The Scripps Research Institute

Gryphon Therapeutics, Inc.

Other

Amylin Pharmaceuticals, Inc.

Gryphon Sciences

The Rockefeller University

University of Toronto

Reflexion Pharmaceuticals, Inc.

Stephen B H Kent

Paolo Botti

Tom W Muir

Philip E Dawson

Christie L Hunter

Gerd G Kochendoerfer

Brian T Chait

Reyna J Simon

Ronald Beavis

Rong Wang

James A Bradburne

Lynne Canne

Martina Schnolzer-Rackwitz

Shiah-yun Chen

Sonya Cressman

Donald W Low

Sachdev Singh Sidhu

Lynne Canne Bannen

Michael A Siani

Steven J Bark

Maruti Uppalapati

Jill Wilken

Dana Ault-Riche

Youhei Soma

Jill G Wilken

Leslie Philip Miranda

Michael C Fitzgerald

Reyna Simon

Gerd Kochendoerfer

Methods and compositions for identifying D-peptidic compounds that specifically bind target proteins are provided. Aspects of the methods include screening libraries of 20 residue or more L-peptidic compounds for specific binding to 40 residue or more D-target proteins. Once a L-peptidic compound has been identified that specifically binds to the D-target protein, the D-enantiomer of that compound may be produced.

Methods and compositions for identifying D-peptidic compounds that specifically bind target proteins

Compositions and methods related to ester insulin or derivatives thereof are provided. The compositions include Glu-Threster insulin, in which side chains of Gluand Throf native human insulin or an insulin analogue such as insulin lispro are covalently linked via a single ester bond. The ester insulin is efficiently folded, forming the desired disulfides. The Thr-Gluester bond can be cleaved in vitro or in vivo to give the desired folded insulin with full biological activity. The ester insulin is readily prepared by total chemical synthesis, and amendable to cost-effective, large scale manufacturing. Also provided are pharmaceutical compositions and kits for use in practicing the subject methods. Also provided are methods of using the subject compositions and kits in the treatment of a variety of different disease conditions, particularly glucose metabolic disorders such as diabetes and obesity.

Ester insulin

Water-soluble thioester and selenoester compounds, their generators, as well as methods for making and using the same, are provided. The subject thioester and selenoester compounds are characterized by including an amino acid synthon having a C-terminal group bonded to a water-soluble polymer through a thioester or selenoester linkage. Solid phase resins and protocols for generating the subject compounds are also provided. The subject water soluble thioester and selenoester compounds and generators find use in a variety of different applications, including thioester or selenoester mediated chemical ligation reactions.

Water-soluble thioester and selenoester compounds and methods for making and using the same

The present invention relates to methods and compositions for lipid matrix-assisted chemical ligation and synthesis of membrane polypeptides that are incorporated in a lipid matrix. The invention is exemplified in production of a prefolded membrane polypeptide embedded within a lipid matrix via stepwise chemoselective chemical ligation of unprotected peptide segments, where at least one peptide segment is embedded in a lipid matrix. Any chemoselective reaction chemistry amenable for ligation of unprotected peptide segments can be employed. Suitable lipid matrices include liposomes, micelles, cell membrane patches and optically isotropic cubic lipidic phase matrices. Prefolded synthetic and semi-synthetic membrane polypeptides synthesized according to the methods and compositions of the invention also permit site-specific incorporation of one or more detectable moieties, such as a chromophore, which can be conveniently introduced during synthesis. The methods and compositions of the invention have multiple uses. For example, they can be used to assay ligand binding to membrane polypeptides and domains comprising a receptor, and thus are extremely useful for structure/function studies, drug screening/selection/design, and diagnostics and the like, including high-throughput applications. The methods and compositions of the invention are particularly suited for FRET analyses of previously inaccessible membrane polypeptides.

Compositions for lipid matrix-assisted chemical ligation

Proteins of moderate size having native peptide backbones are produced by a method of native chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules. The technique of native chemical ligation is employable for chemically synthesizing full length proteins.

Synthesis of proteins by native chemical ligation

This invention provides for novel methods of identifying covalent modifications of an amino acid residue in a polypeptide chain by detecting mass differences.

Method for detecting post-translation modifications of peptides

The present invention relates to methods and compositions for modifying peptides, polypeptides and proteins with polymers, especially glyco-mimetic polymers, so as to improve their biological activity or pharmacokinetic properties. The invention further provides methods and uses for such polymer-modified peptides, polypeptides and proteins. The invention is particularly suitable for use in the synthesis of polymer-modified synthetic bioactive proteins (FIG.D), and of pharmaceutical compositions that contain such proteins.

Polymer-modified synthetic proteins

The present invention provides methods, apparatus and kits for synthesizing assembled peptides and proteins on a solid phase with sequential ligation of three or more unprotected peptide segments using chemoselective and mild ligation chemistries in aqueous solution. Also provided are methods of monitoring solid phase sequential ligation reactions using MALDI or electrospray ionization mass spectrometry of reaction products.

Solid phase native chemical ligation of unprotected or N-terminal cysteine protected peptides in aqueous solution

The present invention concerns methods and compositions for extending the technique of native chemical ligation of a wider range of peptides, polypeptides, other polymers and other molecules via an amide bond (see FIG.). The invention further provides methods and uses for such proteins and derivatized proteins. The invention is particularly suitable for use in the synthesis of optionally polymer-modified, synthetic bioactive proteins, and of pharmaceutical compositions that contain such proteins.

Pseudo native chemical ligation

The invention is directed to nucleophile-stable thioester generating compounds comprising a carboxyester thiol. The compounds have wide applicability in organic synthesis, including the generation of peptide-, polypeptide- and other polymer-thioesters. The invention is particularly useful for generating activated-thioesters from precursors that are made under conditions in which strong nucleophiles are employed, such as peptides or polypeptides made using Fmoc SPPS, as well as multi-step ligation or conjugation schemes that require (or benefit from the use of) compatible selective approaches for directing a specific ligation or conjugation reaction of interest.

Nucleophile-stable thioester generating compounds, methods of production and use

Novel proteins and protein libraries are disclosed. The proteins possess one or more functional protein modules from different parent protein molecules. The proteins and protein libraries are exemplified by the preparation of cross-over chemokines that contain various combinations of peptide segments derived from RANTES, SDS-1 and vMIP-I and to vMIP-II. The proteins and libraries are extremely pure and can be provided in non-limiting high yields suitable for diagnostic and high-throughput screening assays.

Modular protein libraries and methods of preparation

Method is described for sequencing polypeptides by forming peptide ladders comprising a series of polypeptides in which adjacent members of the series vary by one amino acid residue and determining the identity and position of each amino acid in the polypeptide by mass spectroscopy.

Method and product for the sequence determination of peptides using a mass spectrometer

Modified proteins are represented by the formula R-L-R&prime;, wherein R and R&prime; are peptides or pseudopeptides linked to one another by linkage L. Linkage L may be either a thiol ester pseudopeptide backbone linkage or a selenol ester pseudopeptide backbone linkage.

Process for preparing modified proteins

A protein signature analysis is obtained using a peptide ladder library. The molecular signature of a protein is defined to be that subsequence of amino acid positions within the protein which are essential for the protein to bind to a target molecule. The molecular signature may be determined by screening a peptide ladder library which corresponds to the protein against the target molecule. The peptide ladder library is a library of m peptides wherein each peptide has an amino acid sequence of length m corresponding to an amino acid sequence of the protein, with one exception, viz. peptide has a substitute amino acid at position and the substitute amino acid is attached by a labile bond to its neighboring amino acid. Screening the peptide ladder library against the target molecule results in a division of the original mixture into a positive (functional) pool and a negative (non-functional) pool. The pools are separated and subjected to cleavage to obtain cleavage products. Analysis of cleavage products by mass spectrometry identifies the positions that are essential for the protein to bind to its molecular target.

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San Francisco, CA, United States of America

Stephen B H Kent