Volume 13 Supplement 2
Interfacing medicinal chemistry with structural bioinformatics: implications for T box riboswitch RNA drug discovery
© Jentzsch and Hines; licensee BioMed Central Ltd. 2012
Published: 13 March 2012
The T box riboswitch controls bacterial transcription by structurally responding to tRNA aminoacylation charging ratios. Knowledge of the thermodynamic stability difference between two competing structural elements within the riboswitch, the terminator and the antiterminator, is critical for effective T box-targeted drug discovery.
The ΔG of aminoacyl tRNA synthetase (aaRS) T box riboswitch terminators and antiterminators was predicted using DINAMelt and the resulting ΔΔG (ΔGTerminator - ΔGAntiterminator) values were compared.
Average ΔΔG values did not differ significantly between the bacterial species analyzed, but there were significant differences based on the type of aaRS.
The data indicate that, of the bacteria studied, there is little potential for drug targeting based on overall bacteria-specific thermodynamic differences of the T box antiterminator vs. terminator stability, but that aaRS-specific thermodynamic differences could possibly be exploited for designing drug specificity.
We have been investigating the structure-function relationship of the T box antiterminator element and the key recognition features necessary for ligands to specifically bind the antiterminator and disrupt its function. There are few detailed medicinal chemistry studies of ligands targeting RNA  and this project has required an extensive multidisciplinary approach. The solution structure of antiterminator model RNA AM1A  was determined using molecular modelling with NMR-derived constraints . The structure indicated that the seven-nucleotide bulge of the antiterminator was not pre-ordered for tRNA binding, but rather, that binding of the tRNA acceptor end must require a certain extent of tertiary-structure capture and/or an induced fit. Fluorescence life-time studies confirmed that modest antiterminator structural reorganization occurs upon tRNA binding in a magnesium-dependent manner . While some RNAs have specific divalent metal ion binding sites, for the antiterminator RNA, the Mg2+ binds via a diffuse, outer-sphere interaction . In vitro selection studies of both the antiterminator  and tRNA  indicate that there are likely no direct interactions between the tRNA and the antiterminator other than the known base pairing between the acceptor end nucleotides and the first four nucleotides at the 5'-end of the seven-nucleotide bulge. Given this antiterminator structure-function information, ligands could potentially disrupt tRNA binding simply by competing with the base pairing between the tRNA acceptor end and the antiterminator bulge nucleotides.
Aminoglycosides bind AM1A in a structure-specific manner, most likely via electrostatics [13, 14]. In contrast, two novel classes of heterocyclic compounds, triazoles and oxazolidinones, bind AM1A with structure-specificity and high affinity, but without reliance on electrostatics [15–18] and can alter T box transcription antitermination . A computational, quantitative structure activity relationship analysis has shown that hydrophobic interactions play a significant role in the binding of these compounds to AM1A . Furthermore, the small molecule ligands disrupt tRNA binding to the antiterminator in a structure-specific manner .
The thermodynamic stability of T box antiterminator and terminator structural elements was calculated using the DINAMelt server . The DINAMelt server computes the secondary structure and free energy of the folded RNA using a secondary structure folding algorithm and empirically-derived nearest neighbour coefficients . The folding algorithm predicts the minimum energy RNA secondary structure using the available thermodynamic data for base pairing, base stacking and destabilizing energies [22, 23]. The sequences analyzed were predicted to be involved in aaRS T box antiterminator and terminator structural elements from Bacillus cereus (BC), Bacillus subtilis (BS), Clostridium botulinum (CB), Clostridium difficile (CDF), Clostridium perfringens (CPE), Staphylococcus aureus Mu50 (SA), Streptococcus agalactiae (SAG), and, Streptococcus pyogenes (SPY) . The ΔG for each aaRS T box antiterminator and terminator sequence was determined using the Quickfold (RNA 3.0) option on the DINAMelt server and the predicted thermodynamic stability difference calculated from ΔΔG = ΔGTerminator - ΔGAntiterminator. The % suboptimal setting was adjusted as necessary to obtain the lowest free energy antiterminator fold that had the consensus secondary structure of the core seven-nucleotide bulge containing the 5' -UGGN-3' nucleotides that are complementary to the tRNA acceptor end nucleotides .
Results and discussion
The free energy of T box riboswitch antiterminator and terminator elements was predicted and compared for a series of aaRS T box genes. The observed aaRS-specific stability differences between these key riboswitch structural elements could potentially be targeted to effect ligand-specificity in future drug discovery efforts.
Aminoacyl tRNA synthetase
- Mu50 (SA):
Streptococcus agalactiae and, Streptococcus pyogenes (SPY).
We wish to thank the National Institutes of Health (GM073188) for support of this work and the office of the VP for Research, Ohio University and the DAAD for support of FJ on an Internationale Studien- und Ausbildungspartnerschaften (ISAP) grant.
This article has been published as part of BMC Bioinformatics Volume 13 Supplement 2, 2012: Proceedings from the Great Lakes Bioinformatics Conference 2011. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcbioinformatics/supplements/13/S2
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