Patent Application
20080118489
Many antimicrobial proteins are cationic amphiphiles that can interact strongly with the anionic surfaces of microbes, thereby facilitating destabilization of microbial walls and membranes. These binding events are largely electrostatic interactions and are therefore dependent on the ionic environment. Disease states that are characterized by a large concentration of charged polyelectrolytes can present special challenges to effective administration of antimicrobials because these polyelectrolytes can sequester antimicrobials. Such sequestration can limit the efficacy of antimicrobials, as well as other cationic proteins (e.g., antibiotics). An example of one disease state characterized by a large concentration of polyelectrolytes is cystic fibrosis (CF).
Cystic fibrosis is the most common fatal, inherited disease in the United States. It is a genetic disorder resulting from the inheritance of a defective autosomal recessive gene. The average life expectancy of the 30,000 CF patients currently alive in the U.S. is under 30 years. The gene responsible for CF codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP regulated Cl− ion channel found in the apical membranes of secretory epithelial cells. Mutations in CFTR disrupt epithelial ion transport and can lead to thick airway secretions, respiratory failure, as well as a range of other defects. Although CF is a systemic disease affecting a range of epithelial tissues, the major cause of mortality is lung disease associated with the accumulation of viscous mucus in pulmonary airways. Progressive destruction of the lung parenchyma and respiratory failure are attributed to persistent bacterial infections and the accumulation of viscous, infected mucus in pulmonary airways. There is, at present, no cure for the disease.
Although there has been much progress in gene therapy, complexities in the biology of the diseased lung still pose significant problems. Moreover, there is no technology to reconstitute precisely the normal expression of the CF gene, since CFTR is expressed in cells throughout the superficial epithelia as well as cells in the submucosal glands. Current treatment involves recombinant human Dnase I (rhDNase) aerosol combined with antibiotics to control infections. The enzyme rhDNase (Pulmozyme®), cuts entangled DNA molecules in CF mucus and reduces the viscosity of the mucus, thus reducing respiratory distress. However, clinically observed improvement is often moderate at best, and does not act against the infections that ultimately kill the patient. In addition, clinical evidence suggests that treatment with rhDNase is effective only to certain groups. Pulmozyme® treatment is also expensive (on the order of about $1000 per month). In fact, out-patient treatment by rhDNase is often more expensive than in-patient treatment. In addition, it is unclear whether enough CFTR genes can be introduced into enough epithelial cells to sufficiently impact the disease before the immune system or other mitigating factors interfere with vector delivery. There is a need for parallel or independent therapeutic strategies, including regimes to improve antimicrobial activity, to address infections that ultimately result in death of the CF patient.
One of the contributing factors to the occurrence of long-term infections in CF is the inactivation of native airway defense. The inflammatory response to infections leads to the deposition of high concentrations of negatively charged polymers in the airways, including cytoskeletal proteins such as F-actin, DNA, and other cellular debris within the viscous mucus of the pulmonary airways. These negatively charged polymers can bind to and sequester naturally-occurring positively-charged antimicrobial and antibacterial proteins or other introduced pharmaceutical agents such as antibiotics, so that they can no longer fulfill their normal or desired antimicrobial function, contributing to patient debilitation or death from infection.
Other techniques have attempted to improve antimicrobial activity by using charged polymers to dissociate DNA and actin bundles in CF mucus (Tang et al., 2005). That work, however, does not involve modification of antibacterial proteins so as to obtain non-stick versions of the proteins, nor does it involve pacifying the charged surfaces with surfactants. The present invention is associated with substantial recovery of activity; non-stick antibacterial proteins are engineered to reduce sequestration and to demonstrate activity.
The present invention provides several solutions, for example, compositions and methods to improve antimicrobial activity by reducing sequestration of charged antimicrobial proteins by the oppositely charged polymers in the airways. The invention provides methods to engineer non-stick, charge-reduced versions of antibacterial proteins by analyzing and understanding the underlying electrostatic binding process. These charge-reduced antibacterial proteins can be utilized within a suite of aerosol-deliverable therapeutics that can remain active in the unusual electrostatic environment of the airway surface liquid (ASL), and at least partially restore antimicrobial function in the CF airway. By the application of this strategy, compositions and methods of the present invention can also be utilized in other infected biological systems, including for example chronic infections with a prolonged inflammatory response. The present invention also provides compositions and methods for improving efficacy of antibiotics; this can be accomplished by pacifying the charged surfaces that normally function to sequester antibiotics. Further aspects of the invention are also described.
The following abbreviations are applicable. Cystic fibrosis (CF); Airway Surface Liquid (ASL); synchrotron small angle x-ray scattering (SAXS); cystic fibrosis transmembrane conductance regulator (CFTR); Pseudomonas aeruginosa (PA01); Charge-Coupled Device (CCD); 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP); 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE).
Without wishing to be bound by any particular theory, there can be discussion herein of beliefs or understandings of underlying principles relating to the invention. It is recognized that regardless of the ultimate correctness of any mechanistic explanation or hypothesis, an embodiment of the invention can nonetheless be operative and useful.
The invention comprises charge-modified antimicrobials and methods of potentiating antimicrobials by modifying the charge of an antimicrobial. In an embodiment, the charge-modified antimicrobial is a derivative of a reference antimicrobial wherein the derivative has a reduction of a net charge relative to the reference antimicrobial. The reduction in net charge level can be obtained by modifying one or more of the amino-acids of the reference antimicrobial so as to reduce the net charge of the derivative relative to the reference antimicrobial. The modification can be by amino acid deletion, substitution and/or alteration. A single amino acid substitution can effect a net 2 reduction in charge (e.g. substituting lysine or arginine with glutamic acid). Substitutions involving aspartic acid can also effect a net 2 reduction in charge. An amino acid deletion can effect a net 1 reduction in charge. Modification techniques as known in the art, including site-directed mutagenesis, can be used to modify one or more amino acids, thereby reducing the charge of a reference antimicrobial so long as measurable antimicrobial activity remains.
The reference antimicrobial can include any protein, peptide, or functional fragments thereof that kill and/or inhibit growth of microbes, including bacteria. Antimicrobials can be lysozyme, β-defensins, lactoferrin, and functional fragments thereof. In one embodiment the antimicrobial is a lysozyme obtained from any lysozyme-producing source. The antimicrobial source can be mammalian, bacterial or of other species origin. In an embodiment the antimicrobial is a lysozyme. In an embodiment the antimicrobial origin is mammalian, and more preferably human in origin. Antimicrobials such as human lysozyme can be expressed and isolated from recombinant systems, e.g., using bacteria or other expression hosts as known in the art. In a particular embodiment, the invention provides a charge-modified human lysozyme.
The invention comprises charge-modified antimicrobials, wherein the reduction of net charge is 1 or greater. In an embodiment, the reduction of net charge is selected from the group consisting of about 2, about 3, about 4, about 5, about 6, about 7, and about 8. In an embodiment the reduction of net charge is at least about 2. In an embodiment the reduction of net charge is at least about 4. In an embodiment the reduction in net charge is such that the charge-modified antimicrobial has a measurable improvement in antimicrobial activity compared to the reference antimicrobial when administered to a patient suffering from a disease. Preferably, the improvement is at least 10%, at least 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% relative to the reference antimicrobial. The reference antimicrobial can have a relative antimicrobial activity selected from the group consisting of at least about 50%, at least about 60%, at least about 70%, at least about 80%, and at least about 90% in comparison with a reference antimicrobial activity of the reference lysozyme protein.
The invention encompasses methods for potentiating the antimicrobial activity of a protein, a peptide, or functional fragment thereof, by modifying the net charge level of the protein, the peptide, or the functional fragment thereof. In one embodiment the method is for modifying the net charge level of lysozyme. In an embodiment the lysozyme's net charge level is modified to a less positive net charge level.
The invention encompasses methods of treating a microbial infection, comprising administering to a patient in need of the composition any of the compositions of the present invention. In an embodiment, the administration occurs by aerosol delivery. In an embodiment the administration is to the air passage of a patient, including administration to the upper respiratory tract region. In an embodiment the patient is a cystic fibrosis patient. In addition to the context of cystic fibrosis, compounds and methods of the invention are suitable as therapies in chronic infection conditions including such with a prolonged inflammatory response. Furthermore, charge-modified lysozymes in particular are applicable in compositions and methods relating to artificial tears, artificial saliva, and infant formulas, milks, and supplements thereto.
The invention encompasses methods of generating a non-stick, charge-modified form of an antimicrobial protein comprising: (a) providing a candidate antimicrobial protein or sequence information corresponding to nucleic acids or amino acids thereof; (b) developing at least one charge-modified version of said candidate antimicrobial protein; (c) screening said charge-modified version for antimicrobial activity; and (d) selecting an active charge-modified version; thereby generating said non-stick, charge-modified form of an antimicrobial protein. In one embodiment the charge-modified version is developed by substituting at least one positively-charged amino acid with at least one negatively-charged amino acid.
The invention provides surfactant compositions. The invention encompasses methods of potentiating an antibiotic treatment, comprising the steps of (a) administering a surfactant composition; and (b) administering the antibiotic to a patient in need of treatment. In an embodiment, the surfactant composition is administered before antibiotic treatment. In an embodiment, the surfactant and antibiotics are administered substantially simultaneously. The surfactant composition can comprise natural (e.g. lipids) and/or artificial (e.g. artificially synthesized) components and/or amphiphilic molecules. The surfactant composition can comprise a cationic lipid composition. The surfactant composition can comprise a combination of cationic lipid and neutral lipid. The surfactant composition can comprise essentially pure cationic lipid. The surfactant composition can comprise about 100% nominally neutral lipids. The nominally neutral lipids can have both positive and negative charges that cancel. In an embodiment, the surfactant composition is at least partially cationic.
In an embodiment, the surfactant composition is selected from the group consisting of: Didodecyldimethylammonium bromide (DDAB); Cetyltrimethylammonium bromide (CTAB); Cetyltrimethylammonium bromide (CTAB); 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHGPC) from 20:80 to 80:20; DLTAP:DLPC, DOTAP:DOPC, and DNTAP:DNPC (dilauryl trimethyl ammonium propane: dilauryl trimethyl phosphatidylcholine, dioleoyl trimethyl ammonium propane: dioleoyl trimethyl phosphatidylcholine, and dinervonyl trimethyl ammonium propane: dinervonyl trimethyl phosphatidylcholine, respectively) from 100:0 to 10:90.
In an embodiment, the surfactant composition is selected from the group consisting of: 1,2-Diarachidonoyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Didocosahexaenoyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Dielaidoyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Dihexanoyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Dioctanoyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Dihexanoyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Dilauroyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Dipalmitoleoyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Diheptadecanoyl-sn-Glycero-3-Phosphoethanolamine; 1,2-Dicapryl-sn-Glycero-3-Phosphoethanolamine; 1,2-Dimyristoyl-3-Trimethylammonium-Propane; 1,2-Dipalmitoyl-3-Trimethylammonium-Propane; 1,2-Dimyristoleoyl-sn-Glycero-3-Phosphocholine; 1,2-Dimyristelaidoyl-sn-Glycero-3-Phosphocholine; 1,2-Dipalmitoleoyl-sn-Glycero-3-Phosphocholine; 1,2-Dipalmitelaidoyl-sn-Glycero-3-Phosphocholine; 1,2-Dieicosenoyl-sn-Glycero-3-Phosphocholine; 1,2-Dierucoyl-sn-Glycero-3-Phosphocholine; 1,2-Dinervonoyl-sn-Glycero-3-Phosphocholine; 1,2-Dipetroselinoyl-sn-Glycero-3-Phosphocholine; and 1,2-Dielaidoyl-sn-Glycero-3-Phosphocholine.
In an embodiment, the surfactant composition comprises DOTAP and DOPE.
In an embodiment, a surfactant composition is provided in a formulation capable of forming a lamellar or inverse hexagonal phase with a complexing agent such as DNA. In an embodiment, a cationic lipid composition is provided. In an embodiment the surfactant composition comprises DOTAP and DOPE. In an embodiment the DOTAP:DOPE ratio is from about 100:0 to about 10:90. In a particular embodiment, this ratio is from about 70:30 to about 25:75. In an embodiment, the antibiotic is from the aminoglycoside family of antibiotics, wherein the aminoglycoside is positively-charged. In an embodiment, the antibiotic is selected from the group consisting of tobramycin, gentamycin, kanamycin, streptomycin, neomycin, amikacin, and ampramycin. In an embodiment, the antibiotic is tobramycin. In an embodiment the patient has a chronic microbial infection. In an embodiment the patient is at risk for a chronic microbial infection. In an embodiment the patient is a cystic fibrosis patient. In an embodiment, the composition is a medicament for treatment of an infection.
In an embodiment the surfactant composition is positively charged. In an embodiment, the surfactant composition is at least partially cationic. In an embodiment the surfactant composition comprises amphiphilic molecules, including but not limited to block copolymers. In an embodiment the invention comprises compositions and methods relating to amphiphilic molecules. In a particular embodiment, the amphiphilic molecules are surfactants. In a particular embodiment, the amphiphilic molecules are lipids. In a particular embodiment, the amphiphilic molecules are not surfactants or lipids. In a particular embodiment, the amphiphilic molecules are amphiphilic polymers. In a particular embodiment, the amphiphilic polymers are block copolymers. In a particular embodiment, the amphiphilic molecules are at least partially cationic.
In an embodiment the invention is a method of generating a positively-charged surfactant formulation for therapeutic use in connection with a positively-charged antimicrobial agent, wherein said therapeutic use involves an electrostatic environment with at least one anionic component, comprising: (a) identifying said at least one anionic component; (b) providing a positively charged surfactant formulation candidate; (c) adapting said formulation candidate so as to at least partially optimize one or more properties for interaction with said anionic component; (d) measuring an entropic parameter of said candidate upon binding said anionic component; and (e) selecting a formulation candidate exhibiting an entropy gain from said measuring step; thereby generating a positively-charged surfactant formulation for therapeutic use in connection with the positively-charged antimicrobial agent. In an embodiment, the properties in step (c) comprise a charge level and a structural conformation property.
The invention encompasses a method of generating a positively-charged surfactant formulation for therapeutic use in connection with a positively-charged antimicrobial agent, wherein said therapeutic use involves an electrostatic environment with at least one anionic component, comprising: (a) identifying said at least one anionic component; (b) providing a positively charged surfactant formulation candidate; (c) maximizing an entropic gain of said candidate upon binding said anionic component by optimizing one or more of charge density and surfactant curvature of said surfactant formulation candidate; and (d) selecting a formulation candidate exhibiting an entropy gain from said maximizing step; thereby generating a positively-charged surfactant formulation for therapeutic use in connection with the positively-charged antimicrobial agent.
The invention encompasses a charge-modified antimicrobial lysozyme, wherein the charge-modified lysozyme is a derivative of a reference lysozyme protein and has one or more charge decreases relative to the reference lysozyme protein.
The invention encompasses any of the charge-modified lysozymes disclosed herein, excepting a mutant T4 bacteriophage lysozyme as described herein and those other lysozymes which may be known in the art that do qualify as prior art.
In an embodiment, a composition of the invention is isolated or purified.
In an embodiment, the invention provides a method of potentiating an antibiotic treatment, comprising the steps of (a) administering an amphiphilic molecule composition; and (b) administering the antibiotic to a patient in need of treatment.
In an embodiment, the invention provides a method of generating an amphiphilic molecule formulation for therapeutic use in connection with a positively-charged antimicrobial agent, wherein said therapeutic use involves an electrostatic environment with at least one anionic component, comprising: (a) identifying said at least one anionic component; (b) providing an amphiphilic molecule formulation candidate; (c) maximizing an entropic gain of said candidate upon binding said anionic component by optimizing one or more of charge density and curvature of said candidate; and (d) selecting a formulation candidate exhibiting an entropy gain from said maximizing step; thereby generating an amphiphilic molecule formulation for therapeutic use in connection with the positively-charged antimicrobial agent.
In an embodiment, the amphiphilic molecule is at least partially cationic.
In an embodiment, the invention provides a charge-modified mammalian lysozyme comprising a first segment having of an amino acid sequence of a mammalian lysozyme, and a second segment having from about two to about ten negatively charged amino acids. In an embodiment, the second segment comprises six negatively charged amino acids. In an embodiment, the second segment comprises six glutamate residues.
In an embodiment, the charge-modified mammalian lysozyme further comprises a third segment of a spacer, wherein said spacer is positioned between said first segment and said second segment. In an embodiment, the spacer is a peptide comprising from about two to about ten amino acids. In an embodiment, the spacer comprises seven alanine residues. In an embodiment, the lysozyme is human. In an embodiment, the lysozyme has the amino acid sequence of SEQ ID NO:4.
In an embodiment, the invention provides a nucleic acid sequence capable of encoding a charge-modified lysozyme.
In an embodiment, the invention provides a method of treating an infection condition involving a prolonged inflammatory response, comprising administering to a patient in need the composition of the invention. In an embodiment, the infection is a chronic microbial infection.
The invention provides pharmaceutical compositions and formulations of antimicrobial compositions described herein.
In general the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of the invention.
When used herein, the term “charge-modified” refers to an alteration in a charge level of a compound relative to that of a reference compound. A charge-modified compound can have an increased positive charge or an increased negative charge relative to the reference compound. In an embodiment, the charge-modified compound is derived from the reference compound, e.g. a native protein or variant thereof. For example, a charge-modified lysozyme is derived from a native mammalian (preferably human) lysozyme. More widely, a charge-modified lysozyme is a derivative of a reference lysozyme such as any lysozyme whether native, mutant, or other variant (preferably of mammalian origin, and more preferably of human origin), which may be relatively suboptimal with respect to a charge parameter for applications pertinent to the invention. In a preferred embodiment, a charge level is reduced from a greater net charge level to a lesser net charge level, wherein, greater and lesser can refer to a simple mathematical relationship. For example, the terminology can describe a change from an initial +9 charge level for wild-type bacteriophage T4 lysozyme to a resulting +5 charge level for a mutant T4 lysozyme. Alternatively, a resulting charge level that is a negative number is encompassed because mathematically it can have a lesser net charge level than an initial charge level of a positive number, zero, or a negative number which is greater than the resulting charge level.
When used herein, the term “non-stick” refers to a property of a compound to have a tendency not to stick to another compound or a mixture of compounds. The term is not absolute in requiring a complete absence of stickiness. In an embodiment, the term refers to an at least partially reduced ability to interact in electrostatic binding. In an embodiment, a non-stick protein has a decreased positive charge level and has a lesser susceptibility of attraction to and/or sequestration within an environment of an airway surface liquid. In a particular embodiment, the airway surface liquid is in a cystic fibrosis context and contains one or more of anionic polyelectrolytes, and includes biopolymers such as DNA, F-actin, glycoproteins such as mucin, and other cellular debris. The invention encompasses other diseases involving liquids enriched in anionic polyelectrolytes, for example lung diseases, pneumonias, and other severe infections. Any disease characterized by a large concentration of inflammatory polymers (DNA, actin, cell debris, etc.) can be treated using the compositions and methods of the present invention.
When used herein, the term “derivative” in the context of a protein refers to a mutant or variant version relative to a reference protein. For example, in an embodiment the mutant or variant has one or more of at least one changed natural amino acid (substitution), truncation or other deletion, or addition relative to a native or reference sequence. In an embodiment, the mutant or variant has a modification such as a non-natural amino acid substitution, addition, or chemical modification to an amino acid or the protein molecule as would be understood in the art. A derivative can be prepared by synthetic and/or recombinant techniques.
When used herein, the term “positively charged” indicates the presence of at least one positive charge in a molecule and therefore can be synonymous with a description of being at least partially cationic. A subset of positively charged molecules can have a net overall positive charge, for example if the total number of plus charges is greater than the total number of minus charges for a given molecule.
When used herein, the term “potentiating” refers to enhancing or improving a performance property relative to a reference level. For example, an antibiotic activity can be potentiated by administration of a positively-charged surfactant composition. In an embodiment, the surfactant administration can be before, after, or simultaneous with (co-administration) introduction of the antibiotic.
When used herein, the term “surfactant composition” is used broadly to refer to surface-active agents. The surfactant composition can be lipids, wherein the lipids can be biological, artificial (e.g. chemically synthesized) or partially biological and partially artificial in original. The surfactant composition can comprise nominally “neutral” lipids, comprising both positive and negative charges in amounts such that the net charge on the lipid is neutral. In an embodiment the surfactant composition is at least partially cationic. In an embodiment the surfactant composition is positively charged. The surfactant composition can comprise mixtures of multiple lipids and/or multiple surfactants.
When used herein, the term “unsequestered” or “not sequestered” generally refers to a state of a substance being not bound or less bound to another moiety or complex. Regarding an individual substance or population of substances, the term can indicate that there is less than a complete degree of sequestration, e.g., of an individual molecule or population of molecules. The term is consistent with a state of being released or liberated, or remaining free or resistant to being bound, whether from a previously bound state or without having previously been bound.
The invention may be further understood by the following non-limiting examples.
The electrostatic behavior of polyelectrolytes such as DNA and F-actin is considerably more complex than uncharged polymer fluids. In the presence of oppositely charged multivalent cations, both DNA and F-actin can overcome their mutual electrostatic repulsion and attract one another and organize into new self-assembled phases. Examples from nature include the hierarchical ordering of DNA chains via histones within chromosomes, and the high-density liquid crystalline DNA packaging by multivalent protamines in bacteria and viral capsids. We have experimentally established a microscopic mechanism for cation-mediated attraction between F-actin, and find that the cations organize into density waves that induce nanomechanical twist distortions of the actin helix (
The behavior of these condensed polyelectrolyte phases becomes even richer with increasing complexity in the condensing cations. The CF airway is ‘littered’ with both anionic and cationic components that can interact electrostatically. These electrostatic complexes can be understood. For example, gene delivery systems such as DNA-cationic polymers or DNA-cationic membrane complexes have recently received extensive experimental and theoretical scrutiny. See Raedler et al. (1997); Feigner et al. (1987,1991); Gustafsson et al. (1995); Sternberg et al. (1994); Lasic et al. (1997); May et al. (1997); Bruinsma (1998); Koltover et al. (1998). A polymorphism of different structures of these DNA-membrane complexes (such as the lamellar and hexagonal phases) with different transfection efficiencies can exist, as elucidated by recent synchrotron x-ray scattering experiments (
Condensed bundles comprised of F-actin and of DNA occur in CF sputum (Sheils et al. 1996), since these polymers arise in the ASL when neutrophils and other cells lyse as the result of the inflammatory response. It has been suggested that cationic antibacterial polypeptides constitute at least a portion of the ligands holding these polyelectrolytes together. Weiner et al. (2003). We have examined F-actin-lysozyme complexes and determined that lysozyme close-packs into a 1-D column in between a hexagonal arrangement of F-actin filaments. More importantly, the F-actin-lysozyme binding is enhanced at elevated NaCl and KCl concentrations. This is consistent with experimental results in which antibacterial activity is increased as the salt level of the ASL was artificially lowered using an osmolyte. Zabner et al. (2000). By directly measuring the structure and relative stability of these complexes across a wide range of buffer conditions (salt, pH, temperature) using synchrotron x-ray diffraction, and using the data synergistically with computer simulations, rational design dissolution strategies are obtained. From the present invention, one skilled in the art can tailor-design, using biophysical considerations, charge-reduced mutant antibacterial peptides that bind to F-actin and DNA much less than endogenous antibacterial peptides, and therefore, can be active in the electrostatic environment of the airway. Biophysical tools such as synchrotron x-ray diffraction and computer simulations are used to elucidate the nature of the binding and the structure of the resultant complexes (e.g. polymer sequestration of native versus charge reduced antimicrobials).
In order to assess the relative stabilities of the different polyelectrolyte complexes that occur in CF mucus, it is necessary to know their structures. Actin can condense into bundles with model cationic ligands. We have solved the structure of the lysozyme-actin complex at a wide range of monovalent salt concentrations (
Monomeric G-actin (MW 42,000) was prepared from a lyophilized powder of rabbit skeletal muscle purchased from Cytoskeleton, Inc. (Denver, Col.). The non-polymerizing G-actin solution contained a 5 mM TRIS buffer at pH 8.0, with 0.2 mM CaCl2, 0.5 mM ATP, and 0.2 mM DTT and 0.01% NaN3. G-actin (2 mg/ml) was polymerized into F-actin (linear charge density λA˜−1 e /2.5 Å at pH 7) upon the addition of salt (100 mM KCl). Human plasma gelsolin (Cytoskeleton, Inc., Denver, Col.) was used to control the average F-actin length between 0.1 μm to 10 μm. The filaments were treated with phalloidin (Sigma Aldrich, St. Louis, Mo., MW 789.2) to prevent actin depolymerization and resuspended to a final concentration of ˜26.67 mg/ml using Millipore (Billerica, Mass.) H2O (18.2MΩ). Hen Egg White Lysozyme (MW 14,300, Sigma Aldrich, St. Louis, Mo.) was dissolved in Millipore H2O (18.2MΩ) to a final concentration ˜25 mg/ml. Lysozyme carries a pH dependent charge of +9 or +10 and has dimensions of approximately ˜26Å×26Å×45 Å.
The F-actin—lysozyme isoelectric point is found at a concentration ratio of ˜2.5:1 F-actin:lysozyme. X-ray samples were prepared at various F-actin:lysozyme concentration ratios on either side of the isoelectric point, including 2:1, 2.5:1, 3:1, and 5:1. The effect of monovalent salts (KCl & NaCl) on F-actin-lysozyme condensation was investigated by preparing samples at a range of concentrations from 0 mM to 500 mM, so that no matter the ASL ionic concentration, we have the structural solution.
X-ray samples were prepared by sealing F-actin, lysozyme, and monovalent salt solution into 1.5 mm diameter quartz capillaries, followed by mixing and centrifugation. Small angle x-ray scattering (SAXS) experiments were performed at both at Beamline 4-2 of the Stanford Synchrotron Radiation Laboratory (SSRL) as well as at an in-house x-ray source. The incident synchrotron x-rays from the 8-pole Wiggler were monochromatized to 8.98 KeV (λ=1.3806 Å) using a double-bounce Si(111) crystal, focused using a cylindrical mirror. The scattered radiation was collected using a MAR Research charged coupled device (CCD) camera (pixel size=79 μm). For the in-house experiments, incident CuKa radiation (λ=1.54 Å) from a Rigaku rotating-anode generator is monochromatized and focused using Osmic (Auborn Hills, Mich.) confocal multilayer optics, and scattered radiation is collected on a Bruker 2D wire detector (pixel size=105 μm). The 2D SAXS data from both systems are mutually consistent.
A 2-D diffraction pattern for partially aligned F-actin-lysozyme bundles and its associated 1-D integrated slices along the qz and qr directions are shown in
To investigate the effects of monovalent salt on F-actin condensation, a series of high-resolution synchrotron SAXS measurements were performed on F-actin solutions (average length ˜3000 Å). The SAXS data (
Such “in vitro” data can be related to CF mucus in patients by conducting similar experiments on sputum from CF patients. Cystic Fibrosis patients from Carle Clinic in Urbana who have not been treated with DNase voluntarily expectorate approximately 5-10 ml of sputum during respiratory therapy. After collection, the sputum samples are rapidly frozen and stored at −20° C. for later structural analysis experiments.
Obtaining structural information on the supramolecular organization of CF sputum is difficult, since it is highly inhomogeneous, with structures that change over short length-scales (˜10 μm). This problem is solved by using microdiffraction techniques from 3rd generation synchrotron x-ray sources. We performed microdiffraction experiments at beamline 2D-1D-D at the Advanced Photon Source of the Argonne National Laboratory. Monochromatized x-rays at 8.05 keV (λ=1.5498 Å) were focused to a beam size of 0.5 μm×0.5 μm using a Fresnel Au/Si Zone Plate followed by an order sorting aperture of 10 pm diameter. Scattered x-rays were measured using a LN2 cooled Charge-Coupled Device (CCD) area detector. Samples were mounted on a sample stage with submicron translation control necessary for scanning the samples.
Representative results of the microdiffraction data from CF sputum samples are shown in
The techniques discussed herein identify which antimicrobials are capable of forming bundles with the various anionic polyelectrolytes in the ASL. It is known empirically that in general trivalent ions are required to generate attractions and ‘bundle’ DNA, while only divalent ions are required to ‘bundle’ F-actin and microtubules, while monovalent ions do not ‘bundle’ any of them. We have developed a general biophysical criterion for whether a given cation will induce bundle formation in a wide range of biological polyelectrolytes, based on the cation size and valence relative to the Gouy-Chapman screening length. Butler et al. (2003). These results suggest that below a charge threshold, a cationic ligand can not bind to and condense anionic polyelectrolytes such as DNA and F-actin.
A continued inflammatory response to chronic and/or repeated infections in the airways leads to the pathological release of cytoskeletal proteins, DNA and other polyelectrolytes in the airways of CF patients. This release of polyelectrolytes cause the electrostatic assembly of large aggregates stabilized by cationic ligands in CF mucus, and results in the sequestration of endogenous antibacterial polypeptides and contributes to the loss of antimicrobial function. In this example, the charge of a native antimicrobial is reduced to minimize sequestration, thereby rescuing antimicrobial efficacy.
The ionic environment of CF mucus is complex. Electrostatics in complex fluids have recently received extensive attention both theoretically and experimentally. Although there is still an unresolved debate on the ionic strength of the ASL (a ˜5 μm thick liquid layer on the surface of the airway epithelium), it is clear that the ASL in CF is enriched in anionic polyelectrolytes. The primary constituents of normal mucus are water, salts, and the glycoprotein mucin. While production of mucus glycoproteins appears to be normal in CF patients, its degree of hydration, which determines the mucus viscosity, is not. This is, at present, not well understood. A number of different mechanisms have been proposed, based on CFTR induced changes in the salt concentrations in the ASL. In addition to the anionic glycoproteins comprising normal mucus, CF mucus contains highly anionic polyelectrolytes such as extracellular filaments produced by colonizing bacteria, as well as F-actin and DNA released from lysed inflammatory cells. The concentration of DNA in CF sputum can be as high as 20 mg/ml, and comprises 4-10% of the dry weight of the sputum. Likewise, F-actin comprises ˜10% of total leukocyte protein, with concentrations reported to be 0.1-5 mg/ml. There is evidence that the altered salt environment in the airways diminishes the activity of native antibacterial proteins. It has also been observed that these anionic polyelectrolytes can bind to and completely inactivate cationic antibacterial proteins.
The increased ionic strength in the ASL of CF patients inhibits the antimicrobial activities of a number of proteins, including lysozyme, β-defensins, and lactoferrin-derived fragments. Bals et al. (1998ab); Smith et al. (1996); Weinberg et al. (1988). For example, although β-defensins are constitutively expressed and induced in CF (Singh et al. (1998)), they do not function properly in the diseased ASL. One common, unifying feature of a wide range of antibiotic peptides is that they are cationic amphiphiles that can interact strongly with the anionic surfaces of microbes, and destabilize their membranes via binding events that are to a large extent electrostatic, and therefore depend on the ionic environment. Therefore, it is important to understand electrostatic interactions in the ASL.
Charge-reduced antibacterial proteins or peptides have a lower binding affinity to anionic polyelectolytes in the ASL and, therefore, lower sequestration levels. Lower sequestration levels correspond to increased overall antibacterial activity in the airway. Although the method of charge-reduction to improve antimicrobial or antibacterial activity in the CF airway is generally applicable to a broad range of antibacterial proteins or peptides including, for example, lysozyme, β-defensins, and lactoferrin-derived fragments, lysozyme is used in the present examples.
Lysozyme is an antibacterial protein found in high concentrations in the CF airway. We have demonstrated how cationic antibacterial proteins such as lysozyme can self-assemble with the anionic polyelectrolytes in the airways such as F-actin into a stable complex and consequently be sequestered. Such sequestration reduces the effectiveness of antibacterial proteins by limiting contact with the target organism, e.g., with the bacterial cell wall. By using synchrotron small angle x-ray scattering (SAXS), we have determined the structure of actin-lysozyme complexes in a wide range of salt concentrations, and now understand the interactions that govern their self-assembly (
The binding and self-assembly between F-actin and lysozyme can be controlled by modifying the lysozyme charge, as demonstrated by binding studies comparing wild type T4 lysozyme and charge-reduced T4 lysozyme mutants. Wild-type lysozyme has a charge of +9. Using site-directed mutagenesis at two different cationic residues, it is possible to obtain charge +5 mutants at neutral pH. This is accomplished by mutating K16E (lysine to glutamic acid) and R119E (arginine to glutamic acid) (
The SAXS data (
Plasmids for bacteriophage T4 wild-type (SEQ ID NO:1) and mutant lysozymes were provided by Professor Brian Matthews at the University of Oregon. The mutant lysozymes included two single mutants, K135E and R154E; four double mutants: K16E/R119E; K16E/K135E; K16E/R154E; K135E/K147E; and one triple mutant: K16E/K135E/K147E. (K=lysine; E=glutamic acid; R=arginine; where, for example, K135E indicates that a lysine residue at position 135 was substituted with glutamic acid). Proteins were expressed and purified according to previously published methodology. Dao-pin, et al. (1991). Purified lysozymes are diluted to working concentrations using ultrapure H2O (18.2 MΩ; Millipore Corporation, Billerica, Mass.).
Monomeric actin (G-actin) (MW 42 000) was prepared from a lyophilized powder of rabbit skeletal muscle. (Cytoskeleton, Inc., Denver, Col.). The non-polymerizing G-actin solution contained a 5 mM Tris buffer at pH 8.0, with 0.2 mM CaCl2, 0.5 mM ATP, 0.2 mM DTT, and 0.01% NaN3. G-actin (2 mg/ml) was polymerized into F-actin (linear charge density λA≈1e/2.5 Å at pH 7.0) by the addition of monovalent salt (100 mM Nacl final concentration). Human plasma gelsolin, an actin severing and capping protein (Janmey et al., 1986) (Cytoskeleton, Inc.), was added at a gelsolin:actin monomer molar ratio of 1:370 to restrict the length of the F-actin polymers to approximately 1 μm. The filaments were treated with phalloidin (MW 789.2; Sigma Aldrich, St. Louis, Mo.) to prevent actin depolymerization. F-actin gels were ultracentrifuged at 100 000 g for 1 h to pellet the filaments. After the removal of the supernatant buffer solution, the F-actin was resuspended in ultrapure H2O (18.2 MΩ; Millipore Corporation).
Stock solutions of NaCl and KCl were prepared by mixing in ultrapure H2O. In order to ensure uniform mixing and to decrease error associated with pipetting small volumes, lysozyme was pre-mixed with monovalent salt solutions in 100 μl aliquots. The isoelectric actin-lysozyme complexes were prepared by flame sealing F-actin with lysozyme-monovalent salt solutions in 1.5 mm quartz capillaries (Hilgenberg GmbH; Malsfeld, Germany) and mixing thoroughly by centrifugation. The approximate sample volume in the capillary was 30 μl. The final F-actin concentration was ˜4.3 mg/ml while the final wild-type lysozyme concentration was ˜4.2 mg/ml and double mutant concentration was ˜2.3 mg/ml. A series of samples are prepared with the final monovalent salt concentration ranging from 0 mM to 500 mM.
SAXS measurements were performed using the in-house x-ray source located in the Frederick Seitz Materials Research Laboratory (Urbana, Ill.), beamline 4-2 at the Stanford Synchrotron Radiation Laboratory (SSRL; Palo Alto, Calif.), and BESSERC beamline 12-ID-C at the Advanced Photon Source (APS; Argonne National Laboratory, Argonne, Ill.). For the in-house experiments, incident Cu Ka radiation (λ=1.54 Å) from a Rigaku (The Woodlands, Tex.) rotating-anode generator was monochromatized and focused using Osmic (Auborn Hills, Mich.) confocal multilayer optics, and scattered radiation was collected on a Bruker AXS (Madison, Wis.) 2D wire detector (pixel size=105×105 μm2). For the SSRL experiments, incident synchrotron x-rays from the eight-pole wiggler were monochromatized to 8.98 keV using a double-bounce Si(111) crystal (λ=1.3806 Å) and focused using a cylindrical mirror. The scattered radiation was collected using a MAR Research (Evanston, Ill.) charge-coupled device camera (pixel size=79×79 μm2). For the APS experiments, incident x-ray wavelength was set at λ=1.033 Å by a double-crystal Si(111) monochromator and focused using a flat-focusing monochromatic mirror. The scattered x-rays were collected using a two-dimensional mosaic CCD detector (pixel size=79×79 μm2; MAR Research). The sample-to-detector distances were set such that the detecting range is 0.03<q<0.25 Å−1, where q=(4πsinθ/λ, λ is the wavelength of the incident beam, and 2θ is the scattering angle. The 2D SAXS data from all set-ups have been checked for mutual consistency.
Typically, the precipitated F-actin—lysozyme complex is compacted into a dense pellet during mixing. These pellets consist of many coexisting domains of actin bundles locally oriented along different random directions, as indicated by the “powder averaging” of the diffraction pattern and the associated loss of orientational information. In order to minimize these effects, a small (300×300 μm2) x-ray beam is used to obtain diffraction information on locally aligned domains within the pellet.
To further explore the extent of lysozyme sequestration by actin, equilibrium dialysis experiments (
The release of charge-reduced antimicrobials was further verified by antibacterial activity assays similar to that shown in
The antimicrobial activity of wild-type lysozyme decreases with increasing salt (Travis, et al., 1999); this decrease can be compensated with larger lysozyme concentrations. The influence of salt on the activity of the charge-reduced mutants can be assessed as disclosed herein, and compared to that of the wild-type. Data (not shown) indicate that while the antimicrobial activity of wild type lysozyme is higher than that of the charge-reduced mutant at low salt levels (˜10 mM NaCl), the difference in activity between the two decreases dramatically as the salt level is increased. The activity of the two types of lysozyme is comparable at physiological salt levels, so that the mutant lysozyme will show a significant increase in antimicrobial activity due to the lower level of mutant sequestration in the airway relative to the level of wild-type sequestration. Those of ordinary skill in the art will recognize that this approach can be utilized to manufacture human-recombinant versions of such mutants for delivery to the airways, e.g., in aerosolized form, to counter long-term infections.
The aminoglycosides are a family of potent antibiotics that are made of highly cationic sugars, and are used for a variety of biomedical conditions, such as cystic fibrosis. Due to their high positive charge, however, they often become sequestered via electrostatic interactions with oppositely charged molecules preventing them from reaching their intended target, thus decreasing their efficiency. This is illustrated in
We have developed a general strategy to use rationally-designed positively-charged surfactants (similar to those which are currently being used in for gene-therapy), to coat the negatively charged polymers, thus freeing the aminoglycoside antibiotics and allowing them to fulfill their antibacterial function. This is accomplished by designing surfactant formulations that have the appropriate amount of charge and curvature, so that they can maximize counterion entropy gain upon binding, and thereby optimally coat anionic polymers in the airway, thus preventing antibiotic binding. These surfactants can be used to also inhibit binding of the aminoglycoside antibiotics to the glycoproteins present in the airway. This method can also be applied to other highly infected systems as well as for other antimicrobials and antibiotics.
One of the most common bacterial infections in CF patients is that of Pseudomonas aeruginosa which is primarily treated by administration of aminoglycoside antibiotics, specifically tobramycin. It has been observed, however, that the anionic polyelectrolytes present in CF sputum can bind to and completely inactivate these cationic antibiotics (see
Tobramycin is a multivalent cationic anti-pseudomonal antibiotics (charge of +5) currently used to treat bacterial infections in the airways of CF patients. As discussed above, multivalent cations can self-assemble with the anionic polyelectrolytes in the airways (e.g. F-actin and DNA) to form a stable complex (
We demonstrate that the binding and self-assembly between DNA and a charged antimicrobial such as tobramycin can be limited by adding a solution of cationic lipids to the DNA-tobramycin suspension. Cationic lipids are surfactants that are known to form complexes with many different types of anionic polymers including DNA and F-actin. Cationic lipid vesicles are used in other applications, e.g., as gene carriers in clinical trials of non-viral gene therapy. Ewert et al. (2004). In one embodiment, a mixture of two lipids is used: DOPE, which is one of the main neutral lipids in use in gene therapy applications, and DOTAP, which has a positively charged hydrophilic head. A mixture of the two lipids creates an appropriate ratio of charge and lipid curvature in order to wrap the lipid membranes around the DNA. It has been shown that by mixing these lipids together in a ratio from 100:0 to 35:65 of DOTAP:DOPE, the lipids form a lamellar complex with DNA. Raedler et al. (1997). By mixing the lipids together in a ratio from 35:65 to 10:90 of DOTAP:DOPE, the lipids form an inverted-hexagonal phase with the DNA because of the curvature of the DOPE. Koltover et al. (1998). For our experiments we chose ratios within the range of each of the two phases, 70:30 DOTAP:DOPE and 25:75 DOTAP:DOPE, to identify the behavior of the lamellar and inverted hexagonal phases.
A series of SAXS experiments were conducted at physiological salt and pH conditions in which the cationic lipid mixtures were added to DNA+tobramycin suspensions.
This basic strategy of liberating tobramycin bound to DNA complexes by adding lipid-based surfactants can function at many different DNA and tobramycin concentration ranges to restore partial tobramycin antimicrobial activity in the airways. For example,
This method can adequately function even if not all the DNA or actin or mucin in the airways is bound. In one embodiment, these surfactant formulations are administered as an aerosol to pacify the charged surfaces immediately before the administration of antibiotics. Such a method increases the efficacy of the administered antibiotic compared to when an antibiotic is administered without these surfactant formulations.
In vitro bactericidal assays can be used to quantify the accessibility of the tobramycin to work as an antibacterial in the presence of the lipid+DNA complexes (
For our studies of the sequestration of aminoglycoside antibiotics by model CF sputum and subsequent release by the administration of surfactants we used the following materials. The aminoglycoside antibiotic used in our experiments was Tobramycin (Sigma Aldrich), commonly used to treat infections of Pseudomonas aeruginosa in CF patients. Tobramycin has been shown to be sequestered by DNA, F-Actin, and mucin present in CF mucous. For the DNA+tobramycin experiments we used Calf thymus (CT) DNA (USB Corp. Cleveland Ohio), purified it using standard techniques, and resuspended it in an aqueous solution of 100 mM NaCl 5 mM Tris and 5 mM PIPES adjusted to pH 7. DNA has a charge of approximately −2/base pair.
For the F-actin+tobramycin experiments, lyophilized rabbit skeletal muscle monomeric G-actin (Cytoskeleton, Inc., Denver, Col.), was resuspended in non-polymerizing G-actin solution containing 5 mM Tris buffer at pH 8.0, with 0.2 mM CaCl2, 0.5 mM ATP, 0.2 mM DTT, and 0.01% NaN3. G-actin (2 mg/ml) was polymerized into F-actin (linear charge density λA=1 e/2.5 Å at pH 7.0) by the addition of monovalent salt (100 mM KCl final concentration). Human plasma gelsolin, an actin severing and capping protein (Janmey et al. (1986) (Cytoskeleton, Inc.), was added at a gelsolin:actin monomer molar ratio of 1:370 to restrict the length of the F-actin polymers to approximately 1 μm. The filaments were treated with phalloidin (MW 789.2; Sigma Aldrich, St. Louis, Mo.) to prevent actin depolymerization. F-actin gels are ultracentrifuged at 100 000 g for 1 h to pellet the filaments. After the removal of the supernatant buffer solution, the F-actin was resuspended in ultrapure H2O (18.2 MΩ; Millipore Corporation). For the mucin+tobramycin experiments we used porcine stomach mucin (Sigma Aldrich). Similar studies can be conducted using human respiratory mucous. Porcine mucin was resuspended in aqueous solution and autoclaved for 5 minutes to ensure sterility. Mucin is found in respiratory mucous at concentrations of 1-5 mg/ml and is highly negatively charged, though the exact charge is unknown due to the polydispersity of the structure of the polymer.
Surfactant materials used in the present experiments were a mixture of two lipids DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane) and DOPE (1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine) (Avanti Polar lipids, Alabaster Al.) in mass ratios of about 70:30 and about 25:75. DOTAP is a cationic lipid (charge of +1) with no curvature, and DOPE is a neutral lipid with negative curvature. Briefly, the lipids were first dissolved in chloroform, then dried under nitrogen and redissolved in an aqueous solution. The aqueous salt solution was then sonicated to form unilamellar lipid vescicles, and filtered through 0.2 micron pores.
Aminoglycoside antibiotic sequestration by negatively charged bio-polymers in respiratory mucous of CF patients was measured via two techniques. The first method was to use x-ray diffraction techniques to discern the structure of suspensions of mixtures of biopolymers and antibiotic. Specific x-ray diffraction studies were done to determine the structure of mixtures of the DNA with tobramycin and the lipid mixture. The isoelectric DNA-lipid-monovalent salt solutions were sealed in 1.5 mm quartz capillaries (Hilgenberg GmbH; Malsfeld, Germany) and mixed thoroughly by centrifugation. The approximate sample volume in the capillary was 30 μl-60 μl. SAXS experiments were performed as described previously. Tobramycin has charge of approximately +5 at physiological pH, whereas DNA is negatively charged, thus when the ratio of net DNA charge is approximately equal to the net tobramycin charge (T/D˜1), the tobramycin condenses the DNA. The resulting structure is a bundle of DNA. The structures observed via x-ray diffraction indicate whether or not the tobramycin is sequestered with the DNA or free in suspension. The present invention uses positively charged lipids to replace the tobramycin in these bundles, thereby increasing the efficacy of the antibiotic. Consequently, our experiments used a fixed DNA concentration of 3 mg/ml (x-ray experiments) and a varied concentration of tobramycin and/or lipid. Concentrations are varied such that the ratio of positive to negative charges is known. Similar x-ray diffraction studies can be conducted wherein mucin, instead of DNA, is the sequestration agent to examine mucin+tobramycin+lipid complexes and kinetics.
Tobramycin sequestration was also measured via bacteriological killing assays. In these bacterial killing assay experiments Pseudomonas aeruginosa (PA01), the primary bacteria responsible for infection in CF patients, was incubated in the presence of mucin, DNA, tobramycin, lipid or various mixtures of these four materials. Killing assays were performed in the following manner: PA01 was grown in cation adjusted Mueller-Hinton media to a concentration greater than 5×108, sedimented by centrifugation and resuspended in a buffered solution of 5 mM Tris 5 mM PIPES and 100 mM NaCl at pH 7.2. A quantity of ×105 c.f.u. of PAO1 were incubated for 3.5 hours in the buffered salt solution with varied concentrations of tobramycin, DNA, mucin, and lipids. The mucin concentrations were either 0 mg/ml (control) or 10 mg/ml which is near the physiologically measured concentrations of mucin. Lipids added to the bacterial solution were added in varying concentrations in order to determine the amount needed to release tobramycin from its strong interaction with mucin and/or DNA. The bacterial solution was serially diluted and plated on MH-agar plates. Plates were incubated overnight and the number of bacterial colonies counted. The minimal inhibitory concentration (MIC) of tobramycin for these assays is approximately 5 μg/ml.
Based on the disclosure in this application, we demonstrate the applicability of using lipids of any size chain length to interact with substances that can act as sequestering agents such as DNA and F-actin. Thus in addition to DOTAP:DOPE lipid compositions, other lipids of are adaptable for use in embodiments of the invention. For example, PC, PE and TAP lipids of any chain length can be employed to prevent antimicrobial sequestration.
When DNA is mixed with similar cationic lipid mixtures of DOTAP and DOPC, 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine, and when the DOTAP:DOPC ratio varies from 100:0 to 10:90, a lamellar lipid-DNA complex identical to that exhibited by DOTAP:DOPE mixtures at a ratio of 70:30 is measured. Correspondingly, a positively-charged antimicrobial such as tobramycin can be excluded from this DOTAP: DOPC phase in the same way that it is excluded from the DOTAP:DOPE lamellar phase due to the similarity of structural attributes.
SAXS experiments have also shown that cationic lipid mixtures of DLTAP:DLPC, DOTAP:DOPC, and DNTAP:DNPC (dilauryl trimethyl ammonium propane: dilauryl trimethyl phosphatidylcholine, dioleoyl trimethyl ammonium propane: dioleoyl trimethyl phosphatidylcholine, and dinervonyl trimethyl ammonium propane: dinervonyl trimethyl phosphatidylcholine, respectively) for ratios from 100:0 to 10:90 will all form complexes with F-actin. These complexes show that the F-actin is coated by lipids (
Other specific cationic surfactant compositions which display the same interaction with DNA, and can decrease sequestration of antimicrobials, include the following: Didodecyldimethylammonium bromide (DDAB); Cetyltrimethylammonium bromide (CTAB); Cetyltrimethylammonium bromide (CTAB): 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHGPC) from 20:80 to 80:20; and DLTAP:DLPC, DOTAP:DOPC, and DNTAP:DNPC (dilauryl trimethyl ammonium propane: dilauryl trimethyl phosphatidylcholine, dioleoyl trimethyl ammonium propane: dioleoyl trimethyl phosphatidylcholine, and dinervonyl trimethyl ammonium propane: dinervonyl trimethyl phosphatidylcholine, respectively) from 100:0 to 10:90.
Further lipid compositions that can be utilized are listed in Table 1. These lipids have the same hydrophilic head structure but differ in hydrocarbon chain length.
The human body produces a variety of peptides and proteins, such as lysozyme and lactoferrin, to fight infection such as bacterial infection in the lung. While these molecules are very effective antibacterial agents, they carry high number of positive charges and tend to be sequestrated by negatively charged polyelectrolytes naturally produced from the inflammatory response, such as actin and DNA from lysed cells. A desirable “non-stick” version of an antibacterial peptide/protein will retain its native level of activity but would have reduced electrostatic interaction with the polyelectrolytes. We have developed approaches for making charge-engineered human lysozymes. Two different protein engineering approaches have been used. The first is to engineer reduced-charged mutants of human lysozyme; this is analogous to our efforts described herein regarding T4 lysozyme. The second approach is to reduce charge via a tag. A tag is connected to the lysozyme, e.g. by direct conjugation or by employing an optional spacer. A spacer can be used to prevent the charge-balancing tag from folding back to further associate with the protein itself. Preferably the tag is introduced sufficiently far from the enzyme active site so that the protein activity is not affected. This second approach has an advantage of allowing for facile scale-up in production due to a simpler protocol for purification.
As depicted in
Human lysozyme was selected as a suitable candidate for developing novel compositions and methods. The native molecule is a small protein with 130 amino acids and a net charge of +9. Charge reversal mutants of T4 lysozymes as described herein can lead to reduced bacterial killing activity for gram-negative bacteria such as E. Coli and Pseudomonas aeruginosa, even though minimal reduction or even enhancement of killing activity has been observed for gram-positive bacteria for certain mutants. We have investigated the alternate strategy of a ‘charge balancing’ approach with the goal of reducing the net positive charge on human lysozyme while preserving antimicrobial activity. The expression charge-balancing does not necessarily imply an equality of balance in overall net charge such as in a state of neutrality.
Several aspects of engineering charge balancing lysozyme mutants have been incorporated into this strategy. One aspect is the selection of a preferred location for attaching the negatively charged substance(s). The core of lysozyme's bacteria killing activity lies in its active site, which forms a cleft that hydrolyzes the bond between N-acetyl muramic acid (NAM) and N-acetylglucosamine (NAG) in bacteria cell walls, or the bond between NAG and NAG in fungi cell walls. As shown in
A second consideration for the charge balancing mutant engineering is a spacer between the attached charge-balancing moiety/tag and the lysozyme. A particular factor is how close two charges must be on a given macroion before it generates electrostatic attractions between polyelectrolytes (Butler et al., Phys. Rev. Lett. 2003). We therefore designed a tag of an optimized length with an optimized pattern of charges for attachment to a given antimicrobial so that it will disrupt packing into polyelectrolyte complexes while retaining its full bacteria killing activity.
For a particular analog, a third aspect is the possible necessity to prevent the negatively charged amino acid tag from folding back onto the protein itself. Under such requirements a rigid spacer is desirable, and we have designed a spacer sequence of seven alanine residues. Among the 20 amino acids, alanine has the highest propensity to form alpha-helical structure, which is highly rigid. An (Ala)7 sequence is expected to form about two turns of alpha-helix, making it a rigid spacer with a length of about 10 Å. The other advantage of an alanine spacer is that it is neither hydrophobic nor hydrophilic. As a result, this spacer sequence is less likely to interact with the lysozyme or the tag. Other tags are developed based on the potential for alanine-rich sequences to subject the labeled proteins to intracellular degradation in bacteria; this may influence the choice of expression system. A further consideration is protein purification, which can facilitate production scale-up. We add a tandem of six histidines (6×His) to the C- or N-terminus of the new mutant lysozymes, and then use a Ni2+loaded matrix to purify the histidine-tagged protein based on this affinity tag. At pH 7-7.5, this 6×His tag is neutral and unlikely to impart additional positive charges on the constructed proteins. Based on these considerations, we have constructed three charge-balanced human lysozyme variants, designated HLYH, HLYAH, and HLYAEH (HLYH: A six-histidine (6×His) tag is attached to the C-terminal end of human lysozyme; HLYAH: a spacer sequence of seven alanines (7×Ala) is attached to the C-terminal end of human lysozyme, followed by a 6×His tag; HLYAEH: a 7×Ala spacer is attached to the C-terminal end of human lysozyme, followed by a charge-balancing sequence of six negatively charged glutamates (6×Glu), and a 6×His tag).
The human lysozyme variants are constructed with confirmation of correctly engineered sequences. The proteins are expressed and tested for the ability to renature and fold into soluble proteins. Human lysozyme variants are used in pharmaceutical compositions and methods for antimicrobial therapies.
Although T4 lysozyme has been successfully expressed and purified using E. coli bacteria, it is difficult to express human lysozyme using this system due partly to the property of human lysozyme's greater bacteria killing activity, and partly to the property of human lysozyme's involving the formation of four disulfide bonds, which is hindered by the reducing environment inside bacteria. At present, the standard practice in human lysozyme expression is to use a yeast expression system, even though it is much less efficient compared to the E. coli system. We have devised an expression and purification strategy that uses an E. coli system to express human lysozyme variants.
Bacterial Expression and purification of the charge-balanced human lysozyme variants. When newly synthesized protein chains are not folded properly, they tend to aggregate and form the bulk of inclusion bodies inside the bacteria, especially when the protein over-expression level is high. This is generally not desired in protein expression since it can produce inactive protein. However, hen egg-white lysozyme can be refolded into active form from a denatured state (Li et al., J. Chrom. A., 2002). We therefore determine whether human lysozyme can be successfully prepared from bacterial expression. Since the reducing bacterial cytosol will probably prevent the correct folding of human lysozme, we expect that when we express our lysozyme variants in bacteria, these proteins will aggregate, form inclusion bodies, and not be active, thus avoiding inhibition of bacterial growth. The inclusion body can be dissolved in denaturing reagents such as 8 M urea solution. In their denatured state, the human lysozyme variants can be immobilized on a cation exchange matrix due to their net positive charge. The immobilized lysozymes are subjected to a gradient of renaturing buffer supplement with redox reagents to encourage disulfide formation. Afterwards the lysozymes can be eluted and again immobilized on nickel-NTA matrix through the 6×His tags, and other proteins from bacteria can be further removed by washing of the matrix. Subsequently the lysozyme variants are eluted from the Ni2+-NTA matrix with low pH or imidazole, a histidine analog.
A plasmid containing the human lysozyme gene, Cat. No. TC125273, was purchased from Origene (Rockville, Md.). This plasmid was used as a template for the PCR amplification of the human lysozyme DNA sequence (SEQ ID NO:3). Primers used for constructing HLYH, HLYAH, and HLYAEH (SEQ ID NO:4) are the following, with restriction sequences underlined, and segments relating to
The PCR products were purified and digested with restriction enzymes Nco I and Hind lIl. The digested and purified DNAs are ligated onto pQE-60 vector (Qiagen, Valencia, Calif.; Cat. No. 32169). E. coli strain M15 with preloaded pREP4 plasmids is transformed with the ligation reaction mixtures. Resulting colonies are sequenced. To express the proteins, transformed M15 cultured were grown in LB, and protein expression was induced by the addition of 1-2 mM IPTG to the culture media.
To purify the proteins, bacteria were harvested with centrifugation and lysed with denaturing buffer A (50 mM Tris, pH 8.7, 8 M urea, 3 mM GSH, 0.3 mM GSSG). The lysates were clarified using ultracentrifugation to remove insoluble materials before loading onto a HiTrap SP XL 1 ml column (GE Healthcare Bio-Sciences, Piscataway, N.J.). A gradient from buffer A to buffer B (100 mM Tris, pH 8.7, 1 M urea, 3 mM GSH, 0.3 mM GSSG) was run through the column using an AKTAFPLC chromatography system (GE Healthcare Bio-Sciences, Piscataway, N.J.). After the refolding, the HiTrap column was directly connected to a HisTrap 1 ml with Ni2+-NTA matrix (GE Healthcare Bio-Sciences, Piscataway, N.J.), and the proteins on the cation exchange column were eluted with 300 mM NaCl onto the HisTrap column. The HisTrap column was washed with wash buffer (200 mM NaCl, 50 mM Tris, pH 7.5, and 60 mM imidazole), then eluted with elution buffer (200 mM NaCl, 50 mM Tris, pH 7.5, 200 mM imidazole).
All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; unpublished patent applications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).
Any appendix or appendices hereto are incorporated by reference as part of the specification and/or drawings.
Where the terms “comprise”, “comprises”, “comprised”, or “comprising” are used herein, they are to be interpreted as specifying the presence of the stated features, integers, steps, or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component, or group thereof. Separate embodiments of the invention are also intended to be encompassed wherein the terms “comprising” or “comprise(s)” or “comprised” are optionally replaced with the terms, analogous in grammar, e.g.; “consisting/consist(s)” or “consisting essentially of/consist(s) essentially of” to thereby describe further embodiments that are not necessarily coextensive.
The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. It will be apparent to one of ordinary skill in the art that compositions, methods, devices, device elements, materials, procedures and techniques other than those specifically described herein can be applied to the practice of the invention as broadly disclosed herein without resort to undue experimentation. All art-known functional equivalents of compositions, methods, devices, device elements, materials, procedures and techniques described herein are intended to be encompassed by this invention. Whenever a range is disclosed, all subranges and individual values are intended to be encompassed as if separately set forth. This invention is not to be limited by the embodiments disclosed, including any shown in the drawings or exemplified in the specification, which are given by way of example or illustration and not of limitation. The scope of the invention shall be limited only by the claims.
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This application claims the benefit under 35 USC 119(e) of U.S. Provisional Patent Application Ser. No. 60/729,376 filed Oct. 21, 2005, which is incorporated by reference in its entirity.
This invention was made with government support under NSF Grant DMR04-09769 and NIH Grant PHS-1R21-DK068431A awarded by the National Science Foundation and the National Institutes of Health, respectively. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60729376 | Oct 2005 | US |
