acid fraction and a peptide fraction.

 Peptide: oligomer made up of 2 to 50 amino acids linked together by amide bonds known as peptide bonds.

 RPMI: Roswell Park Memorial Institute (Cellular Medium)

 t-Boc: Terbutoxycarbonyl

 TFA: Trifluoroacetic Acid

 TFE: 2,2,2-Trifluoroethanol

 TIS: Triisopropylsilane

Antimicrobial Peptides (AMPs)

Innate immune response, or non-adaptive immunity, is the natural defense form of living organisms against a broad spectrum of pathogenic microorganisms. This response involves the production of various molecules, within which are the antimicrobial peptides (AMPs, or Antimicrobial Peptides) or also known as host defense peptides (HDPs, Host Defense Peptides). (Boman HG. 1995. Peptide antibiotics and their role in innate immunity. Annual Review of Immunology; l3: 6l-92). MPAs present biological activities such as antimicrobial, antiviral, anticancer, immunomodulatory, among others, for which they have been extensively studied (Marcellini L, et al. 2010. Fluorescence and electron microscopy methods for exploring antimicrobial peptides mode (s) of action. : Antimicrobial peptides: methods and protocols p.249-266).

The AMPs contain between 2 and 50 amino acid residues, are cationic in nature (net positive charge that generally varies from +1 to +6 at physiological pH), and exhibit between 40% and 70% hydrophobicity in the amino acid sequence , which gives them an amphipathic character (Laverty G, et al. 2010. Antimicrobial activity of short, synthetic cationic lipopeptides. Chemical Biology and Drug Design; 75: 563-569; Conlon JM, et al. 2014. Potential therapeutic applications of multifunctional host-defense peptides from frog skin as anti-cancer, anti-viral, immunomodulatory, and anti-diabetic agents. Peptides; 57: 67-77).

The AMPs have linear or cyclic molecular structures, with defined secondary structures, which occur by folding between the amino acids of the sequence, through the formation of intermolecular bonds (hydrogen bridges), between the carbonyl group (C = 0) , the amino group (NH) and hydroxyl groups (OH) of the side chains. The types of secondary structures are, a-helices, b-leaves, mixed or extended structures in the membranous environments (De La Luente-Núñez C, et al. 2014. Synthetic antibiofilm peptides. Biochimica et Biophysica Acta; l858: l06l-l069 ).

The techniques to identify the structures of the AMPs can be high resolution such as Nuclear Magnetic Resonance (NMR), X-ray diffraction or low resolution such as Circular Dichroism (DC), which is one of the most used to confirm the structure of peptides. and proteins, for their easy access and for being a non-destructive technique (Marcellini L, et al. 2010. Fluorescence and electron microscopy methods for exploring antimicrobial peptides mode (s) of action. In: Antimicrobial peptides, Op cit. p. 249 -266; Kelly SM, al. 2005. How to study proteins by circular dichroism. Biochimica et Biophysica Acta - Proteins and Proteomics; 1975: 119-139). Identifying the secondary structure of the MPAs is important, because this is fundamental to the mechanism of action, which is based on the damage to the microorganism's cell membrane, caused by a distribution in the hydrophobic and hydrophilic regions of the secondary structure of the peptide that results in increased membrane permeability (Ong ZY, al. 2014. Strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials. Advanced Drug Delivery Reviews; 78: 28-45) .

The AMPs interact electrostatically between the charged hydrophilic region and the phosphate groups on the surface of the bacterial membrane, followed by the hydrophobic interaction with the lipid bilayer, forming pores and causing the penetration or disruption of the cell membrane leading to loss of membrane potential, the escape of cytoplasmic material and finally cell death. This mechanism differs from other antimicrobial compounds that have activity in intracellular targets (Ageitos JM, al. 2017. Antimicrobial peptides (AMPs): Ancient compounds that represent t novel weapons in thefight against bacteria. Biochemical Pharmacology; 133: 117-138) .

MPAs have multiple advantages, such as high selectivity, good efficacy, and standardized synthesis protocols. However, compared to other antimicrobials that are commercially available, they are prone to hydrolysis or oxidation, present physical and chemical instability, and are usually not available for oral use. For this reason, mechanisms are proposed to obtain new peptides, such as: protein fragmentation, in silico designs and / or rational designs, substituting amino acids from the sequence of a precursor peptide or conjugation with other macromolecules (Fosgerau K and Hoffmann T. 2015. Peptide therapeutics: current status and future directions. Drug Discovery Today; 20: 122-128).

Some of the most outstanding modifications in order to increase antimicrobial activity and selectivity towards the target cell are: conjugation of antibiotics, binding of nanoparticles and polymers, formation of organometallic complexes and conjugation of lipids with AMPs. In particular, the conjugation of lipids with peptides leads to the formation of lipopeptides, which have been shown to have greater interaction with the surface of the bacterial membrane (Fosgerau K. 2015. Op cit Reinhardt A and Neundorf I. 2016. Design and application of antimicrobial peptide conjugates. International Journal of Molecular Sciences; l7: l-2l).

Lipopeptides

Lipopeptides are natural or synthetic molecules that have a fatty acid conjugated to the N-terminus of a cyclic or linear peptide sequence, by means of a covalent bond. Natural lipopeptides have a fatty acid in their molecular structure, which gives them hydrophobic characteristics, together with a cyclic peptide sequence of hydrophilic character that has between 7 and 10 amino acid residues. They are synthesized by the non-ribosomal route in some bacteria and fungi (Nasompag S, et al. 2015. Effect of acyl chain length on therapeutic activity and mode of action of the CX-KYR-NH2 antimicrobial lipopeptide. Biochimica et Biophysica Acta; l848: 235l-2364).

Natural lipopeptides are resistant to degradation by enzymes, since in their structure they contain non-native amino acids and the peptide sequence is cyclic. There are three reported families of natural lipopeptides: surfactins, iturins, and phengicines.

Some lipopeptides are used as last resort antibiotics approved by the United States Food and Drug Administration (FDA) for the treatment of infections caused by bacteria highly resistant to drugs. Examples are daptomycin, (anionic lipopeptide active against Gram-positive bacteria) and polymyxin B, a cationic lipopeptide against Gram-negative bacteria.

However, natural lipopeptides have harmful side effects, such as low selectivity and high toxicity in mammalian cells, such as nephrotoxicity, in the case of polymyxin B (Jerala R. 2007. Synthetic lipopeptides: a novel class of anti-infectives . Expert Opinion on Investigational Drugs; 16: 1159-1169; Osorio J, et al. 2017. Factors associated with polymyxin B nephrotoxicity in a university hospital in Neiva, Colombia. 2011-2015. Rev Chilena Infectol; 34: 7-l3 ). Therefore, synthetic lipopeptides generate high expectations as an alternative to MPAs and lipopeptides of natural origin, since reports indicate that they have low cytotoxicity, high retention in alive, greater stability to proteases and lower manufacturing costs (Nasompag S et al, 2015. Op cit).

Synthetic lipopeptides

Short synthetic lipopeptides are molecules that have an amino acid sequence between 3 and 5 residues (Figure 1), can contain unnatural or non-proteinogenic amino acids, a net positive charge and a fatty acid with a chain length of 8 to 16 units of carbon, attached to the N-terminus of the peptide sequence through an amide bond (Makovitzki A, et al. 2006. Ultrashort antibacterial and antifungal lipopeptides. Proceedings of the National Academy of Sciences of the United States of America; 103: 15997 - 16002; Nasompag S et al., 2015. Op cit).

Several studies show that a hydrophobicity threshold is required, which is determined by the length of the fatty acid, to a lesser extent by hydrophobic amino acids and a hydrophilic peptide sequence defined in almost all cases by cationic amino acids, for the lipopeptide to have antimicrobial activity. It should be noted that despite the fact that the peptide sequence is very short compared to traditional AMPs, its mode of action is very similar, and consists of the electrostatic interactions between phospholipids and the net positive charge of lipopeptide, the penetration of the membrane, the formation of aggregates and the depolarization and / or destabilization of the cell membrane, causing the death of the microorganism (Laverty G, et al. 2010. Antimicrobial activity of short, synthetic cationic lipopeptides. Chemical Biology and Drug Design; 75: 563-569 ; Makovitzki A, et al. 2006. Op cit; Nasompag S, et al. 2015. Op cit; Straus SK and Hancock REW. 2006. Mode ofaction of the new antibiotic for Gram-positive pathogens daptomycin: Comparison with cationic antimicrobial peptides and lipopeptides Biochimica et Biophysica Acta; l758: l2l5-l223).

Other studies are looking for an option to improve antimicrobial activity, reaction yields and decrease the high costs of AMP synthesis, using a minimalist approach and a de novo lipopeptide design (Dawgul M, et al. 2017. In vitro evaluation of cytotoxicity and permeation study on lysine- and arginine-based lipopeptides with proven antimicrobial activity. Molecules; 22: 2l73; Jerala R. 2007. Op cit .; Koh JJ, et al. 2017. Recent advances in synthetic lipopeptides as anti-microbial agents : designs and synthetic approaches. Amino Acids; 49: 1653-1677; Konai MM, et al. 2017. Design and solution phase synthesis of membrane targeting lipopeptides with selective antibacterial activity. Chemistry - A European Journal; 23: 12853-12860).

Synthesis of lipopeptides in solid phase by the Fmoc methodology

Amino acids are molecules that have two highly reactive functional groups, a carboxyl group (R-COOH) and an amino group (R-NH ₂ ). The synthesis of the peptide sequence occurs by the formation of amide bonds, between the carboxyl group and the amino group of two adjacent amino acids. Repeating this reaction gives rise to a polymeric sequence known as a peptide (Etchegaray A and Machini MT. 2013. Antimicrobial lipopeptides: in vivo and in vitro synthesis. Microbial Pathogens and Strategies for Combating Them. Science, Technology and Education; 2: 951 -959). For the peptide formation reaction to be selective with only one of the groups where the amide bond is to be formed, the use of protecting groups is necessary. Additionally, some amino acids also have side chains with amino or carboxyl groups, which must be protected, but with a different protective group than the main chain. The most common protecting groups are 9-fluorenylmethoxycarbonyl (Lmoc) and tert-butoxycarbonyl (t-Boc) (Etchegaray A and Machini MT. 2013. Op cit).

The most used methodologies for SPPS are t-Boc and Lmoc. The first requires trifluoroacetic acid (TEA) washes to remove protectors from amino groups and a strong acid such as hydrofluoric acid (HE) for unpinning or cleavage, which can cause loss of sequence and degradation of some amino acids. On the contrary, the Lmoc methodology uses weak bases such as pyridine to remove the protecting group and TLA for unpinning the resin without presenting adverse effects (Lields GB. And Noble RL. 1990. So lid phase peptide synthesis utilizing 9- fluorenylmethoxycarbonyl amino acids.International Journal of Peptide and Protein Research; 35: 161-214, Kimmerlin T and Seebach D. 2005. “100 years of peptide synthesis”: ligation methods for peptide and protein synthesis with applications to beta-peptide assemblies. of Peptide Research; 65: 229-260).

The lipopeptides are obtained by the standardized Lmoc methodology, on a resin or solid polymeric support with a linker, which is a functionalized group that it allows the first amino acid to be coupled by means of a peptide bond, and from this the other amino acids necessary for the formation of the sequence are coupled; finally, a fatty acid is conjugated to the N-terminus of the synthesized sequence. Through an amide bond. The detachment or cleavage of the lipopeptides from the solid support is performed with 95% TFA. With this treatment, all the protecting groups of the side chains such as t-Boc are unprotected (Amblard M, al. 2006. Methods and protocols of modern solid phase peptide synthesis. Molecular Biotechnology; 33: 239-254, Mende F and Seitz O . 2011. 9-Fluorenylmethoxycarbonyl-based solid-phase synthesis of Peptide a-Thioesters. Angewandte Chemie - International Edition; 50: 1232-1240. The procedure for the synthesis of lipopeptides by this methodology can be seen in Figure 2.

The present invention offers short lipopeptides with antimicrobial activity, better retention in vivo and that do not undergo proteolytic degradation.

The relevant design criteria were:

 • A distribution of amphipathicity in the molecules, through the conjugation of a fatty acid of 12 to 16 carbon atoms (hydrophobic end) to a net positively charged peptide sequence (hydrophilic end).

 • An increase in the hydrophobicity of the molecule through the addition of aliphatic amino acids such as leucine.

 • An increase in the hydrophilicity of the molecule through the addition of positively charged amino acids such as lysine or omitin.

 • The use of non-essential or non-proteinogenic amino acids with hydrophilic characteristics in the peptide sequence, to decrease the susceptibility to proteolytic degradation of the molecule.

 • Flexibility of lipopeptides between the fatty acid (hydrophobic region) and the peptide sequence (hydrophilic region) through the amino acid glycine.

Design of lipopeptides

The sequences of the lipopeptides were designed by means of a model that is based on improving or simulating the main characteristics of the AMPs such as cationicity, amphipathicity and / or formation of secondary structures. The de novo design was made based on the reports of synthetic lipopeptides and using the following structural pattern: C „-X _I -X ₂ -X3-NH ₂ (Figure 1).

The criteria used in the construction of the de novo design of the short lipopeptides are summarized in Table 1:

Table 1. Criteria used for the de novo design of short lipopeptides using the structural pattern C _n -Xi-X ₂ -X3-NH ₂ .

Pattern

 Criterion used Molecule used

 Structural

 Lauric acid

 C „Hydrophobicity

 Myristic acid

X i ^. Glycine flexibility

 Omitin

 Positive net charge

X ₂ Lysine

 Non-proteinogenic amino acids Omitin

x ₃ Increased hydrophobicity Leucine

The criteria used for the structural pattern of de novo design are defined below:

• C _n is a fatty acid with a length of 12 to 16 carbon atoms (lauric, tridecanoic, myristic, pentadecanoic, palmitic). The fatty acid gives each molecule the hydrophobic character, by conjugation to the X _I -X ₂ -X ₃ -NH ₂ peptide sequence through an amide bond. The carbon chain length between 12 and 16 carbon atoms provides the best antimicrobial activity.

• Xi corresponds to at least one molecule of glycine (Gly), as a connector between the fatty acid and the amino acids adjacent to it. The use of the amino acid Gly gives each molecule greater flexibility, since being the only amino acid that has a hydrogen atom in its side chain, gives it greater freedom of movement. • X ₂ corresponds to at least two net positively charged and / or non-proteinogenic natural amino acids, which give the molecule the hydrophilic and positively charged character. Conventionally, AMPs exclusively contain arginine (Arg) or lysine (Lys) residues in amino acid sequences, positively charged under physiological conditions. For the de novo design of short lipopeptides, the use of Arg was ruled out, since it increases hemolytic activity compared to Lys residues.

The incorporation of unnatural or non-proteinogenic amino acids in the peptide sequences is a tool to improve the stability to proteases. An example is the amino acid omitin (Om), which is a structural analogue of Lys and is not easily recognized by the binding sites of human proteases, therefore, its incorporation in the designs of lipopeptides is expected. Shorts reduce or stop proteolytic degradation.

• X ₃ represents, when present, at least one aliphatic amino acid, hydrophobic amino acid residues, in order to increase the interaction of the molecule with the membrane of the pathogenic microorganism. The most used in AMP designs are aromatic amino acids, such as tryptophan (Trp), tyrosine (Tyr) and phenylananine (Phe), but they were discarded because they generate greater toxicity in mammalian cells and, additionally, it is known that are susceptible to degradation by the enzyme chymotrypsin, present in the human intestine. Therefore, hydrophobic amino acids with aliphatic chains such as leucine (Leu) were used, which has shown good antimicrobial results and stability against trypsin, chymotrypsin and aureolysin.

Four peptide sequences were designed and the fatty acids used in the de novo design were conjugated to each, for a total of eight short lipopeptides, according to the proposed structural pattern: C ₁₁ -X1-X2-X3-NH2.

The LIP 1, LIP 2, LIP 3 and LIP 4 molecules contain lauric acid and the LIP 5, LIP 6, LIP 11 and LIP 12 molecules contain myristic acid in their structure. The first designed peptide sequence is GOO-NH ₂ , it contains in its structure one glycine and two omitins, it is flexible and with a net charge of +2, this sequence is incorporated into LIP 1 and LIP 5.

The second peptide sequence is GGOO-NH2 _, incorporated into the LIP 2 and LIP 6 lipopeptides _, unlike the previous sequence, there is an additional glycine molecule, in order to evaluate differences in the results of antimicrobial activity, attributed to a increase in the flexibility of the molecule.

The third peptide sequence is GKOO-NH2 _{, it} contains the amino acid lysine in order to increase the net charge of the molecule to +3 and determine if, by increasing the electrical interaction with the microorganism's membrane, it also increases activity, this sequence is contained in it lipopeptides LIP 3 and LIP 11.

The fourth peptide sequence is GOOLL-NH2 and the lipopeptides that contain it are LIP 4 and LIP 12, compared to the initial sequence, has two additional molecules of the amino acid leucine, increasing the hydrophobicity of the lipopeptides, with a net charge of +2.

The structures of the designed lipopeptides can be seen in Figure 3.

Other embodiments of the present invention include, but are not limited to, peptides of formulas GOOA, GOOV, and GOOI.

The lipopeptides of the present invention can be presented in the form of pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt that possesses the efficacy of the parental lipopeptide and that is not biologically or otherwise undesirable (eg, is neither toxic nor otherwise harmful to the recipient thereof). Suitable salts include acid addition salts that can be formed, for example, by mixing a solution of a lipopeptide of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, or benzoic acid. .

Examples of acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphor sulfonates, fumarates, hydrochlorides, hydrobromides, iohydrates, lactates, maleates, methanesulfonates ("mesylates"), naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulphates, tartrates, thiocyanates, toluene sultanates (also known as tosylates).

Basic nitrogen-containing groups can be quaternized with agents such as methyl, ethyl, propyl, and butyl lower alkyl halides (for example, chlorides, bromides, and iodides), dialkyl sulphates (for example, dimethyl, diethyl, and dibutyl sulphates) ), long chain halides (for example decyl, lauryl and stearyl chlorides, bromides and iodides), aralkyl halides (for example benzyl and phenethyl bromides) and others.

The present invention also relates to pharmaceutical compositions, in particular to medicaments, comprising at least one lipopeptide according to the invention, together with one or more pharmaceutically suitable excipients, and to their use for the treatment of infections caused by Gram microorganisms. -positives and Gram-negatives.

Pharmaceutically acceptable excipients include, but are not limited to: fillers such as cellulose, microcrystalline cellulose, lactose, mannitol, starch, etc .; ointment bases such as petroleum jelly, paraffins, triglycerides, waxes, wool wax, wool wax alcohols, lanolin, hydrophilic ointments, polyethylene glycols, etc .;

 suppository bases such as polyethylene glycols, cocoa butter, hard fats, etc .; solvents such as water, ethanol, isopropanol, glycerol, propylene glycol, fatty acids of medium chain length triglycerides, liquid polyethylene glycols, paraffins, etc .;

surfactants, emulsifiers, dispersants or humectants such as sodium dodecyl sulfate, lecithin, phospholipids, fatty alcohols, fatty acid esters of sorbitan, polyoxyethylene glycols of fatty acids, polyoxyethylene glycol esters of fatty acids, polyoxyethylene glycol esters of fatty acids fatty acid esters, poloxamers, etc .; buffer solutions and also acids and bases such as phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, tromethamol, triethanolamine, etc .;

isotonicity agents such as glucose, sodium chloride, etc .;

adsorbents such as highly dispersed silicon;

viscosity increasing agents, gel formers, thickeners and / or binders such as polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, sodium carboxymethylcellulose, starch, carbomers, polyacrylic acids, alginates, gelatin, etc .;

disintegrants such as modified starch, sodium carboxymethyl cellulose, sodium glycol starch, cross-linked polyvinylpyrrolidone, croscarmellose sodium, etc .; flow regulators, lubricants, glides and release agents such as magnesium stearate, stearic acid, talc, highly dispersed silicon, etc .; coating materials such as sugar, shellac, and film formers for diffusion or film membranes that dissolve rapidly or in a modified manner such as polyvinylpyrrolidones, polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate, cellulose, polyacrylates, polymethacrylates, etc .;

capsule materials such as gelatin, hydroxypropyl methylcellulose, etc .;

synthetic polymers such as polylactides, polyglycolides, polyacrylates, polymethacrylates, polyvinylpyrrolidones, polyvinyl alcohols, polyvinyl acetates, polyethylene oxides, polyethylene glycols and their copolymers and block copolymers;

plasticizers such as polyethylene glycols, propylene glycol, glycerol, triacetin, triacetyl citrate, dibutyl phthalate, etc .;

penetration enhancers;

stabilizers, antioxidants such as ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate, etc .;

preservatives such as parabens, sorbic acid, thiomersal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate, etc .;

dyes such as inorganic pigments: iron oxides, titanium dioxide, etc .; flavorings, sweeteners, flavor and / or odor maskers. The lipopeptides of the invention can be formulated for use in agriculture. Compositions for use in agriculture that comprise at least one lipopeptide according to the invention, can be applied in various ways in various formulations, selecting in each case the most appropriate acceptable vehicles for the form of application.

Acceptable agricultural aids include, but are not limited to: diluents, coating polymers, surfactants, pH regulators, pigments, dyes, clays, starches, cellulose derivatives, stearates, natural polymers, synthetic polymers, polyesters, etc.

The compositions of the invention can be solid in the form of powders, granules, tablets or pellets, or they can be liquid in the form of a suspension or emulsion and they can be foliar applied, applied to the soil, by dusting, by irrigation and / or by spraying. and they can be mixed with other bio-inputs, plant extracts and agrochemicals.

EXAMPLES

The invention is illustrated by the following examples, which should not be construed as limiting.

Example 1. Synthesis of lipopeptides

The synthesis was carried out according to the lipopeptides designed based on the criteria described in Table 1 and was carried out by adapting the Fmoc solid phase synthesis methodology of AMPs. The stages of the synthesis are described below.

Example 1.1. Activation and deprotection of the resin

A Rink Amide AM resin (4- (2 ', 4'-Dimethoxyphenyl-Fmoc-aminomethyl) - phenoxyacetamido-aminomethyl) 100-200 mesh, with a degree of substitution of 0.74 mmol / g, was used in a reactor with a porous frit. For its activation, washes were carried out with dichloromethane (DCM) for 5 minutes, and then with N, N-dimethylformamide (DMF) for 2 hours, with constant stirring on an orbital shaker. The step of deprotection of the resin was carried out by removing the protecting group (Fmoc), by means of two successive washes with a solution of 4-methyl-piperidine 25% (v / v) in DMF, with stirring for 15 minutes. each. Washes were performed with DMF, isopropanol (IPA) and DCM to remove 4-methyl-piperidine residues. It was verified that the resin had the available amino groups by means of the positive ninhydrin test, where obtaining the blue color confirms the deprotection of the amino group.

In the initial stage of the reaction mechanism of the deprotection of the Fmoc group, the free electron pair of 4-methyl-piperidine attacks the proton of the Fmoc group, which causes the amide bond to be broken, and subsequently the amino group of the resin or amino acid is free in the second stage, and finally, in the last stages, the formation of the dibenzofulvene adduct of 4-methyl-piperidine that is soluble in DMF is detailed, removing it easily by means of washes.

Example 1.2. Amino acid coupling

For the coupling reaction, the necessary mass of amino acids and the activators dicyclohexylcarbodiimide (DCC) and O-hydroxybenzotriazole (HOBt) were calculated with a ratio of 5 to 1 with respect to the polymeric resin, to guarantee an excess that would react with all the sites resin active ingredients, which were dissolved in DMF and the solution was added by suction to the reactor ensuring that all the resin was submerged. It was stirred for 4 hours at room temperature and then washes with DMF, IPA and DCM were carried out. It was verified that the couplings were carried out by means of the ninhydrin test, where obtaining the light yellow color confirms the reaction of the amino group.

The Fmoc group deprotection steps and the coupling steps were repeated until having the complete amino acid sequence.

Example 1.3. Fatty Acid Coupling

After ensuring deprotection, the coupling reaction occurs via the carboxylic group of the amino acid that has the amino protected with the protecting group Fmoc and DCC to form the Oacilsourean ester that slowly forms the A-acylurea compound, the which is insoluble and not reactive; To avoid this by-product, the nucleophile HOBt was added, generating the active benzotriazole ester that reacts specifically with the free amino group of the resin to form the amide bond.

The deprotection of the N-terminus of the peptide sequence was performed and the fatty acid coupling was carried out for six hours. Successive washes with DMF, IPA, DCM were performed and the resin was allowed to dry with the sequence for 24 hours in a desiccator. Coupling was verified by the ninhydrin test (light yellow color).

Example 1.4. Unpinning

A mixture consisting of trifluoroacetic acid (TFA) / Triisopropylsilane (TIS) / water (H ₂ 0) was added in a volume ratio (95: 2.5: 2.5) to the reactor containing the lipopeptide resin, during two hours in constant agitation. The mixture was expelled from the reactor in a falcon tube and washes with cold ethyl ether (approximately -4 ° C) so that the lipopeptide precipitated. It was centrifuged at 4500 rpm and -4 ° C and washed again with ether to remove impurities. Finally, they were dissolved in water and frozen at -20 ° C to dry by lyophilization.

 The dry lipopeptides were weighed to calculate the percentage of synthesis reaction yield, by means of the following equation and stored in a desiccator until carrying out the respective characterization and biological activity tests.

Yield obtained (g)

% Reaction Yield = - - -—: - —— ^x 100

 Theoretical yield (g)

The yields obtained in the synthesis reaction for each lipopeptide are shown in Table 2, with their respective mass and sequence.

Table 2. Sequence of the synthesized lipopeptides, mass obtained and percentage of reaction yield.

 Mass obtained from

 Lipopeptide Sequence Yield (%)

 lipopeptide (g)

 LIP 1 C12-GOO-NH2 0.1013 93.1

 LIP 2 C12-GGOO-NH2 0.1176 97.6

LIP 3 C12-GKOO-NH2 0.1298 93.1 LIP 4 C12-GOOLL-NH2 0.1551 97.0

 LIP 5 C14-GOO-NH2 0.1111 97.1

 LIP 6 C14-GGOO-NH2 0.1292 99.4

 LIP 11 C14-GKOO-NH2 0.1428 97.6

 LIP 12 C14-GOOLL-NH2 0.1522 92.2

Example 1.5. Lipopeptide purification

The purity of the synthesized lipopeptides was verified by Reverse Phase High Efficiency Liquid Chromatography (RP-HPLC) on an Agilent Technologies 1200 Series chromatograph, with a UV-VIS detector at a wavelength of 220 nm and a column of reverse phase, analytical Zorbax Eclipse RP-18 XDBC18 with dimensions of 4.6 x 150 mm and a pore diameter of 5 pm.

The lipopeptides were eluted using a linear gradient of ACN: TFA (0.1%) and H2O: TFA (0.1%) with a flow of 1 mL / min and an injection volume of 20 pL.

Example 2. Structural characterization

Example 2.1. High-efficiency liquid chromatography

The lyophilized lipopeptides were reconstituted in water to determine purity by high-efficiency liquid chromatography with a UV / VIS detector at a wavelength of 220 nm, because at this length the maximum absorption of the peptide bond is found.

In all the chromatograms (Figure 4) a major peak corresponding to each lipopeptide with a purity greater than 90% and small peaks possibly attributed to impurities from the synthesis are observed.

Retention times are related to the hydrophobicity of each molecule, in which lapoic acid-containing lipopeptides have a retention time between 8.4 and 11.9 minutes and those containing myristic acid have a retention time between 10 , 6 and 14.2 minutes, due to the length of the alkyl chain, which has greater hydrophobic interactions with the stationary phase of the column. Additionally, a relationship can be made between the retention times of the chromatograms of each lipopeptide and the Kyte-Doolittle hydrophobicity scale (hydrophobicity values of amino acid residues) of the peptide sequences, in which the GKOO sequences ( LIP 3 and LIP 11) are observed with the shortest retention time due to greater hydrophilicity due to the presence of 3 positively charged amino acids; in the GOO and GGOO sequences they present very similar times between LIP 1 with LIP 2 and LIP 5 with LIP 6, because they differ only by a glycine that has a low hydrophobicity value. Finally, the GOOLL sequences (LIP 4 and LIP 12) are the ones with the longest retention time due to the additional hydrophobicity provided by the pair of leucines (see Table 3).

Example 2.2. Mass spectroscopy

The mass of the lipopeptides was confirmed by determining the molecular weights generated by an electrospray ionization (ESI) source in positive mode. The characterization was carried out in a high efficiency liquid chromatography equipment coupled to a Shimadzu model 2020 mass spectrometer, using a linear gradient from 5 to 100% (v / v) of acetonitrile and an injection of 10 pL.

The spectral results of each lipopeptide (Figure 5) show that the molecular ion values correspond to the expected or theoretically calculated mass.

ESI ionization in positive mode is a gentle ionization technique, which ionizes protonation-prone functional groups.

In the mass spectra it is observed that most of the lipopeptides tend to form an adduct with acetonitrile (molecular mass 41.05 g / mol) because it is the solvent used in the elution, presenting a peak of molecular ion M + 41 + 2H with z = + 2, it can also be seen that lipopeptides LIP 1, LIP 2, LIP 5 and LIP 6 form dimers with z = + l, which are subunits or monomers of lipopeptide, generally formed by intermolecular forces such as hydrogen bridges, which is very likely to cause the formation of these species in peptides. Analysis of the spectra for each lipopeptide is detailed in Table 4.

Table 4. Assignment of signals in the mass spectra of the synthesized lipopeptides.

Example 2.3. Circular dichroism

The circular dichroism analysis was performed by diluting the lipopeptides in a H ₂ 0: TFE solution in a Jasco model spectropolarimeter, model J-810, with a quartz cell and 1.0 mm optical length. The lipopeptides were prepared at a concentration of 5.0 mM in a 30% (v / v) solution of 2,2,2-trifluoroethanol (TFE), in a reading range of 190 to 260 nm at room temperature, with a bandwidth of 0.5nm and a scanning speed of 50nm / min. Fluorinated alcohol (TFE) is used in order to stabilize the formation of secondary structures such as propellers, b-blades or twists, because it has a low dielectric constant and a high dipole moment, favoring the formation of intramolecular hydrogen bonds. with the amide bond and therefore promoting the folding of the peptide sequence. The circular dichroism spectra are seen in Figure 6.

Taking into account the short sequence length of the lipopeptides, it is expected that they present a disordered structure or also called a random coil, which is theoretically characterized by having an intense negative band around 195-200 nm (p ~ ^ p *) and another positive, but of less intensity at 220 nm (h- p *).

It is observed that in the eight circular dichroism (CD) spectra of the synthesized lipopeptides, there are two negative bands, one with a higher intensity close to 200 nm and another lower intensity around 220 nm. The low intensity band corresponding to the h- p * transitions is negative, but to be characteristic of a disordered structure it must be positive.

This qualitative analysis led to predict that the secondary structures of the eight synthesized lipopeptides are disordered or random coil, presenting similar behaviors in the CD spectra with other reported lipopeptides.

Example 2.3. hydrophobicity index

The hydrophobicity of the peptides can be expressed as a hydrophobicity index (IH) corresponding to the percentage of acetonitrile in which the molecule was eluted by RP-HPLC, because it is the solvent in common use for peptide chromatography. Table 5 shows the calculated values of the hydrophobicity index (IH) of each lipopeptide according to the chromatograms of Figure 6, with a value in the range of hydrophobicity of the AMPs between 40 and 60%.

LIP 4 and LIP 12 lipopeptides have a molecular weight of 710.5 Da and 738.5 respectively, being smaller than natural lipopeptides polymyxin B with 1301.5 Da and daptomycin with 1619.7 Da, but similar in size to synthetic lipopeptides reported as C ₁₂ -OOWW-NH ₂ with 799.5 Da (Laverty et al. 2010. Op cit), C ₁₄ -KYR- NH ₂ with 674.9 Da and others designed and synthesized by Lohan el al., Which have a molecular weight around 700 Da (Lohan et al. 2014. Op cit).

Table 5. Hydrophobicity index of the lipopeptides designed and synthesized in this study. _

Lipopeptide tu ¹ (min) IH ² (% ACN)

 LIP 1 9,427 43.6

 LIP 2 9,489 43.7

 LIP 3 8,353 40.9

 LIP 4 11,872 49.7

 LIP 5 11,851 49.6

 LIP 6 11,959 49.9

 LIP 11 10,648 46.6

 LIP 12 14,220 55.6

¹ retention time determined by RP-HPLC.

² hydrophobicity index calculated with the percentage of ACN.

Example 3. Biological activity

Example 3.1. Antimicrobial activity

Staphylococcus aureus, S. saprophyticus, S. haemolyticus, S. salivarius, Bacillus cereus and Enterococcus faecalis were taken as bacterial models of bacteria and Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Acinetobacter baumannii Additionally, Candida albicans was evaluated as yeast, being all species human pathogens and that generate serious problems in health systems worldwide. Useful strains are found in Table 6.

Table 6. Microorganisms evaluated in antimicrobial activity. Bacterial and yeast species are ATCC (American Type Culture Collection) reference strains with genotypic and phenotypic characteristics defined for each species.

 As a first measure, the bacterial growth curve of each of the evaluated strains was performed, alone and in the presence of the developed lipopeptides, at different concentrations.

The microbial strains used (Table 6) were activated in Luria-Bertani (LB) culture broth for bacteria, in Sabouraud yeast broth at 37 ° C for 18 hours each, to seed 10 pL of suspension in the respective culture medium and obtain individual colonies. A suspension conforming to a 0.5 standard on the McFarland scale was prepared by measuring at a wavelength of 600 nm, which should be between 0.08-0.13 corresponding to lXlO ⁸ CFU / mL. A 10 mL dilution equivalent to a concentration of ¹⁰ CFU / mL was prepared.

It was determined whether the effect is bactericidal or bacteriostatic at the concentration of <50 mM. The test results are shown in Table 7.

Based on the results in Table 7, it can be seen that LIP 12 has a bactericidal effect at concentrations less than 12.5 mM in the Gram positive and Gram negative bacteria tested in this study, with the exception of K. pneumoniae and A. baumannii, comparing its broad-spectrum action with the modified bactericidal lipopeptide C ₁₄ KKC ₁₂ K and unlike the natural lipopeptides that are selective such as Polymyxin B and Daptomycin, used to treat Gram negative and Gram bacteria positive respectively. All lipopeptides that inhibited the growth of S. aureus and E. cotí in this study have a bactericidal effect, whereas in P. aeruginosa the majority of lipopeptides had a bacteriostatic effect, except for LIP 12. The test results Antimicrobials in E. faecalis showed that LIP 1 and LIP 6 have a bacteriostatic effect and LIP 4, LIP 5, LIP 11 and LIP 12 have a bactericidal effect.

Table 7. Bactericidal or bacteriostatic effect of the lipopeptides designed in this study and which were active against the evaluated microorganisms.

Example 3.2. Determination of the minimum inhibitory concentration (MIC)

According to the results obtained in the evaluation of the antimicrobial activity, the lipopeptides that inhibited the growth of the microorganisms at a concentration less than or equal to 25.0 mM were evaluated again and using other reference strains, to determine the MIC. The results obtained were recorded in Table 8 (Annex 1).

It is clearly observed that the inhibition of microbial growth is a factor dependent on hydrophobicity, since the first group of lapoic acid-containing lipopeptides (LIP 1, LIP 2, LIP 3 and LIP 4) have higher MICs than the group that It contains myristic acid (LIP 5, LIP 6, LIP 11 and LIP 12), which is more hydrophobic than lauric acid. It should be noted that LIP 4, LIP 5, LIP 6, LIP 11 and LIP 12 lipopeptides were all active against Gram positive bacteria, possibly being more hydrophobic than LIP 1, LIP 2 and LIP 3, said selectivity is similar to that of the natural lipopeptide daptomycin and the glycopeptide vancomycin, which are peptides conjugated with macromolecules and selective for Gram positive bacteria.

By increasing the flexibility of the lipopeptides by adding two glycine molecules in the sequence, the results were not significantly different between LIP 1 and LIP 2 and between LIP 5 and LIP 6, but the lipopeptides to which the hydrophobicity was increased by adding the pair of leucines in the peptide sequence (LIP 4 and LIP 12) showed bactericidal results at low concentrations since LIP 4 and LIP 12 inhibit both Gram positive and Gram negative bacteria, so they could be considered as broad spectrum biologically active molecules.

In the present study it was shown that some of the designed and synthesized lipopeptides inhibit bacterial growth and that this activity depends on a subtle balance between the hydrophobicity of the molecule provided by fatty acid and leucines and positively charged amino acids, although they present a disordered structure in the solution H ₂ 0: TFE (70:30).

Example 3.3. Hemolytic activity

Hemolytic activity was analyzed on a human blood sample from a healthy fasting donor. The blood sample was centrifuged at 1000 x g, at room temperature for 7 minutes, to separate human erythrocytes. The pellet that formed was washed three times with sterile 0.9% (m / v) saline of NaCl, centrifuging at 1000 x g and discarding the supernatants for each wash. A 1: 10 suspension of erythrocytes was prepared in saline and then 90 pL of the diluted erythrocyte suspension and 10 pL of a solution of each lipopeptide were added to eppendorf tubes for a final concentration of 50.0 pM, 25.0 pM , 12.5 pM, 6.25 pM and 3.13 pM.

Suspensions were incubated at 37 ° C for 3 hours at 90 rpm and centrifuged at 1000 xg at room temperature for 5 minutes. 50 pL of the supernatant were taken and added to a 96-well plate and the absorbance was read at 545 nm. A 0.1% (v / v) solution of Triton X-100 in the erythrocyte suspension was used as a positive control, This corresponds to 100% hemolysis and as a negative control, sterile 0.9% NaCl saline in the erythrocyte suspension (Evans et al., 2013). The tests were carried out in triplicate for each lipopeptide.

The hemolysis percentage was calculated using the following equation: 100

Figure 7 presents the results of the hemolytic activity of lipopeptides in different concentrations.

LIP 4 and LIP 12 lipopeptides showed a low percentage of hemolysis (<5%) in a concentration range between 3.13 mM and 50 pM compared to the positive control used for the assay (Triton X-100 0.1% ) which produced 100% hemolysis, indicating that LIP 4 and LIP 12 have a selectivity towards prokaryotic cells (bacterial models) but not towards the eukaryotic cells evaluated in this study (yeast and human erythrocytes).

The selectivity of lipopeptides can be measured in terms of the therapeutic index (TI), which was determined in Table 9 by relating the concentration that causes 10% hemolysis (CHio) with the MIC values of the 4 bacterial strains tested. Because the 2 lipopeptides present a hemolysis percentage of less than 10% at the maximum concentration evaluated (50.0 pM), this value was taken as CHio. The therapeutic index results indicate that LIP 4 is selective for P. aeruginosa and that LIP 12 has a broader antimicrobial activity compared to LIP 4, although both have low hemolytic activity, from which it can be concluded that LIP 12 may have a high potential for use in the development of antimicrobial drugs.

Table 9. LIP 4 and LIP 12 therapeutic index. The therapeutic index indicates the selectivity of lipopeptides towards human erythrocytes. The higher the therapeutic index a molecule has, the greater the possibility of developing an antimicrobial agent. Therapeutic index ^b

Lipopeptide CHio ^a (mM)

 S. aureus E. faecalis P. aeruginosa E. coli

 LIP 4> 50 1.7 1.0 5.3 0.5

LIP 12> 50 5.0 5.3 5.9 5.9

Concentration of lipopeptide that induces 10% hemolysis in human erythrocytes

 therapeutic index calculated as CHio / CMI.

Example 3.4. Effect of lipopeptides on bacterial morphology

According to the MIC results, the effect of LIP4 and LIP 12 on bacterial morphology was evaluated at 2 times the MIC (2xCMI), using the scanning electron microscopy (SEM) technique. In this way it shows the damage they cause to bacteria in a short time, from 30 minutes to 2 hours.

Samples for SEM were prepared as follows: a bacterial suspension was prepared at a concentration of lXlO ⁶ CFU / mL and 400 pL of the suspension were added to a 24-well plate, to which 400 pL of the solution was added of each lipopeptide so that it remains at a final concentration of 2 times the MIC. The suspensions with the treatment were incubated at 37 ° C with a constant shaking of 90 rpm.

It was filtered through a 1.3 mm diameter and 0.2 µm pore size cellulose membrane at 30 min, 120 min and 20 hours after adding each lipopeptide. An untreated bacterial suspension was used as growth control and filtered after 20 hours. A 2.5% (v / v) glutaraldehyde solution in 0.1 mM phosphate buffer was added to another 24-well plate to fix the sample and the membranes to which the bacterial suspension was filtered were left to incubate for 12 hours at 4 ° C.

The membranes were removed from the glutaraldehyde solution and washed 3 times with phosphate buffer solution and dehydrated 2 times with ethanol solutions of increasing concentration at 30, 50, 70, 80, 96 and 100% (v / v), and leaving to act for 2 minutes. Finally, the membranes were dried for 1 day at room temperature. For SEM observation the samples were gold plated forming a layer around 6 to 9 nm, with a Quorum Technologies brand coater, model Q150. The micrographs have an electron beam voltage level of 15 kV. The relevant details of the damage caused by the lipopeptides on the bacterial membrane that are observed in the micrographs (Figures 8 to 12) are indicated in white ovals.

LIP 4 lipopeptide was shown to be selective at low concentrations for P. aeruginosa, therefore, the morphological change of the bacterial membrane was observed at a concentration of 19 mM; the growth control was kept in incubation at 37 ° C for 20 hours without treatment, in which abundant number of cells is observed and the surfaces of the membranes are integral, smooth, shiny and the cells turgid (Figure 8A); when exposing the bacterial culture for 30 minutes to LIP 4, there is no proliferation of the microorganism and a slight deformation is observed on the surface of the membrane (Figure 8B), finally, in the bacterial culture treated during 2 and 20 hours of incubation, no cells are seen defined, indicating that there was cell lysis and cytoplasmic material is observed on the cellulose membrane (Figures 8C and D), therefore it can be inferred that LIP 4 has a bactericidal effect at 2 times the MIC (19 uM) between 2 and 20 hours of treatment.

LIP 12 lipopeptide caused morphological effects on the membrane of the 4 bacteria tested, in MICs less than or equal to 20 pM. The control of cell growth of each bacterial strain indicates that it is in a stationary phase after 20 hours of growth and it is observed that the surface of the cells is defined and smooth in P. aeruginosa, S. aureus, E. faecalis, and E coli (Figures 9A, 10A, 11A and 12A respectively). The cell membrane of P. aeruginosa presents roughness on the surface between 30 minutes and 2 hours of treatment with LIP 12, observing the formation of vesicles of approximately 100 to 200 nm in diameter, and cytoplasmic material is also observed on the cellulose membranes, indicating that there was cell lysis (Figures 9B and C) at a concentration of 17 pM; and at 20 hours of incubation, no cells are observed, but there are residues of cytoplasmic material (Figure 9D); confirming a bactericidal effect.

The effect on the membrane of S. aureus treated with LIP 12 at a concentration of 20 pM showed that during the first 30 minutes the formation of small blisters or vesicles occurs on the cell surface, which are between 100 and 120 nm (Figure 10B); after 2 hours of exposure to lipopeptide the ampoules become more abundant and traces of cytoplasmic material and dispersion of the vesicles are observed (Figure 10C). At 20 hours no complete cell is observed (Figures 10D), indicating that after 2 hours of exposure to LIP 12 the bacterial membrane has disintegrated, confirming the bactericidal effect.

Damage to the membrane of E. faecalis treated with LIP 12 at a concentration of 19 mM was similar to that observed in S. aureus with treatment with LIP 12 lipopeptide. During the first 30 minutes, vesicle formation was observed and deformation of the cell surface (Figure 11B), after 2 hours of exposure to LIP 12 the membrane is observed with an increase in the formation of vesicles (Figure 11C) and finally after 20 hours cytoplasmic material is observed, indicating that there was lysis cell (Figure 11D), confirming the bactericidal effect.

Damage to the cell membrane of E. cotí is evident after treatment with LIP 12 at a concentration of 17 mM in the first 30 minutes, since there is blistering on the cell and a large number of vesicle aggregates of approximately 100 nm of diameter, possibly derived from damage to the cell membrane (Figure 12B), damage that progresses after 2 hours of exposure, time in which it is observed that the surface of the cells becomes rough and depressed and, in addition, a large amount of residues corresponding to cytoplasmic material (Figure 12C) and finally, at 20 hours of incubation, some cells with a large number of vesicles and depressions (small circle), loss of the outer membrane and formation of vesicles (Figure 12D) are observed, confirming that the effect that lipopeptide LIP 12 has on E. cotí is bactericidal.

According to the SEM micrographs of the 4 bacterial strains evaluated for antimicrobial activity, it was observed that the designed lipopeptides have a high biological potential and present a surfactant effect or also called a micellar model as a mechanism of action.

As observed in all micrographs, the morphological changes of the bacteria with respect to growth control show the presence of vesicles or blisters produced at different times of exposure to lipopeptides. Damage to the cell membrane in Gram negative bacteria is possibly due to electrostatic interaction with phospholipids and in Gram positive bacteria it can be attributed to electrostatic interactions of lipopeptide with wall peptidoglycans, which are formed by alternating polymers of N-acetylglucosamine and N-acetylmuramic acid linked by b-1,4 bond.

In conclusion, it can be inferred that the designed and synthesized lipopeptides cause damage to the bacterial membrane with an action time between 30 and 120 minutes, caused by damage and later structural disruption causing cell death, confirming the mode of action of lipopeptides according to their amphiphilic properties.

Example 3.5. Evaluation of in vitro stability to proteases

To determine the stability of lipopeptides, attributed to the presence of the amino acid ornithine in the sequence of LIP 12, the lipopeptide with the highest antibacterial activity, was treated with human blood serum.

1.5 mg of lipopeptide was added to a 1000 pL solution of RPMI supplemented with 25% (v / v) with blood serum and stabilized at 37 ° C for 15 minutes. An aliquot of 100 pL of the solution was taken and transferred to eppendorf tubes at times 0, 1, 2, 3, 4, 8 and 24 hours and 200 pL of 96% ethanol were added. It was allowed to cool to 4 ° C for 15 minutes and it was centrifuged at 18000 x g for 2 minutes. The supernatant was analyzed by RP-HPLC.

It was observed that LIP 12 does not show signs of degradation in its structure, at a temperature of 37 ° C for 24 hours, in the presence of proteases from blood plasma, since the areas of the peak corresponding to the lipopeptide in the chromatograms (t _R : approximately 15.9 minutes), they did not have significant changes during the test, nor was the formation of new peaks corresponding to other species observed, as illustrated in Figure 13.

APPENDIX 1

Table 8. Minimum inhibitory concentration of the lipopeptides designed and synthesized in this study (mM).

Claims

1. Synthetic lipopeptides of formula

C „-XI-X ₂ -X3-NH ₂

 where:

C _n is a fatty acid selected from the group consisting of Ci ₂ to CI _Ó ;

 Xi is at least one glycine molecule;

X ₂ is at least two net positively charged and / or non-proteinogenic natural amino acids; Y,

X ₃ can be present or absent and when present it is at least one aliphatic amino acid,

2. The synthetic lipopeptide of Claim 1, wherein C _n is a fatty acid selected from Ci ₂ and Ci ₄ .

3. The synthetic lipopeptide of Claim 1, wherein C _n is a fatty acid selected from C13, C15 and CI _Ó .

4. The synthetic lipopeptide of Claim 1, wherein Xi is a glycine molecule.

5. The synthetic lipopeptide of Claim 1, wherein Xi are two glycine molecules.

6. The synthetic lipopeptide of Claim 1, wherein X ₂ corresponds to at least two amino acids selected from the group consisting of K or O, or a mixture thereof.

7. The synthetic lipopeptide of Claim 1, wherein X ₃ is at least one amino acid selected from the group consisting of A, V, I, L.

8. The synthetic lipopeptide of Claim 1, wherein the lipopeptide is 3 to 5 amino acids in length.

9. The synthetic lipopeptide of any one of Claims 1 to 8 that inhibits bacterial growth in genera of Gram positive and Gram negative bacteria.

10. A composition comprising the synthetic lipopeptide of any one of Claims 1 to 8 and an acceptable carrier.

11. Use of the lipopeptide of any one of Claims 1 to 8 or the composition of Claim 10 for the manufacture of a medicament for the treatment of infections caused by Gram positive and Gram negative bacteria.

12. Use of the lipopeptide of any one of Claims 1 to 8 or the composition of Claim 10 for the manufacture of an agricultural product to inhibit the microbial growth of Gram-positive and Gram-negative bacteria.