In our November 27th blog post, we discussed an innovative new technology, stapled peptides, for use in targeting intracellular protein-protein interactions. In the example we gave, the target was a transcription factor complex in the Notch pathway. As we stated, protein-protein interactions are deemed to be “undruggable”, since they cannot be readily addressed with small molecule drugs.

Nevertheless, in some cases, small molecules have been discovered that do address key protein-protein interactions, and which may become clinical candidates.

Back in February 2006, Decision Resources published our report, “Protein-Protein Interactions: Are They Now Druggable Targets?” Among the case studies we discussed in that report was one in which researchers were attempting to discover small-molecule agents that targeted the Wnt pathway. The researchers discovered small-molecule agents that, as with the stapled-peptide example we discussed in our previous blog post, targeted a transcription factor complex. As of late 2009, two of these compounds are in preclinical development for treatment of various cancers.

Mutations that mediate deregulation of the Wnt pathway are causative factors in several types of cancer, most notably colorectal cancer, as well as multiple myeloma (MM), hepatocellular carcinoma (HCC), and B-cell chronic lymphocytic leukemia B-CLL). In the canonical Wnt pathway, soluble extracellular factors that are members of the Wnt family activate the pathway. A complex that includes the protein adenomatous polyplosis coli (APC) is central to the Wnt pathway. When Wnt receptors are not engaged by their ligands, kinases in the APC complex phosphorylate β-catenin, a multifunctional protein that is involved both in signal transduction and in adhesion between cells. Phosphorylation targets β-catenin for degradation.

When Wnt proteins bind to their receptors, the kinase activity of the APC complex is inactivated. This results in the accumulation of β-catenin, which moves into the nucleus. There it binds to proteins of the T cell factor (Tcf) family. β-catenin binding changes Tcf from a transcriptional repressor into a transcriptional activator. Downstream genes controlled by the β-catenin/Tcf complex include the oncogene Myc and other genes that mediate cell proliferation.

In precancerous colonic adenomas or the colorectal cancers that they may evolve into, APC is usually mutated. This results in constitutive stabilization of β-catenin and constitutive activation of Tcf and its downstream genes. In other types of cancer that involve constitutive Wnt pathway activation, β-catenin also becomes stabilized, via other means. This makes the Tcf/β-catenin a tempting target for drug discovery. However, it is a protein-protein interaction, and is thus deemed “undruggable”.

In 2004, A group led by Ramesh Shivdasani (Harvard Medical School, Dana-Farber Cancer Institute, and Brigham and Women’s Hospital, Boston MA), including researchers from the Novartis Institutes for BioMedical Research (Cambridge, MA), discovered several small-molecule inhibitors of the interaction between human Tcf4 and human β-catenin.

Dr. Shivdasani’s group, among others, had previously determined crystal structures of Tcf-β-catenin complexes. The interaction between the two proteins occurs over a large surface area. It is the large, and usually hydrophobic, interface between proteins in protein-protein interactions that forms the theoretical basis for the difficulty of addressing these interactions with small molecules. However, there is a small hydrophobic pocket that is critical for binding (as also confirmed by site-specific mutation studies), which might accommodate a small molecule inhibitor.

Therefore, the researchers screened approximately 7,000 purified natural products from public and proprietary libraries using an enzyme-linked immunosorbent (ELISA) assay involving release of a labeled Tcf4 binding fragment from its complex with a β-catenin fragment absorbed to an ELISA plate. Eight compounds were found that gave reproducible, concentration-dependent release of the Tcf4 fragment at less than 10 micromolar concentration. The structures and purity of these compounds (most of which are complex, multi-ringed planar compounds with multiple hydroxy groups) were then determined. The sources of these compounds include fungi, actinomycetes, and a marine sponge.

The researchers performed several additional biochemical assays to confirm the compounds’ specific disruption of the Tcf/β-catenin complex, and also performed cellular assays and an in vivo assay in the Xenopus (frog) embryo to study the activities of these compounds against β-catenin-mediated cellular effects. Each of the eight compounds shows different levels of potency in the different assays used in this study, and the compounds differ from each other in their activities in the different assays.

Two fungal-derived compounds, PKF115-854 and CGP04909, gave the best results in all the assays. It is those compounds that have been tested in preclinical studies as potential oncology drug candidates. In a study published in PNAS in 2007, researchers at the Dana-Farber and at Brigham and Women’s Hospital tested PKF115-584 in human MM cells in vitro and in xenograft models. The compound blocked expression of Wnt target genes, induced cytotoxicity in MM cells in vitro, and inhibited tumor growth and prolonged survival in the xenograft model. In a study in HCC at the Asian Liver Center at Stanford University School of Medicine, PKF115-584, CGP049090, and another of the Shivdasani group’s compounds, PKF118-310, also induced cytotoxicity in human HCC cell lines in vitro, and suppressed tumor growth and induced apoptosis in tumor cells in a human HCC xenograft model. Finally, in an abstract presented at the American Society of Hematology (ASH) meeting in December 2009, researchers at the Novartis Institute for Biomedical Research in Basel and their academic collaborators presented data that showed that CGP04090 and PKF115-584 potently inhibited the survival of primary human B-CLL cells in vitro and in vivo. In all three cases, the compounds showed no significant cytotoxicty against normal cells.

In the conclusion of the ASH meeting abstract, the authors stated that further investigations are warranted to determine the feasibility of testing these compounds in human clinical trials.

Many medicinal chemists remain skeptical about the ability of researchers to develop small-molecule drugs that target protein-protein interactions, which have satisfactory pharmacokinetics and can advance through clinical trials and reach the market. However, at least one nonpeptide small-molecule compound that targets a protein-protein interaction, the thrombopoietin receptor agonist eltrombopag (Ligand/GSK’s Promacta), has reached the market. (The FDA approved it in November 2008.) Several other small-molecule drugs that target protein-protein interactions are in clinical development. And Cambridge Healthtech Institute will be sponsoring a conference on this subject, which is scheduled for April 2010. This conference is in its third year. Thus, as also shown by the development of stapled peptides, there is renewed interest in discovering and developing drugs that address these “hard targets”.

In the 12 November issue of Nature, there was a research article and a News and Views minireview about targeting an intracellular signaling pathway with a novel type of compound called a stapled peptide.

Signaling pathways are crucial for cellular physiology, and in the pathobiology of important diseases ranging from metabolic diseases to cancer. In many cases, signaling proteins that work by binding to other proteins in protein-protein interactions are key control points in signaling pathways. However, protein-protein interactions in all but a few cases cannot be readily addressed with small molecule drugs. These targets are therefore called “undruggable”. Some signaling pathways consist entirely of these “undruggable” targets, and can only be addressed indirectly (if at all) via targeting other pathways that interact with them.

Several small-molecule drugs that do address protein-protein interactions are natural products. The best known of these is the immunosuppressant FK506 (tacrolimus, Astellas’ Prograf). This is one reason for the new interest in natural products by some companies and researchers, as we discussed in a previous blog post.

However, the 12 November Nature article, authored by James E Bradner (Chemical Biology Program, Broad Institute of Harvard and MIT, Cambridge, MA, and the Dana-Farber Cancer Institute, Boston, MA), Gregory Verdine (Department of Chemical Biology, Harvard University, Cambridge MA, and the Dana-Farber Cancer Institute), and their colleagues, takes a different approach. The researchers target specific intracellular protein-protein interactions by designing special types of peptides known as stapled peptides.

The signaling pathway that is the focus of this article is the Notch pathway. In normal physiology, this pathway regulates various aspects of cell-cell communication, cellular differentiation, cell proliferation, and cellular survival or death. Deregulated Notch pathway function is involved in diseases including cancers of the lung, ovary and pancreas, and in T-cell acute lymphoblastic leukemia (T-ALL), which is a cancer of immature T cells.

Notch is a cell-membrane receptor. Binding of one of its ligands (on the surface of an adjacent cell) to the extracellular domain of Notch triggers sequential cleavage of the Notch intracellular domain by a metalloproteinase known as TACE (tumor necrosis factor alpha converting enzyme) and by γ-secretase (an enzyme which is also involved in the amyloid pathway that is implicated in Alzheimer’s disease). The free intracellular domain of Notch, called ICN, migrates to the nucleus, and docks with the DNA-bound transcription factor CSL. The interaction between CSL and ICN creates a groove along the interface of the two proteins, which serves as a docking site for the mastermind-like protein MAML1. The resulting trimolecular complex initiates specific transcription of Notch-dependent target genes.

The binding domain of MAML1 that engages the elongated groove formed by the ICN-CSL complex is in the form of an α-helix. The researchers therefore designed a series of peptides derived from portions of the sequence of the MAML1 binding domain. These were stapled peptides in which hydrocarbon moieties are used to constrain, or “staple”, MAML1 binding-domain mimetic sequences into an α-helical conformation. One such stapled peptide, SAMH1, gave the highest affinity binding to ICN and CSL, and competitively inhibited binding of wild-type MAML1 to these proteins.

SAMH1 was cell-penetrant, and inhibited intracellular Notch pathway signaling in cultured T-ALL cell lines. Moreover, SAMH1 reduced the proliferation of a variety of T-ALL cell lines in vitro, but was inactive against T-cell tumor lines that were not dependent on the Notch pathway for their proliferation. In SAMH1-sensitive T-cell tumor lines, SAMH1 treatment activated caspases, which are involved in apoptosis. In a mouse model of T-ALL, intraperitoneally injected SAMH1 inhibited leukemic progression, and inhibited Notch pathway signaling in leukemic cells in vivo.

Stapled peptides are not conventional “drug-like” compounds. Their molecular weights are several times greater than the 500-dalton maximum prescribed by Lipinski’s rules (developed by the leading medicinal chemist Chris Lipinski), which are used to define “drug-like” properties of small molecule compounds. Moreover, peptides are usually subject to protease degradation in vivo, and thus have short serum half-lives. In most cases, peptides do not enter into cells efficiently, except for those peptides that have specific cell-membrane receptors.

However, stapled α-helical peptides, in addition to their improved binding activities to their specific targets, are protease-resistant, have improved serum half-lives, and are cell penetrant. Researchers attribute these properties to the constrained conformation of these molecules, and to the hydrocarbon staples themselves. For example, the hydrocarbon staples may confer lipophilic properties to these molecules, and thus render them membrane-penetrant.

In an earlier study, Dr. Verdine and researchers at the Dana-Farber Cancer Institute and Children’s Hospital in Boston designed a stapled α-helical peptide that initiated apoptosis by specifically binding to and activating a member of the Bcl-2 family, and that inhibited the grown of leukemic cells in a mouse model. The researchers have been continuing to develop and to determine the mechanisms of action of their Bcl-2 family-targeting stapled peptides.

The discovery-stage biotechnology company Aileron Therapeutics was founded in 2005 to develop and commercialize stapled peptides. The company’s scientific founders include Dr. Verdine, Loren Walensky (Dana-Farber Cancer Institute), and the late Stanley J. Korsmeyer (Dana-Farber Cancer Institute, a pioneer in the study of the Bcl-2 family and its role in apoptosis and in the biology of cancer). It has a pipeline of stapled peptides that it is developing for the treatment of solid and hematological tumors, the most advanced of which are in the preclinical stage. Aileron has managed to attract venture capital despite the current adverse conditions–in June 2009, the company closed a $40 million Series D financing.

Stapled peptides represent an exciting and innovative technology with the potential to address “undruggable” protein-protein interactions, and thus to treat diseases that represent major unmet medical needs. However, this technology is in an early stage, and the therapeutic value of stapled peptides has not yet been confirmed in the clinic.