Hits validation using protein based ¹H-¹⁵N HSQC.

<p>(A) Spectrum of full length HuR (green) (B) Spectrum of full length HuR with Compound C11 (red) (C) Spectral overlay of free (green) and C10 bound (blue).</p

Identification of Small-Molecule Inhibitors of the HuR/RNA Interaction Using a Fluorescence Polarization Screening Assay Followed by NMR Validation

<div><p>The human antigen R (HuR) stabilizes many mRNAs of proto-oncogene, transcription factors, cytokines and growth factors by recognizing AU-rich elements (AREs) presented in their 3’ or 5’ untranslated region (UTR). Multiple lines of experimental evidence suggest that this process plays a key role in cancer development. Thus, destabilizing HuR/RNA interaction by small molecules presents an opportunity for cancer treatment/prevention. Here we present an integrated approach to identify inhibitors of HuR/RNA interaction using a combination of fluorescence-based and NMR-based high throughput screening (HTS). The HTS assay with fluorescence polarization readout and Z’-score of 0.8 was used to perform a screen of the NCI diversity set V library in a 384 well plate format. An NMR-based assay with saturation transfer difference (STD) detection was used for hits validation. Protein NMR spectroscopy was used to demonstrate that some hit compounds disrupt formation of HuR oligomer, whereas others block RNA binding. Thus, our integrated high throughput approach provides a new avenue for identification of small molecules targeting HuR/RNA interaction.</p></div

C10 displaces the RNA from RRM1-2/RNA complex.

<p>(A) Overlay of free RRM1-2 (blue) and RRM1-2/RNA (red). (B) Overlay of free RRM1-2 (blue) and RRM1-2/RNA/C10. (C) Zoom-in region of (A). (D) Zoom-in region of (B). When RNA binds to RRM1-2, many peaks disappeared as indicated in the cycle and arrow. The peaks come back once adding C10 to RRM1-2/RNA complex, indicating that C10 does block the RRM1-2/RNA interaction.</p

Secondary screening as hits validation using NMR STD approach.

<p>4 out of 12 compounds from our primary screening showed direct interactions with HuR. The data were processed and plotted using the shell script developed for automation in our lab, making STD screening faster, robust and competitive to HTS.</p

CHARLES data

CHARLES dat

MOESM6 of Improving chemical similarity ensemble approach in target prediction

Additional file 6.  Kinase test data set

The Mechanism by which 146-N-Glycan Affects the Active Site of Neuraminidase

<div><p>One of the most conserved glycosylation sites of neuraminidase (NA) is 146-N-glycan. This site is adjacent to the 150-cavity of NA, which is found within the active site and thought to be a target for rational drug development against the antiviral resistance of influenza. Here, through a total of 2.4 μs molecular dynamics (MD) simulations, we demonstrated that 146-N-glycan can stabilize the conformation of the 150-loop that controls the volume of the 150-cavity. Moreover, with 146-N-glycan, our simulation result was more consistent with crystal structures of NAs than simulations conducted without glycans. Cluster analysis of the MD trajectories showed that 146-N-glycan adopted three distinct conformations: monomer-bridged, dimer-bridged and standing. Of these conformations, the dimer-bridged 146-N-glycan was the most stable one and contributed to stabilization of the 150-loop conformation. Furthermore, our simulation revealed that various standing conformations of 146-N-glycan could block the entrance of the binding pocket. This result was consistent with experimental data and explained the relatively low activity of inhibitors with flexible substituents toward the 150-cavity. Together, our results lead us to hypothesize that rigid and hydrophobic substituents could serve as better inhibitors targeting the 150-cavity.</p></div

Bovine Mammary Gene Expression Profiling during the Onset of Lactation

<div><p>Background</p><p>Lactogenesis includes two stages. Stage I begins a few weeks before parturition. Stage II is initiated around the time of parturition and extends for several days afterwards.</p><p>Methodology/Principal Findings</p><p>To better understand the molecular events underlying these changes, genome-wide gene expression profiling was conducted using digital gene expression (DGE) on bovine mammary tissue at three time points (on approximately day 35 before parturition (−35 d), day 7 before parturition (−7 d) and day 3 after parturition (+3 d)). Approximately 6.2 million (M), 5.8 million (M) and 6.1 million (M) 21-nt cDNA tags were sequenced in the three cDNA libraries (−35 d, −7 d and +3 d), respectively. After aligning to the reference sequences, the three cDNA libraries included 8,662, 8,363 and 8,359 genes, respectively. With a fold change cutoff criteria of ≥2 or ≤−2 and a false discovery rate (FDR) of ≤0.001, a total of 812 genes were significantly differentially expressed at −7 d compared with −35 d (stage I). Gene ontology analysis showed that those significantly differentially expressed genes were mainly associated with cell cycle, lipid metabolism, immune response and biological adhesion. A total of 1,189 genes were significantly differentially expressed at +3 d compared with −7 d (stage II), and these genes were mainly associated with the immune response and cell cycle. Moreover, there were 1,672 genes significantly differentially expressed at +3 d compared with −35 d. Gene ontology analysis showed that the main differentially expressed genes were those associated with metabolic processes.</p><p>Conclusions</p><p>The results suggest that the mammary gland begins to lactate not only by a gain of function but also by a broad suppression of function to effectively push most of the cell's resources towards lactation.</p></div

Conformation of Inhibitor 20l bind to N1 with 146-glycan.

<p>Conformation of N1 protein (green) come from crystal structure (PDB ID: 2HU0). 146-glycan, which was from 2HTY by superposition, was colored orange. Important residues V116, I117, Q136, A138 and V149, which construct the hydrophobic zone of 150-cavity were colored cyan. The long rigid substituent of 20l avoids binding to blocking zone of the 146-glycan and binds to the hydrophobic zone of 150-cavity.</p

Differentially expressed tags at the onset of lactation.

<p>(A) Differentially expressed tags at −7 d compared with −35 d. (B) Differentially expressed tags at +3 d compared with −7 d. (C) Differentially expressed tags at +3 d compared with −35 d. The “x” represents the fold-change of differentially expressed unique tags. The “y” axis represents the number of unique tags (log10). The red region represents differentially accumulating unique tags with a 5-fold difference between libraries. The green and blue regions represent unique tags that are upregulated and downregulated for more than 5 fold.</p