Design and synthesis of 5-aryl-pyridone-carboxamides as inhibitors of anaplastic lymphoma kinase.

Anaplastic lymphoma kinase (ALK) is a promising new target for therapy of certain cancers such as anaplastic large-cell lymphoma (ALCL) and inflammatory myofibroblastic tumor (IMT). We have identified a series of novel pyridones as kinase inhibitors of ALK by application of a stepwise process involving in vitro screening of a novel targeted library followed by iterative template modification based on medicinal chemistry insights and computational ranking of virtual libraries. Using this process, we discovered ALK-selective inhibitors with improved potency and selectivity. Herein the details of the design process and synthesis of these novel pyridones, along with their enzymatic and cell-based activity, are discussed.

Structural biology and structure-based inhibitor design of cholera toxin and heat-labile enterotoxin.

Structural biology studies on cholera toxin and the closely related heat-labile enterotoxin from enterotoxigenic Escherichia coli over the past decade have shed light on the mechanism of toxin action at molecular and atomic levels. Also, components of the extracellular protein secretion apparatus that translocate the toxins across the outer membrane are being investigated. At the same time, structure-based design has led to various classes of compounds targeting different toxin sites, including highly potent multivalent inhibitors that block the toxin receptor-binding process.

Nonspanning bivalent ligands as improved surface receptor binding inhibitors of the cholera toxin B pentamer.

A series of bivalent ligands of varying length were synthesized to inhibit the receptor-binding process of cholera toxin. Competitive surface receptor binding assays showed that significant potency gains relative to the constituent monovalent ligands were achieved independently from the ability of the extended bivalent ligands to span binding sites within the toxin pentamer. Several models that could account for the unexpected improvement in IC(50) values are examined, taking into account crystallographic analysis of each ligand in complex with the toxin pentamer. Evidence is presented that steric blocking at the receptor binding surface may play a role. The results of our study suggest that the use of relatively short, "nonspanning" bivalent ligands, or monovalent ligands of similar topology and bulk may be an effective way of blocking the interaction of multimeric proteins with their cell surface receptors.

Solution- and solid-phase syntheses of guanidine-bridged, water-soluble linkers for multivalent ligand design.

[reaction: see text] Efficient syntheses of guanidine-bridged poly(ethylene glycol) linkers of various lengths in fully protected form are reported for both solution- and solid-phase protocols. The application of such linkers in the construction of water-soluble and high-affinity multivalent ligands against cholera toxin is demonstrated. Synthetic intermediates for multivalent ligands as large as 20 kDa in molecular weight have been assembled using presynthesized linkers. The final ligands are highly water-soluble, thus enabling proper biophysical characterization.

3,5-Substituted phenyl galactosides as leads in designing effective cholera toxin antagonists; synthesis and crystallographic studies.

With the aim of developing high-affinity mono and multivalent antagonists of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) we are using the galactose portion of the natural receptor ganglioside GM1 as an anchoring fragment in structure-based inhibitor design efforts. In order to establish a better structure-activity relationship for guiding these studies, we designed and prepared a small focused library of twenty 3,5-substituted phenylgalactosides based on two previous leads. The compounds were tested for their ability to block CTB(5) binding to immobilized ganglioside receptor and compared to the two previous leads. The crystal structures of the most promising compounds bound to either CTB(5) or LTB(5) were then determined in order to understand the basis for affinity differences. The most potent new compound yielded a six-fold improvement over our benchmark lead m-nitrophenyl-alpha-d-galactopyranoside (MNPG), and a two-fold improvement in IC(50) over a newer MNPG derivative. These results support the notion that the m-nitrophenyl moiety of MNPG and its derivatives is an important element to retain in future optimization efforts. Additionally, a consensus binding-pocket for the alkylmorpholine or piperazine moiety present in all of the designed antagonists was established as an important area of the GM1 binding site to target in future work.

Characterization and crystal structure of a high-affinity pentavalent receptor-binding inhibitor for cholera toxin and E. coli heat-labile enterotoxin.

Multivalent ligand design constitutes an attractive avenue to the inhibition of receptor recognition and other biological events mediated by oligomeric proteins with multiple binding sites. One example is the design of multivalent receptor blockers targeting members of the AB(5) bacterial toxin family. We report here the synthesis and characterization of a pentavalent inhibitor for cholera toxin and Escherichia coli heat-labile enterotoxin. This inhibitor is an advance over the symmetric pentacyclen-derived inhibitor described in our earlier work in that it presents five copies of m-nitrophenyl-alpha-D-galactoside (MNPG) rather than five copies of beta-D-galactose. The approximately 100-fold higher single-site affinity of MNPG for the toxin receptor binding site relative to galactose is found to yield a proportionate increase in the affinity and IC50 measured for the respective pentavalent constructs. We show by dynamic light scattering that inhibition of receptor binding by the pentavalent inhibitor is due to 1:1 inhibitor:toxin association rather than to inhibitor-mediated aggregation. This 1:1 association is in complete agreement with a 1.46 A resolution crystal structure of the pentavalent inhibitor:toxin complex, which shows that the favorable single-site binding interactions of MNPG are retained by the five arms of the 5256 Da pentavalent MNPG-based inhibitor and that the initial segment of the linking groups interacts with the surface of the toxin B pentamer.

Anchor-based design of improved cholera toxin and E. coli heat-labile enterotoxin receptor binding antagonists that display multiple binding modes.

The action of cholera toxin and E. coli heat-labile enterotoxin can be inhibited by blocking their binding to the cell-surface receptor GM1. We have used anchor-based design to create 15 receptor binding inhibitors that contain the previously characterized inhibitor MNPG as a substructure. In ELISA assays, all 15 compounds exhibited increased potency relative to MNPG. Binding affinities for two compounds, each containing a morpholine ring linked to MNPG via a hydrophobic tail, were characterized by pulsed ultrafiltration (PUF) and isothermal titration calorimetry (ITC). Crystal structures for these compounds bound to toxin B pentamer revealed a conserved binding mode for the MNPG moiety, with multiple binding modes adopted by the attached morpholine derivatives. The observed binding interactions can be exploited in the design of improved toxin binding inhibitors.