Chemical biology of antigen presentation by MHC molecules
van Kasteren, S.I.; Overkleeft, H.S.; Ovaa, H.; Neefjes, J.J.

MHC class I and MHC class II molecules present peptides to the immune system to drive proper T cell responses. Pharmacological modulation of T-cell responses can offer treatment options for a range of immune-related diseases. Pharmacological downregulation of MHC molecules may find application in treatment of auto-immunity and transplantation rejection while pharmacological activation of antigen presentation would support immune responses to infection and cancer. Since the cell biology of MHC class I and MHC class II antigen presentation is understood in great detail, many potential targets for manipulation have been defined over the years. Here, we discuss how antigen presentation by MHC molecules can be modulated by pharmacological agents and how chemistry can further support the study of antigen presentation in general. The chemical biology of antigen presentation by MHC molecules shows surprising options for immune modulation and the development of future therapies.

Structure-Based Design of beta1i or beta5i Specific Inhibitors of Human Immunoproteasomes
de Bruin, G.; Huber, E. M.; Xin, B. T.; van Rooden, E. J.; Al-Ayed, K.; Kim, K. B.; Kisselev, A. F.; Driessen, C.; van der Stelt, M.; van der Marel, G. A.; Groll, M.; Overkleeft, H. S.

Mammalian genomes encode seven catalytic proteasome subunits, namely, beta1c, beta2c, beta5c (assembled into constitutive 20S proteasome core particles), beta1i, beta2i, beta5i (incorporated into immunoproteasomes), and the thymoproteasome-specific subunit beta5t. Extensive research in the past decades has yielded numerous potent proteasome inhibitors including compounds currently used in the clinic to treat multiple myeloma and mantle cell lymphoma. Proteasome inhibitors that selectively target combinations of beta1c/beta1i, beta2c/beta2i, or beta5c/beta5i are available, yet ligands truly selective for a single proteasome activity are scarce. In this work we report the development of cell-permeable beta1i and beta5i selective inhibitors that outperform existing leads in terms of selectivity and/or potency. These compounds are the result of a rational design strategy using known inhibitors as starting points and introducing structural features according to the X-ray structures of the murine constitutive and immunoproteasome 20S core particles.

Acylazetine as a Dienophile in Bioorthogonal Inverse Electron-Demand Diels-Alder Ligation
Engelsma, S. B.; Willems, L. I.; van Paaschen, C. E.; van Kasteren, S. I.; van der Marel, G. A.; Overkleeft, H. S.; Filippov, D. V.

A new bioorthogonal N-acylazetine tag, suitable for tetrazine mediated inverse electron-demand Diels-Alder conjugation, is developed. The tag is small and achiral. We demonstrate the usefulness of N-acylazetine-tetrazine based bioorthogonal chemistry in two-step activity-based protein profiling. The performance of the new tetrazinophile in the labeling of catalytically active proteasome subunits was comparable to that of the more sterically demanding norbornene tag.

Natural product proteomining, a quantitative proteomics platform, allows rapid discovery of biosynthetic gene clusters for different classes of natural products
Gubbens, J.; Zhu, H.; Girard, G.; Song, L.; Florea, B. I.; Aston, P.; Ichinose, K.; Filippov, D. V.; Choi, Y. H.; Overkleeft, H. S.; Challis, G. L.; van Wezel, G. P.

Information on gene clusters for natural product biosynthesis is accumulating rapidly because of the current boom of available genome sequencing data. However, linking a natural product to a specific gene cluster remains challenging. Here, we present a widely applicable strategy for the identification of gene clusters for specific natural products, which we name natural product proteomining. The method is based on using fluctuating growth conditions that ensure differential biosynthesis of the bioactivity of interest. Subsequent combination of metabolomics and quantitative proteomics establishes correlations between abundance of natural products and concomitant changes in the protein pool, which allows identification of the relevant biosynthetic gene cluster. We used this approach to elucidate gene clusters for different natural products in Bacillus and Streptomyces, including a novel juglomycin-type antibiotic. Natural product proteomining does not require prior knowledge of the gene cluster or secondary metabolite and therefore represents a general strategy for identification of all types of gene clusters.

Discovery of Glycine Sulfonamides as Dual Inhibitors of sn-1-Diacylglycerol Lipase alpha and alpha/beta-Hydrolase Domain 6
Janssen, F. J.; Deng, H.; Baggelaar, M. P.; Allara, M.; van der Wel, T.; den Dulk, H.; Ligresti, A.; van Esbroeck, A. C.; McGuire, R.; Di Marzo, V.; Overkleeft, H. S.; van der Stelt, M.

sn-1-Diacylglycerol lipase alpha (DAGL-alpha) is the main enzyme responsible for the production of the endocannabinoid 2-arachidonoylglycerol in the central nervous system. Glycine sulfonamides have recently been identified by a high throughput screening campaign as a novel class of inhibitors for this enzyme. Here, we report on the first structure-activity relationship study of glycine sulfonamide inhibitors and their brain membrane proteome-wide selectivity on serine hydrolases with activity-based protein profiling (ABPP). We found that (i) DAGL-alpha tolerates a variety of biaryl substituents, (ii) the sulfonamide is required for inducing a specific orientation of the 2,2-dimethylchroman substituent, and (iii) a carboxylic acid is essential for its activity. ABPP revealed that the sulfonamide glycine inhibitors have at least three off-targets, including alpha/beta-hydrolase domain 6 (ABHD6). Finally, we identified LEI-106 as a potent, dual DAGL-alpha/ABHD6 inhibitor, which makes this compound a potential lead for the discovery of new molecular therapies for diet-induced obesity and metabolic syndrome.

Assessing Subunit Dependency of the Plasmodium Proteasome Using Small Molecule Inhibitors and Active Site Probes
Li, H.; van der Linden, W. A.; Verdoes, M.; Florea, B. I.; McAllister, F. E.; Govindaswamy, K.; Elias, J. E.; Bhanot, P.; Overkleeft, H. S.; Bogyo, M.

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the beta5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the beta2 subunit did not affect viability. Interestingly, coinhibition of both the beta2 and beta5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

A Two-Armed Lanthanoid-Chelating Paramagnetic NMR Probe Linked to Proteins via Thioether Linkages
Liu, W. M.; Skinner, S. P.; Timmer, M.; Blok, A.; Hass, M. A. S.; Filippov, D. V.; Overhand, M.; Ubbink, M.

Paramagnetic NMR probes provide valuable long-range structural information on proteins and protein complexes. A new, stable, two-armed lanthanoid probe is reported that can be attached to a protein site-specifically via chemically inert thioether linkages.

Oncogenic human papillomavirus-infected immature metaplastic cells and cervical neoplasia
van der Marel, J.; van Baars, R.; Alonso, I.; del Pino, M.; van de Sandt, M.; Lindeman, J.; ter Harmsel, B.; Boon, M.; Smedts, F.; Ordi, J.; Torne, A.; Jenkins, D.; Quint, W.

Persistent cervical high-risk human papillomavirus (HR-HPV) infection results in high-grade cervical intraepithelial neoplasia (CIN2/3) and cervical carcinoma. The susceptibility of the cervix to HPV carcinogenesis and the importance of HPV18 in cervical carcinoma despite relative infrequency in CIN2/3 could be linked to HR-HPV infection of immature metaplasia (IM) at the squamocolumnar junction. Atypical IM (AIM) is an equivocal category used to describe changes in IM suggestive of high-grade neoplasia, which causes diagnostic and management problems. We used laser capture microscopy combined with polymerase chain reaction in 24 women with HPV18, HPV16, or other HPV infections on cytologic analysis and a cervical loop electrosurgical excision procedure to locate HR-HPV in cervical tissue. HPV18-positive AIM and CIN2/3 were present in 7/12 cases with HPV18 on cytologic analysis. In 2 cases with HPV18 and other HPV types, HPV18 was only present in AIM and not in CIN2/3. HPV16-positive AIM was present in 3/7 and HPV16-positive CIN2/3 in 5/7 cases with HPV16. No cases had HPV16 AIM without CIN2/3. Other HR-HPV-positive AIM and CIN2/3 cases were present, respectively, in 1/6 and 5/6 cases positive for HR-HPV types other than HPV16/18. In a subset, 94% HPV18 AIM regions showed CK17 and p16 positivity, and 41% were CK7 positive. CIN2/3 and AIM with other HR-HPVs showed similar patterns. AIM was a particular feature of HPV18 infection in women with CIN2/3. HR-HPV infection of CK7/17-positive AIM expressing p16 was particularly seen for HPV18 with and without classical CIN2/3 and should be regarded as a high-grade precancer.

Current developments in activity-based protein profiling
Willems, L. I.; Overkleeft, H. S.; van Kasteren, S. I.

Activity-based protein profiling (ABPP) has emerged as a powerful strategy to study the activity of enzymes in complex proteomes. The aim of ABPP is to selectively visualize only the active forms of particular enzymes using chemical probes termed activity-based probes (ABPs). These probes are directed to the active site of a particular target protein (or protein family) where they react in a mechanism-based manner with an active site residue. This results in the selective labeling of only the catalytically active form of the enzyme, usually in a covalent manner. Besides the monitoring of a specific enzymatic activity, ABPP strategies have also been used to identify and characterize (unknown) protein functions, to study up- and down-regulation of enzymatic activity in various disease states, to discover and evaluate putative new enzyme inhibitors, and to identify the protein targets of covalently binding natural products. In this Topical Review we will provide a brief overview of some of the recent developments in the field of ABPP.

N-Tetradecylcarbamyl Lipopeptides as Novel Agonists for Toll-like Receptor 2
Willems, M. M.; Zom, G. G.; Khan, S.; Meeuwenoord, N.; Melief, C. J.; van der Stelt, M.; Overkleeft, H. S.; Codee, J. D.; van der Marel, G. A.; Ossendorp, F.; Filippov, D. V.

New analogues (UPam) of triacylated lipopeptide Pam3CysSK4, a popular agonist of Toll-like receptor 2 (TLR2), were designed making use of the cocrystal structure of a TLR2 heterodimer (with TLR1) with Pam3CysSK4. Twenty-two UPam derivatives that feature an N-tetradecylcarbamyl chain to mimic the native N-palmitoyl moiety and various small amino acids residues at the penultimate N-terminal position were prepared via solid-phase synthesis. In vitro evaluation of immunostimulatory properties revealed new potent TLR2 ligands.