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Review Article

Vol. 5, Núm. 2 Mayo-Agosto 2016 Properties and design of pp 96-115 as potential tools against pathogens and malignant cells Propiedades y diseño de péptidos antimicrobianos como herramientas potenciales contra patógenos y células malignas

Jazmín Huerta-Cantillo,* Fernando Navarro-García*

* Department of Cell Biology, Abstract Centro de Investigación y de Estudios Avanzados del IPN, In the last years, the indiscriminate use of conventional has generated a worrisome CINVESTAV-IPN, México D.F. increase of resistant pathogens. (AMPs) are considered a plausible alterna- Mailing address: tive therapy against pathogens due to their structural and functional characteristics, as well as their Fernando Navarro-García low toxicity against eukaryotic cells and their broad spectrum of action against different pathogens, Department of Cell Biology, including Gram-negative and Gram-positive bacteria, fungi, parasite and virus. Interestingly, AMPs CINVESTAV-IPN. also have the capability to recognize certain types of plasma membranes, and this selectivity allows Ap. Postal 14-740, 07000, México City, México. differential recognition of normal cells, non-malignant tumor cells and malignant tumor cells; thereby Tel: (52-55)57473990 the use of these AMPs could be a viable alternative for cancer treatment. These peptides can be Fax: (52-55)57473393 isolated from different organisms, such as microorganisms, plants and animals. Such peptides are E-mail: [email protected] amphipathic and cationic molecules of low molecular weight and they have a low probability to generate resistance. Therefore these natural peptides have been utilized as the base for synthetizing Recibido: 08 de marzo de 2016. Aceptado: 30 de marzo de 2016. new analog peptides with chemical or structural modifications for improving their antimicrobial stability and efficiency. In this review, we focused on an overview of the AMPs: properties, mecha- Este artículo puede ser consultado nisms of action, and their different applications for combating pathogens in diverse fields, as well as en versión completa en: http://www.medigraphic.com/rid their use due to the anticancer activity. We also focused on some strategies for the design of new peptides, and finally, we discussed some drawbacks to overcome their use as therapeutic agents.

Key words: Infection, Resumen cancer, peptides, molecular structure, En los últimos años, el uso indiscriminado de antibióticos convencionales ha generado un au- anticancer activity, bacteriocins, mento inquietante de patógenos resistentes. Los péptidos antimicrobianos (PAMs) se consideran tumor cell, una terapia alternativa plausible contra los patógenos debido a sus características estructurales chemical modifications, y funcionales, así como a su baja toxicidad frente a células eucariotas y a su amplio espectro clinical studies, designed peptides. de acción contra diferentes patógenos, incluyendo bacterias Gram-negativas y Gram-positivas, hongos, parásitos y virus. Curiosamente, los PAMs también tienen la capacidad de reconocer Palabras clave: Infección, ciertos tipos de membranas plasmáticas, y esta selectividad permite el reconocimiento de células cáncer, péptidos, estructura normales, células tumorales no malignas y células tumorales malignas; así el uso de estos PAMs molecular, actividad podría ser una alternativa viable para el tratamiento del cáncer. Estos péptidos se pueden aislar anticancerígena, bacteriocinas, de diferentes organismos, tales como microorganismos, plantas y animales. Tales péptidos son células tumorales, www.medigraphic.org.mxmoléculas anfipáticas y catiónicas de bajo peso molecular y tienen baja probabilidad de generar modificaciones químicas, resistencia. Por lo tanto estos péptidos naturales se han utilizado como base para la síntesis de estudios clínicos, diseño de péptidos. nuevos péptidos análogos con modificaciones químicas o estructurales para mejorar su esta- bilidad y su eficacia antimicrobiana. En esta revisión, nos centramos en una visión general de los PAMs: propiedades, mecanismos de acción, y sus diferentes aplicaciones para combatir a los patógenos en diversos campos, así como su uso derivado de su actividad contra el cáncer. También nos enfocamos en algunas estrategias para el diseño de nuevos péptidos, y por último, discutimos algunos inconvenientes a superar para su uso como agentes terapéuticos.

96 Investigación en Discapacidad Jazmín Huerta-Cantillo et al.

Introduction The role of the AMPs on the innate immune response AMPs have been isolated from a great variety of organ- isms, including animals, plants, , bacteria, fungi, AMPs exist in virtually all multicellular organisms and and even from viruses.1 These peptides are part of the have been studied in insects and other invertebrate innate immunity that is an ancestral defense mecha- organism that lack an adaptive . AMPs nism used by organisms for controlling the natural also play an important role in the immune system of fl ora and for combating pathogens.2 These peptides mammals, including humans. AMPs are present at all are the fi rst in recognizing foreign particles and they human body sites normally exposed to microbes such show specifi city to components of bacterial as the skin and mucosa.12 such as (LPS) from Gram-negative AMPs are part of the innate immune response bacteria, from Gram-positive bacteria, with a relevant role in the fi rst line of defense against glycolipids from mycobacteria, β-glycan from yeast and microorganisms. Peptides can be inducible or consti- RNA strands from viruses; they are able to recognize tutive, they are nonspecifi c and respond before the to most of the microorganisms.2-4 acquired immune response had been initiated, they do Peptides are of low molecular weight (2-50 kDa), not generate immune memory, they are not catalytic although some of them can be heavier. The main char- but provide economy to cells by being small effector acteristics of the AMPs are based on what they are (i) molecules.9,10,12 net positive charge molecules (generally +2 to +9) due In addition to their direct antimicrobial activity, to their high amount of positive amino acids (aa) such some AMPs possess different immunomodulatory as Lys and Arg, (ii) well-defi ned amphipathic molecules functions.12,13 Some of the most relevant are: 1) Che- (with ≥ 30% of hydrophobic residues), stable in aque- motactic activity: in a direct manner, AMPs act as a ous and hydrophobic solutions, (iii) they can undergo chemo-attractant capable of recruit immune cells to the post-translational modifi cations.5,6 site of infection. In indirect manner, AMPs induce the AMPs are mainly coded in the genome as one or expression of a broad range of chemokines such as several copies. All AMPs are derived from larger pre- cursors, including a signal sequence. Post-translational modifi cations include proteolytic processing, and in α-helical some cases glycosylation, carboxyl-terminal amida- β-sheet tion and amino-acid isomerization, halogenations and cyclization. Some peptides are derived by proteolysis from larger , such as buforin II from histone 2A and lactoferricin from .5 The AMPs show different secondary structures, but most of them fall into four types (Figure 1): (i) α-helix, (ii) β-pleated sheet, due to two or more disulfi de bridges, (iii) mix of β-sheet and α-helix, due to a disulfi de bridge and/or circularization, and (iv) lineal or ran- Mixed α/β Lineal or Random dom.7 Most of these peptides do not show a particular structure when they are free in solution and they are folded to their fi nal conformation when directly bind to the membrane.8 The AMPs are generally synthesized in exposed tis- sues, such as skin, intestine, lungs and red blood cells. These peptides are synthesizedwww.medigraphic.org.mx 100 times more rapid than an immunoglobulin and at low metabolic cost, but they can also be stored as a reserve into cells and be released when cells are stimulated by the contact with Figure 1. Representative secondary structure of AMPs. pathogens.9,10 These peptides are a fast non-specifi c α-helix (Moricin), β-sheet (Hepcidin-20), mixed α-helix/ way to combat a broad spectrum of microorganisms β-sheet (Hydramacin-1) and lineal or random (Lf11). and it has been discovered in the last decade that these α-helix structures are shown in magenta, β-sheet in yel- AMPs are effi cient to combat tumor cells as well.4,11 low, and green lines represent the disulfide bridges.

Volumen 5 Número 2 Mayo-Agosto 2016 97 Use of antimicrobial peptides

CXCL8/IL8, CCL2/MCP1 by , monocytes (iii) peptidoglycanase type colicins: they digest the and other immune cells.14 2) Anti-endotoxin activity: precursors.20 2) The other group is that AMPs possess the capacity to dampen production of of the microcins, which are molecules of low molecular endotoxin-induced pro-infl ammatory mediators such weight (< 10 kDa) produced as precursor peptides as tumor necrosis factor alpha (TNF-α) by blocking (amino-terminal signal peptide and core peptide) and or modulating toll like receptor (TLR) signaling path- are secreted to the extracellular milieu through the type ways.15 3) Immune cell differentiation: AMPs appear I system. Microcins are divided in two cat- to have a direct link inducing cell differentiation and egories depending on their molecular weight and their activation, bridging the innate and adaptive immune post-translational modifi cations: (i) Class I microcins: responses.16 4) Wound healing and angiogenesis: they are of < 5 kDa and are post-translational modifi ed, wound healing involves the re-growth of epithelial and (ii) Class II microcins: they are of 5-10 kDa and layers and the formation of new blood vessels (an- can or can not have post-translational modifi cations. giogenesis). AMPs act directly on epithelial and en- The mechanism of action of microcins is varied and dothelial cells, inducing promoting re-epithelialization includes pore formation, nuclease activity, interference and angiogenesis. AMPs also induce wound healing with energetic processes and inhibition of syn- indirectly through their chemotactic effects.17 Due to thesis, and DNA replication.21 these immunomodulatory and antibacterial properties, The bacteriocins produced by Gram-positive bacte- AMPs are excellent candidates for infections treatment ria have a major diversity of characteristics regarding since they can control infl ammation in the infection site. to size, structure and physicochemical properties. These bacteriocins are divided in three classes: (i) Distribution of AMPs Class I: they are post-translational modifi ed peptides, lantibiotics (contain lanthionine and dehydrated amino AMPs are conserved from primitive organisms to mam- acids), of < 5 kDa and can be lineal o globular; lineal mals as humans with a phylogenetic relation relatively peptides are membrane disruptors and the globular close indicating their presence since thousands of peptides have enzymatic activity. (ii) Class II: they are years in the earth, without suffering marked changes unmodifi ed peptides, without lanthionine, of < 10 kDa, during evolution. Since the fi rst AMP was discovered, positively charged and with great heat resistance. (iii) more than 1400 AMPs have been isolated from bac- Class III: they are high molecular weight peptides (> teria, plants, insects and other invertebrates, including 30 kDa), very sensible to heat and can or not have vertebrates such as amphibians, birds, fi shes and bacteriolytic function.19 mammals.18 Plants. Plants produce small cysteine-rich AMPs Bacteria. Bacteria produce some AMPs as strat- and are produced in all organs, but they are more egy for maintaining the control of population and for abundant in the outer layer. Most of plant AMPs have a combating other microorganisms competing by space molecular weight between 2-10 kDa and are classifi ed and nutrients of their environment. AMPs produced based on the identity of their amino acid sequence and by bacteria are known as bacteriocins.19 Bacteriocins the number and position of cysteines forming disulfi de produced by Gram-negative bacteria are classifi ed in bonds.22 There are twelve families described for plant two large groups: 1) Colicins, which are high molecular AMPs: (i) thionins are synthesized in leafs, fl owers and weight molecules (25-80 kDa), encoded on plasmids seeds and have activity against fungi and bacteria, (ii) and possessing three functional domains: a transloca- denfesins are synthesized in seeds during germination tion amino-terminal domain, a central domain for recep- and have activity against fungi and bacteria, (iii) cy- tor binding and a cytotoxic carboxyl-terminal domain clotides are macrocyclic peptides, resistant to protease (possessing the antibacterial activity). Colicins are degradation and have activity against bacteria, virus simultaneously synthesized with specialized proteins and insects, (iv-vi) knottin-like, Ib-AMPs and Hevein- to maintain them inactive andwww.medigraphic.org.mx in that way avoid the like are effective only against Gram-positive bacteria damage to their own producer bacteria. Colicins are and fungi, (vii-xi) shepherdins, MBP-1, LTP, Snakins divided in three categories depending on their mecha- and 2S albumins have antibacterial and antifungal nism of action: (i) pore-forming colicins: they form pores activity and (xii) β-barrelins are only actives against in the plasma membrane of the target cell causing fungi. Thionins, and cyclotides have been ions and cytoplasmic components loss, (ii) nuclease shown to have anticancer activity.3,22,23 type colicins: DNAases, rRNAases 16s, tRNAases Additionally, there are peptides in plants whose and for unspecifi c digestion of DNA and RNA, and main function is the inhibition of proteases, such AMPs

98 Investigación en Discapacidad Jazmín Huerta-Cantillo et al. are found in tubers and seeds and they are stored as rial and antifungal activity. These peptides are: (i) energy source and as defense mechanism. Tomatoes Porifera (sponges): discodermins, halicylindramides, produce inhibitors of trypsin and chymotrypsin and theonellamides, cyclolithistides and phoriospongins; potatoes produce inhibitors of -proteases and (ii) Cnidaria (anemones, corals and medusas): au- carboxypeptidases; these inhibitors are involved in the relins, hydralysins, styholysins; (iii) Mollusca (snails, response induced by wounds and UV radiations. The clams, mussels, squids and octopi): mytilus, mytilins importance of these inhibitor peptides is due to they and myticins; (iv) Annelida (segmented worms): he- can be used in a safe way in humans because they distins, arenicins, theromyzins and theromacins; (v) are not toxic and can avoid the growth of tumor cells, Arthropoda (crustaceous): penaedins, callinectins, helping to reduce the food ingest and to inhibit most astacidins, thachyplesin and tachystatins; and (vi) of the intestinal proteases; however, their specifi city Echinodermata (sea stars): strongyolocins.26,27 The and potency depend on the inhibition sites and on peptides derived from marine organism have been the organism from the target proteinase was derived.4 shown to have inherent ability to sustain under high salt concentrations, so they may have high probability Invertebrates of success under physiological salt concentrations.26,28

Insects. AMPs are synthesized in fat bodies and Vertebrates constitutive AMPs are accumulated in blood cells and salivary glands, in the presence of microbes the AMPs Amphibians. The amphibian skin is characterized are secreted into the hemolymph. Induced AMPs are by its remarkable cutaneous exocrine apparatus with synthesized after the microbial infection takes place.24 numerous granular and mucous glands.29 Additionally, Some of the peptides produced by insects are: (i) Ce- amphibian skin possesses the greater variety and con- cropins are linear α-helical peptides of 3-4 kDa, which centration of AMPs that can be found in a vertebrate have a high content of proline but no cysteine. Ce- or invertebrate, allowing them a great capability to cropins have activity against Gram-negative bacteria, heal wounds and prevent infections. Frogs and toads fungi, viruses and also have insecticidal and antitumor (Anura) skins produce different AMPs, which present activities. (ii) Insect defensins are cyclopeptides of 4-5 a broad spectrum of action against Gram-positive and kDa with three disulfi de bridges and they can control Gram-negative bacteria, fungi and protozoa.30 Some most Gram-positive bacteria and a few fungi and yeast. examples of the families of peptides in Anura are: (iii) Drasomycin is cyclopeptide of 5 kDa which has a brevinins, esculetins, japonicins, mellitin-like peptides, strong antifungal activity. (iv) Thanatin is a cyclopeptide nigrocins, palustrins, ranacyclins, ranatuerins, tempo- of 2.5 kDa with one disulfi de bridge and has antimicro- rins, aureins, caerins, citropins, dermasepins, phillo- bial activity against multidrug resistant clinical isolates septins, plasticins, phylloxins, alyteserins, bombinins, of Enterobacter aerogenes and . and signiferins.31 (v) Apidaecin and abaecin from bees are peptides Birds. For avian species, three main classes of of 4 kDa and have a high content of proline and are AMPs have been described. These are β-defensins, effective mainly against Gram-negative bacteria. (vi) (CATH) and liver-expressed antimicro- Metchnikowin is a proline-rich peptide of 2 kDa and bial peptide-2 (LEAP-2). These peptides protect the has activity against Gram-positive bacteria, fungi and avian embryo and young hatchling from infections. insects. (vii) Formaecin, drosocin, pyrrhocoricin and Post-hatch, AMPs protect chickens, turkeys, ostriches, lebocin are proline-rich peptides (2-3.5 kDa), which ducks, gooses, quails and penguins from Gram- have activity against Gram-negative bacteria. (viii) At- negative and Gram-positive bacteria, mycoplasm and tacin, gloverin, diptericin, hymenoptaecin, coleoptericin fungi infections.32 and hemiptericin are glyicine-rich peptides of 8-30 kDa Mammals. Within mammalian species, defensins and these peptides are mostlywww.medigraphic.org.mx active against Gram- and cathelicidins are the two principal antimicrobial negative bacteria. (ix) Attacin and sarcotoxin also have peptide families. Defensins are 18-45 amino acid long insecticidal activity.3,24,25 (2-4.5 kDa), cysteine-rich, cationic peptides charac- Marine invertebrates. AMPs are important for terized by three conserved disulfi de bridges without survival of marine invertebrates because they lack glycosyl- or acyl- side-chain modifi cation, a β-sheet acquired immune response mediated by B and T structure and both hydrophobic and cationic amino lymphocytes.26 Several AMPs have been detected in acids. Based on the site of expression, size, structure marine invertebrates and mainly possess antibacte- and pattern of disulphide bridge, the family

Volumen 5 Número 2 Mayo-Agosto 2016 99 Use of antimicrobial peptides can be subdivided into three main groups: the α, β and lungs, bronchus, colon and rectus of bovine produce θ-defensins.33,34 β-defensins with activity against E. coli, K. pneumoniae, α-defensins are 29-35 residues long with a disulfi de S. aureus, P. aerugionosa and C. albicans.43 alignment pattern of 1-6, 2-4 and 3-5 and only present in mammalian species. α-defensins are either predomi- Mechanism of action nantly synthesized in neutrophils at high concentration and in a constitutive way in its active form or stored as The classifi cation of the mechanisms of action of the propeptides in paneth cells.35 β-defensins are 36-42 AMPs can be divided in two classes: membrane dis- residues long with a disulfi de alignment pattern of 1-5, ruptors and non-membrane disruptors; however, the 2-4 and 3-6 and can be found in all vertebrate species. initial common target for both is their interaction with β-defensins are mainly produced by epithelial cells, at the plasma membrane through electrostatic charges. low concentration and are generally produced in an Membrane disruptor peptides are the most predomi- inducible way.36,37 The θ-defensins are 18 residues nant and much of them have α-helix structures, which cyclic defensins with a disulfi de alignment patter of 1-6, directly act at the plasma membrane level, altering the 2-5 and 3-4 and they are formed by post-translational cell permeability or lysing cells through pores forma- ligation of two 9-residue sequences derived by het- tion. Membrane disruptor peptides can be subclassi- erodimeric splicing of α-defensin-related precursors. θ-defensins are found in neutrophils and leukocytes from certain non-human primates, whereas only pseudogenes for θ-defensins are present in the human 38 genome. Since human do not produce θ-defensins, A the peptides corresponding to pseudogenes have been recreated using solid-phase synthetic approaches and are called retrocyclins.39 Retrocyclins and other θ-defensins are also known as minidefensins.40,41 Cathelicidins form the second largest group of B AMPs produced by mammals, and are characterized by far N-terminal end named cathelin domain, a central conserved region and a variable C-terminal region.42 Like defensins, cathelicidins are synthesized as pro- State S peptides, which are cleaved in a two-step process to release the active peptides. To date, cathelicidins have been found in fi sh, birds, snakes and mammals. There is only one type of in humans (hCAP18/ C LL37).18 The also produces a great amount of cathelicidin and defensins, which are effective against Gram-positive and Gram-negative bacteria, as well as State I against yeasts. The intestine produces β-defensins that control the enteric fl ora and pathogens, while granules from leucocytes contain a high concentration Figure 2. Schematic model of action mechanism of of α-defensins that control Gram-positive and Gram- α-helix AMPs. A. Peptides in aqueous solution have lin- negative bacteria, fi lamentous fungi, enveloped viruses eal conformation. B. At the contact with the membrane, and some mycobacteria.18,43,44 the peptides acquire a helix structure, where are po- Unlike humans, which havewww.medigraphic.org.mx α- and β-defensins larized the hydrophobic and hydrophilic amino acids; genes, the bovine genome contains only the β-defensin the electrostatic interaction between the phospholipids subfamily. Cattle have the most diverse repertoire of the membrane and the cationic peptides allows that of β-defensin genes so far identifi ed and they are peptides get a parallel position respect to the mem- expressed mainly in epithelial mucosa but also in brane (state S). C. At high concentration, the peptides neutrophils and . Bovine β-defensin are are perpendicularly inserted into the membrane (state I) often related to the reproductive success and immu- forming pores. Hydrophobic regions are represented in nity of cattle to diverse diseases.45 Mouth, trachea, black and the hydrophilic regions in blue.

100 Investigación en Discapacidad Jazmín Huerta-Cantillo et al.

fi ed in two groups based on their biological activity: (i) acids, (ii) interference with the synthesis of nucleic selective, which are active against bacteria but inac- acids, (iii) inhibition of the synthesis of proteins, (iv) tive against mammalian cells, and (ii) non selective, inhibition of the enzymatic activity, (v) inhibition of the which have activity against both type of cells.46 The synthesis of cell wall, (vi) cell damage by the accumu- Estemechanism documento of esAMPs elaborado with porα-helix Medigraphic structure is given lation of peptides inside the cell, (vii) fl occulation of by the following steps (Figure 2): (i) binding of AMPs intracellular components, (viii) alteration of the septum to acid phospholipids through electrostatic forces. formation, and (ix) blocking some virulence factors, Monomers are accommodated by their amphipathic such as fl agella, proteases, secretion systems, effector regions in a way that their positive amino acids match proteins, etc. The blocking of these factors represses with the negative heads of phospholipids, when the the bacterium motility or reduces its infectivity, which local relative concentration of the peptide is low, these allows to the host controlling the infection.47-49 are parallel oriented on the membrane and they stay inactive in a state known as state S «surface»; (ii) Importance and specific when the local relative concentration of the peptide applications of AMPs increases, it tends to take a perpendicular orienta- tion respect to the membrane, in that way it becomes Due to their structural and functional characteristics, active and is inserted into the membrane, known as as well as by their low toxicity to eukaryotic cells, their state I «inserted». The different susceptibility grades broad spectrum of action against different pathogens of the membrane depend on their composition and and their immumodulatory properties, the AMPs are properties, which confer determined acceptation of the being evaluated as therapeutic and prophylactic peptide; (iii) fi nally, the insertion of peptides alters the agents.13 permeability of the lipid bilayer or the cell occurs The main interest of the use of AMPs in clinical ap- by the pore formation.47 Actually, three models have plications is based on the great concern on resistance been proposed by which the peptides are inserted in and multi-drug resistance of pathogens to antibiotics, the membrane by (Figure 3): type barrel-stave, toroidal which has increased dramatically over the past few pore and type carpet or detergent.3,11,47 years. A promising solution is the production of new Non-membrane disruptor AMPs use some of the antibiotics, which must be completely different to those following mechanisms of action: (i) binding to nucleic that have loss the effectively and whose resistance has

ABC

Figure 3. Schematic models ofwww.medigraphic.org.mx action mechanisms of membrane disruption by AMPs. A. Barrel stave pore. Peptides on the membrane surface are aggregated and inserted inside the membrane; the hydrophobic regions are aligned with the central lipid region of the bilayer, while the hydrophilic regions form the pore interior. B. Toroidal pore. Pep- tides are aggregated and induce that the lipid monolayer are continuously curved forming a pore; the pore interior is covered of peptides and also of polar heads of lipids. C. Carpet or detergent type. Peptides cover the membrane surface forming a carpet; toroidal pore are transitorily formed, which permit the entry of peptides and the membrane is starting to be disaggregated by the formation of micelles. Hydrophobic regions are represented in black and the hydrophilic in blue.

Volumen 5 Número 2 Mayo-Agosto 2016 101 Use of antimicrobial peptides not been easy to reach. Unlike the current antibiotics, tion. Bacteriocins are considered as natural innocuous the action of AMPs depends not only on the neutraliza- preservatives since due to their sensibility to protease, tion of the chemical agent but also on the alteration once inside the gastrointestinal tract, they can be of the membrane or blocking of a cellular component degraded to small peptides or to amino acids. In the that is not possible to mutate or replace. Peptides livestock trade, diverse colicins have been used as food have the advantage that there is not possibility of additives for pigs and chicken to fi ght infections and resistance transmission by horizontal transfer or in an reducing the use of antibiotics in animals.50,55 interspecies way. Additionally, unlike the traditional antibiotics, these peptides, used in a therapeutic way, Peptides with anticancer activity will not affect to the natural fl ora of patients during the treatment of an infection. Currently, the cancer is a serious health problem Currently, there is a wide fi eld of applications for worldwide. According to the World Health Organiza- the AMPs. In the treatment of diseases associated to tion (WHO) there were 8.2 millions of deaths caused pathogens, bacteriocins have been used to inhibit im- by cancer and 14.5 millions of new cases worldwide in portant pathogens of animals, humans and plants, such 2012.56 In spite of many investigations on cancer, it is as Shiga-toxin producing E. coli (STEC), enterotoxi- not completely understood due to the complexity of its genic E. coli (ETEC), -resistant Staphylococ- origins, which makes more diffi cult to fi nd alternatives cus aureus (MRSA), -resistant Enterococci for its treatment. The feature to all types of cancer is (VRE), Agrobacterium and Brenneria spp.19,50 the abnormal growth of cells, which results from dif- In the combat to sexually transmitted diseases, ferent genetic or external factors. The current treat- defensins, minidefensins and cathelicidins have ment against located cancers remains surgery and/ been studied as peptides with activity against the or chemotherapy, which are associated with severe human immunodeficiency virus (HIV). Some of damage to surrounding normal cells; furthermore, the these anti-viral peptides have been tested as topical number of cancers resistant to chemotherapy has in- microbicides against other sexually transmitted dis- creased.57 For these reasons, it is needed to develop eases.39,41,51,52 Dermaseptins and magainins, which a new class of anticancer drugs, which do not cause are AMPs from the frog skin, have spermicide activity toxicity, with low risk for inducing multi-drug resistance and can be used as intravaginal contraceptives, in but being more effective and specifi c to attack cancer the same way, they are being studied as candidates cells. In last years, the research on the use of AMPs to protect against HIV and other sexually transmit- as an alternative for cancer treatment has started and ted pathogens.53 Subtilosin has demonstrated being until today great advances have been achieved against effective for combating bacterial vaginitis caused by cancer in different systems, in vivo and in vitro.11,22,58 multi-drug resistant Gardnerella vaginalis without af- The basis of this application lies in the capability of fecting lactobacilli of the normal vaginal microbiota. these peptides to selectively recognize the membranes Subtilosin is safe, remains viable for long periods and with certain characteristics. Such selectivity depends also completely eliminates the spermatozoa motility on the difference in the membrane composition, and on in a dose dependent way.54 the electric charge in the extracellular side of the mem- In the development of resistant plants to several brane. The cationic AMPs are selectively attracted to pathogens, an ecologically responsible alternative the cell membrane depending on their negative charge; could be the genetic introduction of AMPs that serve a reason why which the peptides have a strong affi n- for protecting plants from pests and pathogen micro- ity for bacterial membranes, since they have a large organisms. This approach could reduce the use of amount of negative phospholipids on their exterior.5,59 harmful pesticides and could improve the production of The same selectivity allows recognizing the difference vegetable species with major resistance to pathogens.4 among normal cells, non-malignant tumor cells and In the production of marine www.medigraphic.org.mxspecies of commercial malignant tumor cells, since the plasma membrane interest, the study of defense mechanisms in com- characteristics of the cells change as the cancer pro- mercial species of crab, white shrimp and mussel has gresses. In a natural way, normal mammalian cells opened the possibility of the manipulation of genes that have mostly zwitterionic phospholipids in the external encode for AMPs allowing to obtain cultivable organ- side of the plasma membrane, thus they have a neutral isms more resistant to diseases.3 In the food industry, net charge. As the neoplasm advances, these phos- bacteriocins are widely used to avoid contamination by pholipids acquire a net negative charge, like normal microorganisms and then to extend the food preserva- bacteria have; this change in charge makes them more

102 Investigación en Discapacidad Jazmín Huerta-Cantillo et al. susceptible for the attack by some AMPs and antican- of large scarlet runner beans and phaseococcin from cer agents.60 Differences in the membrane composi- the seeds of small scarlet runner beans, both inhibit tion, fl uidity, charge and surface area among the cell proliferation in the leukemia cell lines and does not types of cancer affect the effi ciency of each anticancer affect the proliferation of mouse splenocytes.69,70 Cyclo- peptide. Other changes in the membrane composition, violacin O2 (CyO2), a cyclotide from Viola odorata has during a cancerous process that favors its susceptibility antitumor effects and causes cell death by membrane to the peptides, are a variation in glycosylation patterns, permeabilization in breast cancer cells, but it does not the amount of cholesterol and the amount and type produce signifi cant membrane disruption in primary of proteins present in the membrane.61 Drosocin is a human brain endothelial cells. It also can work as potential anticancer peptide since it highly recognizes chemosensitizing agent against drug resistant breast glycosylated membranes, which are present in breast cancer cells.71 Cn-AMP2 from green coconut water cancer.62 Anticancer peptides have a broad spectrum is an anionic peptide with anti-proliferative activity of action, ability to rapidly kill cancer cells, to destroy against 1321N1 and U87MG human glioma cell lines.72 primary tumors, to prevent metastasis, and at adequate Cyclosaplin from somatic seedlings of sandalwood has concentrations do not damage normal cells or vital antiproliferative activity against human breast cancer organs.58,63 Antitumor AMPs effects may generally cells but not against normal fi broblast cells by induc- occur either by membranolytic or non-membranolytic ing apoptosis.73 mechanisms. Beside of the membrane disruption, Insects. Cecropins A and B exert selective cytotoxic membranolytic events involve the permeation and and antiproliferative effi cacy in bladder cancer cells swelling of mitochondria with release of cytochrome c, without damaging the normal human fi broblast by in- apoptosis events or also acting by necrotic pathways. ducing an increase in LDH release that decompensate Non-membranolytic activities involve the perturbation and kill the cells.74 CB1a, a cecropin derived peptide, of angiogenesis process.64 show high cytotoxic activity against leukemia and stom- Nowadays, there are different peptides with tested ach carcinoma with low hemolysis.75 CecropinXJ be- anticancer activity.1,11,22,64,65 In fact, there are various longs to cecropin-B family; it could kill cancer cells but examples of anticancer peptides from different organ- have no toxicity to some normal cells. The mechanism isms, some examples are: of action occurs by inducing damage to microtubules Bacteria. Some bacteriocins have activity against and actin of human esophageal carcinoma cells, and different tumor cells. Colicins A and E1 form pores and inhibits migration and invasion of tumor cells.76 CopA3, inhibit the growth of 11 tumor cell lines, while colicins an analog of coprisin, an insect defensin from dung D, E2 and E3 inhibit the viability of leukemia cells. beetle, shows cytotoxicity against three human leuke- is a lantibiotic that induce DNA fragmentation mia cell lines by induction of a caspase-independent and apoptosis in three tumor cell lines of head and signaling pathway mediated by apoptosis inducing neck squamous cell carcinomas (HNSCC). Lantibio- factor (AIF). CopA3 also causes necrosis of gastric tics and colicins could be an alternative therapy for cancer cells, probably through interactions with phos- the treatment against cancer, since they are safe due phatidylserine.77,78 HaA4, the synthetic analogue of to the properties of these bacteriocins.19,66 The AMPs harmoniasin from ladybug, is a homodimeric peptide, m2163 and m2386 from a lactic acid bacterium Lac- which has cytotoxic effect against two human leukemia tobacillus casei induce apoptosis in human colorectal cell types by induction of necrosis and apoptosis.79 cancer cell by both extrinsic and intrinsic apoptosis Anoplin is a recently discovered AMP isolated from the pathways.67 venom sac of a spider wasp; it could inhibit the prolif- Plants. Of the 12 plant AMP families, 3 contain eration of murine erythroleukemia cells via disrupting members with cytotoxic and anticancer properties: the the integrity of cell membrane and additionally arrests 22 80 thionins, defensins, and cyclotides. Unfortunately, the cell cycle in the G0/G1 phase. MP1-1 is an analog most of the thionins and cyclotideswww.medigraphic.org.mx with anticancer of the AMP polybia-MPI, a peptide isolated from the activity are unselective or un-tested in their ability to venom of wasp; it binds to human prostate and liver damage normal cells; nevertheless they could be use cancer cells causing injury, swelling, bursting, and fi nal for the design of new anticancer molecules. Defensin cell death by necrosis.81 from the purple pole bean inhibit the proliferation of four Myriapods. It is recently reported that the AMP colo- cancer cell lines and do not affect human embryonic pendrasin VII derived from a centipede has anticancer liver cells or human erythrocytes under de same con- activity against leukemia cell lines by decreasing their ditions.68 Coccinin, a peptide purifi ed from the seeds viability and inducing necrosis mediated by specifi c

Volumen 5 Número 2 Mayo-Agosto 2016 103 Use of antimicrobial peptides interaction with phosphatidylserine, which is enriched variants and with decreased affinity toward human in the membrane of cancer cells.82 healthy cells even by intratumoral administration.91 Arachnids. MAP-04-03, which is derived from Piscidin-1, isolated from mast cells of a hybrid striped Ixosin-B, an AMP found in the salivary glands of the bass, at low doses induces both apoptosis, necrosis hard tick shows antiproliferative effects on breast and also inhibits the migration of fi brosarcoma cells.92 cancer cells and also inhibits cancer cells migration.83 Pardaxin, another fi sh AMP exhibits antitumor activ- Marine invertebrates. Tachyplesin I restricts pros- ity toward murine fi brosarcoma in vitro and in vivo; it tate cancer cells by activating the classical complement inhibits the proliferation of MN-11 cells and induces pathway and cell lysis mediated by tachyplesin also apoptosis through the death receptor/nuclear factor activates the alternative complement pathway; all these (NF)-κB signaling pathway in tumor-bearing mice.93 events provoke a decrease of the proliferative capacity Mammals. BMAP-27 and BMAP-28 that are derived of several types of cancerous cells.84 Penaeidin-2 (Pen- of bovine cathelicidin have cytotoxic effects against dif- 2) is a derived-AMP from the Pacifi c white shrimp that ferent leukemia cells by altering the infl ux of Ca2+, DNA induce apoptosis specifi cally on kidney cancer cell.85 fragmentation and reducing the mitochondrial potential Amphibians. Magainins and other peptides derived provoking a permeabilization that kills the cancer cells from amphibian have cytotoxic activity in cell lines of without having hemolytic activity.65 Defensins have bladder cancer, permeabilize membranes from cervi- cytotoxic effects on different human and mouse cancer cal cancer lines, and induce apoptosis by releasing cell lines from lymphomas, oral carcinoma, teratocarci- cytochrome c from the mitochondria of leukemia cells. noma and renal cancers through an interaction with the Magainins have been intratumorally injected and are membrane.11 In fact, the β-defensin 2 (BD2) has been able to completely eradicate the melanoma growth.11 proved in studies of cancer gene therapy, BD2 exhib- MG2A ( II conjugated to the N-terminus of its chemotactic activity in dendritic cells (DCs) both in the cell-penetrating peptide [CPP]) causes enhanced vitro and in vivo models. Recruitment and activation of cytotoxicity against tumor cells, at least 30 times higher DCs in tumor niches result in signifi cant tumor growth than mangainin unconjugated and it is about three to fi ve inhibition.94 Bovine lactoferricin (LfcinB), like defensins, times less cytotoxic to normal cells.86 Temporin-1CEa is has a cytotoxic activity on leukemia, fi brosarcoma, an antimicrobial peptide isolated from the skin neuroblastoma and breast carcinoma cells and its of the Chinese brown frog; this AMP triggers a rapid activity involves the sequential generation of reactive cytotoxicity in breast cancer cells through membrane- oxygen species, loss of mitochondrial potential and destruction and intracellular mechanisms involving activation of the caspase cascade, culminating in cell mitochondria, such as intracellular calcium release and death by apoptosis.95 LL-37/hCAP-18 is a cathelicidin ROS over production.87 B2 is found in skin that induces DNA break and mitochondrial damage in secretions of the Amazonian tree frog, inhibits the pro- leukemia cells and inhibits proliferation and induces G0/ 96 liferation of various human tumor cell types in vitro and G1 phase cell cycle arrest in gastric cancer cells. An inhibit tumor growth of the human prostate adenocarci- analog of the LL-37 peptide, FF/CAP18, induces apop- noma cell line in a mice model in vivo inducing necrosis totic cell death, via mitochondrial membrane depolariza- by increasing lactate dehydrogenase (LDH) release.88 tion and DNA fragmentation, in the oral squamous cell Birds. GLI13-8, one of AMP from linear avian carcinoma cells and in human colon carcinoma cells. β-defensin-4 (RL38) analogs, has cytotoxicity in three FF/CAP18 induces apoptotic cell death via changes human carcinoma cells (HepG2, SGC7901, and A375) in the metabolic profi le in colon cancer cells.97 In fact, in a dose-dependent manner. GLI13-8 induces apop- LL37 is one of few AMPs with anticancer properties that tosis and loss of mitochondrial membrane potential in has currently been approved to start clinic phase I and HepG2 cells, and in the appropriate doses it had no II assays. The goal of this clinical research study is to toxicity towards the normal human fi broblasts.89 fi nd the appropriate dose of LL37 that can be given to Fishes. Currently, there arewww.medigraphic.org.mx several studies on patients with melanoma by intra-tumoral injections and AMPs from fi shes with anticancer properties. Tilapia also learn if LL37 can stimulate the immune system to hepcidin TH2-3 specifi cally inhibits human fi brosar- help control cancer (NCT02225366). coma cell proliferation and migration in a concentration- dependent manner causing lethal membrane disrup- Challenges to overcome about the AMPs tion.90 NRC-03 and NRC-07 are two peptides from the AMP pleurocidin family from fl atfi sh with activity against In spite of the great potential of the AMPs, it is still human breast cancer cells including drug-resistant needed to overcome several challenges and to improve

104 Investigación en Discapacidad Jazmín Huerta-Cantillo et al. several factors to obtain safe and functional products results of interest and clinical importance. The de- capable to hit the market. These challenges consist on sign of novel peptides is based on conserving the fi nding optimal doses and routes of administration to characteristics and fundamental properties of the achieve the high concentrations required so the peptides AMPs, such as their cationic character, amphipathic can work, to reduce their hemolytic activity to avoid region and tridimensional structure, which are crucial any kind of toxicity, to improve their specifi city to avoid characteristics that determine their antimicrobial activ- their broad spectrum of action, which affect the normal ity.103 In the same way, it is known that the hemolytic fl ora and to improve their stability for reducing their activity and therefore their toxicity correlate with the lability to proteases in vivo. Additionally, it is important high hydrophobicity, amphipathicity and helicity of the to improve other factors as the effi ciency of synthesis, peptides. The electrostatic interactions are respon- and production costs for that the AMPs can really be sible for inducing a stable secondary structure, so the a viable alternative for their use as drugs.98 There are binding process requires that the peptide conforma- many theoretical advantages and disadvantages of tion is adapted in response to these interactions to development of peptides as anti-infective drugs, such maximize the attraction and minimize the repulsion as those mentioned above and in other reviews.99 forces.46 Currently, it has been envisaged different strate- Peptides can also be designed base on the previ- gies to overcome some of these disadvantages. Since ous knowledge of proteins and types of membranes; anticancer peptides require constant administration for instance all D-amino acid analogues are highly for maintaining determined systemic levels for their resistant to proteolytic degradation without negative effectiveness, an alternative can be the introduction effects in the peptide or protein functionality; also by of genes encoding for the peptides directly to the knowing that the chemical composition of different cancer cells. To reduce the toxicity of the peptides, it types of membranes contributes to the selectivity of the has been proposed optimizing their sequences using AMPs, which confers the specifi city for attacking to a computational approaches for decreasing their he- determined type of pathogen or certain type of cells.11,46 molytic activity but keeping the antibacterial activity.98 The following strategies are used for the design of To diminish the susceptibility to peptide proteolysis, these novel peptides: the use of D-amino acids for their synthesis has been (i) Synthesis de novo by the selective incorpo- proposed.46 Finally, to improve the specifi city of the ration of unusual or non-natural amino acids. The peptides, it has been proposed to design peptides incorporation of D-amino acids with different hydropho- directed to a specifi c binding sequence of factors of bicity can reduce the resultant hemolytic activity, but it pathogen agents.100,101 can also modify their microbicidal activity by favoring Regarding to the economic viability, the main the attack to some types of pathogens but decreasing problem is the high cost of the synthesis for produc- the affi nity by others. This strategy consists in deleting ing the AMPs. It is possible to synthesize most of the or inserting natural amino acids or exchanging them peptides including defensins with their complex disul- for unusual amino acids having distinct functional fi de bridges, but the cost is very high, which prevents groups to the originals. This change generates larger their production at large scale. To avoid this problem, or smaller spaces within the amino acid sequence it has been currently developed biological expression provoking a change in the tertiary structure of peptides. systems, as recombinant systems in baculovirus ex- Distinct parameters such as the hemolytic activity, pressing active defensins, which are pure, structurally selectivity, microbicidal activity or resistance to prote- correct and in milligram amounts. The AMPs can be ases can be evaluated or improved by changing the produced in transgenic plants, in bacteria and in milk tertiary structure.46 from transgenic cows; however, it is still needed to Synthetic anticancer diastereoisomer peptides have improve the purifi cation process to avoid the contami- been designed, with the ability to permeabilize only nation by host proteins, which couldwww.medigraphic.org.mx induce an immune membranes with phospholipids with negative charges 102 response in the patient. as the D-K4R2L9, that has a third of its sequence composed by D-amino acids. This peptide binds and Design of novel peptides lyses cells from melanoma, and when intravenously injected prevents the formation of lung tumors. The

The design of novel peptides is looking for more ef- D-K6L9 diastereoisomer, containing D-Lys and D-Leu fi cient peptides and stable in different environments in a third of its sequence, has anticancer activity and and conditions but mostly, looking to fi nd particular synergically acts with other drugs, it has the capacity

Volumen 5 Número 2 Mayo-Agosto 2016 105 Use of antimicrobial peptides to stop, reduce and inclusive disappear tumors in vivo. with the insertion positions, since they can dramatically

The D-K6L9 diastereoisomer avoids the metastasis affect the biological activity. The same methodology dissemination in immunosuppressed mice, and it is can be used to test the toxicity in erythrocytes varying resistant to the degradation by proteases, while the the type of descriptors used to build the QSAR mod- 106-108 L-K6L9 (that does not contain D-amino acids) does not el. Several studies of QSAR have been done using have activity and it is degraded by proteases once it is different descriptors and models to fi nd lactoferricin inoculated to mice.104 derivatives, protegrin analogues, bactenecin-derived In control of plant pathogens, an alternative to the peptides or similar de novo peptides.109-111 QSAR mod- currently used pesticides, AMPs with non-natural eling of AMPs to date has been based on predicting amino acids such as triazolyl, biaryl and D-amino differences between peptides that are highly similar. acids into their sequence with activity against plant The mathematical models used to relate the QSAR de- pathogenic bacteria and fungi are a great promise.105 scriptors to biological activity have been linear models (ii) Design by quantitative structure-activity rela- such as principle component analysis or multivariate tionship (QSAR). This strategy is used starting from a linear regression. However, with the development of model of proven effectiveness, from which the peptide high-throughput peptide synthesis and an antibacterial analogues are generated by making small modifi ca- activity assay, the numbers of peptides and sequence tions in the sequence. As in the previous strategy, diversity able to be studied have increased dramati- the modifi cations provoke signifi cant structural and cally. Also, «inductive» QSAR descriptors have been biophysical changes that correspond to very marked recently developed to accurately distinguish active from effects in the antimicrobicidal activity and the toxicity of inactive drug-like activity in small compounds. «Induc- the peptides. Thus, a peptide with proven effectiveness tive» QSAR in combination with more complex math- is used as a base sequence, then a library of variants ematical modeling algorithms such as artifi cial neural or analogues can be created by making point mutations networks (ANNs) may yield powerful new methods for and combination of two or three particular mutations, in silico identifi cation of novel AMPs.112,113 such mutations are introduced at different positions (iii) Design of selective peptides for Gram- along the base sequence, and fi nally the microbicide negative bacteria. Gram-negative bacteria as Pseu- potential for each analog is evaluated. Based on certain domonas aeruginosa, Acinetobacter baumannii and number of analogues and their corresponding activity, enterobacteria are the main agents causing hospital- a quantitative model is developed making a structure- acquired infections.114 Gram-negative bacteria have as activity relationship (QSAR), which is used for predict- a particular feature the presence of ing variants with improved activity. The construction of (LPS) on their cell surface, which are responsible for the QSAR model requires a set of surface descriptors septic shock in the patients; for this reason, it has as: surface and volume (VolSurf) and the charged been proposed the design of peptides that specifi cally partial surface area (CPSA). The VolSurf descriptor recognize to Gram-negative bacteria. Cyclic peptides is based on interaction regions specifi cally designed mimicking the binding sites of LPS-binding proteins for the optimization of the pharmacokinetic properties have been designed and they show 200 times more of the protein, while CPSA descriptor is based on the activity than on Gram-positive bacteria.115 In fact, some partial charges of the proteins according to the fi eld derivatives of cathelicidins, polymixins and bactericidal/ force used. In the QSAR model, these descriptors permeability-increasing protein (BPI) display an ability mimic the interaction of the peptide with the membrane, to strongly bind LPS and break its aggregates, but they as well as the surrounding environmental; fi nally the have also endotoxin-neutralizing activity which can reliability of the model is validated by predicting and potentially suppress LPS-induced hyper-infl ammatory testing the new peptides.106 responses that leads to multiple organ failure and le- The knowledge that the net positive charge of thality.116-118 Similarly, peptides, having other cellular peptides must be maintainedwww.medigraphic.org.mx during the design was components of Gram-negative bacteria as target, can obtained from this type of model, and that a big polar be designed. area will not provide a signifi cant increase in their po- (iv) Design of specifically targeted AMPs tency. The hydrophobic area is extremely needed, and (STAMP). One of the complications of AMPs is that the increased hydrophobicity allows a major insertion they must reach certain concentrations on the mem- within the membrane, but causes a loss of selectivity. brane to become actives, and thereby it is needed that Consequently, a balance between charges and the peptides reach and accumulate on their target sites. hydrophobic amino acids is required and to be careful To help on this, a technology to smartly design selec-

106 Investigación en Discapacidad Jazmín Huerta-Cantillo et al. tive peptides has been developed, using a technology tive bacteria and fungi as the original peptides, but known as specifi cally targeted AMPs (STAMP). This they had neither hemolytic, nor toxicity in in technology involves the construction of a peptide for vitro and in vivo experiments.124 The hybrid peptides binding to two functionally independent components: melittin-LL37 combining the hydrophobic N-terminal a region for target localization and an antimicrobial fragment of melittin with the core antibacterial frag- region; both joined by a short and fl exible connector. ment of LL37, had an even more potent antibacterial The target localization region provides selectivity to activity again bacteria than melittin and LL37 alone, the antimicrobial region since it specifi cally binds to whereas did not exhibit hemolytic activity to sheep determinants of the pathogen. This binding increases erythrocytes.125 Hybrid design also helps to achieve the selective accumulation of the peptide and sig- short length peptides with improved potency and nifi cantly increases its local concentration, thus the selectivity; a novel short peptide CS-1a (14 residues) antimicrobial region can eliminate the pathogen. The was derived using a sequence hybridization approach STAMP technology is an effective tool for eradicat- on sarcotoxin I (39 residues) and cecropin B (35 ing pathogens without affecting the normal fl ora, and residues). CS-1a shows a good antibacterial activity thereby preventing secondary infections.100,101 against susceptible as well as drug-resistant bacte- The STAMP C16G2 was developed to target the rial strains with non-hemolytic activity against human cariogenic oral pathogen mutans. red blood cells even at very high concentration.126 In C16G2 specifi cally kills S. mutans in a mixed-culture the same manner, hybrid peptide design also can be environment, having minimal effects on noncariogenic used to develop salt resistant peptides. The human oral fl ora, which is benefi cial to prevent exogenous S. β-defensin-1 (HBD-1) antibacterial activity is consid- mutants colonization. C16G2 has a truncated version erably impaired by elevated ionic strength meanwhile of the S. mutans competence stimulating peptide retrocyclins (synthetic human θ-defensins) are salt

(CPSC16) pheromone, as the STAMP targeting do- resistant, non-hemolytic and non-cytotoxic in vitro. main for effective accumulation on the S. mutants cell The hybrid peptide of the C-terminal region of HBD-1 surface.119,120 Other new STAMP recently reported is and the nonapeptide sequence of a retrocyclin exhibits CDAK, which is composed of a cyclic isoDGR motif, enhanced antimicrobial activity against bacteria, fungi which is used as a targeted delivery tool because it and clinical isolated of eye infections and the peptide binds to αvβ3, a type of integrin that overexpresses in retained activity in the presence of NaCl and serum tumor cells and of an AMP region. CDAK shows cyto- and is non-hemolytic in vitro.127 Finally, hybrid peptides - toxic activity in CD13 /αvβ3 breast cancer cells in vitro could serve as enhancer of activity. Lysozyme is highly and in vivo, whereas normal cells are less sensitive active against Gram-positive bacteria but alone is inac- to this peptide.121 Thus, peptides specifi cally directed tive against Gram-negative bacteria because it cannot against overexpressed compounds in tumor cells are reach the peptidoglycan layer and cecropin may disrupt good candidates for being used as anticancer agents the outer membrane. Novel hybrid protein combining since they would have little chance of affecting healthy cecropin with human lysozyme shows improved in vitro tissue. antimicrobial activity and action spectrum being that (v) Design of hybrid peptides. This is another cecropin disrupts the Gram-negative bacteria outer strategy that consists in the fusion of two peptides membrane, giving the access to the peptido- with tested activity but directed to a different target, glycan in cell wall.128 enhancing its action spectrum.122 To date, several new (vi) Peptidomimetics. There is a new class of de- peptides has been designed using this strategy: human signed AMPs that can mimic the structure, function and β-defensins have different intensities of antibacterial mode of action of native AMPs, while being stable to activity; human β-defensin-3 (HBD3) kills Staphylo- enzymatic degradation and conceivably exhibit better coccus aureus with a 4- to 8-fold higher effi ciency pharmacological properties. Peptidomimetics can dis- compared to human β-defensin-2www.medigraphic.org.mx (HBD2) and both play antibacterial activity against drug-resistant strains has the same activity against E. coli but one HBD2/ and are less susceptible to resistance development in HBD3-chimera kill both S. aureus and E. coli with an bacteria.129,130 Peptidomimetics have been prepared by even higher effi cacy compared to the wild type mol- cyclization of linear peptides and/or coupling of stable ecules.123 Hybrid peptides design has also been used unnatural amino acids. Unnatural amino acids can be to reduce the hemolytic activity of the original peptides; generated modifying the native ones by different ways cecropin-melittin hybrids (CAM and CBM) have strong such as: amine alkylation, side chain substitution, antibacterial abilities against Gram-positive and nega- structural bond extension, cyclization and isosteric

Volumen 5 Número 2 Mayo-Agosto 2016 107 Use of antimicrobial peptides replacement within the amino acid backbone. Isosteric virulence factors.99 Many peptides have entered into replacements within a peptide backbone result in im- clinical trials, with varying degrees of success.13,18,135 In proved pharmacokinetic properties. Backbone modifi - Table I are shown some peptides that have reached a cations of amino acids can be categorized as follows: clinical phase and obtained good results. (i) changing the amino functionality; (ii) replacement Regarding to the peptides that failed, the mayor of α-carbon; (iii) extension of the backbone by one or problems are poor distribution, frequent toxicity and two atoms and (iv) atom modifi cation of the carbonyl presence of adverse events, fast descomposition or function.131 Some peptidomimetic types are β-peptides, half-life, or that induce a high level of humoral response peptoids, arylmamide oliogmers, β-turn mimetics and and antibody production.18 However, there are some AA peptides (which contains N-acylated-N-aminoethyl peptides that do not present these inconvenient but amino acids).130 It has been shown that peptidomimet- they have been rejected because they have not demon- ics have more dihedral angles compared to canonical strated any signifi cant advantage over some approved peptides, and this molecular design induces high fl ex- drugs. Based upon the data obtained from clinical tri- ibility. The presence of backbones with certain fl exibility als, no data exist regarding peptides for development leads to a potent and broad-spectrum of antimicrobial of immune response when they are applied topically. activity; this is more important than the secondary In spite of there currently are not drugs based on structures.130,132 approved antimicrobial peptides, in the food industry (vii) Design of peptides sensible to the environ- have had better advances regarding the use and com- ment. The pathogen organisms frequently cause mercialization of AMPs. Nisin is a bacteriocin produced drastic changes in the surrounding environment as a by Lactococcus lactis and the fi rst approved lantibiotics result of their growth and metabolic activities. Thereby, as food preservative and it is commercialized in more a strategy consists on designing peptides sensible than 45 countries (Nisaplin®). The bacteriocin, pediocin to the changes in the environment, such as the pH. PA-1, inhibits Listeria monocytogenes growth in meat Peptides highly sensitive to pH changes in their en- products and it is commercialized as Alta® 2341. For vironment have been designed, these peptides have inhibiting contamination, it has also been used the not activity at normal physiological pH of 7.5 but they enterocin AS-48 in ciders, fruit juices and canned become active at pH of 5.5. Therefore, they will work vegetables, while the enterocin CCM4231 has been only in where they fi nd an acid environment generated used in soy milk and the enterocin EJ97 in puree.136 by a pathogen.133 This strategy has also been used to Although hundreds of AMPs have been identifi ed, obtain an anticancer peptide with less toxicity, since only some ones have entered to clinic phase assays this peptide only can be activated by tumor acidity and and only a few ones have obtained favorable results it is not active or less active under physiological condi- and no one has been approved yet for clinical use. tions. AMitP is an activatable heterodimer formed by Thereby, it is necessary to continue with more research the AMP mitoparan (MitP) fused to its anionic binding to improve AMPs to reach clinic phases. However, we partner peptide (MitPE) (which contains glutamic acids can recognize that in the last years great advances and histidines). AMitP remains inactivated at normal pH have been done and the future appears promising for values; MitP has no lytic activity because is binding to developing and use of AMPs mainly for those that will

MitPE by electrostatic attraction. In contrast, MitP dis- be used in topical way. sociates from MitPE and recovers its lytic activity when the anionic charges of glutamate residues in MitPE are Conclusions and perspectives neutralized at acidic pH values.134 The AMPs have a completely different mechanism of Developments in clinic phase action to common antibiotics, and during the evolution and commercial application of AMPs they have been functioning as a chemical shield of www.medigraphic.org.mxdefense over millions of years in plants and animals, There are different strategies of therapeutic applica- thereby they could become in an alternative for com- tion for AMPs: (i) as simple anti-infective agents, (ii) bating diseases, mainly by the increase of multi-drug in combination with conventional antibiotics or anti- resistant pathogens or superbugs. The mechanism by virus to promote an additive or synergistic effect, (iii) which these peptides act, make them very diffi cult to as immunostimulatory agents to improve the innate generate bacterial resistance, becoming a promising immune response, and (iv) as neutralizing agents of future as more durable therapeutic agents. Besides, endotoxins to prevent complications associated with knowing the role of peptides as modulator of the in-

108 Investigación en Discapacidad Jazmín Huerta-Cantillo et al. 7 138 138 143 12,144 NCT00509834 References NCT00509938 139,140 141,142 NCT00563394 NCT00563433 NCT01590758 NCT01594762 NCT02066545 cream cream Topical Topical Topical Topical Topical solution topical gel Method of application Intravenous Mouthrinse/ Oral solution NCT00630656 Safety in humans established Intravenous Diminish the severity of disease in subjects with mild to moderate acne/reduce all types of acne lesions Safety in humans established Intravenous Safety and reduces plaque, gingivitis and gingival bleeding Reduce 28-day all-cause mortality in patients with severe sepsis. This reduction was sustained at six months Reduce serious complica- tions, such as amputations Effective treating diabetic pa- tients with a mildly infected foot ulcer and might reduce the risk of selecting antimicrobial-resistant bacteria Recruiting Antimicrobial peptides tested in clinical studies. Table I. Table Study on the tolerability and early efficacy of hLF1-11 in patients with proven candidaemia Safety, tolerability and Safety, prevention of fungal infections in autologous hematopoietic stem cell transplant recipients of experimental Treatment gingivitis of mildly infected Treatment diabetic foot ulcers Treatment of meningitis Treatment Lactoferrin Indolicidin of acne Treatment Parent nameParent IndicationLactoferrin Outcome Lactoferrin of severe sepsis Treatment Magainins of diabetic foot ulcers Treatment Magainins www.medigraphic.org.mxBPI (Bactericid- al/permeabil- ity-increasing protein)

XOMA

21 Antimicrobial/immunomodulatory hLF1-11 Phase I AM-Pharma Omiganan (MBI 594AN) Phase IIa/IIb Cutanea Life Sciences, Inc. Peptide name/ Peptide phase/company hLF1-11 Phase I/II AM-Pharma P-113 Phase I/II Talactoferrin Phase II Agennix eBPI Phase III MSI-78 (Pexiganan) Phase III MacroChem Corporation Pexiganan Phase III Dipexium pharmaceuticals

Volumen 5 Número 2 Mayo-Agosto 2016 109 Use of antimicrobial peptides 145 130 12,144 References NCT02225366 NCT01158235 NCT01211470 NCT02052388 NCT00231153 Topical gel Topical Oral solution NCT00095186 Method of application Intratumoral Injections Topical gelTopical NCT00608959 gelTopical NCT01784133 Intranasal topical gel Intravenous infusion Reduce catheter colonization and local site catheter-related infections Completed, no results posted Effective for killing bacteria (germs) that live on the surface of the skin Reduce lesion count with once-daily at nine weeks of treatment Reduce the bacterial load already after a single day of treatment Decrease of lesion area, and this effect is sustained at least until 28 days

Antimicrobial peptides tested in clinical studies. Table I. Table Staphylococcus aureus Prevention of catheter-related Prevention of catheter-related infections of advanced renal cell Treatment carcinoma Treatment of acute skin infec- Treatment tions Safety and efficacy in subjects with Rosacea of nasal colonization Treatment with (MRSA/MSSA) Indolicidin Lactoferrin Parent nameParent IndicationIndolicidin topical skin antisepsis For Outcome Peptidomi- metic Cathelicidin of melanoma Treatment Approved, no yet recruiting www.medigraphic.org.mxIndolicidin Peptidomi- metic Agennix Omiganan (MX-226, CLS001) Phase III Mallinckrodt Talactoferrin Phase I Peptide name/ Peptide phase/company Omiganan Phase III Mallinckrodt (PMX-30063) Phase II Cellceutix Corporation Anticancer LL37 Phase I/II M.D. Anderson Cancer Center Omiganan (CLS001) Phase II Cutanea Life Sciences, Inc. LTX-109 Phase I/IIa Lytix Biopharma AS

110 Investigación en Discapacidad Jazmín Huerta-Cantillo et al. nate immune response, they can be helpful as guide that allow anticipating that over the next decade some for developing immunomodulatory therapies. AMPs of these results will be used by the industry and will fulfi ll many of the requirements demanded by the contribute to the society in the fi ght against pathogens. pharmaceutical industry, agriculture, aquaculture, and the food production in general. The manipulation of the Acknowledgment chemical structure and fundamental characteristics of AMPs, will allow the creation of synthetic peptides We thank Secretaría de Ciencia, Tecnología e In- specifi cally designed to fi nd responses to different novación de la Ciudad de México for a grant to FNG problems affecting health. (PICSA 12-231), and Alejandra Ochoa and Roberto Vi- Due to that the AMPs differentially interact with dal for their invaluable comments to improve this work. diverse types of membranes, small changes, carefully selected, in the structural and physicochemical prop- Bibliography erties of the amino acids can allow major changes in the potency and selectivity of a particular peptide for 1. Pushpanathan M, Gunasekaran P, Rajendhran J. Anti- a specifi c pathogen or cell. Nowadays, some AMPs microbial peptides: versatile biological properties. Int J are being optimized for a more effective anticancer Pept. 2013; 2013: 675391. activity as well as for a better therapeutic alternative 2. Boman HG. Innate immunity and the normal microfl ora. and economically more viable for generating chemo- Immunol Rev. 2000; 173: 5-16. therapeutic drugs in a close future. 3. Montano-Perez K, Vargas-Albores F. 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