PTG-100, an Oral α4β7 Antagonist Peptide: Preclinical Development and Phase 1 and 2a Studies in Ulcerative Colitis.

Background & Aims: Oral therapies targeting the integrin α4β7 may offer unique advantages for the treatment of inflammatory bowel disease. We characterized the oral α4β7 antagonist peptide PTG-100 in preclinical models and established safety, pharmacokinetic/pharmacodynamic relationships, and efficacy in a phase 2a trial in patients with ulcerative colitis (UC).

Methods: In vitro studies measured binding properties of PTG-100.

Single- and Multiple-Dose Pharmacokinetics and Pharmacodynamics of PN-943, a Gastrointestinal-Restricted Oral Peptide Antagonist of α4β7, in Healthy Volunteers.

PN-943 is an orally stable, gastrointestinal-restricted peptide that binds specifically to α4ß7 integrin on leukocytes, blocking leukocyte trafficking to and activation in the gut, inhibiting colon inflammation and reducing signs and symptoms of active ulcerative colitis. Two pharmacokinetic/pharmacodynamic studies were conducted in healthy volunteers. Study 1 was a first-in-human study with 40 male subjects receiving PN-943, 100 to 1400 mg or placebo, as single doses and 57 male subjects receiving PN-943, 100 to 1000 mg or placebo, as multiple doses.

Discovery of a novel class of potent and orally bioavailable sphingosine 1-phosphate receptor 1 antagonists.

A series of subtype selective sphingosine 1-phosphate receptor 1 (S1P(1)) antagonists are disclosed. Our high-throughput screening campaign revealed hit 1 for which an increase in potency and mouse oral exposure was achieved with minor modifications to the chemical scaffold. In vivo efficacy revealed that at high doses compounds 12 and 15 inhibited tumor growth.

Optical coding of mammalian cells using semiconductor quantum dots.

Cell-based assays are widely used to screen compounds and study complex phenotypes. Few methods exist, however, for multiplexing cellular assays or labeling individual cells in a mixed cell population. We developed a generic encoding method for cells that is based on peptide-mediated delivery of quantum dots (QDs) into live cells.

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.

Assay technologies for screening ion channel targets.

The sequence of the human genome will soon provide researchers with hundreds of new ion channel genes. To create a successful ion channel drug discovery program, it is necessary to quickly develop reliable and robust high-throughput screening (HTS) assays for those ion channels implicated in important diseases. Ion channels are dynamic proteins, and therefore require assays that 'sense' their various functional states.

Expression of cre recombinase as a reporter of signal transduction in mammalian cells.

Background: Cell-based reporter assays, which rely on a reporter gene under the control of a regulated promoter, are widely used to screen chemical libraries for novel receptor ligands. Here, we describe a reporter system that is based on ligand-induced DNA recombination to express the reporter gene. This system converts a transient activation of a signal transduction pathway into an amplified, constitutive and heritable expression of the reporter gene.

Genetic recombination as a reporter for screening steroid receptor agonists and antagonists.

Reporter cell lines are often used for high throughput screening of chemical libraries to identify new receptor ligands. Here we show how Cre recombinase can be used in mammalian cells to screen for steroid receptor ligands. A translational fusion of Cre recombinase and the ligand binding domain of the human glucocorticoid receptor was transfected into mammalian cells with a loxP/luciferase reporter gene.

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.

Selection of diphtheria toxin active-site mutants in yeast. Rediscovery of glutamic acid-148 as a key residue.

Saccharomyces cerevisiae was transformed with expression plasmids carrying the DTA gene under control of the GAL1 promoter; colonies that formed under inducing conditions were selected; and plasmids from these colonies were screened for mutations in DTA that failed to block expression of the protein. Substitutions at three sites were identified, all of which are in the active-site cleft; and each of the substitutions reduced ADP-ribosyltransferase activity by > 10(5). The substitutions include a charge reversal mutation of a catalytically important residue (Glu148Lys) and replacements of either of two glycines (Gly22 and Gly52) with bulky residues.

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.

Post-transcriptional regulation of the str operon in Escherichia coli. Ribosomal protein S7 inhibits coupled translation of S7 but not its independent translation.

The str operon of Escherichia coli consists of the genes for ribosomal proteins S12 (rpsL) and S7 (rpsG) and elongation factors G (fusA) and Tu (tufA). Previous studies have shown that S7 is a translational feedback repressor and inhibits the synthesis of itself and of elongation factor G. We have now shown that induction of S7 synthesis from the S7 gene fused to the arabinose promoter on a plasmid also leads to inhibition of the synthesis of S12 from the chromosomal S12 gene, and that this regulation takes place using the same target site as that used for distal gene regulation, i.

Diphthamide synthesis in Saccharomyces cerevisiae: structure of the DPH2 gene.

A gene involved in diphthamide biosynthesis, DPH2, was cloned from Saccharomyces cerevisiae by complementation of a diphthamide mutant. DPH2 exists as a single-copy gene in the yeast genome and is located on the left arm of chromosome XI. Sequence analysis of the DPH2 locus predicts that the DPH2 gene product is a 534-amino acid (aa) protein, with a calculated M(r) of 59,772.

DNA sequence analysis of the YCN2 region of chromosome XI in Saccharomyces cerevisiae.

A 6.8 kbp DNA fragment localized to the left arm of chromosome XI from Saccharomyces cerevisiae was sequenced and analysed (EMBL accession no. X69765).

DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases.

Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B.

Retroregulation of the synthesis of ribosomal proteins L14 and L24 by feedback repressor S8 in Escherichia coli.

Previous studies on regulation of the spc operon containing genes for ribosomal proteins have shown that S8, encoded by the fifth gene of the operon in Escherichia coli, is a translational repressor and regulates the synthesis of the third gene product (L5) and distal gene products by acting at a site near the L5 mRNA translation initiation site. We have now shown that S8 also regulates the synthesis of the first and second gene products (L14 and L24) of the operon by acting at the same mRNA target site--that is, the site located distal to sites coding for L14 and L24--and that mRNA degradation is involved in this retroregulation. It was shown that single base substitutions in the target site, which abolish repression of the synthesis of L5 and L5-distal gene products by S8, also cause derepression of L14-L24 synthesis.

Translational regulation of the spc operon in Escherichia coli. Identification and structural analysis of the target site for S8 repressor protein.

The spc ribosomal protein operon of Escherichia coli is feedback-regulated by ribosomal protein S8, a translational repressor. We have analyzed the region of the spc mRNA that is responsible for this regulation. First, we have established that the S8 target site on the mRNA is near the translation start site of the third gene encoding ribosomal protein L5 in the operon.

Feedback regulation of the spc operon in Escherichia coli: translational coupling and mRNA processing.

The spc operon of Escherichia coli encodes 10 ribosomal proteins in the order L14, L24, L5, S14, S8, L6, L18, S5, L30, and L15. This operon is feedback regulated by S8, which binds near the translation start site of L5 and inhibits translation of L5 directly and that of the distal genes indirectly. We constructed plasmids carrying a major portion of the spc operon genes under lac transcriptional control.