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Compositions and methods for inhibiting the interaction between cftr and cal

a technology of cftr and cal, which is applied in the direction of drug compositions, peptide/protein ingredients, metabolic disorders, etc., can solve the problems of insufficient pancreatic and digestive function, abnormally concentrated sweat, and chronic pulmonary disease and respiratory infections, so as to prevent or increase the cell surface expression of the degradation-prone cftr protein. , the effect of increasing the cell surface expression of the degradation-prone cftr protein

Inactive Publication Date: 2013-03-07
DUKE UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In CF, unusually thick mucus leads to chronic pulmonary disease and respiratory infections, insufficient pancreatic and digestive function, and abnormally concentrated sweat.
Lowered temperature results in the appearance of CFTR protein and channel activity at the cell surface, suggesting an intrinsic thermodynamic instability in ΔF508-CFTR at 37° C. that leads to recognition of the mutant protein by the ER quality control mechanism, prevents further trafficking, and results in protein degradation.
However, even with such treatments, frequent hospitalization is often required as the disease progresses.
However, only a limited number of “corrector” drugs has been described for the treatment of CF.
However, no high-affinity and selective inhibitor compounds have been identified for PIST, nor have PIST reporter sequences been identified that would permit small-molecule screening, nor have any such compounds been shown to have efficacy in stabilizing mutant, degradation-prone CFTR.

Method used

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  • Compositions and methods for inhibiting the interaction between cftr and cal

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example 1

Materials and Methods

[0056]Protein Expression and Purification.

[0057]CALP (UniProt Accession No. Q9HD26-2) was expressed and purified (Cushing, et al. (2008) Biochemistry 47:10084-10098). TIP-1 (Accession No. 014907) was expressed and purified similarly except that an N-terminal His10 tag was used with a HRV 3C protease recognition sequence (LEVLFQ*G; SEQ ID NO:35) upstream of the full-length protein sequence. Following TIP-1 purification via immobilized metal-affinity chromatography, the protein was injected onto a SUPERDEX S75 gel filtration column (GE Healthcare) equilibrated in 50 mM Tris pH 8.5, 150 mM NaCl, 0.1 mM TCEP, 0.02% NaN3. Human rhinovirus 3C protease (Novagen) was added to the protein at a 1:30 mass ratio and incubated at 4° C. for 48 hours. Following cleavage, the protein was passed through a 1 mL HISTRAP HP column (GE Healthcare) equilibrated in 20 mM imidazole, 25 mM Tris pH 8.5, 150 mM NaCl, 0.1 mM TCEP, 0.02% NaN3. The protein was further purified on a SUPERDEX ...

example 2

Identification of Selective Inhibitors of the CAL and CFTR Interaction

[0098]Using peptide-array screening and fluorescence-polarization binding assays, a series of peptide sequences were identified that bind CAL progressively more tightly than CAL binds to CFTR, and that in parallel bind NHERF1 and NHERF2 progressively more weakly than these proteins bind to CFTR.

[0099]To test the ability of CAL inhibitors to rescue CFTR, cultured airway epithelial cells (cell line CFBE410-, derived from a CF patient's Bronchial Epithelium) were grown on filters, permitting formation of polarized cell monolayers similar to those found in epithelial tissues. The CFBE41o- cell line is well-recognized as an airway epithelial model system for the study of CF processes. These cells express the most common disease mutant associated with CF, ΔF508-CFTR, which is characterized by the loss of a single amino acid codon at position 508 of CFTR. Roughly 50% of CF patients are homozygous for ΔF508-CFTR, and anot...

example 3

Assays for Assessing Activity of Selective Inhibitors

[0103]Agents of the present invention can be assayed for their ability to stimulate chloride transport in epithelial tissues. Such transport may result in secretion or absorption of chloride ions. The ability to stimulate chloride transport may be assessed using any of a variety of systems. For example, in vitro assays using a mammalian trachea or a cell line, such as the permanent airway cell line Calu-3 (ATCC Accession Number HTB55) may be employed. Alternatively, the ability to stimulate chloride transport may be evaluated within an in vivo assay employing a mammalian nasal epithelium. In general, the ability to stimulate chloride transport may be assessed by evaluating CFTR-mediated currents across a membrane by employing standard Ussing chamber (see Ussing & Zehrahn (1951) Acta. Physiol. Scand. 23:110-127) or nasal potential difference measurements (see Knowles, et al. (1995) Hum. Gene Therapy 6:445-455). Within such assays, ...

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Abstract

The present invention features compositions and methods for increasing the cell surface expression of degradation-prone CFTR proteins and preventing or treating cystic fibrosis. The invention provides peptides and peptidomimetics that selectively inhibit the interaction between CAL and mutant CFTR proteins, thereby stabilizing the CFTR and facilitating transport of the same to the cell surface.

Description

[0001]This application is a continuation application of U.S. patent application Ser. No. 13 / 292,151 filed Nov. 9, 2011, which is a continuation-in-part application of U.S. patent application Ser. No. 13 / 124,470, filed Apr. 15, 2011, which claims the benefit of priority of PCT / US2009 / 061246, filed Oct. 20, 2009, and U.S. Provisional Application No. 61 / 107,438, filed Oct. 22, 2008, which are incorporated herein by reference in their entireties.[0002]This invention was made with government support under grant numbers R01—DK075309 and R01 GM-78031 awarded by the National Institutes of Health. The government has certain rights in the invention. Work on this invention was also supported by grants from the Cystic Fibrosis Foundation.BACKGROUND OF THE INVENTION[0003]CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) is the target of mutations that cause cystic fibrosis (CF). CF is characterized by abnormal endocrine and exocrine gland function. In CF, unusually thick mucus leads to ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K7/06A61K38/08
CPCA61K38/00A61K38/04A61K38/08C07K5/1013C07K14/4703A61K38/10A61P11/00A61P3/00
Inventor MADDEN, DEAN R.CUSHING, PATRICK R.BOISGUERIN, PRISCAVOLKMER, RUDOLFVOUILLEME, LARSDONALD, BRUCE R.
Owner DUKE UNIV
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