David C. Fry

Targeting protein-protein interactions for cancer therapy

An increasing number of protein–protein interactions have been identified as potential intervention points for the development of anticancer agents. However, such systems have historically been considered high-risk targets due to the relatively large interaction surfaces involved in protein–protein binding. This characterization has to be reexamined as progress has been made recently in identifying small-molecule inhibitors of several protein–protein systems in oncology including the p53–MDM2 interaction. This review presents a survey of protein–protein interactions that have been identified as potential oncology targets and evaluates their attractiveness in terms of drug discovery. The analysis focuses primarily on the structural characteristics of the participating binding sites, particularly the dimensions of the sites. Known ligands are also examined, especially with regard to their druglikeness.

A Selective Phosphatase of Regenerating Liver Phosphatase Inhibitor Suppresses Tumor Cell Anchorage-Independent Growth by a Novel Mechanism Involving p130Cas Cleavage

The phosphatase of regenerating liver (PRL) family, a unique class of oncogenic phosphatases, consists of three members: PRL-1, PRL-2, and PRL-3. Aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice. We have now shown that small interfering RNA-mediated depletion of PRL expression in cancer cells results in the down-regulation of p130Cas phosphorylation and expression and prevents tumor cell anchorage-independent growth in soft agar. We have also identified a small molecule, 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (thienopyridone), which potently and selectively inhibits all three PRLs but not other phosphatases in vitro. The thienopyridone showed significant inhibition of tumor cell anchorage-independent growth in soft agar, induction of the p130Cas cleavage, and anoikis, a type of apoptosis that can be induced by anticancer agents via disruption of cell-matrix interaction. Unlike etoposide, thienopyridone-induced p130Cas cleavage and apoptosis were not associated with increased levels of p53 and phospho-p53 (Ser(15)), a hallmark of genotoxic drug-induced p53 pathway activation. This is the first report of a potent selective PRL inhibitor that suppresses tumor cell three-dimensional growth by a novel mechanism involving p130Cas cleavage. This study reveals a new insight into the role of PRL-3 in priming tumor progression and shows that PRL may represent an attractive target for therapeutic intervention in cancer.

NMR characterization of interleukin-2 in complexes with the IL-2Rα receptor component, and with low molecular weight compounds that inhibit the IL-2/IL-Rα interaction

Nuclear magnetic resonance (NMR) methods were employed to study the interaction of the cytokine Interleukin‐2 (IL‐2) with the α‐subunit of its receptor (IL‐2Rα), and to help understand the behavior of small molecule inhibitors of this interaction. Heteronuclear 1H‐15N HSQC experiments were used to identify the interaction surface of 15N‐enriched Interleukin‐2 (15N‐IL‐2) in complex with human IL‐2Rα. In these experiments, chemical shift and line width changes in the heteronuclear single‐quantum coherence (HSQC) spectra upon binding of 15N‐IL‐2 enabled classification of NH atoms as either near to, or far from, the IL‐2Rα interaction surface. These data were complemented by hydrogen/deuterium (H/D) exchange measurements, which illustrated enhanced protection of slowly‐exchanging IL‐2 NH protons near the site of interaction with IL‐2Rα. The interaction surface defined by NMR compared well with the IL‐2Rα binding site identified previously using mutagenesis of human and murine IL‐2. Two low molecular weight inhibitors of the IL‐2/IL‐2Rα interaction were studied: one (a cyclic peptide derivative) was found to mimic a part of the cytokine and bind to IL‐2Rα; the other (an acylphenylalanine derivative) was found to bind to IL‐2. For the interaction between IL‐2 and the acylphenylalanine, chemical shift perturbations of 15N and 15NH backbone resonances were tracked as a function of ligand concentration. The perturbation pattern observed for this complex revealed that the acylphenylalanine is a competitive inhibitor—it binds to the same site on IL‐2 that interacts with IL‐2Rα.

Deconstruction of a Nutlin: Dissecting the Binding Determinants of a Potent Protein–Protein Interaction Inhibitor

Protein-protein interaction (PPI) systems represent a rich potential source of targets for drug discovery, but historically have proven to be difficult, particularly in the lead identification stage. Application of the fragment-based approach may help toward success with this target class. To provide an example toward understanding the potential issues associated with such an application, we have deconstructed one of the best established protein-protein inhibitors, the Nutlin series that inhibits the interaction between MDM2 and p53, into fragments, and surveyed the resulting binding properties using heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR), surface plasmon resonance (SPR), and X-ray crystallography. We report the relative contributions toward binding affinity for each of the key substituents of the Nutlin molecule and show that this series could hypothetically have been discovered via a fragment approach. We find that the smallest fragment of Nutlin that retains binding accesses two subpockets of MDM2 and has a molecular weight at the high end of the range that normally defines fragments.

Design of Libraries Targeting Protein–Protein Interfaces

TARGETING PPIS: A novel strategy for designing libraries targeting protein-protein interfaces enabled us to identify diverse chemical entry points to interact with therapeutic targets for which conventional screening libraries delivered no or only few hit structures. The concept was experimentally validated by early hit evaluation in biochemical screens and early ADMET profiling.

Biochemical analysis of the transducin-phosphodiesterase interaction

In vertebrate rod cells, the activated α-subunit of rod transducin interacts with the γ (regulatory) subunits of phosphodiesterase to disinhibit the catalytic subunits. A 22-amino acid long region of rod transducin involved in phosphodiesterase activation has recently been identified. We have used peptides from this region of rod transducin and from several other G protein α-subunits to study the nature and specificity of the G protein α-effector interaction. Although peptides derived from rod transducin, cone transducin and gustducin are similar, only the rod peptide is capable of activating rod phosphodiesterase. Using substituted peptides we have identified five residues on one exposed face of rod transducin as important to phosphodiesterase activation. These results disagree with previous models which propose that loop regions of rod transducin interact with phosphodiesterase γ

N-Acyl-l-phenylalanine derivatives as potent VLA-4 antagonists that mimic a cyclic peptide conformation

A series of N-benzylpyroglutamyl-L-phenylalanine derivatives bearing carbamoyl substituents in the 3- or 4-positions was prepared and assayed for inhibition of the interaction between VCAM and VLA-4. Potent inhibition was observed in a number of analogues with substitution in the 4-position favored over the 3-position. A crystal structure of the key intermediate 25 indicates that it accesses a low energy conformation which closely matches key pharmacophores of a structurally characterized cyclic peptide.

The design and synthesis of potent cyclic peptide VCAM–VLA-4 antagonists incorporating an achiral Asp-Pro mimetic

The Asp-Pro sequence of the cyclic peptide Ac-HN-Tyr-Cys*-Asp-Pro-Cys*-OH (1) could be replaced with the achiral dipeptide mimetic 1-(2-aminoethyl)cyclpentylcarboxylic acid with retention of potent inhibition of the VCAM-VLA-4 interaction.

Focused Library Approach for Identification of New N-Acylphenylalanines as VCAM/VLA-4 Antagonists

A structure-based focused library approach was employed in an effort to identify more lipophilic replacements for the N-benzylpyroglutamyl group of the VCAM/VLA-4 antagonist 2. This effort led to the discovery of two new classes of potent antagonists characterized by the N-(alpha-phenylcyclopentanoyl- and the N-(2,6-dimethylbenzoyl)-derivatives 60 and 64.

Development of E3-substrate (MDM2-p53)-binding inhibitors: structural aspects.

Inhibition of E3 ligase-substrate binding is the most direct approach for blocking protein ubiquitylation and degradation. However, protein-protein interactions have proven to be difficult targets for discovery of small molecules that bind at the interface and modulate protein activity in a selective manner. Recently, we developed the first potent and selective small-molecule inhibitors of the binding between MDM2 E3 ligase and its substrate p53 (Vassilev et al., 2004). This process was aided significantly by the acquisition and use of structural information. We describe herein how such information was obtained and used at various stages in the program. These applications included assessment of MDM2 as a target, evaluation of hits from high-throughput screening and the selection of lead molecules, and analysis of binding strategies used by the inhibitors as a basis for guiding studies of similar systems. These tools are likely to be useful in any attempt to find and develop druglike compounds that modulate the function of a protein-protein interaction.