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Biomedical subjects

Peter A Beal

Publications and source records attributed to Peter A Beal.

At least 19 recordsLinked to original sources
PubMed2006

Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification.

The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3- to 5-fold by siRNA duplexes with 19 bp and 2 nt 3'-overhangs, whereas the maximum activation observed for poly(I)*poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N2-benzyl-2'-deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.

Binding Sites↗
PubMed2006

C6-substituted analogues of 8-azanebularine: probes of an RNA-editing enzyme active site.

[reaction: see text] We describe the synthesis of derivatives of 8-azanebularine, a known inhibitor of adenosine deaminases including the RNA-editing enzyme ADAR2. 6-Methyl, 6-hydroxymethyl, 6-cyano, and 6-mercapto derivatives were obtained from 6-bromo precursors using different cross-coupling or substitution reactions. The C6-methyl derivative was incorporated into an RNA substrate for ADAR2 via the phosphoramidite. Quantitative gel mobility shift experiments with the resulting RNA indicate that methylation at C6 dramatically reduces the affinity of 8-azanebularine for ADAR2.

Adenosine Deaminase↗
PubMed2006

RNA binding and thiolytic stability of a quinoline-containing helix-threading peptide.

Helix-threading peptides (HTPs) bind selectively to sites predisposed to intercalation in folded RNA molecules placing peptide functional groups into the dissimilar grooves of the duplex. Here we report the design and synthesis of new HTPs with quinoline as the intercalation domain. A quinoline-containing HTP is shown to bind selectively to duplex RNA binding sites. Furthermore, the affinity cleavage pattern generated using an EDTA.Fe modified derivative is consistent with minor groove localization of its N-terminus. This compound binds base-pair steps flanked by single nucleotide bulges on the 3' side on both strands, whereas bulges on the 5' side of the intercalation site do not support binding. Furthermore, unlike acridine HTPs, the quinoline compound is resistant to thiolytic degradation that leads to loss of RNA-binding activity. The RNA-binding selectivity and stability observed for quinoline-containing HTPs make them excellent candidates for further development as regulators of intracellular RNA function.

Base Sequence↗
PubMed2005

Inhibitory properties of nucleic acid-binding ligands on protein synthesis.

The use of small molecule inhibitors in the study of cellular processes is a powerful approach to understanding gene function. During the course of a high throughput screen for novel inhibitors of eukaryotic translation, we identified a number of nucleic acid binding ligands that showed activity in our assay. When tested on a panel of mRNA transcripts displaying different modes of translation initiation, these ligands showed a range of biological activities--with some inhibiting both cap-dependent and internal initiation and others preferentially blocking internal initiation. We used this information to identify a novel threading intercalator that inhibits Hepatitis C virus internal initiation.

Acridines↗
PubMed2005

Binding of helix-threading peptides to E. coli 16S ribosomal RNA and inhibition of the S15-16S complex.

Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.

Binding Sites↗
PubMed2004

A transition state analogue for an RNA-editing reaction.

Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6-N1 double bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate receptor B subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight binding ligand for an ADAR can be generated by incorporation of 8-azanebularine. The observed high-affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2's two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue into the sequence/structural context of a known editing site. These results advance our understanding of substrate recognition during ADAR-catalyzed RNA editing and are important for structural studies of ADAR.RNA complexes.

Adenosine Deaminase↗
PubMed2004

Recognition of duplex RNA by helix-threading peptides.

Many important biological processes, from the interferon antiviral response to the generation of microRNA regulators of translation, involve duplex RNA. Small molecules capable of binding duplex RNA structures with high affinity and selectivity will be useful in regulating these processes and, as such, are valuable research tools and potentially therapeutic. In this paper, the synthesis and duplex RNA-binding properties of EDTA.Fe-modified peptide-intercalator conjugates (PICs) are described. Peptide appendages at the 4- and 9-positions of the planar acridine ring system render these PICs threading intercalators, directing the substituents into both grooves of double helical RNA simultaneously. Directed hydroxyl radical cleavage experiments conducted with varying RNA stem-loop structures indicate a preferred binding polarity with the N- and C-termini of the PIC in the minor and major grooves, respectively. However, this binding polarity is shown to be dependent on both the structure of the PIC and the RNA secondary structure adjacent to the intercalation site. Definition of the minimal RNA structure required for binding to one of these PICs led to the identification of an intercalation site in a pre-microRNA from Caenorhabditis elegans. Results presented will guide both rational design and combinatorial approaches for the generation of new RNA binding PICs and will continue to facilitate the identification of naturally occurring RNA targets for these small molecules.

Edetic Acid↗
PubMed2004

Peptide quinoline conjugates: a new class of RNA-binding molecules.

[reaction: see text] A synthesis of 4,8-disubstituted 2-phenylquinoline amino acids is reported with the incorporation of one example into a peptide by solid-phase synthesis. The phenylquinoline-containing peptide binds an RNA target with nanomolar affinity (K(D) = 208 nM). The strategy can be used to prepare a variety of 2-substituted quinoline amino acids for alteration of affinity in intercalator peptides. Since quinolones represent an important class of antibacterials, these compounds may be useful in the discovery of new antibacterial agents.

Amino Acids↗
PubMed2004

The binding selectivity of ADAR2's dsRBMs contributes to RNA-editing selectivity.

ADAR2 is an RNA editing enzyme that deaminates adenosines in certain duplex structures. Here, we describe the role of its RNA binding domain, consisting of two copies of a common dsRNA binding motif (dsRBM), in editing site selectivity. ADAR2's dsRBMs bind selectively on a duplex RNA that mimics the Q/R editing site in the glutamate receptor B-subunit pre-mRNA. This selectivity is different from that of PKR's dsRBM I, indicating that dsRBMs from different proteins possess intrinsic binding selectivity. Using directed hydroxyl radical cleavage data, molecular models were developed that predict important recognition surfaces on the RNA for identified dsRBM binding sites. Blocking these surfaces by benzyl modification of guanosine 2-amino groups impeded RNA-editing, demonstrating a correlation between deamination efficiency by ADAR2 and selective binding by its dsRBMs. In addition, the editing activity of a mutant of ADAR2 lacking dsRBM I on N(2)-benzylguanosine-modified RNA suggests the location of the dsRBM I binding site that leads to editing at the GluR-B Q/R site.

Adenosine Deaminase↗
PubMed2003

Substrate analogues for an RNA-editing adenosine deaminase: mechanistic investigation and inhibitor design.

ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that occur in eukaryotic mRNAs, including the mRNAs of glutamate and serotonin receptors. ADARs capable of editing biologically relevant RNA substrates have been identified. In addition, the consequence of the RNA-editing reaction on the function of the gene product is known in several cases. However, our understanding of the chemical mechanism of the ADAR-catalyzed adenosine deamination in RNA is lagging. By studying analogues of a naturally occurring substrate for ADAR2, we infer features of the enzyme's active site and reaction mechanism. 8-Aza substitution at adenosine in various RNA substrates accelerates the rate of deamination at these sites by ADAR2 (2.8-17-fold). The magnitude of this "aza effect" depends on the RNA structural context of the reacting nucleotide. N(6)-Methyladenosine in RNA is a slow substrate for ADAR2 (rate is 2% that of adenosine), with no product observed with N(6)-ethyladenosine, suggesting a limited size of the leaving group pocket. 2,6-Diaminopurine ribonucleoside in RNA is not a substrate for ADAR, in contrast to adenosine deaminase (ADA), which catalyzes a similar reaction on nucleosides. This and other results indicate that ADAR2 uses a base recognition strategy different from that of ADA. Consistent with the large 8-aza effect observed for the ADAR2 reaction, we find that 8-azanebularine, as the free nucleoside, inhibits the ADAR2 reaction (IC(50) = 15 +/- 3 mM) with no inhibition observed with nebularine or coformycin.

Adenosine↗
PubMed2003

Small molecule inhibitors of the RNA-dependent protein kinase.

The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase that phosphorylates the alpha subunit of the eukaryotic initiation factor 2 in response to viral infection. Classical genetic approaches for studying the role of PKR in cell signaling have their limitations due to overlapping but non-redundant pathways. Small molecule inhibitors of PKR will be useful in this regard. We report here, the discovery of a small molecule inhibitor of the kinase reaction of PKR. The inhibitor was discovered by screening a library of 26 different ATP-binding site directed inhibitors of varying structure. We also describe the development of a high-throughput assay for screening a large number of compounds for a PKR inhibitor using a rabbit reticulocyte lysate system and luciferase mRNA. The assay takes advantage of the fact that the reticulocyte lysate is rich in components of the translational machinery, of which PKR is an integral part. This assay can be carried out with added exogenous human PKR to study the effect of various compounds in their ability to rescue the translational block imposed by human PKR.

Enzyme Inhibitors↗
PubMed2003

Recognition of double-stranded RNA by proteins and small molecules.

Molecular recognition of double-stranded RNA (dsRNA) is a key event for numerous biological pathways including the trafficking, editing, and maturation of cellular RNA, the interferon antiviral response, and RNA interference. Over the past several years, our laboratory has studied proteins and small molecules that bind dsRNA with the goal of understanding and controlling the binding selectivity. In this review, we discuss members of the dsRBM class of proteins that bind dsRNA. The dsRBM is an approximately 70 amino acid sequence motif found in a variety of dsRNA-binding proteins. Recent results have led to a new appreciation of the ability of these proteins to bind selectivity to certain sites on dsRNA. This property is discussed in light of the RNA selectivity observed in the function of two proteins that contain dsRBMs, the RNA-dependent protein kinase (PKR) and an adenosine deaminase that acts on dsRNA (ADAR2). In addition, we introduce peptide-acridine conjugates (PACs), small molecules designed to control dsRBM-RNA interactions. These intercalating molecules bear variable peptide appendages at opposite edges of an acridine heterocycle. This design imparts the potential to exploit differences in groove characteristics and/or base-pair dynamics at binding sites to achieve selective binding.

Acridines↗
PubMed2003

Preferred RNA binding sites for a threading intercalator revealed by in vitro evolution.

In pursuit of small molecules capable of controlling the function of RNA targets, we have explored the RNA binding properties of peptide-acridine conjugates (PACs). In vitro evolution (SELEX) was used to isolate RNAs capable of binding the PAC Ser-Val-Acr-Arg, where Acr is an acridine amino acid. The PAC binds RNA aptamers selectively and with a high degree of discrimination over DNA. PAC binding sites contain the base-paired 5'-CpG-3' sequence, a known acridine intercalation site. However, RNA structure flanking this sequence causes binding affinities to vary over 30-fold. The preferred site (K(D) = 20 nM) contains a base-paired 5'-CpG-3' step flanked on the 5' side by a 4 nt internal loop and the 3' side by a bulged U. Several viral 5'- and 3'-UTR RNA sequences that likely form binding sites for this PAC are identified.

Acridines↗
PubMed2002

Selection of small-molecule mediators of the RNA regulation of PKR, the RNA-dependent protein kinase.

The RNA-dependent protein kinase (PKR) is a component of the interferon antiviral response and a member of the class of RNA-binding proteins with a double-stranded RNA binding motif. PKR is activated when it binds to double-stranded RNA (dsRNA) or viral replicative intermediates that comprise dsRNA and this activation results in the inhibition of protein synthesis. Some viruses circumvent this activity through the synthesis of highly structured decoy RNAs that bind PKR and block activation. Small-molecule mediators of the binding of PKR to these RNA inhibitors would be useful tools to further define the importance of specific PKR-RNA complexes in vivo and may possess antiviral activity. Here we investigate the ability of a library of structurally diverse peptide-acridine conjugates (PACs) to target a complex formed between the dsRNA binding domain (dsRBD) of PKR and a viral RNA inhibitor. We used a novel screening method based on the cleavage of RNA ligands with ethylenediaminetetraacetic acid.Fe modified protein. The selection revealed a PAC (9-anilinoacridine-4-Hyp-Nap-Nap, where Hyp is trans-4-hydroxyproline and Nap is 1-napthylalanine), able to inhibit the binding of the PKR dsRBD to RNA with an IC(50) value of 10 +/- 5 microM. Furthermore, the structural requirements for inhibition by the selected PAC were substantiated in an independent PKR activation assay. We found that the potency of inhibition by an intercalating ligand can be increased by the introduction of a substituent that does not increase the overall charge of the molecule. This result is important for the design of inhibitors of PKR-RNA binding that function inside living cells.

Acridines↗
PubMed2002

Point of attachment and sequence of immobilized peptide-acridine conjugates control affinity for nucleic acids.

Screening of combinatorial libraries by spatial arraying strategies requires library members to be solid-phase immobilized. However, for nucleic acid ligands that bind via intercalation, immobilization may inhibit binding if the tethering functionality is present at the edge of the heterocyle that approaches the duplex during the binding reaction. We report here a method for immobilizing peptide-acridine conjugates (PACs) via either their C- or their N-terminus, corresponding to functionalization at either the 4- or the 9-position of acridine, respectively, and for assaying the nucleic acid binding properties of the resulting resins. We find that both the amino acid sequence of the PAC as well as its point of attachment to the solid support are important in determining affinity for duplex nucleic acids. These results have implications for the design of future on-bead and microarray-based selections and in understanding the nucleic acid binding of functionalized intercalators.

Amino Acids↗