Area(s) of interest
In July 2016 the Ovaa lab has started at Leiden University Medical Center, where his lab together with the lab of Jacques Neefjes has initiated the new division of Chemical Immunology. This division that is equipped to the state-of-the-art for chemical synthesis, immunology and cell biology and has its own screening center basis on accoustic dispensing. The department will be part of a new inter-faculty multidisciplinary research institute with a focus on chemical biology in which Leiden University Medical Center and Leiden Institute of Chemistry participate.
Areas of interest
The Ovaa lab is active researching ubiquitin-mediated proteasomal proteolysis and antigen presentation. The lab carries out drug target validation and drug discovery projects. The lab uses a multidisciplinary approach combining organic synthesis, cell biology, genetics and immunology.
In 2010 we have developed routes for a high yielding linear synthesis of ubiquitin as well as method for the efficient construction of ubiquitin conjugates. Using these two findings it is now possible to make virtually any ubiquitin conjugate of ubiquitin-based assay for use in research.
Building on our experience in the synthesis we started making non-hydrolyzable ubiquitin chains and ubiquitin conjugates to probe target specificity of deubiquitinating enzymes. Although this concept proves to work (such conjugates act as potent inhibitors) we found by accident that one of the reactants for making *click* conjugates also act as inhibitors. These reactants called alkynes proved to react covalently with active site cysteine nucleophiles to provide a quarternary thiovinyl ether adduct. This finding is remarkable because no direct addition of thiols to alkynes is known in the chemistry textbooks. This finding is important because alkynes are generally considered biochemically inert. Thus alkyne moieties provide a route for the generation of covalent drugs that are intrinsically unreactive until hitting the intended target with great precision. Reactive molecules are usually circumvented by industry because of the inherent danger of unforeseen side reactions and alkyne-based inhibitors may circumvent this issue all together.
The third most interesting finding is MHC exhange technology, developed together with the lab of Ton Schumacher at the NKI. In this technique MHC complexes loaded with photocleavable peptide ligands are irradiated in the presence of desired peptides resulting in a net exchange reaction. This allows the routine production of peptide-loaded MHC complexes that can for example be used to map T-cell epitopes.
Three most important papers since 2010
Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. El Oualid F, Merkx R, Ekkebus R, Hameed DS, Smit JJ, de Jong A, Hilkmann H, Sixma TK, Ovaa H. Angew Chem Int Ed Engl. 2010: 49(52); 10149-10153
Abstract: An efficient linear solid-phase peptide synthesis of ubiquitin (Ub) has been developed. This approach allows the incorporation of desired tags and mutations (see picture; blue denotes a pseudoproline dipeptide, red a dimethoxybenzyl dipeptide) as well as specific C-terminal modification and the construction of all diubiquitin conjugates in high yields and purities in a straightforward manner.
On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. Ekkebus R, van Kasteren SI, Kulathu Y, Scholten A, Berlin I, Geurink PP, de Jong A, Goerdayal S, Neefjes J, Heck AJ, Komander D, Ovaa H. J Am Chem Soc. 2013: 135(8); 2867-2870
Abstract: Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was obersved. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub. as highlighted by the selective reaction between caspase-1 (interleukin convering enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.
Altered peptide ligands revisited: vaccine design through chemically modified HLA-A2-restricted T cell epitopes. Hoppes R, Oostvogels R, Luimstra JJ, Wals K, Toebes M, Bies L, Ekkebus R, Rijal P, Celie PH, Huang JH, Emmelot ME, Spaapen RM, Lokhorst H, Schumacher TN, Mutis T, Rodenko B, Ovaa H. J Immunol. 2014: 193(10); 4803-4813
Abstract: Virus or tumor Ag-derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation on nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligangs (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A-*0201 revealed the molecular interactions likely responsible for improved binding. The best CPSs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.
Leiden University Medical Center
2333 ZC Leiden
Tel: +31 71 5268721
P.O. Box 9600
2300 RC Leiden