A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy.

Interleukin (IL)-7 is broadly active on T-cell populations, and modified versions have been clinically evaluated for a variety of therapeutic applications, including cancer, lymphopenia, and infectious diseases; and found to be relatively well-tolerated and biologically active. Here we describe novel IL-7R agonists that are unrelated in structure to IL-7, bind to the receptor subunits differently from IL-7, but closely emulate IL-7 biology. The small size, low structural complexity, and the natural amino acid composition of the pharmacologically active peptide MDK1472 allows facile incorporation into protein structures, such as the IgG2-Fc fusion MDK-703.

Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human.

While P-glycoprotein (PGP, ABCB1) is known to play an important role in drug exclusion at the blood brain barrier (BBB), less is known about the contribution of other members in the ATP-binding cassette (ABC) transporter family to BBB drug efflux, or whether these transporters are expressed differently in humans and in mammalian species of pharmacological interest. We used quantitative real-time PCR to determine mRNA expression levels for the majority of ABC family members in brain and in isolated brain microvessel endothelial capillary cells (BMEC) from human, rat, mouse, pig and cow. We confirmed BBB expression of several well-characterized ABC family members that are implicated in xenobiotic exclusion from the brain, including ABCB1 (PGP), ABCG2 (BCRP), ABCC1 (MRP1), ABCC4 (MRP4), and ABCC5 (MRP5).

Synthetic compound libraries displayed on the surface of encoded bacteriophage.

We describe a technology for attaching libraries of synthetic compounds to coat proteins of bacteriophage particles such that the identity of the chemical structure is encoded in the genome of the phage, analogous to peptides displayed on phage surfaces by conventional phage-display techniques. This format allows a library of synthetic compounds to be screened very efficiently as a single pool. Encoded phage serve as extremely robust reporters of the presence of each compound, providing exquisite sensitivity for identification of active compounds engaged in complex biological processes such as receptor-mediated endocytosis and transcytosis.

In vitro selection as a powerful tool for the applied evolution of proteins and peptides.

New in vitro methods for the applied evolution of protein structure and function complement conventional cellular and phage-based methods. Strategies employing the direct physical linkage of genotype and phenotype, and the compartmental association of gene and product to select desired properties are discussed, and recent useful applications are described. Engineering of antibodies and other proteins, selection from cDNA libraries, and the creation of functional protein domains from completely random starting sequences illustrate the value of the in vitro approaches.

Peptide agonists of the thrombopoietin receptor.

We have screened a variety of L-amino acid peptide libraries against the extracellular domain of the human thrombopoietin (HuTPO) receptor, c-Mpl. A large number of peptide ligands were recovered and categorized into two families. Peptides from each family compete with the binding of HuTPO and with the binding of peptides from the other familiy.

Targeting growth factor and cytokine receptors with recombinant peptide libraries.

The single-transmembrane-spanning receptors of cytokines and growth factors have historically proven resistant to the small-molecule screening efforts of the pharmaceutical industry. Advances in combinatorial library approaches to ligand discovery have begun to show success with these targets. There are several recent reports of peptides, derived from randomly assembled collections of L-peptides expressed in recombinant display vectors, that are high-affinity antagonists and even agonists of these receptors.

Identification of a 13 amino acid peptide mimetic of erythropoietin and description of amino acids critical for the mimetic activity of EMP1.

To obtain information about the functional importance of amino acids required for effective erythropoietin (EPO) mimetic action, the conserved residues of a peptide mimetic of EPO, recently discovered by phage display, were subjected to an alanine replacement strategy. Further, to identify a minimal mimetic peptide sequence, a series of truncation peptides has been generated. One EPO mimetic peptide sequence, EMP1, was targeted and more than 25 derivatives of this sequence were evaluated for their ability to compete with [125I]EPO for receptor binding and for their ability to support the proliferation of two EPO-responsive cell lines.

Increased potency of an erythropoietin peptide mimetic through covalent dimerization.

We have synthesized a chemically defined, dimeric form of an erythropoietin mimetic peptide (EMP) that displays 100-fold increased affinity for the erythropoietin receptor (EPOR) and correspondingly elevated potency in cell-based assays and in mice. The dimeric EMP1 was synthesized using a C-terminal lysine residue as a branch point. A beta-alanine residue was coupled to the main-chain (alpha) amino group of the lysine residue in order to provide a pseudosymmetrical scaffold where both the side-chain and main-chain were of approximately equal length.

Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine.

Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar.

Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A.

The functional mimicry of a protein by an unrelated small molecule has been a formidable challenge. Now, however, the biological activity of a 166-residue hematopoietic growth hormone, erythropoietin (EPO), with its class 1 cytokine receptor has been mimicked by a 20-residue cyclic peptide unrelated in sequence to the natural ligand. The crystal structure at 2.

Small peptides as potent mimetics of the protein hormone erythropoietin.

Random phage display peptide libraries and affinity selective methods were used to isolate small peptides that bind to and activate the receptor for the cytokine erythropoietin (EPO). In a panel of in vitro biological assays, the peptides act as full agonists and they can also stimulate erythropoiesis in mice. These agonists are represented by a 14- amino acid disulfide-bonded, cyclic peptide with the minimum consensus sequence YXCXXGPXTWXCXP, where X represents positions allowing occupation by several amino acids.

High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries.

Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM).

Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries.

Ideal gene therapy vectors would be delivered intravenously to transfect only specific cells. Existing vectors only transfect cells in vivo in a manner determined by blood flow and the site of introduction. As a general and systematic approach for generating cell-targeting ligands for gene therapy vectors, we have used peptide-presenting phage libraries to select peptides that bind and enter several different cell types.

A generic method for expression and use of "tagged" soluble versions of cell surface receptors.

A general method for expression, purification, immobilization, detection and radiolabeling of extracellular domains (ECD) of type I membrane proteins. The type I interleukin-1 receptor (IL-1RtI), the alpha-subunit of interleukin-2 receptor (IL-2R alpha) and E-selectin are used as illustrative examples of cell surface receptors. DNA encoding the ECD of the proteins are fused at their 3' end to a chimeric DNA which serves to generically "tag" the recombinant ECD.

An in vitro polysome display system for identifying ligands from very large peptide libraries.

We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 10(12) random decapeptides was incubated in an Escherichia coli S30 coupled transcription/translation system. Polysomes were isolated and screened by affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B.

Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressors.

The filamentous phage coat protein pIII has been used to display a variety of peptides and proteins to allow easy screening for desirable binding properties. We have examined the biological constraints that restrict the expression of short peptides located in the early mature region of pIII, adjacent to the signal sequence cleavage site. Many functionally defective pIII fusion proteins contained several positively charged amino acids in this region.

Synthetic peptide ligands of the antigen binding receptor induce programmed cell death in a human B-cell lymphoma.

Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM.

Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP.

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.

Generation and screening of an oligonucleotide-encoded synthetic peptide library.

We have prepared a library of approximately 10(6) different peptide sequences on small, spherical (10-microns diameter) beads by the combinatorial chemical coupling of both L- and D-amino acid building blocks. To each bead is covalently attached many copies of a single peptide sequence and, additionally, copies of a unique single-stranded oligonucleotide that codes for that peptide sequence. The oligonucleotide tags are synthesized through a parallel combinatorial procedure that effectively records the process by which the encoded peptide sequence is assembled.