SUMMARY Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that “high-throughput kinase profiling” is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1/PLK1–3 and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2 and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.

Overexpression of the CXCR4 receptor is a hallmark of chronic lymphocytic leukemia (CLL) and is important for CLL cell survival, migration, and interaction with their protective microenvironment. In acute myelogenous leukemia (AML), PIM1 was shown to regulate the surface expression of the CXCR4 receptor. Here, we show that PIM (proviral integration site for Moloney murine leukemia virus) kinases 1-3 are overexpressed and that the CXCR4 receptor is hyperphosphorylated on Ser339 in CLL compared with normal lymphocytes. Furthermore, CXCR4 phosphorylation correlates with PIM1 protein expression and PIM1 transcript levels in CLL. PIM kinase inhibition with three different PIM kinase inhibitors induced apoptosis in CLL cells independent of the presence of protective stromal cells. In addition, PIM inhibition caused dephosphorylation of the CXCR4 receptor on Ser339, resulting in enhanced ligand-dependent CXCR4 internalization and reduced re-externalization after withdrawal of CXCL12. Furthermore, PIM inhibition in CLL cells blocked CXCR4 functions, such as migration toward CXCL12-or CXCL12-induced extracellular signal-regulated kinase (ERK) phosphorylation. In concordance, pretreatment of CLL cells with PIM kinase inhibitors strongly reduced homing of CLL cells toward the bone marrow and the spleen of Rag2

For studies of RNA structure, folding, and catalysis, site-specific modifications are typically introduced by solid-phase synthesis of RNA oligonucleotides using nucleoside phosphoramidites. Here, we report the preparation of two complete series of RNA nucleoside phosphoramidites; each has an appropriately protected amine or thiol functional group. The first series includes each of the four common RNA nucleotides, U, C, A, and G, with a 2'-(2-aminoethoxy)-2'-deoxy substitution (i.e., a primary amino group tethered to the 2'-oxygen by a two-carbon linker). The second series encompasses the four common RNA nucleotides, each with the analogous 2'-(2-mercaptoethoxy)-2'-deoxy substitution (i.e., a tethered 2'-thiol). The amines are useful for acylation and reductive amination reactions, and the thiols participate in displacement and oxidative cross-linking reactions, among other likely applications. The new phosphoramidites will be particularly valuable for enabling site-specific introduction of biophysical probes and constraints into RNA.

As the canonical image of molecular biology, the DNA double helix exemplifies stability and structural integrity. The persistence length of duplex DNA is several hundred base pairs (1 base pair ) 3.4 Å ) 0.34 nm), 1 suggesting that the DNA double helix may be a useful nanoscale construction element. 2 Previous efforts used DNA as a static framework for assembling other preformed objects 3 or for self-assembly of DNA, 4 and dynamic DNA machines have been constructed. 5 However, any practical nanotechnology must have moving parts not made solely out of DNA. In this report, we describe for the first time the application of DNA to control the conformation of another macromolecule, RNA. Our efforts suggest a more complete DNA nanotechnology in which the structures of a wide variety of molecules are brought under rational control using DNA. Figure 1A schematically illustrates the application of a covalently attached duplex DNA constraint to control macromolecular conformation, as applied to RNA (for a more detailed version, see Figure S2). For the macromolecular RNA, we used the Tetrahymena group I intron P4-P6 domain ( Figure 1B). This RNA folds in a Mg 2+ -dependent manner via formation of interactions among Watson-Crick paired and unpaired secondary structure elements. 7 P4-P6 is structurally well-characterized, 6 and a nondenaturing polyacrylamide gel electrophoresis (native PAGE) approach for monitoring its folding has been described. 8 The two DNA strands that form the duplex constraint were attached to P4-P6 by reductive amination (Scheme 1). This was achieved using a 2′-amino group on each of two P4-P6 nucleotides, which were coupled with complementary nonpalindromic DNA oligonucleotides that each have a 5′-aldehyde created by periodate oxidation. Pairs of RNA nucleotides were chosen for DNA derivatization on the basis of the P4-P6 X-ray crystal structure, 6 which reveals particular 2′-hydroxyl groups that are exposed to solvent ( Figure 1B). Exposed 2′-hydroxyl groups are good sites for appending DNA strands without disrupting the RNA structure.RNA generally requires Mg 2+ for folding. 9 When a 10-base pair (bp) DNA constraint was created using complementary DNA strands attached at P4-P6 nucleotides U107 and C240, a substantial increase in the Mg 2+ requirement for P4-P6 folding was observed by native PAGE (Figure 2, 2 versus b). The increased Mg 2+ requirement when the DNA constraint is attached implies a less favorable free energy of RNA folding (∆G°), and this effect is readily quantified from the shift in the Mg 2+ midpoint of the titration curve. 8 The observed energetic effect of ∆∆G°> 6 kcal/mol is consistent with the P4-P6 X-ray crystal structure. 6 This structure suggests that a 10-bp duplex (length ∼34 Å) is too short to span U107-C240, because the 2′-positions are ∼56 Å apart in the folded RNA. Therefore, the duplex DNA constraint must either fray partially, distort considerably, or melt entirely if the RNA is to fold properly (see Figure S3 for computer modeling). The ∆G°is reduced by the energy ...