Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von hippel-lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities

E3 ubiquitin ligases are attractive targets in the ubiquitin-proteasome system, however, the development of small-molecule ligands has been rewarded with limited success. The von Hippel-Lindau protein (pVHL) is the substrate recognition subunit of the VHL E3 ligase that targets HIF-1α for degradation. We recently reported inhibitors of the pVHL:HIF-1α interaction, however they exhibited moderate potency. Herein, we report the design and optimization, guided by X-ray crystal structures, of a ligand series with nanomolar binding affinities

Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex

Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting

Structural basis of PROTAC cooperative recognition for selective protein degradation

Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase-PROTAC-target species and its impact on target degradation selectivity remain elusive. We solved the crystal structure of Brd4 degrader MZ1 in complex with human VHL and the Brd4 bromodomain (Brd4BD2). The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with Brd4BD2. Structure-based-designed compound AT1 exhibits highly selective depletion of Brd4 in cells. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation

Comparison of user groups' perspectives of barriers and facilitators to implementing electronic health records: a systematic review

<p>Abstract</p> <p>Background</p> <p>Electronic health record (EHR) implementation is currently underway in Canada, as in many other countries. These ambitious projects involve many stakeholders with unique perceptions of the implementation process. EHR users have an important role to play as they must integrate the EHR system into their work environments and use it in their everyday activities. Users hold valuable, first-hand knowledge of what can limit or contribute to the success of EHR implementation projects. A comprehensive synthesis of EHR users' perceptions is key to successful future implementation. This systematic literature review was aimed to synthesize current knowledge of the barriers and facilitators influencing shared EHR implementation among its various users.</p> <p>Methods</p> <p>Covering a period from 1999 to 2009, a literature search was conducted on nine electronic databases. Studies were included if they reported on users' perceived barriers and facilitators to shared EHR implementation, in healthcare settings comparable to Canada. Studies in all languages with an empirical study design were included. Quality and relevance of the studies were assessed. Four EHR user groups were targeted: physicians, other health care professionals, managers, and patients/public. Content analysis was performed independently by two authors using a validated extraction grid with pre-established categorization of barriers and facilitators for each group of EHR users.</p> <p>Results</p> <p>Of a total of 5,695 potentially relevant publications identified, 117 full text publications were obtained after screening titles and abstracts. After review of the full articles, 60 publications, corresponding to 52 studies, met the inclusion criteria. The most frequent adoption factors common to all user groups were design and technical concerns, ease of use, interoperability, privacy and security, costs, productivity, familiarity and ability with EHR, motivation to use EHR, patient and health professional interaction, and lack of time and workload. Each user group also identified factors specific to their professional and individual priorities.</p> <p>Conclusions</p> <p>This systematic review presents innovative research on the barriers and facilitators to EHR implementation. While important similarities between user groups are highlighted, differences between them demonstrate that each user group also has a unique perspective of the implementation process that should be taken into account.</p

The structure of DMSO-treated SOCS2:EloC:EloB.

<p>(A) Anomalous difference map (<i>black mesh</i>) shown at 5σ calculated from As-Peak diffraction data in proximity to SOCS2_C111 residue of Chain J (<i>CPK colored sticks</i>) with the dimethylarsenic modification. (B) Structure of the two most conformationally different complexes of DMSO-treated SOCS2-EloBC (A/B/C and M/N/O), aligned via the backbone atoms of EloB. Protein chains are shown as ribbons, with EloB (<i>cyan</i>), EloC (<i>green</i>), SOCS2 SOCS box (<i>blue</i>) and SOCS2 SH2 domain (<i>red</i>). Chains A/B/C are in darker colors and M/N/O lighter. The linker/hinging region between SOCS2 SOCS box and SH2 domain is shown in black/grey and residue Pro161 as spheres. (C) SH2 domains of DMSO-treated SOCS2-EloBC illustrating the five different conformations observed in the ASU. Chains A/B/C (<i>red</i>), D/E/F (<i>green</i>), G/H/I (<i>blue</i>), J/K/L (<i>yellow</i>) and M/N/O (<i>magenta</i>). Complexes have been aligned via the backbone atoms of EloB from each complex and superposed. Complex P/Q/R is not shown as its conformation is identical to that of D/E/F. (D) Root mean square deviation (RMSD) values (in Å) for the backbone atoms of EloB, EloC, SOCS2 SOCS-box domain (residues 162–198) and SOCS2 SH2 domain (32–134) when complexes are superposed as in <i>C</i>. RMSD values are between each complex and complex A/B/C.</p

Mechanism for dimethylarsenic modification of cysteine residues proposed by Maignan <i>et al</i>. [29].

<p>DTT reduces As(V) of cacodylate in its acid form (p<i>K</i>_a = 6.3) into an As(III)-containing dimethylarsenic-dithiothreitol conjugate. This adduct then reacts with a reduced cysteine sidechain to result in dimethylarsenic cysteine and a replenished molecule of DTT.</p

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

X-ray diffraction data and model statistics for DMSO-treated SOCS2:EloC:EloB.

<p>Values in parentheses are for the highest resolution shell.</p><p>Values for the highest resolution shell are given in parentheses.</p><p>^a<i>R</i>_merge = Σ_<i>hkl</i> Σ_<i>i</i>|(<i>I</i>_<i>i</i>(<i>hkl</i>)–[<i>I</i>(<i>hkl</i>)]|/Σ_<i>hkl</i> Σ_<i>i</i><i>I</i>_<i>i</i>(<i>hkl</i>)</p><p>^b<i>R</i>_work = |<i>F</i>_obs–<i>F</i>_calc|/|<i>F</i>_obs|, where <i>F</i>_obs and <i>F</i>_calc are the observed and calculated structure amplitudes, respectively.</p><p>^c<i>R</i>_free is <i>R</i>_work for the 5% validation set.</p><p>X-ray diffraction data and model statistics for DMSO-treated SOCS2:EloC:EloB.</p

Selective Wee1 degradation by PROTAC degraders recruiting VHL and CRBN E3 ubiquitin ligases

The Ser/Thr protein kinase Wee1 plays a regulatory role at the G 2/M checkpoint by phosphorylating CDK1 when DNA is damaged to allow time for DNA to repair, disruption of which is a key approach to sensitise cancer cells to DNA-damaging therapies. The main selective inhibitor for Wee1 undergoing development in clinical trials, AZD1775, however, has been shown to have off target effects towards other protein kinases with similar potency. Here we describe the synthesis and assessment of a series of Wee1-degrading PROTACs using AZD1775 linked to either the VHL ligand VH032 or to the CRBN ligand pomalidomide using different types and lengths of linkers. The conversion of AZD1775 into a PROTAC induces selective Wee1 degradation for compounds of both series depending on the nature of the linker