Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

The present study reports the discovery of a small-molecule negative allosteric modulator for the β2-adrenergic receptor (β2AR) via in vitro affinity-based iterative selection of highly diverse DNA-encoded small-molecule libraries. Characterization of the compound demonstrates its selectivity for the β2AR and that it negatively modulates a wide range of receptor functions. More importantly, our findings establish a generally applicable, proof-of-concept strategy for screening DNA-encoded small-molecule libraries against purified G-protein–coupled receptors (GPCRs), which holds great potential for discovering therapeutic molecules

Describing knowledge encounters in healthcare: a mixed studies systematic review and development of a classification

This review was self-funded

Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf

Isoprenylcysteine carboxyl methyltransferase (Icmt) methylates the carboxyl-terminal isoprenylcysteine of CAAX proteins (e.g., Ras and Rho proteins). In the case of the Ras proteins, carboxyl methylation is important for targeting of the proteins to the plasma membrane. We hypothesized that a knockout of Icmt would reduce the ability of cells to be transformed by K-Ras. Fibroblasts harboring a floxed Icmt allele and expressing activated K-Ras (K-Ras-Icmt(flx/flx)) were treated with Cre-adenovirus, producing K-Ras-Icmt(Δ/Δ) fibroblasts. Inactivation of Icmt inhibited cell growth and K-Ras–induced oncogenic transformation, both in soft agar assays and in a nude mice model. The inactivation of Icmt did not affect growth factor–stimulated phosphorylation of Erk1/2 or Akt1. However, levels of RhoA were greatly reduced as a consequence of accelerated protein turnover. In addition, there was a large Ras/Erk1/2-dependent increase in p21(Cip1), which was probably a consequence of the reduced levels of RhoA. Deletion of p21(Cip1) restored the ability of K-Ras-Icmt(Δ/Δ) fibroblasts to grow in soft agar. The effect of inactivating Icmt was not limited to the inhibition of K-Ras–induced transformation: inactivation of Icmt blocked transformation by an oncogenic form of B-Raf (V599E). These studies identify Icmt as a potential target for reducing the growth of K-Ras– and B-Raf–induced malignancies

Mice with an isoform-ablating Mecp2 exon 1 mutation recapitulate the neurologic deficits of Rett syndrome

Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT OMIM 312750). Alternative inclusion of MECP2/Mecp2 exon 1 with exons 3 and 4 encodes MeCP2-e1 or MeCP2-e2 protein isoforms with unique amino termini. While most MECP2 mutations are located in exons 3 and 4 thus affecting both isoforms, MECP2 exon 1 mutations but not exon 2 mutations have been identified in RTT patients, suggesting that MeCP2-e1 deficiency is sufficient to cause RTT. As expected, genetic deletion of Mecp2 exons 3 and/or 4 recapitulates RTT-like neurologic defects in mice. However, Mecp2 exon 2 knockout mice have normal neurologic function. Here, a naturally occurring MECP2 exon 1 mutation is recapitulated in a mouse model by genetic engineering. A point mutation in the translational start codon of Mecp2 exon 1, transmitted through the germline, ablates MeCP2-e1 translation while preserving MeCP2-e2 production in mouse brain. The resulting MeCP2-e1 deficient mice developed forelimb stereotypy, hindlimb clasping, excessive grooming and hypo-activity prior to death between 7 and 31 weeks. MeCP2-e1 deficient mice also exhibited abnormal anxiety, sociability and ambulation. Despite MeCP2-e1 and MeCP2-e2 sharing, 96% amino acid identity, differences were identified. A fraction of phosphorylated MeCP2-e1 differed from the bulk of MeCP2 in subnuclear localization and co-factor interaction. Furthermore, MeCP2-e1 exhibited enhanced stability compared with MeCP2-e2 in neurons. Therefore, MeCP2-e1 deficient mice implicate MeCP2-e1 as the sole contributor to RTT with non-redundant functions

Reduced <i>Ppd</i> chromosomes at birth – CZECHII/EiJ cross.

<p>Reduced <i>Ppd</i> chromosomes at birth – CZECHII/EiJ cross.</p

Typical <i>Ppd</i> caudal duplications, among numerous anomalies observed in mutants.

<p>Please see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003967#pgen.1003967-Lehoczky1" target="_blank">[15]</a> for a complete description of other anomalies, listed in the Introduction. Variation in presentation of ectopic legs with or without caudal masses has been observed.</p

<i>Ppd</i> characteristic caudal mass/ectopic limb phenotypes in engineered offspring.

<p>A. (one panel) Bruce4.G9 targeted ES cell clone (9281)-derived chimeric male produced a female heterozygous carrier (Neo⁺/eETn⁺) that was mated to a β-actin FLPe male to produce the newborn (Neo⁻/eETn⁺) shown in the panel here. Genotyping confirmed the proper FLPe recombination fragment demonstrating Neo cassette removal in this mouse. B., C. Typical caudal masses with ectopic limbs in offspring of Neo⁻/eETn⁺ mice. 9 out of 69 offspring from mating between Neo⁻/eETn⁺ males X B6/D2 F₁ females demonstrated typical caudal masses/ectopic limbs (3 panels in B), whereas 8 out of 31 offspring from mating between Neo⁻/eETn⁺ males X FVB females demonstrated typical caudal masses and in one mouse a bifurcated tail seen at birth and later at 3 weeks (5 panels in C).</p

<i>Dusp9</i> mRNA and protein are increased in mutant ES cells.

<p>A, B. Taqman confirmation of increased <i>Dusp9</i> mRNA in mutant ES cells. ES cell steady-state <i>Dusp9</i> mRNA quantitation in ES cell lines (D3, C4, and D5) derived from the original <i>Ppd</i> strain (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003967#s5" target="_blank">Materials and Methods</a>) compared with Bruce4.G9 ES cell RNA. Steady-state <i>Dusp9</i> mRNA levels in engineered eETn targeted ES cell lines (928B, 928D, 9281, 9284, 9287) derived by homologous recombination in Bruce4.G9 cells, compared to <i>Dusp9</i> expression in wild-type Bruce4 ES cells. All targeted ES cell lines also demonstrate a large increase in <i>Dusp9</i> mRNA compared to Bruce4.G9; n = 3 independent RNA preparations. Note variability of RNA expression across different ES cell lines. C. Increased expression of DUSP9 protein in mutant ES cells. DUSP9 protein was quantitated by Western blot with antibody to DUSP9 (top blot), produced and kindly supplied by R. Dickinson, relative to β-actin (lower blot) from normal ES cells (PAT-5, ND-D3, UMB6J-D7, and Bruce4.G9) compared to original <i>Ppd</i> mutant-derived ES cells (Ppd-D3, Ppd-D5, Ppd-C4) and eETn ES cells (as shown on right, blue bars); graph below represents data from two separate protein extraction experiments. Bruce4.G9 is the parent ES cell line for eETn cells. Data with the second antibody (anti-MKP4; Santa Cruz Biotech.) was identical (data not shown). Pre-incubation of each antibody with a synthesized peptide to DUSP9 reduced or eliminated the signal observed in ES cells, and pre-incubation with a control peptide did not visibly alter the DUSP9 signals (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003967#pgen.1003967.s005" target="_blank">Figure S5</a>).</p