M2GlyR derived channel forming peptides

Details M2GlyR derived channel forming peptides M2GlyR derived channel forming peptides

2000-11-09

2004-06-15

Spector, Lorraine (Department: 1646)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S300000, C530S326000, C435S004000, C435S325000, C436S151000, C514S014800

Reexamination Certificate

active

06750200

ABSTRACT:

SEQUENCE LISTING
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 floppy diskette.
BACKGROUND OF THE INVENTION
1. Field of the Invention
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), as well as in the killing of undesirable cells. More particularly, the invention pertains to such channel assembly forming peptides, and corresponding methods of use, wherein the peptides are derived from a segment of a native (i.e., naturally occurring) channel protein and have their water solubilities enhanced by modification of the C- or N-ends thereof modified with a plurality of polar amino acid residues such as lysine. Still more particularly, the invention pertains to derivatives of the M2GlyR sequence which remain predominantly in monomer form when in solution, have a desired amount of helical configuration, and alter the transepithelial electrical resistance of cells to a greater extent than was heretofore possible.
2. Description of the Prior Art
Introduction.
A major problem in CF is the inability of airway epithelia to secrete fluid. The resulting changes in the composition of the mucous coating the airway epithelia result in infection and subsequent inflammation, scarring, and eventual pulmonary destruction. The basis of the problem is the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane of the epithelial cells. This leads to an increase in the absorption of salt and water and an inability to respond to appropriate stimuli by secreting chloride and water. CFTR is a chloride channel; in addition it down-regulates sodium channels and up-regulates another population of chloride channels, the outwardly rectifying chloride channel (ORCC). These properties of CFTR enable the airway cells to secrete chloride and this drives the secretion of sodium and water.
A synthetic-23-residue &agr;-helical peptide designated M2GlyR forms anion-selective channels in phospholipid bilayers. This peptide has the amino acid sequence of the putative transmembrane segment M2 of the strychnine-binding a subunit of the inhibitory glycine receptor.
The origin and properties of M2GlyR.
The glycine receptor is a membrane protein present in post-synaptic membranes. Binding of glycine activates a Cl
−
conducting channel, leading to hyperpolarization of the membrane and inhibition of the synapse. The receptor consists of two major glyco-polypeptides, an a subunit of 48 kd and a &bgr; subunit of 58 kd, and a receptor-associated cytoplasmic protein of 93 kd. Strychnine, an antagonist of the glycine receptor, binds only to the &agr; subunit. Messenger RNA corresponding to this subunit leads to the expression of functional, glycine-activated, Cl
−
channels upon injection into Xenopus oocytes.
The glycine receptor channel in cultures of embryonic mouse spinal cord is selective for monovalent anions, with conductances of 27 and 46 pS in 145 mM Cl
−
solution. Pharmacological studies suggested the presence of two sequentially occupied anion binding sites in the channel. These sites are considered to be the functional correlates of the positively charged amino acids bordering the M2 segment of the &agr; subunits. This finding led to the development of the synthetic peptide with the sequence of the M2 segment of the glycine receptor.
Electrical recordings from phospholipid bilayers containing M2GlyR showed single-channel conductances of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes in the millisecond range. Single channel events occurred in 0.5 M N-methyl-D-glucamine HCl but not in sodium gluconate, indicating that the channel is anion selective. A transference number for anions of 0.85 was calculated from reversal potential measurements under a 5-fold KCl concentration gradient.
After insertion into the lipid bilayers the monomeric peptides self-assemble to form conductive oligomers of different amplitudes. To gain control over the aggregate number, four identical M2GlyR peptide units were tethered to a 9-amino acid carrier template to form a four-helix bundle protein. This tetramer, incorporated into lipid bilayers, formed channels of uniform unitary conductance of 25 pS. The 49 pS conductance described above is presumed to be due to the presence of a pentamer.
The tetrameric channel was blocked by the Cl
−
channel blockers 9-anthracene carboxylic acid (9-AC) and niflumic acid (NFA). It was not blocked by QX-222, an analogue of lidocaine and a blocker of cation-selective channels. Strychnine, an antagonist of the glycine receptor, does not block the channel-forming tetramer. Strychnine is presumed to bind to the ligand-binding domain of the receptor exposed to the extracellular surface but not to the channel domain.
Structure of channel forming peptides.
While great strides have been made in the area of channel function and regulation, using the intact protein or in some cases purified channel proteins reconstituted into model membranes, many aspects of channel function remain unresolved. The K
+
from streptomyces lividans was crystallized and the structure determined at 3.2 Angstroms. This structure has served as a model for other related channels using homology modeling methodologies. This structure however is for a 4 subunit channel as opposed the five subunit channel proposed for the glycine receptor.
Considerable structural data exists for the related class of channel forming peptides (CFPs). These channels are much smaller in size and contain only a ring of short peptide chains organized around the central ion conducting pore in the lipid bilayer. These channels are unique in that they assemble by the oligomerization of a single peptide. These structures are models for studying the structure and function of the various regulated channels that occur in nature. This class of CFPs includes: the &agr;-aminoisobutyric acid-containing channels such as alamethicin and zervamicin, and a number of toxins and venoms such as melittin, cecropins, mast cell degranulating peptides, and the defensins. Melittin is somewhat of a special case because it forms channels only at low concentrations; at higher concentrations it acts as a lytic agent. In some cases CFPs assemble spontaneously upon insertion into the bilayer while in the remaining cases the assembly requires an electrical potential across the membrane (V
m
).
The structure of the channels arising from the assembly of these peptides vary from trimers to hexadecamers associated in the form of helical bundles or &bgr;-barrels. The most widely accepted model which is in accord with the model for channel proteins has the helices arranged with their dipoles all pointing in the same direction (parallel). Since CFP channels, unlike authentic channel proteins, are not generated from the association of large protein subunits, alternative stabilization schemes must be invoked to account for the presence of this higher energy arrangement of parallel segments. These could include aligning the dipoles in response to the presence of the membrane potential and/or an increase in the favorable inter-molecular interactions promoted by the parallel assembly. Most CFPs form multiple size bundles of parallel segments (e.g., n=4, 5, 6) that can spontaneously increase or decrease in size upon the addition or deletion of a peptide monomer from the channel assembly. These observations imply that enough information is contained in a single channel forming polypeptide to drive the correct folding, assembly, and activity of these channels.
The activity of these assembled molecules, the opening and closing of the chann

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