Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • A61K38/1761—Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
  • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

• Dendrimers are complex molecules with very well defined chemical structures. From a chemical perspective, dendrimers are monodisperse macromolecules (of consistent shape and size) with a three-dimensional, regular and highly branched architecture. These dendrimers consist primarily of two parts (listed from its internal center to its surface): 1) a central core with two or more reactive groups, 2) repetitive units covalently bonded to the central core and organized in a series of radially homocentric layers called "generations. "
• the molecule also includes 3) a third layer which corresponds to amino acids or functional groups thereof, and optionally alcohol groups which are conjugated as peripheral terminal groups that will modulate the physical and chemical properties of the surface of the dendrimer, giving these particles surface properties equivalent to those observed in some proteins.
• the dendrimer-based synthetic proteins of the present invention correspond to polymers with repetitive units that generate a tertiary structure with moieties and domains where the amino acids or functional groups of the surface define the electrostatic potential of the dendrimer-based synthetic proteins. This electrostatic potential, rationally designed, is what causes the synthetic proteins to have properties of biological proteins.
• dendrimers size, shape, and surface functionality can be controlled. Dendrimers may be customized through the use of many reactive groups for conjugation, making this a commercially attractive drug delivery method. Moreover, the small size of dendrimers (5-15 nm) allows the molecule to easily diffuse through tissues (Muro 2009). Another advantage is that dendrimers have desirable chemical and physical properties for pharmaceutical applications, including low viscosity (Klajnert, 2001). In terms of dendrimers as vector or carriers for gene delivery, PAMAM (poly(amido amine)) is one of the most prevalent, and currently being tested and produced (Holister, 2003). It is the first complete, commercialized dendrimer family.
• PAMAM is terminated in amino groups that interact with phosphate groups of nucleic acids. This guarantees consistent formation of transfection complexes. Furthermore, PAMAM molecules have the ability to travel across cell monolayers via paracellular and transcellular pathways, with special emphasis on the endocytosis pathway (Saovapakhiran 2009).
• a commercially available reagent for transfection, called Superfect contains activated dendrimers (Klajnert 2001). Activated dendrimers can carry a quantity of genetic material equaling or surpassing that carried by viral vectors. Superfect-DNA complexes are described by high stability and effective and efficient transport of DNA into the nucleus.
• the polyamidoamine (PAMAM) dendrimers represent a new class of artificial macromolecules and are versatile candidates as scaffolding or vehicles for nanomedicine, especially in the field of cancer diagnosis and treatment (Cheng et al., 2011).
• PAMAM polyamidoamine
• synthesis of high generation dendrimers is limited by crowding of the terminal groups, a phenomenon known as the "starburst effect" (Amiji 2007).
• Amiji 2007 With PAMAM it becomes difficult to add new terminal groups beyond the 10 th generation, where the dendrimer has a diameter near 12 nm (Amiji 2007).
• dendrimers can -in some situations- be a weakness; for example, the dendrimer can diffuse into non-favorable tissue regimes because of its small size. Therefore, controlling the dendrimer diameter would be of crucial importance.
• siRNAs are 20-30 nucleotide-long RNA molecules that exhibit sequence-selective inhibition of gene transcription (Hannon 2004). In particular, siRNAs mediate transcriptional gene silencing by disrupting messenger RNAs (mRNAs) through complementary binding (Reischl 2008).
• mRNAs messenger RNAs
• siRNA-based therapeutics are complicated by their low transfection efficiency, poor tissue penetration, and non-specific immune stimulation in vivo tissues (Persengiev 2004, Reischl 2008). Not surprisingly, the greatest challenge in developing siRNA-based therapeutics is developing an efficient delivery system that will specifically target desired cells.
• PEI Polyethyleneimine
• Superfect has been widely used as gene delivery agent due to its cationic nature that allows it to interact with the negative-charged phosphate backbone of nucleotides and the negative-charged elements of cell membranes. These interactions induce endocytotic uptake of the nucleotide into the cells (Sirsi, 2009).
• PEI-complexed particles can cause cytotoxicity (Bivas-Benita 2004).
• PEG polyethylene glycol
• the structure and size for PEI was chosen to be branched 25 kDa PEI because this size has been successfully used in transferring DNA (Bivas-Benita 2004).
• This PAMAM Arg-DNA complex maintained the gene expression for 10 days in mice, which makes this new dendrimer an effective alternative as a gene vector (Kim et al., 2006).
• the same investigators obtained biodegradable dendrimers by conjugating them with lysine or arginine separately (Nam et al., 2009; Nam et al., 2008).
• This new dendrimer exhibited great efficiency in terms of transfection and low cytotoxicity (90% of the viable cells) and provides an efficient method for the transfection of siRNA in cortical cell cultures.
• the cytotoxicity and efficiency of delivery of PAMAM conjugated with amino acids depends a lot on its properties; the anionic ones as well as glutamate and the neutral ones such as phenylalanine and leucine can be less toxic than the cationic ones (lysine or arginine) (Kono et al., 2005; Kim et al., 2006).
• Dendrimers functionalized on their surface with neutral and hydrophobic amino acids show great efficiency due to their ability to pass through membranes (Kono et al., 2005).
• the synthesis of heterobifunctional dendrimers has also been achieved with an amino acid and other chemical groups, such as carboxyls or hydroxyls. These dendrimers have been shown not to be toxic (Navath et al., 2010).
• a functionalized dendrimer were to be used as a carrier for releasing drugs for treating diseases or disorders, it is necessary to evaluate the cytotoxicity of the dendrimer in different types of cell so as to detect early on whether they behave as toxic or biocompatible agents.
• the property of surface charge is likely the primary determining factor for the cytotoxicity of the dendrimers (Cheng et al., 2011).
• Cationic dendrimers with amino groups on the surface exhibit a high level of cytotoxicity in comparison to anionic dendrimers, as has been shown in Caco-2 cells (Jevprasesphant et al., 2003).
• toxicity has been attributed to the introduction of large quantities of DNA into the cell (Andrews et al, 1997).
• dendrimers have been constructed with biocompatible polymers such as PEG, giving them certain advantages such as better biodistribution performance and a reduced cytotoxicity.
• Dendrimers with surface modifications have also been constructed, including the PEGylated, acetylated, glycosylated dendrimers, and those functionalized with amino acids (Cheng et al, 2011).
• dendrimers as release systems for therapeutic agents are difficult to have a rational design which ensures both transfection and efficient release as well as the protection of nucleic acid-based drugs while still being non-toxic to cells.
• the affinity of the dendrimer for the nucleic acids can be very high, which can impede release of drug within the cell effectively.
• dendrimers that have the capacity to overcome disadvantages of dendrimers use, target the cells through cell wall receptors, improve the cell permeation; cross the cell membrane, protect the substance it carries, and release the substances it carries.
• the synthetic proteins based on dendrimers do not necessary cross the cell membrane, because they function as biomarkers recognizing a cell receptor, or function as a blocker or toxin for a cell channel.
• the dendrimer functionality can be modified through the inclusion of side groups and end groups providing multiple functions, which can be distributed on the dendrimer surface.
• the functionality can be dispersed or can comprise functional segments.
• the composition of the dendrimer may comprise a wide range of functional groups, and include dimeric dendrimers, thereby allowing the bulk or surface properties of a material to be tailored to the application. Materials prepared by other processes can be incorporated into the final structure as macromonomers, macroinitiators, or as other functional materials.
• antisense oligonucleotides will be used as a model for interference in the expression of the survivin protein (Carrasco et al., 2011) (See figure 1).
• ODN antisense oligonucleotides
• Figure 1 shows a sketch of the transfection processes using dendrimer-based synthetic proteins (SP).
• SP dendrimer-based synthetic proteins
• FIG. 1 shows the interaction between SP and the antisense oligonucleotide (ODN) when the assembly of the SP-ODN complex is produced
• stage (c) shows the process of the uptake of the SP-ODN complex upon passing through the cell membranes.
• stage (c) the ODN is released by the SP.
• the ODN acts on the survivin mRNA, degrading it, thus inhibiting the expression of this protein.
• apoptosis is activated as a result of the degradation of survivin.
• the processes of assembly (a) and release (c) are regulated by the chemical equilibrium and the type of interaction between the SP and the ODN.
• the nature of this interaction is modulated by the type and the proportion of amino acids that are conjugated to the terminal groups of a dendrimer.
• This ability to modulate the interaction between the SP and the ODN differentiates this type of dendrimer from other known ones, since the rational design of the dendrimer-based synthetic proteins of the present invention makes it possible to modulate the electrostatic potential of the surface of a dendrimer in such a manner that it efficiently emulates proteins that interact with DNA.
• the combinatories of the amino acid proportions is inferred from a bioinformatics analysis done for all of the crystallographic data containing protein-DNA complexes that exist to date. In other studies, dendrimers are developed that are heterogeneously; however, the reason for these proportions and the reason for the selection of the functionalized groups are not derived from a bioinformatic analysis.
• Survivin is an apoptosis inhibitor protein belonging to the family of the IAPs (Inhibitors of Apoptosis Proteins) (Crook et al, 1993). Just like the other IAPs, survivin is associated with multiple functions such as cell cycle progression, cell proliferation, angiogenesis and stress response. The ability of survivin to inhibit apoptosis is a result of a sophisticated mechanism between the dynamic balance of pro-apoptotic and anti- apoptotic factors.
• the present invention is directed to the rationalization of the design and production of synthetic proteins, which are based on a core structure of a dendrimer. These synthetic proteins based on dendrimers can be fabricated in order to provide different functions.
• synthetic proteins based on dendrimers can be fabricated in order to provide different functions.
• This example is related to the generation of a medicinal carrier, which is used for the delivery of a therapeutically effective nucleic acid molecule.
• the disease model addressed in this example corresponds to cancer, and the particular nucleic acid selected for treatment is an antisense oligonucleotide directed against expression of survivin.
• EP2206787 describes functionalized dendrimers for transfection into eukaryotic cells, wherein the functionalization is made by using cationic amino acids.
• This document describes dendrimer functionalization with arginine or lysine, but not both amino acids in the same dendrimer, i.e., this document describes a homogeneous dendrimer, as opposed to the synthetic protein based on dendrimers described in the present invention, which corresponds to a heterogeneous dendrimer functionalized with different proportions of amino acids.
• the proportion of the amino acids is defined through structural bioinformatics tools, computer modeling, and experimental data.
• Another example is the international patent application WO9619240, describing conjugates between nucleic acids and dendrimers, wherein the bond between the nucleic acid and the dendrimer is through an internucleotide bridge to a nucleic acid base or to the oligonucleotide sugar.
• EP2206787 can be considered as the closest document, since its disclosure describes a functionalized dendrimer using a cationic amino acid, such as arginine or lysine. Nevertheless, this document does not mention other amino acids, nor the possibility of a heterogeneously functionalized dendrimer.
• Stach et al. (Stach et al., 2012) mention the use of peptide dendrimers composed primarily of lysine, leucine and phenylalanine in different proportions.
• the dendrimers of this investigation are lineal branched polymers, not dendrimers like in the present invention.
• the polymers of this investigation are constituted by polypeptides synthesized de novo. Due to the differences indicated, the system of the present invention has several advantages, since it is more stable than a polypeptide- based system, it is possible to precisely regulate the size of the final synthetic protein and, since it involves the modification of the surface of a dendrimer and not the complete synthesis of a new structure, it is cheaper and simpler.
• These dendrimers were tested for antimicrobial activity due to their ability to break the bacterial membranes, unlike the dendrimers of the present invention, which enable efficient transfection in the cells.
• Navath et al. (Navath et al, 2010) describes the synthesis of new dendrimers, each functionalized with the same amino acid (serine, cysteine, aspartic acid) and a chemical group (-OH, -SH, -COOH). None of these dendrimers was functionalized with two or more types of amino acid.
• the dendrimers synthesized in this research were conjugated with the drug dexamethasone. Although the use of these dendrimers for transporting genes is proposed, only in vitro cytotoxicity studies on the dendrimer alone are described without transporting any drug or oligonucleotide.
• the present invention describes the use of a library of combinations of different amino acids for the synthesis of the synthetic proteins, designed in a rational manner, for a specific purpose. It is important to note that the mixture of arginine and lysine or arginine and asparagine was not described in the prior art, either.
• Synthetic protein based on dendrimers in the present specification, a synthetic protein must be understood as a synthetic molecule that can have properties similar to a protein and is fabricated by conjugating amino acid groups, or their functional groups, in different proportions, to the termini of a dendrimer.
• synthetic proteins based on dendrimers of the present invention can also comprise alcohol groups. The proportions of the amino acids on the surface of this dendrimer should mimic the electrostatic properties, molecular weight, isoelectric point, and/or hydrogen bounds formation pattern of a protein, and therefore reproduce the affinity with a given specific ligand.
• the functionalization with multiple distinct terminal groups grants biological properties to the synthetic protein based on dendrimers, allowing, among other functions, the transfection and effective release of drugs or nucleic-acid based drugs, with minimal toxic effects to the target cells.
• Synthetic proteins based on dendrimers of the present invention can have other functions, such as, for example, the function of binding to RNA or other oligonucleotides, the function of binding to peptides or oligopeptides, the function of protecting a substance such as a drug or a biomarker during the transport into extracellular or intracellular space, the function of blocking a specific or a nonspecific membrane channel protein in a cell (acting as a synthetic toxin), among others.
• the present invention describes synthetic proteins, which are based on a dendrimer.
• Terminal groups of said dendrimer can be conjugated with diverse functional groups, particularly amino acids or their functional groups, allowing control of the affinity for particular ligands, thus permitting the fabrication of synthetic proteins based on dendrimers mimicking the function of natural proteins.
• the control of the affinity can be focused or directed to specific tasks or functions.
• the functionalization with multiple distinct terminal groups grants biological properties to the synthetic protein based on dendrimers, allowing, among other functions, the transfection and effective release of drugs or nucleic-acid based drugs, with minimal toxic effects to the target cells.
• synthetic proteins based on dendrimers of the present invention can have other functions, such as, for example, the function of binding to RNA or other oligonucleotides, the function of binding to peptides or oligopeptides, the function of protecting a substance, such as a drug or a biomarker during the transport into extracellular or intracellular space, the function of blocking a specific or a non-specific membrane channel protein in a cell (acting as a synthetic toxin), among others.
• the affinity is directed toward nucleic acids using at least two different functional groups, turning the conjugated dendrimer in a heterogeneous functionalized nanoparticle.
• the synthetic proteins based on dendrimers mimic natural proteins in two ways. First, they have an atomically precise primary structure—the molecular topology including all atoms and bonds is specified precisely (although in some cases we may choose to make exceptions to this in the same way that natural proteins may undergo probabilistic modifications). Second, the synthetic proteins based on dendrimers are designed to have specific interactions with particular biomolecules and interactions with other biomolecules are kept to a minimum. Synthetic proteins based on dendrimers differ from drug molecules in that they have a large complex structure with different portions functionalized for different purposes.
• FIG 1 Diagram of transfection processes using dendrimer-based synthetic proteins (SP).
• SP dendrimer-based synthetic proteins
• the synthetic protein transfects an antisense oligonucleotide (ODN).
• ODN antisense oligonucleotide
• (c) Release of the ODN into the cell
• Inhibition of the expression of survivin through the degradation of the survivin mRNA (e) Activation of apoptosis.
• Figure 2 Statistical analysis of the Protein Data Bank (PDB) to determine which amino acids occupy the contact zone of protein-DNA complexes (1813 PDB archives).
• PDB Protein Data Bank
• Analysis includes amino acids less than 10 A from any DNA molecule, (a) Frequencies of amino acids in the contact zone of adenine nucleotides in protein-DNA complexes. Amino acids with the highest frequencies are Arg, Asn and Gin. (b) Frequencies of amino acids in the contact zone of guanine nucleotides in protein-DNA complexes. Amino acids with the highest frequencies are Arg, Lys, and Asn.
• Figure 3 Statistical analysis of the Protein Data Bank (PDB) to determine which amino acids occupy the contact zone protein-DNA complexes (1813 PDB archives). Analysis includes amino acids less than 10 A from any DNA molecule, (a) Frequencies of amino acids in the contact zone of cytosine nucleotides in protein-DNA complexes. Amino acids with the highest frequencies are Arg, Glu, and Lys. (b) Frequencies of amino acids in the contact zone of thymine nucleotides in protein-DNA complexes. Amino acids with the highest frequencies are Arg, Lys, and Asn.
• PDB Protein Data Bank
• Figure 4 Statistical analysis of the Protein Data Bank (PDB) to determine which amino acids occupy the contact zone of protein-DNA complexes (1813 PDB archives). Analysis includes amino acids less than 10 A from any DNA molecule. Frequency of amino acids in the contact zone of any nucleotide in protein-DNA complexes. Amino acids with the highest frequencies are Arg, Lys, Asn, and Gin, followed by Ser, Thr, and Tyr.
• PDB Protein Data Bank
• the present invention corresponds to a molecule that can be fabricated, according to the present invention, having properties similar to a protein.
• the molecule is composed of a dendrimer, whose terminal groups are conjugated with diverse functional groups, particularly amino acids or their side chains, and also optionally alcohol groups.
• the dendrimer has repetitive units that generate a tertiary structure with moieties and domains.
• the conjugated amino acids or functional groups on the surface of the synthetic protein provide an electrostatic potential to the synthetic protein which simulated the properties and functions of biological proteins.
• the dendrimer can be selected from a wide variety of generations, such as for example first generation dendrimers, second generation dendrimers, etc.
• the core molecule is a fourth generation poly(amide- amine) or PAMAM dendrimer.
• the size of the dendrimer used as a core is selected depending on the size of the protein to be mimicked.
• a first or second generation dendrimer with a small surface, can be selected.
• terminal groups of the dendrimer can be realized with amino acids, selected among, but not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine or combinations thereof.
• amino acids selected among, but not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine or combinations thereof.
• the functionalization can be carried out by using the functional groups of amino acids.
• the functionalization when fabricating a synthetic protein which would mimic the function of an arginine residue, the functionalization can be made with an arginine residue or a guanidinium group.
• the properties to mimic were to be those of an asparagine residue, the functionalization would be made with an asparagine residue or an amide group.
• the functionalization can be made with a lysine residue or an amine group.
• Other options are alcohols, in the case of serine, threonine and tyrosine; thiols, carboxilates or aliphatic or aromatic groups.
• the functionalization of terminal groups of the dendrimer can be realized with alcohol groups besides the amino acids residues or the functional groups of amino acids.
• the synthetic protein based on dendrimers can be mono- functionalized, bi-functionalized, tri-functionalized or multi-functionalized if only one, two, three or more different functional groups are included in the functionalization of only one core or dendrimer.
• the ratio between each of the functional groups used to functionalize a single dendrimer is randomly determined.
• the ratio between each of the functional groups used in the functionalization of a dendrimer is determined in order to mimic the distribution of functional groups of a protein whose function is to be replicated.
• the proportions of the amino acids on the surface of this dendrimer should mimic the electrostatic properties, molecular weight, isoelectric point, and/or hydrogen bond formation pattern of a protein, and therefore reproduce the affinity with a given specific ligand.
• the functionalization with multiple distinct terminal groups grants biological properties to the synthetic protein based on dendrimers, allowing, among other functions, the transfection and effective release of drugs or nucleic-acid based drugs, with minimal toxic effects to the target cells.
• Synthetic proteins based on dendrimers of the present invention can have other functions, such as, for example, the function of binding RNA or other oligonucleotides, the function of binding peptides or oligopeptides, the function of protecting a substance such as a drug or a biomarker during the transport into extracellular or intracellular space, the function of blocking a specific or a non-specific membrane channel protein in a cell (acting as a synthetic toxin), among others.
• the synthetic proteins of the present invention have a tertiary structure that provides stability and protection to the ligand that is bound to it, allowing transport of the ligand and/or transfection of the ligand to the target without hydrolysis.
• the ligand bound to the synthetic target undergoes controlled release into the extracellular or intracellular space to interact with its target.
• the synthetic protein surface charge is a key property that modulates the release of the ligand.
• the surface charges interact with the charged groups of the ligand; thus, the charge ratio between the dendrimer and ligand plays an important role in determining their affinity. Therefore, one goal of the present invention is to optimize the surface charge of the synthetic proteins, providing them the capacity to bind the ligand and, under the appropriate conditions, to release the ligand to perform its particular function with the target. This charge balance is provided by the rational design of the synthetic protein, functionalized with defined proportions of defined functional groups, particularly with defined amino acids or their functional groups.
• the ligand is exposed on the surface of the synthetic protein and can interact with its target even if it is bound to the synthetic protein.
• the choice of the residues used for the functionalization is determined by the distribution of the most common amino acid residues found in a set of known nucleic acid binding proteins.
• the distribution of the amino acid residues can be determined based on bioinformatics data and computer modeling as well as experimental data. For example, the distribution of the amino acid residues can be determined by using a statistical analysis of the nucleic acid binding proteins found in the Protein Data Bank (PDB).
• PDB Protein Data Bank
• the distribution of the residues of amino acids or functional groups can also be determined by means of statistical analyses and the selection of a protein-ODN interaction model in order to determine an optimal modulation of the interaction between synthetic protein and drug.
• a protein-ODN interaction model in order to determine an optimal modulation of the interaction between synthetic protein and drug.
• the main amino acids are arginine and asparagine or arginine and lysine.
• the ratio arginine: asparagine can range from 9: 1 to 1 :9, more preferentially from 8 :2 to 2:8, and even more preferentially from 7:3 to 3:7. In a further embodiment, the ratio between arginine and asparagine can range from 6:4 to 4:6 and can also be 1 :1.
• the synthetic protein is functionalized with arginine and lysine residues.
• the ratio arginine: lysine can range from 9:1 to 1 :9, more preferentially from 8:2 to 2:8, and even more preferentially from 7:3 to 3:7.
• the ratio between arginine and lysine can range from 6:4 to 4:6 and can also be 1 :1.
• the synthetic protein is functionalized with arginine, asparagine and alcohol groups.
• the ratio arginine:asparagine:alcohol can range from 80:10:10 to 10:80:10 or 10:10:80, or more preferentially be 60:30:10 or 30:40:30.
• the synthetic protein is functionalized with arginine, lysine and alcohol groups.
• the ratio arginine:lysine:alcohol can range from 80:10:10 to 10:80:10 or 10: 10:80, or more preferentially be 60:30:10 or 30:40:30.
• two or more synthetic proteins can be bound by a linker, mimicking a hetero -dimeric, -trimeric, -polymeric protein with the same or different proportion of functional groups (particularly amino acids or their functional groups.
• the goal of the functionalization of the dendrimer is to achieve nucleic acid binding capabilities, granting the synthetic protein the ability to, among other things, transfect and effectively release nucleic acids, all without causing toxic effects to the cells.
• Figures 2, 3, and 4 show the results of the statistical analysis of the Protein Data Bank (PDB) to determine the amino acids in the contact zone of the protein-DNA complexes (1813 archives pdbs).
• the analysis includes amino acids less than 10 A from any DNA molecule.
• Figure 2a shows the frequencies of amino acids in the contact zone of adenine nucleotides in protein-DNA complexes. Amino acids with the highest frequencies are Arg, Asn and Gin.
• Figure 2b shows the frequencies of amino acids in the contact zone of guanine nucleotides in protein-DNA complexes. Amino acids with the highest frequencies are Arg, Lys, and Asn.
• Figure 3a shows the frequencies of amino acids in the contact zone of cytosine nucleotides in protein-DNA complexes.
• Amino acids with the highest frequencies are Arg, Glu, and Lys.
• Figure 3b shows the frequencies of amino acids in the contact zone of thymine nucleotides in protein-DNA complexes. Amino acids with the highest frequencies are Arg, Lys, and Asn.
• Figure 4 shows the frequencies of amino acids in the contact zone of any nucleotide in protein-DNA complexes. Amino acids with the highest frequencies are Arg, Lys, Asn, and Gin, followed by Ser, Thr, and Tyr.
• the crude product obtained from the reaction between PAMAM G4 or G5 and Boc-aa- NHS was purified using the dialysis method.
• a cellulose dialysis membrane (pore size 1000 Da) was used, to which the reaction mixture was added.
• This membrane with the mixture on its interior was placed in a container with DMSO, which was changed every 8 hours. This was done three times with the purpose of eliminating the byproduct of the reaction and the excess reagent.
• the solvent was removed by freeze-drying in order to obtain only the compound protected with Boc.
• the compound protected by Boc was treated with a solution of TFA/DCM under stirring for 10 minutes. After deprotection, the solution was neutralized (pH 7.0) using a NaOH solution at a concentration of 1 N and was purified in order to obtain the final product, which corresponds to dendrimers functionalized with amino acids.
• Control of the functionalization of the surface of the dendrimers is considered vital within the synthesis method.
• the preferred technique for monitoring the progress of the synthesis of dendrimers, the synthesis of proteins and the purity of the compounds is matrix-assisted laser desorption ionization using a time-of-fiight matrix (MALDI-TOF). This technique offers the advantage of generating fast analytical results and easy interpretation of their data.
• the patterns of dendrimer fragmentation observed by means of MALDI can be interpreted and assigned. It is known that a main path of the decomposition of the dendrimer begins with rupture of the amide bond of terminal chelating groups or close to the nucleus of the molecule. In the latter case, the fragmentation is primarily of type A, i.e., on the N terminal of the amide bond.
• the contrast of the fragmentation of peptide bonds between MALDI and ESI-Ion Trap is that the breaking of peptide bonds generally produces high signals. This is because the dendrimer fragmentation is controlled by a terminal unit of tertiary carbon adjacent to the amide bond.
• the spectra of the synthetic proteins were obtained using a matrix of 2,5- dihydroxybenzoic acid (DHB).
• the specimens were prepared by mixing the diluted solution of the analyte in acetonitrile/water with an approximately equal volume of a solution of DHB in acetonitrile/water, followed by deposition of the specimen in an aluminum well. These specimens were then introduced into the mass spectroscopy equipment to obtain their spectra.
• DHB 2,5- dihydroxybenzoic acid
• Cellular uptake was estimated by fluorescent labeling using the ODN LY2181308 tagged with Alexa-fluor 488 (green fluorescence).
• fluorescently labeled ODN was complexed with synthetic protein and added to tumor and normal cells. The fluorescence distribution within the cell was determined by confocal microscopy or flow cytometry.
• HeLa human cervical cancer cell line
• N/P ratio the charge ratios of dendrimer to nucleic acid
• N the total concentration of charged terminal groups in the dendrimer (PAMAM)
• P the number of (charged) phosphate groups in DNA
• Ratios of nucleic acid to dendrimer were obtained upon the basis of the electrostatic charge present on each component, the number of phosphate groups in the nucleic acid in comparison to the number of positive charged groups on a dendrimer (PAMAM).
• Transfection conditions were optimized for each cell line.
• the optimal final concentration of DNA should be within the range of 50-500 nM.
• 100 nM and the following conditions were used per 24-well in a final volume of 250 ⁇ ⁇ . (Table 2).
• Table 2 Volume used for each charge ratio : charged terminal groups (dendrimer) to phosphate groups (DNA), (N/P).
• a wide range of tumor cell lines were used, such as, human cervical carcinoma (HeLa), non-small cell lung cancer (A549), prostate carcinoma (PC3), breast cancer (MDA MB231).
• Cells were grown at a 50% to 65% density overnight in a 96-well plate or 12-well plate.
• the transfection complex synthetic protein-survivin ODN
• Opti-MEM media was resuspended in Opti-MEM media and preincubated for 15-20 minutes at room temperature. The initial concentration of ODN was 100 nM.
• the transfection reagent and lipofectamine-2000 (invitrogen) were used as positive control.
• the transfection mixture was then removed following 4 to 6 hours of incubation at 37°C and replaced with the complete medium, i.e., a medium that supports the growth of the cells. Cells were incubated in the complete medium for 24 to 72 hours prior to harvest.
• the cDNA synthesis was carried out with 100 ng of RNA with "First strand buffer” (MgCl 2 3mM, KCl 75mM and Tris-HCl 5 mM, pH 8.3, Invitrogen), 2 ⁇ of DTT (0.01 M), 1 ⁇ of dNTPs (0.5 mM), 50 ng of random primers and 20 U RNase-OUT (Invitrogen) in a final volume of 20 ⁇ . Then, 1 ⁇ of M-MLV (200 U/ ⁇ , Invitrogen) was added, and the mix was incubated at 25°C during 10 min, following by 50 min at 37°C. Amplification of survivin cDNA was performed using the following primers: Forward SEQ ID NO: 1 : 5'-GTGAATTTTTGAAACTGGACAG-3' ;
• Reverse SEQ ID NO: 2 5 ' -CCTTTCCTAAGACATTGCTAAG-3 ' .
• GAPDH was amplified using the following primers:
• the PCR product of survivin (aprox. 240-pb) was analyzed by agarose mini-gel.
• Transfected cells were harvested by washing twice with lx cool-PBS and then lysed with RIPA buffer (20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 50 mM NaCl, 10 mM sodium pyrophosphate, 50 mM NaF, and 1% NP40) plus a protease inhibitor cocktail (SIGMA) and 1 mM of PMSF.
• RIPA buffer 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 50 mM NaCl, 10 mM sodium pyrophosphate, 50 mM NaF, and 1% NP40
• SIGMA protease inhibitor cocktail
• Cell lysates were centrifuged at 10000 rpm for 10 minutes at 4°C.
• Bradford reaction was used to determinate protein concentration.
• the protein was resolved by SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with non-fat milk and the incubation was carried out with the human
• Tumor cells were seeded at an approximate density of 5 x 10 4 cells per well in a 96-well plate.
• Cells treated with the SP-ODN complex (ODN-GFP; ODN-survivin); controls (without the complex; ODN control; only SP) were incubated at 37 °C in 5% CO 2 .
• the cell viability was determined using the assay called MTS (CellTiter 96® Assay, PROMEGA), which is based on the cellular conversion of tetrazolium salt to formazan, which can be quantified by measuring the absorbency at 570 nm.
• Table 3 shows the results of uptake performance and cytotoxity assays with different synthetic proteins according to the invention. Table 3: Summary of uptake performance and cytotoxity of Synthetic Proteins
• Table 3 shows that the dendritic systems are more efficient than the two commercial products used as references.
• PAMAM G4 Arg-Lys turned out to have an excellent uptake of ODN and very low cytotoxicity compared to the other systems.
• the 10: 1 charge ratios turned out to be more efficient than the 1 :1 ratios. This may be because a greater number of PSs contributes cooperatively to the uptake of the ODN.
• RNA small interfering RNAs

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Health & Medical Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Medicinal Chemistry (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• Molecular Biology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Genetics & Genomics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Wood Science & Technology (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Hematology (AREA)
• Oncology (AREA)
• Marine Sciences & Fisheries (AREA)
• Gastroenterology & Hepatology (AREA)
• Immunology (AREA)
• Peptides Or Proteins (AREA)
• Medicinal Preparation (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=50150446

Family Applications (1)

Country Status (1)

Cited By (2)

Family Cites Families (4)

• 2013
  • 2013-08-23 WO PCT/IB2013/056845 patent/WO2014030147A2/fr not_active Ceased

Also Published As

Similar Documents

Legal Events