Channel forming peptides

Abstract

The present invention provides a family of peptides based upon the M2GlyR sequence. These peptides are derivatives of the M2GlyR sequence and can be modified at their ends to include a plurality of polar amino acid residues to enhance their solubility. Particularly preferred derivatives include portions of the M2GlyR sequence which are palindromic to another portion of the peptide or to the M2GlyR sequence itself. Preferably these portions are at least 7 amino acid residues in length. Peptides embraced by the present invention are characterized by having greater effects on the transepithelial electrical resistance of cells at lower concentrations. Peptides of the present invention have been shown to increase Isc in MDCK epithelial cell monolayers with half maximal effects observed at or below 30 μM, a nearly 10-fold improvement over any peptide previously characterized in the M2GlyR family. Additionally, peptides of the invention have been shown to increase transepithelial electrical conductance and modulate the permeability of tight junctions in epithelial cells.

Description

RELATED APPLICATIONS[0001]This application is a continuation-in-part of application Ser. No. 09 / 710,419, filed on Nov. 9, 2000 now U.S. Pat. No. 6,750,200 and claims the benefit of Ser. No. 60 / 569,299, filed on May 7, 2004. The content and teachings of each of these applications is hereby incorporated by reference herein.SEQUENCE LISTING[0002]A printed Sequence Listing accompanies this application, and has also been submitted with identical contents in the form of a computer-readable ASCII file on a CD-ROM.BACKGROUND OF THE INVENTION[0003]1. Field of the Invention[0004]The present invention is broadly concerned with multiple-peptide channel assemblies which provide transport of anions through epithelial cell membranes wherein the preferred peptides have from about 16-31 amino acid residues and are soluble in water to a level of at least 5 mM; such channel assemblies can be used in the treatment of diseases such as cystic fibrosis (CF) and adult polycystic kidney disease (APKD). More...

AI Technical Summary

Benefits of technology

[0031]The present invention solves the problems inherent in the prior art and provides a distinct advance in the state of the art. Peptides of the present invention exhibit an improvement in activity that is about 5 fold greater with respect to activity levels and / or a 10 fold increase in effective concentration than was heretofore possible. The present invention is directed to improved 1) multiple peptide channel assemblies for transport of anions (e.g., Cl−) through epithelial cells, 2) synthetic peptides capable of forming such channel assemblies, 3) methods of using the channel assemblies in therapeutic contexts for altering the flux of water across epithelial cell layers, and 4) multiple peptide channel assemblies which alter the transepithelial electrical resistance of cell layers. The peptides of present invention exhibit greater stability and reduced solution aggregation, which lead to increased bio-availability of the peptides, thereby reducing the amount of peptide necessary to affect a desired response.

[0032]In preferred forms, the channel assemblies of the invention comprise multiple peptides each having from about 16-31 amino acid residues, and more preferably from about 22-27 residues. The peptides are characterized by the ability of providing, in an embedded channel assembly, transport of anions through an epithelial cell membrane and modulation (alteration) of the water flux across the epithelium. The peptides are also characterized by their effect on reducing the transepithelial electrical resistance of cell monolayers. Preferred peptides of the present invention will have activity profiles of greater than about 15.0 μA / cm2 in MDCK cells when applied to the MDCK cells at a concentration of about 500 μM. More preferably, peptides of the present invention will have activity profiles of greater than about 15.0 μA / cm2 in MDCK cells when applied to the MDCK cells at a concentration of about 300 μM, and still more preferably at a concentration of about 200 μM, and most preferably at a concentration of less than about 100 μM. Moreover, some preferred peptides are soluble in water to a level of at least about 5 mM, and more preferably at least about 10 mM, and still more preferably at least about 15 mM. The peptides of the invention also should exhibit at least about 50% helical content (advantageously at least about 65% helical content, and still more preferably at least about 75%) when dispersed in a 20% trifluoroethanol / 80% water solution and measured using circular dichroism spectroscopy (CD). Preferred peptides of the present invention are also characterized by greater stability and fewer multimeric forms in solutions. Preferably, the peptides will predominantly form only monomers when dissolved. Monomers are preferred due to their higher binding affinity to the membrane. This increased affinity is due to the non-aggregation of the hydrophobic portions, which are required for membrane binding, and are therefore available for binding. This increases the overall bio-availability of sequences comprising mainly monomers. When sequences include multimeric forms, the hydrophobic portions aggregate, thereby rendering them unavailable for binding and decreasing their bio-availability. For peptide sequences that affect epithelial barrier function, such sequences will induce a reduction in electrical resistivity of cell monolayers, that is transient in nature and allows for the passage of larger molecular weight polymers (up to 40,000 Daltons) that are hydrophilic in character. This effect is thought to reversibly modulate the tight junctions that join adjacent epithelial cells into a confluent layer.

Problems solved by technology

When sequences include multimeric forms, the hydrophobic portions aggregate, thereby rendering them unavailable for binding and decreasing their bio-availability.