No abstract

Probably every cellular process is governed by proteinprotein interaction (PPIs), which are often highly dynamic in nature being modulated by in-or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent ␤-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken A protein's function is largely mediated through its interactions with other proteins, hence the critical importance of protein-protein interaction (PPI) 1 maps for understanding cellular mechanisms of action in health and disease. Whereas many proteins are organized in stable multi-protein complexes, the majority of cellular processes are governed by transient protein encounters, the dynamics of which are directed by a diversity of both intra-and extracellular signals. Our view of protein networks is still, however, mainly a static one (1). Current interactomes consist mainly of data generated by yeast 2-hybrid (Y2H) (2) and (tandem) affinity purification combined with mass spectrometry (3) and should be interpreted as scaffolds of potential PPIs that might occur at a certain time and place in the cell or as snapshots of PPIs taking place under a specific cellular condition. Although very robust and highly efficient, these approaches do not allow

Proteasomes degrade most proteins in mammalian cells and are established targets of anti-cancer drugs. The majority of proteasome inhibitors are composed of short peptides with an electrophilic functionality (pharmacophore) at the C terminus. All eukaryotic proteasomes have three types of active sites as follows: chymotrypsin-like, trypsin-like, and caspase-like. It is widely believed that active site specificity of inhibitors is determined primarily by the peptide sequence and not the pharmacophore. Here, we report that active site specificity of inhibitors can also be tuned by the chemical nature of the pharmacophore. Specifically, replacement of the epoxyketone by vinyl sulfone moieties further improves the selectivity of ␤5-specific inhibitors NC-005, YU-101, and PR-171 (carfilzomib). This increase in specificity is likely the basis of the decreased cytotoxicity of vinyl sulfone-based inhibitors to HeLa cells as compared with that of epoxyketone-based inhibitors.The ubiquitin-proteasome pathway is essential in the maintenance of protein homeostasis in all eukaryotic cells and is involved in the regulation of numerous biologic processes. Proteasome inhibition causes apoptosis of malignant cells (1, 2). The proteasome inhibitor bortezomib (Velcade, PS-341) is used for the treatment of multiple myeloma and mantle cell lymphoma. Four other proteasome inhibitors are at different stages of clinical trials (3-6).The 26 S proteasome is a large (1.6 -2.4 MDa), hollow cylindrical, and multifunctional particle that consists of a 20 S proteolytic core and one or two 19 S regulatory complexes. Each eukaryotic 20 S core particle has three pairs of proteolytic sites with distinct substrate specificities (7-11). The ␤5 proteolytic sites are "chymotrypsin-like," and the ␤2 sites are "trypsin-like." The ␤1 sites cleave after acidic residues (Glu and Asp) and are referred to as "post-acidic," "post-glutamate peptide hydrolase," or "caspase-like." Tissues of the immune system also express immunoproteasomes, in which ␤5, ␤1, and ␤2 catalytic subunits are replaced by their major histocompatibility complex (MHC) locus-encoded counterparts LMP7 (␤5i), LMP2 (␤1i), and MECL-1 (␤2i).The chymotrypsin-like sites have long been considered the only suitable targets for anti-neoplastic agents and are the primary targets of all these agents. However, our recent work indicates that cytotoxicity of proteasome inhibitors correlates poorly with exclusive inhibition of the chymotrypsin-like sites and that co-inhibition of other sites is usually needed to achieve maximal cytotoxicity (12). In this regard, we have considered it of interest to determine whether inhibitors with increased specificity for ␤5 display decreased cytotoxicity.Many structural classes of proteasome inhibitors are known (2, 13). The majority of these are N-terminally capped short peptides (2-4 residues) with an electrophilic trap at the C terminus (e.g. aldehydes, boronates, epoxyketones, and vinyl sulfones). This electrophile reacts with the catalytic N-terminal threonines ...

In recent years, thymidylate kinase (TMPK), an enzyme indispensable for bacterial DNA biosynthesis, has been pursued for the development of new antibacterial agents including against Mycobacterium tuberculosis, the causative agent for the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous efforts toward the exploration of novel and potent Mycobacterium tuberculosis TMPK ( MtTMPK) inhibitors, and reported here the design of a novel series of non-nucleoside inhibitors of MtTMPK. The inhibitors display hitherto unexplored interactions in the active site of MtTMPK, offering new insights into structure-activity relationships. To investigate the discrepancy between enzyme inhibitory activity and the whole-cell activity, experiments with efflux pump inhibitors and efflux pump knockout mutants were performed. The minimum inhibitory concentrations of particular inhibitors increased significantly when determined for the efflux pump mmr knockout mutant, which partly explains the observed dissonance.

We present a scalable synthesis of a versatile MTX reagent with an azide ligation handle that allows rapid γ-selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT, a three-hybrid system that enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest. We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine, and FK506) as model baits. After acetylene functionalization of these baits, MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration-dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non-regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full-length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in luciferase activity. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening.

No abstract

We report on the design of glycosylated nanogels via core-cross-linking of amphiphilic non-watersoluble block copolymers composed of an acetylated glycosylated block and a pentafluorophenyl (PFP) activated ester block prepared by RAFT polymerization. Self-assembly, pH-sensitive corecross-linking and removal of remaining PFP esters and protecting groups is achieved in one-pot yielding fully hydrated sub-100 nm nanogels. Using cell subsets that exhibit high and low expression of the mannose receptor under conditions that suppress active endocytosis, we show that mannosylated but not galactosylated nanogels can efficiently target the mannose receptor (MR) that is expressed on the cell surface of primary dendritic cells (DCs). These nanogels hold promise for immunological applications involving DCs and macrophage subsets.

ABSTRACT. In the wake of the escalating development of resistance to currently available drugs, blocking the vital MEP pathway for isoprenoid biosynthesis in pathogens such as Plasmodia or Mycobacteria offers interesting prospects for inhibiting their growth. Although the natural product retro-hydroxamate fosmidomycin and its homologue FR900098 potently inhibit 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in the MEP pathway, their in vivo activity is compromised due to poor absorption and a suboptimal pharmacokinetic profile. In an effort to facilitate cellular uptake, we introduced aryl or aralkyl substituents at the β-position of the hydroxamate analogue of FR900098. While direct addition of a β-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes, as well as activity against P. falciparum.X-ray structures of the parasite Dxr-inhibitor complexes show that the modes of binding of the two classes of compounds are in fact different. When the "shorter" compounds were bound, the active site flap was fully ordered and similar to that observed for the retro-hydroxamates. The "longer" compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the methyl group on the hydroxamate. Although the most promising new Dxr inhibitors lack activity against E. coli and M. smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth.3