Modulation of protein-protein interactions with small organic molecules


Protein-protein interactions form the basis of most biological processes, but represent very challenging targets for small organic molecules. The overall aim of our research is to devise innovative methods for the efficient design, development, and identification of small organic modulators of protein-protein interactions. Using an interdisciplinary combination of chemical and biological methods, our work has demonstrated that the inhibition of protein-protein interactions actually represents a powerful approach towards expanding the druggable proteome, and towards the selective inhibition of certain protein kinases. We have been able to identify numerous natural products and natural product-derived inhibitors of protein-protein interactions, and have demonstrated that the inhibition of protein–protein interactions is potentially a hitherto unidentified mode of action of existing therapeutic compounds in human use.


Specific research topics include:

1. Small-molecule inhibitors of dimeric transcription factors

Signals originating at the cell surface are conveyed by a complex system of interconnected signaling pathways to the nucleus. They converge at transcription factors, which in turn regulate the transcription of sets of genes which ultimately determine the cellular phenotype. Owing to their lack of enzymatic activities, transcription factors are frequently considered to be “non-druggable”. However, our recent work has demonstrated that dimeric transcription factors can in fact be targeted by inhibitors of protein-protein interactions.

One of our model systems is the oncogenic transcription factor c-Myc. The vast majority of the known biological functions of c-Myc depend on the interaction with its binding partner Max. We have identified three pyrazolo[1,5-a]pyrimidines as the first small molecules which inhibit both dimerization and DNA binding of c-Myc with preference over related transcription factors in vitro (Chem. Biol. 2006; ChemMedChem 2007). Our inhibitors exhibit c-Myc specific effects in various cellular assays. Other model systems include STAT proteins, which belong to a family of latent cytoplasmic transcription factors that transmit signals from the cell membrane to the nucleus. Activities of STAT proteins crucially depend on their functional SH2 domain. Using sensitive assay systems based on fluorescence polarization developed by us (Anal. Biochem. 2004; Anal. Biochem. 2008) we have identified small-molecule inhibitors of the SH2 domains of STAT3 and STAT5 (Chem. Biol. 2006; ChemBioChem 2008). The compounds selectively inhibit the functions of the respective STAT SH2 domain in vitro, and exert potent and selective STAT-dependent effects in mammalian cells. These studies have provided proof of principle that dimerization and DNA binding of transcription factors can be selectively inhibited by cell-permeable, non-peptidic compounds.


2. Targeting protein kinases by the inhibition of protein-protein interactions

The Ser/Thr kinase Plk1 is a key player for multiple stages of mitosis. Most inhibitors of Plk1 target the conserved ATP-binding site. In collaboration with the group of Prof. Klaus Strebhardt at the University of Frankfurt, we have provided proof-of-principle that Plk1 can alternatively be targeted by small-molecules which inhibit the protein-protein interactions required for correct intracellular localization of Plk1. By screening of chemical libraries in a high-throughput assay developed by us (Anal. Biochem. 2008), we have identified the natural product Thymoquinone and a synthetic derivative, dubbed “Poloxin”, as the first known inhibitors of the polo-box domain (PBD), a recently discovered protein domain which mediates intracellular localization of Plk1. Both compounds inhibit the function of the Plk1 PBD in vitro, and cause Plk1 mislocalization, chromosome congression defects, mitotic arrest, and apoptosis in HeLa cells (Chem. Biol. 2008). Subsequently, we could demonstrate that the pan-selective inhibition of the PBDs of Plk1, Plk2, and Plk3 by the small molecule Poloxipan causes the same mitotic phenotype as the selective inhibition of the Plk1 PBD by Poloxin (ChemBioChem 2009).


3. Mode of action studies of natural products and known bioactive compounds

Stimulated by our discoveries of small molecules which inhibit protein-protein interactions and transcription factors, we hypothesized that known bioactive molecules, including natural products, could possess similar activities. In the course of these studies, we have identified fosfosal and dexamethasone-21-phosphate, two phosphate prodrugs in human use as anti-inflammatory agents, as inhibitors of the SH2 domain of the transcription factor STAT5b and the substrate-binding domain of the peptidyl-prolyl isomerase Pin1, respectively (ACS Chem. Biol. 2011). In addition, we were able to identify the pentacyclic triterpenoid betulinic acid as a pan-specific inhibitor of the C/EBP transcription factor family, proteins which play prominent roles in adipogenesis and tumorigenesis (ChemBioChem 2012). Taken together, these studies have revealed hitherto unknown off-target effects of known biologically active substances, and have thereby led to a more complete understanding of their cellular activities.

Most recently, we have demonstrated that chlorhexidine and alexidine, the active ingredients of oral infectants in daily human use, inhibit cancer-relevant protein-protein interactions mediated by the anti-apoptotic Bcl-2 family protein Bcl-xL (Angew. Chem. Int. Ed. 2013). The compounds induce apoptosis in human tumor cell lines derived from tissue exposed during the process of oral disinfection at physiologically-relevant concentrations. We have also presented the first synthesis of both enantiomers of the chiral diastereoisomer of alexidine in high enantiomeric and diastereomeric purities (Bioorg. Med. Chem. 2013).