Targeting Cullin-RING E3 ubiquitin ligases for drug discovery:structure, assembly and small-molecule modulation

In the last decade, the ubiquitin–proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin–RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs

Endonuclease from Gram-Negative Bacteria Serratia marcescens Is as Effective as Pulmozyme in the Hydrolysis of DNA in Sputum

One of the approaches to effective airway cleansing is the degradation of DNA into smaller fragments. For this purpose Pulmozyme® is used with high efficacy because it contains recombinant DNase I as its active component. The aim of the study was to comparatively analyze DNase activity of Pulmozyme® and the nuclease from gram-negative bacteria Serratia marcescens, because at optimal conditions the catalytic efficiency of the nuclease is much higher than the efficiency of DNase I. Highly polymerized DNA and purulent-mucous sputum were used as substrates. The examination showed that both S. marcescens nuclease and Pulmozyme® hydrolyzed DNA in sputum. Also S. marcescens nuclease was found capable of hydrolyzing DNA in conditions that are standard for Pulmozyme® and suitable for its therapeutic application. For manifesting the similar hydrolytic activity the nuclease amount in the assay mixture containing highly polymerized DNA or the sonicated sputum and NaCl together with calcium- or magnesium- cations can be about 10- time lower than that of the recombinant DNase I. In the presence of magnesium cations the DNase activity of both S. marcescens nuclease and Pulmozyme® was higher than in the presence of calcium cations

Small Molecule Modulators of RING-Type E3 Ligases: MDM and Cullin Families as Targets

Ubiquitin–proteasome system (UPS) is a primary signaling pathway for regulation of intracellular protein levels. E3 ubiquitin ligases, substrate-specific members of the UPS, represent highly attractive protein targets for drug discovery. The importance of E3 ligases as prospective targets for small molecule modulation is reinforced by ever growing evidence of their role in cancer and other diseases. To date the number of potent compounds targeting E3 ligases remains rather low and their rational design constitutes a challenging task. To successfully address this problem one must take into consideration the multi-subunit nature of many E3 ligases that implies multiple druggable pockets and protein–protein interfaces. In this review, we briefly cover the current state of drug discovery in the field of RING-type E3 ligases with focus on MDM and Cullin families as targets. We also provide an overview of small molecule chimeras that induce RING-type E3-mediated proteasomal degradation of substrate proteins of interest

Identification of arsenic as a source of experimental phasing power.

<p>(A) The 2<i>F</i>_o−<i>F</i>_c (<i>blue mesh</i>) and <i>F</i>_o−<i>F</i>_c (<i>green mesh</i>) maps around modelled SOCS2_C111 (<i>CPK colored sticks</i>) and adjacent Ni(II) (<i>green sphere</i>) from the PDB structure 2C9W. (B) As in A, with maps recalculated following removal of Ni(II). (C) As in A, with SOCS2_C111 remodelled as dimethylarsenic cysteine. 2<i>F</i>_o−<i>F</i>_c maps are contoured at 1σ and <i>F</i>_o−<i>F</i>_c at 3σ. (D) A fluorescence scan (<i>green line</i>) and measurements of <i>f</i>≠ and <i>f</i>≡ (<i>red</i> and <i>blue lines</i>, respectively) identifying the presence of arsenic and resulting anomalous signals from DMSO-treated SOCS2:EloC:EloB crystals. (E) Observed anomalous signal [Δ<i>F</i>/σ(Δ<i>F</i>)] in diffraction data plotted against resolution for the As-Peak datasets collected. The cut-off of useful signal (1.2) is indicated by a dashed line [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131218#pone.0131218.ref022" target="_blank">22</a>].</p

Crystals of the SOCS2:EloC:EloB complex grown after 7 d.

<p>The crystals shown here are 200–300 μm in the longest dimension.</p

Assessment of Thermal Stability of Mutant p53 Proteins via Differential Scanning Fluorimetry

The p53 protein is a transcription factor that preserves the integrity of the genome. The TP53 gene has inactivating mutations in about 50% of all human cancers. Some missense mutations lead to decreased thermal stability in the p53 protein, its unfolding and aggregation under physiological conditions. A general understanding of the impact of point mutations on the stability and conformation of mutant p53 is essential for the design and development of small molecules that target specific p53 mutations. In this work, we determined the thermostability properties of some of the most common mutant forms of the p53 protein—p53(R273H), p53(R248Q), p53(R248W) and p53(Y220C)—that are often considered as attractive therapeutic targets. The results showed that these missense mutations lead to destabilization of the p53 protein and a decrease in its melting temperature