The Impact of Variable Selection Coverage on Detection of Ligands from a DNA-Encoded Library Screen.

DNA-encoded library (DEL) technology has become a prominent screening platform in drug discovery owing to the capacity to screen billions or trillions of compounds in a single experiment. Although numerous successes with DEL technology have been reported, we are unaware of a rigorous examination of the many different variables that can influence a screen's success. Herein, we explore the impact of variable sample sequencing depth on the detection of tool compounds with known affinities toward a given target while simultaneously probing the effect of initial compound input. Our sequencing data confirm reports that high-affinity compounds can be discovered directly from a DEL screen, but we demonstrate that a mismatch between selection output and sequencing quantity can obscure useful ligands. Our results highlight the importance of selection coverage in grasping the entire picture of a DEL screen where the signal of a weak or underrepresented ligand may be suppressed by the inherent noise of a selection. These potential missed ligands may be critical to the success or failure of a drug discovery program.

Characterization of Ser338 phosphorylation for Raf-1 activation.

Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser338 on Raf-1 is autophosphorylated in response to mitogens.

Identification of beta-cell-specific insulin gene transcription factor RIPE3b1 as mammalian MafA.

Of the three critical enhancer elements that mediate beta-cell-specific and glucose-responsive expression of the insulin gene, only the identity of the transcription factor binding to the RIPE3b element (RIPE3b1) has remained elusive. Using a biochemical purification approach, we have identified the RIPE3b1 factor as a mammalian homologue of avian MafA/L-Maf (mMafA). The avian MafA is a cell-type determination factor that expressed ectopically can trigger lens differentiation program, but no mammalian homologue of avian MafA has previously been identified. Here, we report cloning of the human mafA (hMafA) and demonstrate that it can specifically bind the insulin enhancer element RIPE3b and activate insulin-gene expression. In addition, mMafA has a very restrictive cellular distribution and is selectively expressed in pancreatic beta but not in alpha cells. We suggest that mMafA has an essential role in the function and differentiation of beta-cells and thus may be associated with the pathophysiological origins of diabetes.