Development of SYK NanoBRET Cellular Target Engagement Assays for Gain-of-Function Variants.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK.

Protomer selectivity of type II RAF inhibitors within the RAS/RAF complex.

RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells.

DELs enable the development of BRET probes for target engagement studies in cells.

DNA-encoded libraries (DELs) provide unmatched chemical diversity and starting points for novel drug modalities. Here, we describe a workflow that exploits the bifunctional attributes of DEL ligands as a platform to generate BRET probes for live cell target engagement studies. To establish proof of concept, we performed a DEL screen using aurora kinase A and successfully converted aurora DEL ligands as cell-active BRET probes.

Development of Cell Permeable NanoBRET Probes for the Measurement of PLK1 Target Engagement in Live Cells.

PLK1 is a protein kinase that regulates mitosis and is both an important oncology drug target and a potential antitarget of drugs for the DNA damage response pathway or anti-infective host kinases. To expand the range of live cell NanoBRET target engagement assays to include PLK1, we developed an energy transfer probe based on the anilino-tetrahydropteridine chemotype found in several selective PLK inhibitors. Probe was used to configure NanoBRET target engagement assays for PLK1, PLK2, and PLK3 and measure the potency of several known PLK inhibitors.

Development of Cell Permeable NanoBRET Probes for the Measurement of PLK1 Target Engagement in Live Cells.

PLK1 is a protein kinase that regulates mitosis and is both an important oncology drug target and a potential anti target of drugs for the DNA damage response pathway or anti-infective host kinases. To expand the range of live cell NanoBRET target engagement assays to include PLK1 we developed an energy transfer probe based on the anilino-tetrahydropteridine chemotype found in several selective PLK inhibitors. Probe 11 was used to configure NanoBRET target engagement assays for PLK1, PLK2, and PLK3 and measure the potency of several known PLK inhibitors.

KRAS is vulnerable to reversible switch-II pocket engagement in cells.

Current small-molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket (SII-P), exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the SII-P is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory.

Quantitative Analysis of Multimodal Skeletal SPECT/CT Reconstructions in Diagnosing Medication-related Osteonecrosis of the Jaw.

Aim: Our goal was to assess visual and quantitative aspects of multimodal skeletal SPECT/CT reconstructions (recon) in differentiating necrotic and healthy bone of patients with suspected MRONJ.

Methods: Prior to surgery, 20 patients with suspected MRONJ underwent SPECT/CT of the jaw 3-4 hours after injection of Tc-99m-DPD (622±112.4 MBq).

Differential Regulation of Genes following Binding to Mannose Receptors.

Regulation of the uropathogenic (UPEC) and genes was examined following type 1 pili binding to mannose-coated Sepharose beads. Within 25 min after mannose attachment, expression dropped eightfold, whereas transcription increased about two- to fourfold. Because both genes encode site-specific recombinases that affect the position of the element containing the promoter, the positioning of was also examined.

Photoselective vaporization prostatectomy: experience with a novel 180 W 532 nm lithium triborate laser and fiber delivery system in living dogs.

Purpose: We studied vaporization parameters, and anatomical and histopathological outcomes of photoselective vaporization of the prostate with the novel GreenLight™ XPS™ 180 W, 532 nm lithium triborate laser and MoXy™ fiber in a survival model of living dogs. We compared these findings with those of the existing GreenLight HPS™ 120 W 532 nm lithium triborate laser photoselective vaporization of the prostate in living dogs.

Materials And Methods: Eight dogs underwent antegrade photoselective vaporization of the prostate with the 180 W laser delivered through a new 750 μm (vs the existing 600 μm core diameter), 50% larger, spot sized, side firing fiber.

Down-regulation of the kps region 1 capsular assembly operon following attachment of Escherichia coli type 1 fimbriae to D-mannose receptors.

A differential-display PCR procedure identified the capsular assembly gene kpsD after Escherichia coli type 1 fimbrial binding to mannose-coated Sepharose beads. Limiting-dilution reverse-transcribed PCRs confirmed down-regulation of the kpsD gene, and Northern blot and lacZ fusion analyses showed down-regulation of the kpsFEDUCS region 1 operon. KpsD protein levels fell, and an agglutination test showed less K capsular antigen on the surface following the bacterial ligand-receptor interaction.

Prolactin antagonist-endostatin fusion protein as a targeted dual-functional therapeutic agent for breast cancer.

In previous studies (Chen, W. Y. et al.

Regulation of bcl-2 gene expression in human breast cancer cells by prolactin and its antagonist, hPRL-G129R.

To gain insight into the molecular basis of human prolactin (hPRL) antagonist induced apoptosis, we compared the differential gene expression profile of four human breast cancer cell lines following treatment with hPRL and its antagonist (hPRL-G129R). Among the genes identified, the bcl-2 gene was of particular interest. We found that bcl-2 mRNA was up regulated in three of the four cell lines that were treated with hPRL.

Osmolarity and pH growth conditions regulate fim gene transcription and type 1 pilus expression in uropathogenic Escherichia coli.

A comparative study was performed to determine the effects of pH, osmolarity, and human urine on the transcription of several fim genes, as well as the overall expression of type 1 pili. Several fim-lacZYA fusions were constructed on single-copy plasmids to test a range of pHs and a range of osmolarities. Growth in acidic medium slightly reduced expression from all of the fim promoters (fimA, fimB, and fimE).