Development of Tyrphostin Analogues to Study Inhibition of the Mycobacterium tuberculosis Pup Proteasome System*.

Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds.

Diubiquitin-Based NMR Analysis: Interactions Between Lys6-Linked diUb and UBA Domain of UBXN1.

Ubiquitination is a process in which a protein is modified by the covalent attachment of the C-terminal carboxylic acid of ubiquitin (Ub) to the ε-amine of lysine or N-terminal methionine residue of a substrate protein or another Ub molecule. Each of the seven internal lysine residues and the N-terminal methionine residue of Ub can be linked to the C-terminus of another Ub moiety to form 8 distinct Ub linkages and the resulting differences in linkage types elicit different Ub signaling pathways. Cellular responses are triggered when proteins containing ubiquitin-binding domains (UBDs) recognize and bind to specific polyUb linkage types. To get more insight into the differences between polyUb chains, all of the seven lysine-linked di-ubiquitin molecules (diUbs) were prepared and used as a model to study their structural conformations in solution using NMR spectroscopy. We report the synthesis of diUb molecules, fully 15N-labeled on the distal (N-terminal) Ub moiety and revealed their structural orientation with respect to the proximal Ub. As expected, the diUb molecules exist in different conformations in solution, with multiple conformations known to exist for K6-, K48-, and K63-linked diUb molecules. These multiple conformations allow structural flexibility in binding with UBDs thereby inducing unique responses. One of the well-known but poorly understood UBD-Ub interaction is the recognition of K6 polyubiquitin by the ubiquitin-associated (UBA) domain of UBXN1 in the BRCA-mediated DNA repair pathway. Using our synthetic 15N-labeled diUbs, we establish here how a C-terminally extended UBA domain of UBXN1 confers specificity to K6 diUb while the non-extended version of the domain does not show any linkage preference. We show that the two distinct conformations of K6 diUb that exist in solution converge into a single conformation upon binding to this extended form of the UBA domain of the UBXN1 protein. It is likely that more of such extended UBA domains exist in nature and can contribute to linkage-specificity in Ub signaling. The isotopically labeled diUb compounds described here and the use of NMR to study their interactions with relevant partner molecules will help accelerate our understanding of Ub signaling pathways.

Enhanced Delivery of Synthetic Labelled Ubiquitin into Live Cells by Using Next-Generation Ub-TAT Conjugates.

Proteins and other macromolecules can be delivered into live cells by noninvasive techniques using cell-penetrating peptides. These peptides are easily synthesised by solid-phase peptide synthesis and can be conjugated onto cargo molecules to mediate cellular delivery. We designed a TAT-based cell-penetrating ubiquitin (Ub) reagent by conjugating a dimeric disulfide-linked TAT peptide to the C terminus of a rhodamine-labelled Ub (RhoUb) protein. This reagent efficiently enters the cell by endocytosis and escapes from endosomes into the cytoplasm. Once the conjugate is inside the cytoplasm, the delivery vehicle is proteolytically removed by endogenous deubiquitinases (DUBs), at which point the intrinsic ubiquitination machinery is able to incorporate the RhoUb into ubiquitin conjugates. Our approach enables the controlled delivery of labelled or mutant Ub derivatives into cells, increasing our options for studying the ubiquitin system.

Total Chemical Synthesis of SUMO and SUMO-Based Probes for Profiling the Activity of SUMO-Specific Proteases.

SUMO is a post-translational modifier critical for cell cycle progression and genome stability that plays a role in tumorigenesis, thus rendering SUMO-specific enzymes potential pharmacological targets. However, the systematic generation of tools for the activity profiling of SUMO-specific enzymes has proven challenging. We developed a diversifiable synthetic platform for SUMO-based probes by using a direct linear synthesis method, which permits N- and C-terminal labelling to incorporate dyes and reactive warheads, respectively. In this manner, activity-based probes (ABPs) for SUMO-1, SUMO-2, and SUMO-3-specific proteases were generated and validated in cells using gel-based assays and confocal microscopy. We further expanded our toolbox with the synthesis of a K11-linked diSUMO-2 probe to study the proteolytic cleavage of SUMO chains. Together, these ABPs demonstrate the versatility and specificity of our synthetic SUMO platform for in vitro and in vivo characterization of the SUMO protease family.

Dynamic recruitment of ubiquitin to mutant huntingtin inclusion bodies.

Many neurodegenerative diseases, such as Huntington's disease, are hallmarked by the formation of intracellular inclusion bodies (IBs) that are decorated with ubiquitin, proteasomes and chaperones. The apparent enrichment of ubiquitin and components involved in protein quality control at IBs suggests local ubiquitin-dependent enzymatic activity. In this study, we examine recruitment of ubiquitin to IBs of polyglutamine-expanded huntingtin fragments (mHtt) by using synthesized TAMRA-labeled ubiquitin moieties. We show that intracellular TAMRA-ubiquitin is dynamic at mHtt IBs and is incorporated into poly-ubiquitin chains of intracellular substrates, such as mHtt, in a conjugation-dependent manner. Furthermore, we report that mHtt IBs recruit catalytically active enzymes involved in (de)-ubiquitination processes based on novel activity-based probes. However, we also find that the overexpression of the GFP-ubiquitin reporter, unlike the endogenous ubiquitin and TAMRA-ubiquitin, becomes irreversibly sequestered as a ring-like structure around the mHtt IBs, suggesting a methodical disadvantage of GFP-tagged ubiquitin. Our data provide supportive evidence for dynamic recruitment of ubiquitin and ubiquitin (de)-conjugating activity at mHtt initiated IBs.

A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades.

N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids.

Despite technological advances in metabolomics, large parts of the human metabolome are still unexplored. In an untargeted metabolomics screen aiming to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5), we identified a class of mammalian metabolites, N-lactoyl-amino acids. Using parallel protein fractionation in conjunction with shotgun proteomics on fractions containing N-lactoyl-Phe-forming activity, we unexpectedly found that a protease, cytosolic nonspecific dipeptidase 2 (CNDP2), catalyzes their formation. N-lactoyl-amino acids are ubiquitous pseudodipeptides of lactic acid and amino acids that are rapidly formed by reverse proteolysis, a process previously considered to be negligible in vivo. The plasma levels of these metabolites strongly correlate with plasma levels of lactate and amino acid, as shown by increased levels after physical exercise and in patients with phenylketonuria who suffer from elevated Phe levels. Our approach to identify unknown metabolites and their biosynthesis has general applicability in the further exploration of the human metabolome.

An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens.

Target specificity of the E3 ligase LUBAC for ubiquitin and NEMO relies on different minimal requirements.

The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin.

Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes.

Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.

Synthesis and evaluation of a selective fluorogenic Pup derived assay reagent for Dop, a potential drug target in Mycobacterium tuberculosis.

A litter of pups: The synthesis and in vitro evaluation of new Pup-based fluorogenic substrates for Dop, the mycobacterial depupylase, are described. A full-length Pup-amidomethylcoumarin conjugate as well as an amino-terminus-truncated analogue exhibited high sensitivity and specificity towards hydrolysis by Dop. The substrates developed here might find application as high-throughput screening assay reagents for the identification of Dop inhibitors.

The differential modulation of USP activity by internal regulatory domains, interactors and eight ubiquitin chain types.

Ubiquitin-specific proteases (USPs) are papain-like isopeptidases with variable inter- and intramolecular regulatory domains. To understand the effect of these domains on USP activity, we have analyzed the enzyme kinetics of 12 USPs in the presence and absence of modulators using synthetic reagents. This revealed variations of several orders of magnitude in both the catalytic turnover (k(cat)) and ubiquitin (Ub) binding (K(M)) between USPs. Further activity modulation by intramolecular domains affects both the k(cat) and K(M), whereas the intermolecular activators UAF1 and GMPS mainly increase the k(cat). Also, we provide the first comprehensive analysis comparing Ub chain preference. USPs can hydrolyze all linkages and show modest Ub-chain preferences, although some show a lack of activity toward linear di-Ub. This comprehensive kinetic analysis highlights the variability within the USP family.

Photochemical cleavage of leader peptides.

We report a photolabile linker compatible with Fmoc solid phase peptide synthesis and Cu(I)-catalyzed alkyne-azide cycloaddition that allows photochemical cleavage to afford a C-terminal peptide fragment with a native amino terminus.

Synthesis of DOTA-conjugated multimeric [Tyr3]octreotide peptides via a combination of Cu(I)-catalyzed "click" cycloaddition and thio acid/sulfonyl azide "sulfo-click" amidation and their in vivo evaluation.

Herein, we describe the design, synthesis, and biological evaluation of a series of DOTA-conjugated monomeric, dimeric, and tetrameric [Tyr(3)]octreotide-based analogues as a tool for tumor imaging and/or radionuclide therapy. These compounds were synthesized using a Cu(I)-catalyzed 1,3-dipolar cycloaddition ("click" reaction) between peptidic azides and dendrimer-derived alkynes and a subsequent metal-free introduction of DOTA via the thio acid/sulfonyl azide amidation ("sulfo-click" reaction). In a competitive binding assay using rat pancreatic AR42J tumor cells, the monomeric [Tyr(3)]octreotide conjugate displayed the highest binding affinity (IC(50) = 1.32 nM) followed by dimeric [Tyr(3)]octreotide (2.45 nM), [DOTA(0),Tyr(3)]octreotide (2.45 nM), and tetrameric [Tyr(3)]octreotide (14.0 nM). Biodistribution studies with BALB/c nude mice with subcutaneous AR42J tumors showed that the (111)In-labeled monomeric [Tyr(3)]octreotide conjugate had the highest tumor uptake (42.3 +/- 2.8 %ID/g) at 2 h p.i., which was better than [(111)In-DOTA(0),Tyr(3)]octreotide (19.5 +/- 4.8 %ID/g). The (111)In-labeled dimeric [Tyr(3)]octreotide conjugate showed a long tumor retention (25.3 +/- 5.9 %ID/g at 2 h p.i. and 12.1 +/- 1.3 %ID/g at 24 h p.i.). These promising results can be exploited for therapeutic applications.

'Sulfo-click' for ligation as well as for site-specific conjugation with peptides, fluorophores, and metal chelators.

The 'sulfo-click' reaction, which is a chemoselective amidation reaction involving the reaction of an aminoethane sulfonyl azide with a thio acid, encompasses a new approach for ligation and conjugation. Detailed protocols are provided for decorating biologically active peptides or dendrimers with biophysical tags, fluorescent probes, metal chelators, and small peptides by using this reaction as a novel, metal-free 'sulfo-click' approach.

Resin-bound sulfonyl azides: efficient loading and activation strategy for the preparation of the N-acyl sulfonamide linker.

This paper describes an optimized protocol for the efficient loading of resin-bound aminoethane sulfonyl azides by either Boc- or Fmoc-protected amino thioacids. The resulting N-acyl sulfonamide is a convenient linker for use in Boc- or Fmoc-based solid-phase peptide synthesis. Activation of the N-acyl sulfonamide via a microwave-assisted alkylation procedure and subsequent treatment with functionalized nucleophiles yields C-terminally modified peptides that can be applied in chemoselective (bio)conjugation or ligation reactions.

Efficient microwave-assisted synthesis of multivalent dendrimeric peptides using cycloaddition reaction (click) chemistry.

Multivalent dendrimeric peptides were synthesized via a microwave-assisted Huisgen 1,3-dipolar cycloaddition between azido peptides and dendrimeric alkynes in yields ranging from 46 to 96%.

Highly efficient coupling of beta-substituted aminoethane sulfonyl azides with thio acids, toward a new chemical ligation reaction.

[reaction: see text] A highly efficient coupling of protected beta-substituted aminoethane sulfonyl azides with thio acids is reported. In the case of peptide thio acids, this method encompasses a new chemoselective ligation method. Furthermore, the resulting alpha-amino acyl sulfonamides can be alkylated with suitable electrophiles to obtain densely functionalized sulfonamide scaffolds.

Identification of substituted 6-amino-4-phenyltetrahydroquinoline derivatives: potent antagonists for the follicle-stimulating hormone receptor.

Substituted 6-amino-4-phenyl-tetrahydroquinoline derivatives are described that are antagonists for the G(s)-protein-coupled human follicle-stimulating hormone (FSH) receptor. These compounds show high antagonistic efficacy in vitro using a CHO cell line expressing the human FSH receptor. Antagonist 10 also showed a submicromolar IC(50) in a more physiologically relevant rat granulosa cell assay and was found to significantly inhibit follicle growth and ovulation in an ex vivo mouse model. This compound class may open the way toward a novel, nonsteroidal approach for contraception.