Assessing Specific Oligonucleotides and Small Molecule Antibiotics for the Ability to Inhibit the CRD-BP-CD44 RNA Interaction

<div><p>Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a K_d of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to ³²P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction <i>in vitro</i> correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction <i>in vitro</i>. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions.</p></div

Effect of neomycin and lincomycin on the FLAG-CRD-BP-mRNA interaction in cells.

<p>HeLa cells were plated and transfected with pcDNA-FLAG-CRD-BP as described in Materials and Methods. Twenty-four hours after transfection, cells were treated with 750 μM of neomycin or lincomycin, or received no treatment. Immuno-precipitated FLAG-CRD-BP from cell lysate was subjected to RNA extraction and then quantitative real-time PCR (<b>A</b>) or Western blot analysis using anti-FLAG antibody as shown in (<b>B</b>). In a different set of experiments, HeLa cells transfected with pcDNA-FLAG-CRD-BP were directly subjected to total RNA extraction to assess steady-state levels of the c-<i>myc</i>, β-actin and CD44 mRNAs by quantitative real-time PCR (<b>C</b>). Total cell lysate was also analyzed to detect FLAG-CRD-BP protein expression in all groups, with β-actin used as a loading control (<b>D</b>). Data shown in (<b>A</b>) and (<b>C</b>) were pooled from three biological replicates.</p

Predicted RNA secondary structure of Fragment 4 CD44 and target sites of antisense oligonucleotides.

<p>The predicted secondary structure of Fragment 4 (nts 2861–3055) CD44 was generated using the MFOLD program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091585#pone.0091585-Zuker1" target="_blank">[53]</a>. As shown, the solid lines indicate regions where DD4 (nts 2921–2943), DD7 (nts 2976–2997) and DD10 (nts 3034–3055) antisense oligonucleotides hybridize. The broken line from nts 2980–3009 indicates the missing region of RNA going from P5 to P4 truncated fragments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091585#pone-0091585-g002" target="_blank">Figure 2</a>).</p

Effect of antisense oligonucleotides on CD44 mRNA expression in cells.

<p>HeLa cells were plated and transfected with various concentrations of 2′-<i>O</i>-methyl derivatives of DD1, DD4, DD7, or DD10 as described in the Materials and Methods. Total RNA extracted was subjected to quantitative real-time PCR for measurements of CD44, β-actin, c-<i>myc</i> and APE1 mRNAs. (<b>A</b>) Steady-state levels of CD44 mRNA was measured in cells using β-actin mRNA as a reference gene and as a function of increasing concentrations of DD1, 4, 7 and 10. Both DD4 and DD7 caused a reduction in CD44 mRNA levels in a concentration-dependent manner while DD1 and DD10 had no effect. Both DD4 and DD7 were tested for their effect on c-<i>myc</i> and APE1 mRNA levels as non-specific target RNAs as shown in (<b>B</b>) and (<b>C</b>). Data shown in all panels are expressed as a percentage of the control (non-transfected cells), and were averaged from three biological replicates with S.E. as error bars.</p

CRD-BP binding affinity for CD44 Fragment 4.

<p>(<b>A</b>) Purified His₆-tagged CRD-BP (2000 nM) was incubated with [³²P] CD44 Fragment 4 nts 2862–3055 (40 nM, 40,000 c.p.m) without (lane 2) or with molar excess of unlabeled competitor RNA CD44 Fragment 4 (lanes 3–6), CD44 RNA nts 4236–4566 (lanes 7 and 8), or β–globin RNA nts 1–145 (lanes 9 and 10). Lane 1 has no CRD-BP added. (<b>B</b>) EMSA showing increasing amounts of recombinant CRD-BP binding to [³²P] CD44 Fragment 4. (<b>C</b>) A representative saturation binding curve is shown for CRD-BP binding to CD44 fragment 4. The K_d and Hill coefficient was averaged from replicate binding experiments using three different batches of recombinant CRD-BP.</p

Inhibition of the CRD-BP-CD44 RNA interaction by antisense oligonucleotides.

<p>(<b>A</b>) Purified recombinant CRD-BP (2000 nM) was incubated with [³²P] CD44 Fragment 4 in the presence of antisense oligonucleotides DD8 (lanes 4 and 5, upper panel), DD1 (lanes 6 and 7, upper panel), DD9 (lanes 8 and 9, upper panel), DD7 (lanes 13 and 14, upper panel), DD3 (lanes 3 and 4, lower panel), DD8 (lane 6, lower panel), DD2 (lanes 10 and 11, lower panel), DD5 (lanes 12 and 13, lower panel), or DD6 (lanes 14 and 15, lower panel). Samples in lanes 2 and 10 (labeled None, upper panel) and in lanes 1 and 7 (lower panel) had no CRD-BP added. In contrast, samples in lanes 3 and 11 (upper panel) and in lanes 2 and 8 (lower panel) had CRD-BP added. A fifty-fold excess of unlabeled CD44 Fragment 4 was used as a competitor positive control (lanes 1 and 12, upper panel; lanes 5 and 9, lower panel). Increasing amounts of oligonucleotides DD4 (<b>B</b>), DD7 (<b>C</b>), and DD10 (<b>D</b>) were incubated with [³²P] CD44 Fragment 4 and purified recombinant CRD-BP. Lane 1 in Panel B, C, and D had no CRD-BP added. The average percentage of bound complex, taken from three separate experiments, is expressed on the graphs shown in the right panel of (<b>B</b>), (<b>C</b>), and (<b>D</b>).</p

Both Chemical and Non-Chemical Steps Limit the Catalytic Efficiency of Family 4 Glycoside Hydrolases

The glycoside hydrolase family 4 (GH4) α-galactosidase from <i>Citrobacter freundii</i> (MelA) catalyzes the hydrolysis of fluoro-substituted phenyl α-d-galactopyranosides by utilizing two cofactors, NAD⁺ and a metal cation, under reducing conditions. In order to refine the mechanistic understanding of this GH4 enzyme, leaving group effects were measured with various metal cations. The derived β_lg value on <i>V</i>/<i>K</i> for strontium activation is indistinguishable from zero (0.05 ± 0.12). Deuterium kinetic isotope effects (KIEs) were measured for the activated substrates 2-fluorophenyl and 4-fluorophenyl α-d-galactopyranosides in the presence of Sr²⁺, Y³⁺, and Mn²⁺, where the isotopic substitution was on the carbohydrate at C-2 and/or C-3. To determine the contributing factors to the virtual transition state (TS) on which the KIEs report, kinetic isotope effects on isotope effects were measured on these KIEs using doubly deuterated substrates. The measured ^D<i>V</i>/<i>K</i> KIEs for MelA-catalyzed hydrolysis of 2-fluorophenyl α-d-galactopyranoside are closer to unity than the measured effects on 4-fluorophenyl α-d-galactopyranoside, irrespective of the site of isotopic substitution and of the metal cation activator. These observations are consistent with hydride transfer at C-3 to the on-board NAD⁺, deprotonation at C-2, and a non-chemical step contributing to the virtual TS for <i>V</i>/<i>K</i>

Structure and Mechanism of <i>Staphylococcus aureus</i> TarS, the Wall Teichoic Acid β-glycosyltransferase Involved in Methicillin Resistance

<div><p>In recent years, there has been a growing interest in teichoic acids as targets for antibiotic drug design against major clinical pathogens such as <i>Staphylococcus aureus</i>, reflecting the disquieting increase in antibiotic resistance and the historical success of bacterial cell wall components as drug targets. It is now becoming clear that β-O-GlcNAcylation of <i>S</i>. <i>aureus</i> wall teichoic acids plays a major role in both pathogenicity and antibiotic resistance. Here we present the first structure of <i>S</i>. <i>aureus</i> TarS, the enzyme responsible for polyribitol phosphate β-O-GlcNAcylation. Using a divide and conquer strategy, we obtained crystal structures of various TarS constructs, mapping high resolution overlapping N-terminal and C-terminal structures onto a lower resolution full-length structure that resulted in a high resolution view of the entire enzyme. Using the N-terminal structure that encapsulates the catalytic domain, we furthermore captured several snapshots of TarS, including the native structure, the UDP-GlcNAc donor complex, and the UDP product complex. These structures along with structure-guided mutants allowed us to elucidate various catalytic features and identify key active site residues and catalytic loop rearrangements that provide a valuable platform for anti-MRSA drug design. We furthermore observed for the first time the presence of a trimerization domain composed of stacked carbohydrate binding modules, commonly observed in starch active enzymes, but adapted here for a poly sugar-phosphate glycosyltransferase.</p></div

Structural features of TarS_1-349.

<p>(A) Ribbon representation of TarS_1-349 in complex with UDP-GlcNAc (pink), Mn²⁺ (purple sphere) and sulfates (yellow). (B) Comparison of the position of the CS loop in the superimposed UDP complexed (blue) and UDP-GlcNAc complexed (gold) ribbon structures. (C) Comparison of the disordered and ordered states of the SA loop in surface representations of the UDP complexed (blue) structure (left) and superimposed UDP complexed and UDP-GlcNAc complexed (50% transparent, gold) structures (right) highlighting the partial occlusion of the active site in the ordered state. Only UDP-GlcNAc is displayed for simplicity. Substrates and residues are displayed in stick form and colored according to heteroatom type.</p

Analysis of TarS catalytic activity.

<p>(A) Comparison of activity in the presence and absence of divalent cations by HPLC based UDP detection. A no protein control was included for reference, and reactions proceeded in the presence of 1mM UDP-GlcNAc and 1mM metal/EDTA where indicated. (B) Relative activities of various TarS catalytic site mutants compared to wild-type by HPLC based UDP detection in the presence of 1mM UDP-GlcNAc. For comparative purposes, relative activity is given as a fraction of wild-type activity whose value was adjusted to 1.0. (C) Thermostability of various TarS catalytic site mutants in the presence and absence of UDP-GlcNAc, analyzed by differential static light scattering as a measure of T_agg upon thermodenaturation. (D) Kinetic parameters of full-length and TarS_1-349 constructs. Kinetic parameters were determined using continuous fluorescence-based UDP detection with increasing UDP-GlcNAc concentrations (hydrolysis reaction) or increasing polyRboP concentrations (glycosyltransferase reaction; in presence of 1mM UDP-GlcNAc).</p