Trio-pharmacophore DNA-encoded chemical library for simultaneous selection of fragments and linkers.

The split-and-pool method has been widely used to synthesize chemical libraries of a large size for early drug discovery, albeit without the possibility of meaningful quality control. In contrast, a self-assembled DNA-encoded chemical library (DEL) allows us to construct an m x n-member library by mixing an m-member and an n-member pre-purified sub-library. Herein, we report a trio-pharmacophore DEL (T-DEL) of m x l x n members through assembling three pre-purified and validated sub-libraries. The middle sub-library is synthesized using DNA-templated synthesis with different reaction mechanisms and designed as a linkage connecting the fragments displayed on the flanking two sub-libraries. Despite assembling three fragments, the resulting compounds do not exceed the up-to-date standard of molecular weight regarding drug-likeness. We demonstrate the utility of T-DEL in linker optimization for known binding fragments against trypsin and carbonic anhydrase II and by de novo selections against matrix metalloprotease-2 and -9.

Nonlinear manipulation and analysis of large DNA datasets.

Information processing functions are essential for organisms to perceive and react to their complex environment, and for humans to analyze and rationalize them. While our brain is extraordinary at processing complex information, winner-take-all, as a type of biased competition is one of the simplest models of lateral inhibition and competition among biological neurons. It has been implemented as DNA-based neural networks, for example, to mimic pattern recognition. However, the utility of DNA-based computation in information processing for real biotechnological applications remains to be demonstrated. In this paper, a biased competition method for nonlinear manipulation and analysis of mixtures of DNA sequences was developed. Unlike conventional biological experiments, selected species were not directly subjected to analysis. Instead, parallel computation among a myriad of different DNA sequences was carried out to reduce the information entropy. The method could be used for various oligonucleotide-encoded libraries, as we have demonstrated its application in decoding and data analysis for selection experiments with DNA-encoded chemical libraries against protein targets.

Tyrosine Kinase c-MET as Therapeutic Target for Radiosensitization of Head and Neck Squamous Cell Carcinomas.

The receptor tyrosine kinase c-MET activates intracellular signaling and induces cell proliferation, epithelial-to-mesenchymal-transition and migration. Within the present study, we validated the prognostic value of c-MET in patients with head and neck squamous cell carcinoma (HNSCC) treated with radio(chemo)therapy using the Cancer Genome Atlas database and found an association of increased MET gene expression and protein phosphorylation with reduced disease-specific and progression-free survival. To investigate the role of c-MET-dependent radioresistance, c-MET-positive cells were purified from established HNSCC cell lines and a reduced radiosensitivity and enhanced sphere-forming potential, compared to the c-MET-depleted cell population, was found in two out of four analyzed cell lines pointing to regulatory heterogeneity. We showed that c-MET is dynamically regulated after irradiation in vitro and in vivo. Interestingly, no direct impact of c-MET on DNA damage repair was found. The therapeutic potential of eight c-MET targeting agents in combination with irradiation demonstrated variable response rates in six HNSCC cell lines. Amongst them, crizotinib, foretinib, and Pha665752 exhibited the strongest radiosensitizing effect. Kinase activity profiling showed an association of crizotinib resistance with compensatory PI3K/AKT and MAP kinase signaling. Overall, our results indicate that c-MET is conferring radioresistance in HNSCC through modulation of intracellular kinase signaling and stem-like features.

Influence of the geometry of fluorescently labelled DNA constructs on fluorescence anisotropy assay.

DNA-encoded chemical libraries (DECLs) are powerful tools for modern drug discovery. A DECL is a pooled mixture of small molecule compounds, each of which is tagged with a unique DNA sequence which functions as a barcode. After incubation with a drug target and washing to remove non-binders, the bound molecules are eluted and submitted for DNA sequencing to determine which molecules are binding the target. While the DECL technology itself is ultra-high throughput, the following re-synthesis of identified compounds for orthogonal validation experiments remains the bottleneck. Using existing DNA-small molecule conjugates directly for affinity measurements, as opposed to complete compound resynthesis, could accelerate the discovery process. To this end, we have tested various geometries of fluorescently-labelled DNA constructs for fluorescence anisotropy (FA) experiments. Minimizing the distance between the fluorescent moiety and ligand can maximize the correlation between ligand-protein interaction and corresponding change in fluorophore rotational freedom, thus leading to larger, easier to interpret changes in FA values. However, close proximity can also cause artifacts due to potentially promiscuous interactions between fluorophore and protein. By balancing these two opposite effects, we have identified applicable fluorescently labelled DNA constructs displaying either a single ligand or pairs of fragments for affinity measurement using a FA assay.

Second generation DNA-encoded dynamic combinatorial chemical libraries.

We present a DNA-encoded chemical library, which allows dynamic selection followed by ligation of the encoding strands. As a chemical approach to mimic the genetic recombination process of adaptive immunity, the technology led to an enhanced enrichment factor and signal-to-noise ratio compared to static libraries.

The CD98 Heavy Chain Is a Marker and Regulator of Head and Neck Squamous Cell Carcinoma Radiosensitivity.

PURPOSE: The heavy chain of the CD98 protein (CD98hc) is encoded by the SLC3A2 gene. Together with the light subunit LAT1, CD98hc constitutes a heterodimeric transmembrane amino acid transporter. High SLC3A2 mRNA expression levels are associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC) treated with radiochemotherapy. Little is known regarding the CD98hc protein-mediated molecular mechanisms of tumor radioresistance. EXPERIMENTAL DESIGN: CD98hc protein expression levels were correlated with corresponding tumor control dose 50 (TCD50) in HNSCC xenograft models. Expression levels of CD98hc and LAT1 in HNSCC cells were modulated by siRNA or CRISPR/Cas9 gene editing. HNSCC cell phenotypes were characterized by transcription profiling, plasma membrane proteomics, metabolic analysis, and signaling pathway activation. Expression levels of CD98hc and LAT1 proteins were examined by IHC analysis of tumor tissues from patients with locally advanced HNSCC treated with primary radiochemotherapy (RCTx). Primary endpoint was locoregional tumor control (LRC). RESULTS: High expression levels of CD98hc resulted in an increase in mTOR pathway activation, amino acid metabolism, and DNA repair as well as downregulation of oxidative stress and autophagy. High expression levels of CD98hc and LAT1 proteins were significantly correlated and associated with an increase in radioresistance in HNSCC in vitro and in vivo models. High expression of both proteins identified a poor prognosis subgroup in patients with locally advanced HNSCC after RCTx. CONCLUSIONS: We found that CD98hc-associated signaling mechanisms play a central role in the regulation of HNSCC radioresistance and may be a promising target for tumor radiosensitization.

Evolution of a multi-step phosphorelay signal transduction system in Ensifer: recruitment of the sigma factor RpoN and a novel enhancer-binding protein triggers acid-activated gene expression.

Most Ensifer strains are comparatively acid sensitive, compromising their persistence in low pH soils. In the acid-tolerant strain Ensifer medicae WSM419, the acid-activated expression of lpiA is essential for enhancing survival in lethal acidic conditions. Here we characterise a multi-step phosphorelay signal transduction pathway consisting of TcsA, TcrA, FsrR, RpoN and its cognate enhancer-binding protein EbpA, which is required for the induction of lpiA and the downstream acvB gene. The fsrR, tcrA, tcsA and rpoN genes were constitutively expressed, whereas lpiA and acvB were strongly acid-induced. RACE mapping revealed that lpiA/acvB were co-transcribed as an operon from an RpoN promoter. In most Ensifer species, lpiA/acvB is located on the chromosome and the sequence upstream of lpiA lacks an RpoN-binding site. Nearly all Ensifer meliloti strains completely lack ebpA, tcrA, tcsA and fsrR regulatory loci. In contrast, E. medicae strains have lpiA/acvB and ebpA/tcrA/tcsA/fsrR co-located on the pSymA megaplasmid, with lpiA/acvB expression coupled to an RpoN promoter. Here we provide a model for the expression of lpiA/acvB in E. medicae. This unique acid-activated regulatory system provides insights into an evolutionary process which may assist the adaptation of E. medicae to acidic environmental niches.