Adaptive resistance of myeloma to proteasome inhibition represents a clinical challenge, whose biology is poorly understood. Proteasome mutations were implicated as underlying mechanism, while an alternative hypothesis based on low activation status of the unfolded protein response was recently suggested (IRE1/XBP1-low model). We generated bortezomib- and carfilzomib-adapted, highly resistant multiple myeloma cell clones (AMO-BTZ, AMO-CFZ), which we analyzed in a combined quantitative and functional proteomic approach. We demonstrate that proteasome inhibitor-adapted myeloma cells tolerate subtotal proteasome inhibition, irrespective of a proteasome mutation, and uniformly show an 'IRE1/XBP1-low' signature. Adaptation of myeloma cells to proteasome inhibitors involved quantitative changes in >600 protein species with similar patterns in AMO-BTZ and AMO-CFZ cells: proteins involved in metabolic regulation, redox homeostasis, and protein folding and destruction were upregulated, while apoptosis and transcription/translation were downregulated. The quantitatively most upregulated protein in AMO-CFZ cells was the multidrug resistance protein (MDR1) protein ABCB1, and carfilzomib resistance could be overcome by MDR1 inhibition. We propose a model where proteasome inhibitor-adapted myeloma cells tolerate subtotal proteasome inhibition owing to metabolic adaptations that favor the generation of reducing equivalents, such as NADPH, which is supported by oxidative glycolysis. Proteasome inhibitor resistance may thus be targeted by manipulating the energy and redox metabolism.

A procedure is presented for copper(I)-catalyzed [3+2] cycloaddition of nucleosides and nucleotides in near-quantitative yield. Azido-alkyne cycloaddition was applied for the preparation of a range of adenosine dimers and derivatives with versatile functionality, as well as for the smooth condensation of two oligonucleotide strands. The described technology may find valuable application in the synthesis of oligonucleotide dimers and conjugates.

Diacylglycerol lipase-a (DAGL-a) is an intracellular, multidomain protein responsible for the formation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system. [1] 2-AG is an endogenous signaling lipid that interacts with the cannabinoid CB1 and CB2 receptors. [2] Little is known about the regulation of its biosynthetic pathway and it is largely unclear to what extent 2-AG is responsible for distinct cannabinoid CB1 receptor mediated biological processes. Selective inhibitors of DAGL-a may contribute to a more fundamental understanding of the physiological role of 2-AG and may serve as potential drug candidates for the treatment of obesity and neurodegenerative diseases. [3] Currently, there are no selective inhibitors and activity-based probes available for the study of DAGL-a. [4] The identification of selective DAGL-a inhibitors is hampered by a lack of structural knowledge of the target, and a lack of assays that make use of endogenous DAGL-a activity in proteomes. No crystal structures are available and no homology models have been reported to aid hit identification and to guide optimization of the inhibitors. Determination of the selectivity of the inhibitors in native tissues is important because DAGL-a belongs to the serine hydrolase family, which contains more than 200 members with various physiological functions. [5] Fluorophosphonate (FP)based probes are routinely employed in competitive activitybased protein profiling (ABPP) experiments to determine the selectivity of serine hydrolase inhibitors in complex proteomes. DAGL-a, however, does not react with these activitybased probes. [6] Therefore, a new probe that can label native DAGL-a would be of value for studying the potency and selectivity of novel DAGL-a inhibitors in brain proteomes.Here we present a strategy that combines a knowledge-based in silico design approach and the development of a novel activity-based probe (ABP), based on the nonselective DAGL-a inhibitor tetrahydrolipstatin (THL; also known as Orlistat, a drug used for the treatment of obesity). This strategy resulted in the rapid identification of DAGL-a inhibitors with a new chemotype and high selectivity in the brain proteome.To identify novel DAGL-a inhibitors, we built a pharmacophore model based on THL using Discovery Studio Software Suite from Accelrys. Since THL can assume many different conformations, we searched the protein crystallographic database for crystal structures with a bioactive conformation for THL. A cocrystal structure of THL with fatty acid synthase (pdb-code: 2PX6) was identified (Figure 1 A) [7] that contains the same Ser-His-Asp catalytic triad and typical a/b hydrolase fold motif as DAGL-a. In this cocrystal structure, the nucleophilic Ser of the enzyme is covalently attached to the carbonyl moiety of the lactone. We reconstituted the ester to form the b-lactone to recover the active warhead of THL. After optimization of the geometry of the lactone, the resulting conformation was used to generate two pharmacophore models (Figure...

The post-translational modification of proteins that is known as adenosine diphosphate ribosylation (ADPr) regulates a wide variety of important biological processes, such as DNA-damage repair and cellular metabolism. This modification is also involved in carcinogenesis and the process of aging. Therefore, a better understanding of the function of ADP-ribosylation is crucial for the development of novel therapeutics. To facilitate the elucidation of the biology of ADPr, the availability of well-defined fragments of poly(ADP-ribose) is essential. Herein we report a solid-phase synthetic approach for the preparation of ADP-ribose oligomers of exactly defined length. The methodology is exemplified by the first reported synthesis of an ADP-ribose dimer and trimer.

Chemical conjugates comprising synthetic Toll-like receptor ligands (TLR-L) covalently bound to antigenic synthetic long peptides (SLP) are attractive vaccine modalities, which can induce robust CD8þ T-cell immune responses. Previously, we have shown that the mechanism underlying the power of TLR-L SLP conjugates is improved delivery of the antigen together with a dendritic cell activation signal. In the present study, we have expanded the approach to tumor-specific CD4 þ as well as CD8 þ T-cell responses and in vivo studies in two nonrelated aggressive tumor models. We show that TLR2-L SLP conjugates have superior mouse CD8 þ and CD4