High-throughput chemical modification of oligonucleotides for systematic structure-activity relationship evaluation.

Chemical modification of siRNA is achieved in a high-throughput manner (96-well plate format) by copper catalyzed azide-alkyne cycloadditions. This transformation can be performed in one synthetic operation at up to four positions with complete specificity, good yield, and acceptable purity. As demonstrated here, this approach extends the current synthetic options for oligonucleotide modifications and simultaneously facilitates the systematic, rapid biological evaluation of modified siRNA.

mRNA knockdown by single strand RNA is improved by chemical modifications.

While RNAi has traditionally relied on RNA duplexes, early evaluation of siRNAs demonstrated activity of the guide strand in the absence of the passenger strand. However, these single strands lacked the activity of duplex RNAs. Here, we report the systematic use of chemical modifications to optimize single-strand RNA (ssRNA)-mediated mRNA knockdown. We identify that 2'F ribose modifications coupled with 5'-end phosphorylation vastly improves ssRNA activity both in vitro and in vivo. The impact of specific chemical modifications on ssRNA activity implies an Ago-mediated mechanism but the hallmark mRNA cleavage sites were not observed which suggests ssRNA may operate through a mechanism beyond conventional Ago2 slicer activity. While currently less potent than duplex siRNAs, with additional chemical optimization and alternative routes of delivery, chemically modified ssRNAs could represent a powerful RNAi platform.

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and in vivo.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.

Nucleoside optimization for RNAi: a high-throughput platform.

The RNA induced silencing complex (RISC) contains at its core the endonuclease Argonaute (Ago) that allows for guide strand (GS)-mediated sequence-specific cleavage of the target mRNA. Functionalization of the sugar/phosphodiester backbone of the GS, which is in direct contact with Ago, presents a logical opportunity to affect RISC's activity. A systematic evaluation of modified nucleosides requires the synthesis of phosphoramidites corresponding to all four canonical bases (A, U, C, and G) and their sequential evaluation at each position along the 21-nucleotide-long GS. With the use of a platform approach, the sequential replacement of canonical bases with inosine greatly simplifies the problem and defines a new activity baseline toward which the corresponding sugar-modified inosines are compared. This approach was validated using 2'-O-benzyl modification, which demonstrated that positions 5, 8, 15, and 19 can accommodate this large group. Application of this high-throughput methodology now allows for hypothesis-driven rational design of highly potent, immunologically silent and stable siRNAs suitable for therapeutic applications.

Selective kinase inhibition by exploiting differential pathway sensitivity.

Protein kinase inhibitors are optimized to have high affinity for their intended target(s) to elicit the desired cellular effects. Here, we asked whether differences in inhibitory sensitivity between two kinase signaling pathways, controlled by the cyclin-dependent kinases Cdk1 and Pho85, can be sufficient to allow for selective targeting of one pathway over the other. We show the oxindole inhibitor GW297361 elicits a Pho85-selective response in cells despite having a 20-fold greater biochemical potency for Cdk1 in vitro. We provide evidence that partial inhibition of Pho85 is sufficient to activate Pho85-dependent signaling, but partial inhibition of Cdk1 is not sufficient to block Cdk1-dependent cell proliferation. Identification of highly sensitive kinases may provide a means to achieve selective perturbation of kinase signaling pathways complementary to efforts to achieve maximal differences between in vitro IC50 values.

Phosphospecific proteolysis for mapping sites of protein phosphorylation.

Protein phosphorylation is a dominant mechanism of information transfer in cells, and a major goal of current proteomic efforts is to generate a system-level map describing all the sites of protein phosphorylation. Recent efforts have focused on developing technologies for enriching and quantifying phosphopeptides. Identification of the sites of phosphorylation typically relies on tandem mass spectrometry to sequence individual peptides. Here we describe an approach for phosphopeptide mapping that makes it possible to interrogate a protein sequence directly with a protease that recognizes sites of phosphorylation. The key to this approach is the selective chemical transformation of phosphoserine and phosphothreonine residues into lysine analogs (aminoethylcysteine and beta-methylaminoethylcysteine, respectively). Aminoethylcysteine-modified peptides are then cleaved with a lysine-specific protease to map sites of phosphorylation. A blocking step enables single-site cleavage, and adaptation of this reaction to the solid phase facilitates phosphopeptide enrichment and modification in one step.

In vivo activity and duration of short interfering RNAs containing a synthetic 5'-phosphate.

Endogenous and exogenous short interfering RNAs (siRNAs) require a 5'-phosphate for loading into Ago2 and cleavage of the target mRNA. We applied a synthetic 5'-phosphate to siRNA guide strands to evaluate if phosphorylation in vivo is rate limiting for maximal siRNA knockdown and duration. We report, for the first time, an in vivo evaluation of siRNAs with a synthetic 5'-phosphate compared to their unphosphorylated versions. siRNAs that contained a 5'-phosphate had the same activity in vivo compared with unphosphorylated siRNAs, indicating phosphorylation of an siRNA is not a rate limiting step in vivo.

siRNA-optimized Modifications for Enhanced In Vivo Activity.

Current modifications used in small interfering RNAs (siRNAs), such as 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), improve stability, specificity or immunogenic properties but do not improve potency. These modifications were previously designed for use in antisense and not siRNA. We show, for the first time, that the siRNA-optimized novel 2'-O modifications, 2'-O-benzyl, and 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), are tolerated at multiple positions on the guide strand of siRNA sequences in vivo. 2'-O-benzyl and 2'-O-CH2Py(4) modifications were tested at each position individually along the guide strand in five sequences to determine positions that tolerated the modifications. The positions were combined together and found to increase potency and duration of siRNAs in vivo compared to their unmodified counterparts when delivered using lipid nanoparticles. For 2'-O-benzyl, four incorporations were tolerated with similar activity to the unmodified siRNA in vivo, while for 2'-O-CH2Py(4) six incorporations were tolerated. Increased in vivo activity was observed when the modifications were combined at positions 8 and 15 on the guide strand. Understanding the optimal placement of siRNA-optimized modifications needed for maximal in vivo activity is necessary for development of RNA-based therapeutics.

Somatic mutations in p85alpha promote tumorigenesis through class IA PI3K activation.

Members of the mammalian phosphoinositide-3-OH kinase (PI3K) family of proteins are critical regulators of various cellular process including cell survival, growth, proliferation, and motility. Oncogenic activating mutations in the p110alpha catalytic subunit of the heterodimeric p110/p85 PI3K enzyme are frequent in human cancers. Here we show the presence of frequent mutations in p85alpha in colon cancer, a majority of which occurs in the inter-Src homology-2 (iSH2) domain. These mutations uncouple and retain p85alpha's p110-stabilizing activity, while abrogating its p110-inhibitory activity. The p85alpha mutants promote cell survival, AKT activation, anchorage-independent cell growth, and oncogenesis in a p110-dependent manner.

Chemical genetic engineering of G protein-coupled receptor kinase 2.

G protein-coupled receptor kinases (GRKs) play a pivotal role in receptor regulation. Efforts to study the acute effects of GRKs in intact cells have been limited by a lack of specific inhibitors. In the present study we have developed an engineered version of GRK2 that is specifically and reversibly inhibited by the substituted nucleotide analog 1-naphthyl-PP1 (1Na-PP1), and we explored GRK2 function in regulated internalization of the mu-opioid receptor (muOR). A previously described method that conferred analog sensitivity on various kinases, by introducing a space-creating mutation in the conserved active site, failed when applied to GRK2 because the corresponding mutation (L271G) rendered the mutant kinase (GRK2-as1) catalytically inactive. A sequence homology-based approach was used to design second-site suppressor mutations. A C221V second-site mutation produced a mutant kinase (GRK2-as5) with full functional activity and analog sensitivity as compared with wild-type GRK2 in vitro and in intact cells. The role of GRK2-as5 activity in the membrane trafficking of the muOR was also characterized. Morphine-induced internalization was completely blocked when GRK2-as5 activity was inhibited before morphine application. However, inhibition of GRK2-as5 during recycling and reinternalization of the muOR did not attenuate these processes. These results suggest there is a difference in the GRK requirement for initial ligand-induced internalization of a G protein-coupled receptor compared with subsequent rounds of reinternalization.

Chemical genomic profiling to identify intracellular targets of a multiplex kinase inhibitor.

The identification of the kinase or kinases targeted by protein kinase inhibitors is a critical challenge in validating their use as therapeutic agents or molecular probes. Here, to address this problem, we describe a chemical genomics strategy that uses a direct comparison between microarray transcriptional signatures elicited by an inhibitor of unknown specificity and those elicited by highly specific pharmacological inhibition of engineered candidate kinase targets. By using this approach, we have identified two cyclin-dependent kinases, Cdk1 and Pho85, as the targets of the inhibitor GW400426 in Saccharomyces cerevisiae. We demonstrate that simultaneous inhibition of Cdk1 and Pho85, and not inhibition of either kinase alone, by GW400426 controls the expression of specific transcripts involved in polarized cell growth, thus revealing a cellular process that is uniquely sensitive to the multiplex inhibition of these two kinases. Our results suggest that the cellular responses induced by multiplex protein kinase inhibitors may be an emergent property that cannot be understood fully by considering only the sum of individual inhibitor-kinase interactions.

A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases.

Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.

Engineering a "methionine clamp" into Src family kinases enhances specificity toward unnatural ATP analogues.

A single alanine or glycine mutation in the ATP binding site of a protein kinase allows unique use of an unnatural analogue of ATP (N(6)-(benzyl) ATP) as a phosphodonor, which is not accepted by wild-type kinases. Addition of [gamma(32)P] N(6)-(benzyl) ATP to a cell lysate containing an ATP analog-specific kinase allele (as1 allele) results in the exclusive radiolabeling of bona fide substrates of the mutant kinase. Here we report efforts to engineer kinase alleles that have enhanced selectivity for ATP analogues and decreased catalytic activity with ATP, thus increasing the signal-to-noise ratio of substrate labeling. Two conserved leucine residues that contact each face of the adenine ring of ATP were mutated to methionine. The introduction of this "methionine clamp" resulted in Src and Fyn kinase alleles that have markedly improved specificity for unnatural N(6)-substituted ATP analogues over the natural substrate, ATP. This preference for unnatural nucleotides is reflected in more efficient labeling of protein substrates in cell extracts using the new analogue-specific v-Src allele. Kinase alleles with enhanced selectivity for unnatural ATP analogues should greatly facilitate the ultimate goal of labeling kinase substrates in intact cells, where concentrations of ATP and other competing nucleotides are high.

Isoform-specific phosphoinositide 3-kinase inhibitors from an arylmorpholine scaffold.

Phosphoinositide 3-kinases (PI3-Ks) are an ubiquitous class of signaling enzymes that regulate diverse cellular processes including growth, differentiation, and motility. Physiological roles of PI3-Ks have traditionally been assigned using two pharmacological inhibitors, LY294002 and wortmannin. Although these compounds are broadly specific for the PI3-K family, they show little selectivity among family members, and the development of isoform-specific inhibitors of these enzymes has been long anticipated. Herein, we prepare compounds from two classes of arylmorpholine PI3-K inhibitors and characterize their specificity against a comprehensive panel of targets within the PI3-K family. We identify multiplex inhibitors that potently inhibit distinct subsets of PI3-K isoforms, including the first selective inhibitor of p110beta/p110delta (IC(50) p110beta=0.13 microM, p110delta=0.63 microM). We also identify trends that suggest certain PI3-K isoforms may be more sensitive to potent inhibition by arylmorpholines, thereby guiding future drug design based on this pharmacophore.