Since the discovery of ubiquitin-mediated protein degradation, scientists have tried to translate knowledge of this important system into new medicines. Indeed, there are currently four approved drugs that work by targeting enzymes in the ubiquitin system. However, when compared to the rate of drug discovery for other enzyme classes, such as kinases, progress in the ubiquitin area has been slow.
In 2004, the same year that ubiquitin pioneers were awarded the Nobel Prize in Chemistry, an alternative was suggested - targeting ubiquitin itself. Small molecules called ‘ubistatins’ were discovered in a screen for inhibitors of protein degradation.1 Ubistatins were found later to bind Lys48-linked ubiquitin chains, the critical post-translational modification which tags proteins for degradation. Weak binding and poor specificity halted progress of ubistatins, but the idea of binding and modulating ubiquitin chains remained with Prof. Ashraf Brik (Technion). A meeting between Brik and Hiroaki Suga (University of Tokyo) presented an opportunity to revisit this approach. Published in Nature Chemistry, Brik and Suga introduce a new drug discovery strategy to tackle challenging targets like ubiquitin chains.
Ubiquitin chains come in many forms. They can be different lengths and assembled using different lysine linkages. Extracting a particular ubiquitin chain from the mixtures made in human cells is difficult, as is assembling homogenous chains in vitro using purified enzymes. This poses a problem for target-based drug discovery, which requires a pure target to screen against. The Brik lab overcame this problem by assembling particular ubiquitin chains by total chemical synthesis.2 Built from the amino acids up, the Brik lab were able to prepare pure K48-linked ubiquitin chains of defined lengths, dimers and tetramers, ready for screening.
Ubiquitin is a small protein lacking ‘grooves’ for small molecule binding, and the various ubiquitin chains have only subtle structural differences. This makes them problematic to bind tightly and specifically using traditional drug-like molecules, such as the ubistatins. However, the Suga lab has a method to screen molecules from a different structural class, one with superior protein-binding abilities - cyclic peptides.3 Brik’s chemically-made ubiquitin chains were fed into this method, where huge numbers (trillions, >1012) of unique cyclic peptides can be screened in parallel to isolate specific, high-affinity cyclic peptide binders.
In collaboration with Prof. Aaron Ciechanover (Technion) and Prof. David Fushman (Maryland University) the specific binding of these new cyclic peptides to ubiquitin chains was investigated, and their inhibition of cellular protein degradation demonstrated. These cyclic peptides are available now as tools to study and understand the complexities of the ubiquitin system, and might form the basis of new drugs. Cancer cells, which are extremely sensitive to interference of the ubiquitin system, undergo apoptosis upon expose to these cyclic peptides.
The combination of total chemical synthesis of a complex protein target, and screening using huge cyclic peptide libraries, opens up many other drug discovery opportunities. Different ubiquitin chains, or other elaborate post-translationally modified proteins can now be targeted using this protein synthesis and cyclic peptide approach.
1. Verma, R. et al. Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain. Science 306, 117–120 (2004).
2. Mali, S. M., Singh, S. K., Eid, E. & Brik, A. Ubiquitin signaling: chemistry. comes to the rescue. J. Am. Chem. Soc. 139, 4971–4986 (2017).
3. Jongkees, S. a. K., Hipolito, C. J., Rogers, J. M. & Suga, H. Model foldamers: applications and structures of stable macrocyclic peptides identified using in vitro selection. New J. Chem. 39, 3197–3207 (2015).