Inhibitors of proteases and related enzymes have versatile applications in medicine and other areas. They are used in the clinic e.g. for the treatment of cancer, hypertension, thrombosis, diabetes as well as viral and bacterial infections. Most of these drugs are produced synthetically but a substantial part of them has been developed from or are inspired by natural products. Protease inhibitors typically mimic the peptide substrates of the target enzymes and feature specialized moieties that specifically interact with catalytic residues in the active centre. Such moieties may consist of electrophilic warheads e.g. β-lactones, Michael systems, epoxyketones or β-lactams that are attacked by active site nucleophiles and generate covalent adducts. Metalloproteinase inhibitors often contain functional groups which facilitate the chelation of active site metal ions e.g. hydroxamates, carboxylates or phosphoramidates. Other reversible protease inhibitors feature γ-amino acids or ketomethylene pseudopeptides to form stable substrate or transition state analogs. The discovery of such functional groups in natural products has been extensively exploited by medicinal chemistry to generate synthetic protease inhibitors. However, the biosynthetic principles for many of these moieties have remained obscure. Understanding the genetic basis that directs the formation of the specialized, activity-conferring moieties in protease inhibitors will allow targeted genome mining for the discovery of new derivatives. Moreover, in the age of synthetic biology understanding the enzymatic formation of the warhead moieties and their coupling to the (pseudo)peptide backbones will greatly facilitate the generation of tailor-made (unnatural) bioactive molecules.
Studies on new biosynthetic mechanisms in the formation of protease inhibitors
The basis of our research approach is a detailed understanding about the formation of small-molecule protease inhibitors and their warheads. We are currently studying the biosynthetic pathways of various protease inhibitors using comparative bioinformatics, in-frame gene deletion experiments, heterologous gene cluster expression, feeding of isotope-labelled precursors, in vitro biochemistry and protein crystallography (in collaboration). Thereby we have found a number of intriguing biosynthetic features. One example is the single-enzyme-transformation generating the epoxyketone warhead of epoxomicin and eponemycin proteasome inhibitors via decarboxylation, dehydrogenation and epoxidation. Remarkably, a similar mechanism generates the chemical diversity in the matlystatin family of metalloproteinase inhibitors. We also found that the hydroxamic acid warhead of the matylstatins is assembled by an unprecedented variation of the ethylmalonyl-CoA pathway. Moreover, we discovered that construction of the β-lactone moiety of belactosin and cystargolides is reminiscent of leucine biosynthesis. Recently, we described the first N-P-bond forming kinase in natural product biosynthesis involved in the formation of phosphoramidon and talopeptin.
Biosynthesis-guided discovery of novel protease and proteasome inhibitors
An important focus of our research is the discovery of new protease inhibitors from bacterial sources. To this end, we are pursuing a targeted genome-guided approach, which takes advantage of the tremendous progress made in the fields of bioinformatics and analytical chemistry. We use the information from our biosynthetic studies to identify orphan gene clusters with analogous organization. We make use of our long standing expertise on heterologous systems to express known and orphan biosynthetic pathways in surrogate host organisms. LC-MS-assisted comparative metabolic profiling is employed to detect the new heterologous compounds. Structural characterization of isolated molecules is done in collaboration with natural product chemists. To address the problem that some of the compounds or their gene clusters are difficult to access, we have established a number of synthetic biology tools, including yeast-assisted direct cloning techniques, de novo synthesis and assembly of reconstructed pathways and use genome-optimized heterologous host strains.
Generation of new protease inhibitors by biosynthetic engineering
Warheads are defined structural features that allow small-molecule bioactive compounds to specifically bind to the catalytic centre of their target enzymes. Protease inhibitors usually consist of a (pseudo)peptidic core structure in which the warhead is incorporated on a distinct moiety. This structural organization implicates the possibility to engineer the warhead moieties into other peptides. This is especially appealing as many of these warhead moieties are installed by single enzymes which can be altered to accept different substrates. With the advancement of synthetic biology strategies, Red/ET-recombineering and yeast-assisted recombination the technologies for such an approach are now available and are routinely used in my laboratory.
Protease inhibitors and other secondary metabolites of Nocardia spp. and their role in pathogenicity
Recently, we have started to explore another research area that is centred on Nocardia spp., filamentous Actinobacteria of the order Corynebacteriales and opportunistic pathogens that can cause localized and severe systemic nocardiosis infections. It has been shown that virulent species are able to resist phagocytosis by inhibiting phagosomal-lysosomal fusion and induce host cell apoptosis and immunosuppressive microenvironments. However, the molecular strategies how these facultative intracellular pathogens can escape from microbiocidal mechanisms developed by the host are poorly understood. It is also unclear what triggers the different progression and diverse symptoms induced by different Nocardia strains. Intriguingly, recent genome sequencing showed an extraordinary secondary metabolism for most Nocardia strains raising the question of the role of these compounds in bacterial virulence and disease development. Two recent grants and our in-house S2 facility enable us to address this fascinating issue. We have established the required molecular tools for the genetic engineering of Nocardia and Nocardia-derived pathways and set up a lab for the work with human cell cultures. We are now able to analyse the effect of small molecules in cell-based assays e.g. mixed lymphocyte reaction, cell proliferation and cytokine expression. In our approach to explore the secondary metabolism of Nocardia spp. and its physiological function we are focussing primarily on proteasome inhibitors and other known or predicted compounds with cytotoxic or immunosuppressive properties.