A Pyrazolo[3,4‑<i>d</i>]pyrimidin-4-amine Derivative Containing an Isoxazole Moiety Is a Selective and Potent Inhibitor of RET Gatekeeper Mutants

Aberrant RET kinase signaling plays critical roles in several human cancers such as thyroid carcinoma. The gatekeeper mutants (V804L or V804M) of RET are resistant to currently approved RET inhibitors such as cabozantinib and vandetanib. We, for the first time, report a highly selective and extremely potent RET inhibitor, <b>6i</b> rationally designed. Compound <b>6i</b> inhibits strongly RET gatekeeper mutants and other clinically relevant RET mutants as well as wt-RET. This substance also significantly suppresses growth of thyroid cancer-derived TT cell lines and Ba/F3 cells transformed with various RET mutants. Docking studies reveal that the isoxazole moiety in <b>6i</b> is responsible for binding affinity improvement by providing additional site for H-bonding with Lys758. Also, <b>6i</b> not only substantially blocks cellular RET autophosphorylation and its downstream pathway, it markedly induces apoptosis and anchorage-independent growth inhibition in TT cell lines while having no effect on normal thyroid Nthy ori-3-1 cells

High-Throughput Profiling of Peptide–RNA Interactions Using Peptide Microarrays

A rapid and quantitative method to evaluate binding properties of hairpin RNAs to peptides using peptide microarrays has been developed. The microarray technology was shown to be a powerful tool for high-throughput analysis of RNA–peptide interactions by its application to profiling interactions between 111 peptides and six hairpin RNAs. The peptide microarrays were also employed to measure hundreds of dissociation constants (<i>K</i>_d) of RNA–peptide complexes. Our results reveal that both hydrophobic and hydrophilic faces of amphiphilic peptides are likely involved in interactions with RNAs. Furthermore, these results also show that most of the tested peptides bind hairpin RNAs with submicromolar <i>K</i>_d values. One of the peptides identified by using this method was found to have good inhibitory activity against TAR–Tat interactions in cells. Because of their great applicability to evaluation of nearly all types of RNA–peptide interactions, peptide microarrays are expected to serve as robust tools for rapid assessment of peptide–RNA interactions and development of peptide ligands against RNA targets

Effects of OCLI-023 on RANKL-induced mRNA expression during osteoclast differentiation and resorption pit formation.

<p>(A) BMMs were cultured in the presence of M-CSF (10 ng/mL) and RANKL (20 ng/mL) with vehicle or OCLI-023 (5 μM). The mRNA expression of TRAP (<i>Acp5</i>), cathepsin K (<i>Ctsk</i>), DC-STAMP (<i>Dcstamp</i>), and NFATc1 (<i>Nfatc1</i>) was analyzed using real-time quantitative PCR. (B) BMMs were seeded on bone slices and incubated with M-CSF (10 ng/mL) and RANKL (20 ng/mL) to induce osteoclast differentiation. After 3 days, the cells were incubated with or without OCLI-023 (5 μM) for an additional 2 days. Resorption pits were observed by hematoxylin staining (upper) and scanning electron microscopy (lower). **<i>p</i> < 0.01 versus the vehicle-treated control.</p

Effects of OCLI-023 on RANKL-stimulated signaling.

<p>BMMs were serum-starved for 5 h, then pretreated with OCLI-023 (5 μM) or vehicle for 1 h before RANKL (50 ng/mL) stimulation for the indicated times. Phosphorylation of JNK and IκBα was assessed by western blot. JNK or β-actin was used as the loading control.</p

Histological analysis of alveolar bones in an experimental periodontitis model.

<p>(A) Both nonligated and ligated mice were injected with either vehicle or OCLI-023 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170159#pone.0170159.g004" target="_blank">Fig 4</a>. The fixed maxillae were decalcified, sectioned, and stained with H&E (upper and middle) and with TRAP (lower). Scale bars, 100 μm. P, pulp; D, dentin; IDS, interdental septum of the alveolar process; IRS, interradicular septum of the alveolar process. (B) The osteoclast number per bone surface was assessed. n = 5 in each group. **<i>p</i> < 0.01 versus the ligated, vehicle-treated group.</p

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties-5

<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties"</p><p>http://www.biomedcentral.com/1472-6807/7/47</p><p>BMC Structural Biology 2007;7():47-47.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1936996.</p><p></p> and EstE1(▯), in 20 mM potassium phosphate buffer (pH 7.0) were incubated at 80°C for the indicated times. Residual activities were then determined by measuring the amount of -nitrophenol released by esterase-catalyzed hydrolysis. The activity of a non-incubated sample was defined as 100

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties-2

<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties"</p><p>http://www.biomedcentral.com/1472-6807/7/47</p><p>BMC Structural Biology 2007;7():47-47.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1936996.</p><p></p>from archaeon and the mesophilic brefeldin A esterase (BFAE) from (PDB code ). The regions encompassing EstE1 dimerization motifs and the sequence blocks showing the amino acids involved in the formations of the catalytic triad and oxyanion hole are presented. Identical and similar residues have a grey background. Symbols: ●, amino acids forming a catalytic triad; ○, amino acids involved in oxynion hole formation; □ and ▯, amino acid residues involved in hydrophobic and ionic interactions at 1JJI dimeric interface, respectively; ▯, amino acid residues involved in ionic interactions at 1JKM dimeric interface; ■ and ▲ amino acid residues involved in hydrophobic and ionic interactions at EstE1 dimeric interface, respectively. Amino acid sequence alignment was performed as described previously [5]

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties-1

<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties"</p><p>http://www.biomedcentral.com/1472-6807/7/47</p><p>BMC Structural Biology 2007;7():47-47.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1936996.</p><p></p>ck representation. Val274, Phe276, and Leu299 are involved in the hydrophobic interactions. Arg270-Asp291 and Lys177-Glu295 form salt bridges. (B) The centrosymmetric conformation of the interface between the two monomers, consisting of centric hydrophobic interactions (red circle) and salt bridges. (C) A detailed view of the salt bridges that support the dimeric conformation of EstE1. The side chain of Arg270on the loop between the α8 helix and the β8 strand forms a salt bridge with Asp291on the α9 helix. An additional salt bridge is formed between the side chains of Lys177on the β6 strand and Glu295on the α9 helix. (D) A detailed view of the hydrophobic interaction interface observed in the EstE1 dimer. The hydrophobic core residues (Leu299 on the α9 helix, and Phe276 and Val274 on the β8 strand) are indicated. (E) A detailed view of the interface observed in a current AFEST dimer model [19]. Dimeric interactions of AFEST are supported by hydrogen bonds between Tyr280 and Gln303, and by a weak hydrophobic interaction through Val278

Discovery of a novel potent peptide agonist to adiponectin receptor 1

<div><p>Activation of adiponectin receptors (AdipoRs) by its natural ligand, adiponectin has been known to be involved in modulating critical metabolic processes such as glucose metabolism and fatty acid oxidation as demonstrated by a number of <i>in vitro</i> and <i>in vivo</i> studies over last two decades. These findings suggest that AdipoRs’ agonists could be developed into a potential therapeutic agent for metabolic diseases, such as diabetes mellitus, especially for type II diabetes, a long-term metabolic disorder characterized by high blood sugar, insulin resistance, and relative lack of insulin. Because of limitations in production of biologically active adiponectin, adiponectin-mimetic AdipoRs’ agonists have been suggested as alternative ways to expand the opportunity to develop anti-diabetic agents. Based on crystal structure of AdipoR1, we designed AdipoR1’s peptide agonists using protein-peptide docking simulation and screened their receptor binding abilities and biological functions <i>via</i> surface plasmon resonance (SPR) and biological analysis. Three candidate peptides, BHD1028, BHD43, and BHD44 were selected and confirmed to activate AdipoR1-mediated signal pathways. In order to enhance the stability and solubility of peptide agonists, candidate peptides were PEGylated. PEGylated BHD1028 exhibited its biological activity at nano-molar concentration and could be a potential therapeutic agent for the treatment of diabetes. Also, SPR and virtual screening techniques utilized in this study may potentially be applied to other peptide-drug screening processes against membrane receptor proteins.</p></div

Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties-6

<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties"</p><p>http://www.biomedcentral.com/1472-6807/7/47</p><p>BMC Structural Biology 2007;7():47-47.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1936996.</p><p></p>elical segments and β-strands are shown in blue and yellow, respectively. G2 and G3 represent 3-helices. Helix α1 is not shown because of its disordered electron map. The catalytic triad containing residues Ser154, Asp251, and His281, are shown in stick representation. N and C denote the N and C termini, respectively