Chemical biology

A Notch above other inhibitors

Article metrics

A tenet of drug discovery states that molecules greater than a certain size don't enter cells. But not only do certain synthetic peptides refute this idea, they also inhibit 'undruggable' biological targets.

A basic limitation of drug development is the inability of traditional 'small-molecule' pharmaceuticals to target large protein interfaces, many of which are desirable drug targets. On page 182 of this issue, Moellering et al.1 report that the solution could be to work with bigger molecules. They describe the design and synthesis of stabilized peptide helices that bind to a protein–protein interface in the Notch signalling pathway — a system that governs fundamental aspects of cell development, proliferation and death. Their remarkable results highlight the potential of molecules that mimic the secondary structures of proteins to target normally intractable protein–protein interactions.

Most drugs are small molecules that target molecular pockets in enzymes or protein receptors. In general, such molecules fail to bind to the large, often flat, interfaces that hold protein partners together — or bind with insufficient specificity and affinity to be useful for pharmaceutical applications2. A broad effort is therefore under way in chemical biology to develop ligand molecules that target protein–protein interfaces. The focus for this is on screening libraries of synthetic compounds in the hope of finding weakly binding ligands that might be good starting points for drug discovery.

A complementary strategy is to rationally design synthetic ligands by adapting nature's protein-recognition principles. This approach is based on the fact that the secondary structures of proteins have vital roles in their interactions with other biomolecules3, so that stable mimics of these structures may provide potential ligands for protein interfaces. Moellering and colleagues' stabilized peptide helices1 offer further proof-of-principle for this approach: they bind to the interface between the gene-regulatory protein (transcription factor) and the multi-protein co-activator that is required for transcription of genes in the Notch signalling pathway.

Inappropriate Notch function has been implicated in several human diseases, including cancers of the lung, ovary, pancreas and — of particular relevance to Moellering and colleagues' study — T-cell acute lymphoblastic leukaemia (T-ALL, a cancer of immature T cells). Unlike typical transcription factors, Notch is a transmembrane receptor that is displayed at cell surfaces, and binds to protein ligands on the surfaces of adjacent cells. Ligand binding to the extracellular domain of Notch triggers intramembrane cleavage of the receptor, mediated by the γ-secretase enzyme. The liberated intracellular domain of Notch (NotchICN) then enters the cell nucleus, where it docks with the DNA-bound transcription factor CSL and helps to bind a co-activator, MAML1 (Fig. 1a). Recruitment of the co-activator and its associated transcriptional machinery initiates the expression of the Notch-targeted genes4.

Figure 1: Inhibition of the Notch signalling pathway.
figure1

Notch proteins are transmembrane receptors found in cell membranes. When Notch's extracellular region binds to a ligand, the intracellular domain (NotchICN) is released, and enters the cell nucleus. a, NotchICN docks with CSL, a DNA-bound transcription factor. The CSL–NotchICN complex is recognized by an α-helical structure in the binding site of MAML1, a co-activator protein. Binding of MAML1 to the CSL–NotchICN complex initiates the expression of Notch-targeted genes. b, Moellering et al.1 prepared peptide segments of the MAML1 binding site, constrained into α-helical conformations by hydrocarbon 'staples'. The stapled helices bind to the CSL–NotchICN complex, preventing MAML1 from binding and so downregulating the expression of Notch-targeted genes.

The binding domain of MAML1 adopts an α-helical conformation to target the protein–protein interface of CSL–NotchICN. Moellering et al. constrained various peptide segments from this binding domain in stable helical conformations by crosslinking side chains of the amino acids that reside on the same face on the helix5,6. The authors found that a 'stapled helix' obtained from the wild-type sequence of the co-activator competitively inhibits MAML1 binding to CSL–NotchICN, and represses Notch-mediated gene expression dose dependently in a panel of T-ALL cell lines (Fig. 1b). More significantly, the stapled helix reduces leukaemic cell proliferation in a mouse model of T-ALL.

Strikingly, Moellering and colleagues' stapled helix bypasses some of the strict limitations that have been placed on drug discovery. When designing potential drug candidates, most medicinal chemists adhere to the Lipinski rules, which stipulate that the molecular mass of a drug should not exceed 500 daltons. However, the stapled peptides1 are several times that size, and yet still traffic to the nucleus and efficiently compete with cellular transcription factors, contrary to the Lipinski rules' predictions. Several arginine amino-acid residues in Moellering and colleagues' helices might facilitate cellular uptake of the peptides, because polyarginines are cell permeable7. But there is accumulating evidence that the hydrocarbon crosslinks in stapled peptides might also favourably influence the cellular uptake of this class of compounds8.

One of the main challenges for drugs that target transcriptional complexes is that they have to disrupt the cooperative assembly of several proteins on DNA. Cells fine-tune the balance of a transcription factor complex's concentration with its affinity for specific DNA sites, coordinating many weak individual interactions to stabilize the optimal assembly of proteins that regulates the expression of target genes. Targeting any single surface within a complex is therefore likely to have a limited effect on the function of the complex as a whole.

Another daunting hurdle is imposed by the ability of several physiologically unrelated transcription factors to interact with a common surface on a co-activator4. A minimally sized peptide that mimics such a surface is therefore likely to perturb the function of many transcription factors. The fact that Moellering and colleagues' stapled helix1 selectively blocks the Notch–CSL complex is particularly intriguing in this context. Its selectivity may derive from the ability of the stapled helix to dock into the Notch–CSL surface groove, thus inhibiting the binding of these transcription factors to MAML1, but not that of other transcription factors that bind to MAML1.

Moellering and colleagues' report1 highlights the potential of synthetic ligands to target gene-specific transcription factors in general. The inappropriate activation of these key regulators of cell fate is linked to the aetiology of numerous genetic diseases, making them attractive drug targets. But although some of the most successful therapeutics act on a handful of transcription factors, including p53 and oestrogen receptors, the majority of transcription factors (more than 2,000 of them) have so far proved to be 'undruggable'9,10.

Nevertheless, tantalizing hints are emerging about how to tackle these recalcitrant proteins. Genetically engineered peptides can associate with targeted transcription factors to block their function, both in vitro and in vivo11. An improved understanding of the recognition principles underlying protein–protein interactions is paving the path towards the rational design of protein–interface mimetics that are highly target-specific, potentially lowering the number of off-target effects in cells2,6. And, as Moellering et al. show, the ability to make relatively large agents that are cell-permeable, such as stapled helices, offers the tangible prospect of targeting the evasive regulators of gene networks that dictate cell fate and function.

References

  1. 1

    Moellering, R. E. et al. Nature 462, 182–188 (2009).

  2. 2

    Wells, J. A. & McClendon, C. L. Nature 450, 1001–1009 (2007).

  3. 3

    Jones, S. & Thornton, J. M. Proc. Natl Acad. Sci. USA 93, 13–20 (1996).

  4. 4

    Mapp, A. K. & Ansari, A. Z. ACS Chem. Biol. 2, 62–75 (2007).

  5. 5

    Schafmeister, C. E., Po, J. & Verdine, G. L. J. Am. Chem. Soc. 122, 5891–5892 (2000).

  6. 6

    Henchey, L. K., Jochim, A. L. & Arora, P. S. Curr. Opin. Chem. Biol. 12, 692–697 (2008).

  7. 7

    Wender, P. A. et al. Proc. Natl Acad. Sci. USA 97, 13003–13008 (2000).

  8. 8

    Walensky, L. D. et al. Science 305, 1466–1470 (2004).

  9. 9

    Darnell, J. E. Jr Nature Rev. Cancer 2, 740–749 (2002).

  10. 10

    Vazquez, A., Bond, E. E., Levine, A. J. & Bond, G. L. Nature Rev. Drug Discov. 7, 979–987 (2008).

  11. 11

    Oh, W. J., Rishi, V., Orosz, A., Gerdes, M. J. & Vinson, C. Cancer Res. 67, 1867–1876 (2007).

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.