A Bayesian machine learning approach for drug target identification using diverse data types.

Drug target identification is a crucial step in development, yet is also among the most complex. To address this, we develop BANDIT, a Bayesian machine-learning approach that integrates multiple data types to predict drug binding targets. Integrating public data, BANDIT benchmarked a ~90% accuracy on 2000+ small molecules. Applied to 14,000+ compounds without known targets, BANDIT generated ~4,000 previously unknown molecule-target predictions. From this set we validate 14 novel microtubule inhibitors, including 3 with activity on resistant cancer cells. We applied BANDIT to ONC201-an anti-cancer compound in clinical development whose target had remained elusive. We identified and validated DRD2 as ONC201's target, and this information is now being used for precise clinical trial design. Finally, BANDIT identifies connections between different drug classes, elucidating previously unexplained clinical observations and suggesting new drug repositioning opportunities. Overall, BANDIT represents an efficient and accurate platform to accelerate drug discovery and direct clinical application.

Highly conserved base A55 of 16S ribosomal RNA is important for the elongation cycle of protein synthesis.

Accurate decoding of mRNA requires the precise interaction of protein factors and tRNAs with the ribosome. X-ray crystallography and cryo-electron microscopy have provided detailed structural information about the 70S ribosome with protein factors and tRNAs trapped during translation. Crystal structures showed that one of the universally conserved 16S rRNA bases, A55, in the shoulder domain of the 30S subunit interacts with elongation factors Tu and G (EF-Tu and EF-G, respectively). The exact functional role of A55 in protein synthesis is not clear. We changed A55 to U and analyzed the effect of the mutation on the elongation cycle of protein synthesis using functional assays. Expression of 16S rRNA with the A55U mutation in cells confers a dominant lethal phenotype. Additionally, ribosomes with the A55U mutation in 16S rRNA show substantially reduced in vitro protein synthesis activity. Equilibrium binding studies showed that the A55U mutation considerably inhibited the binding of the EF-Tu·GTP·tRNA ternary complex to the ribosome. Furthermore, the A55U mutation slightly inhibited the peptidyl transferase reaction, the binding of EF-G·GTP to the ribosome, and mRNA-tRNA translocation. These results indicate that A55 is important for fine-tuning the activity of the ribosome during the elongation cycle of protein synthesis.

Steric complementarity in the decoding center is important for tRNA selection by the ribosome.

Accurate tRNA selection by the ribosome is essential for the synthesis of functional proteins. Previous structural studies indicated that the ribosome distinguishes between cognate and near-cognate tRNAs by monitoring the geometry of the codon-anticodon helix in the decoding center using the universally conserved 16S ribosomal RNA bases G530, A1492 and A1493. These bases form hydrogen bonds with the 2'-hydroxyl groups of the codon-anticodon helix, which are expected to be disrupted with a near-cognate codon-anticodon helix. However, a recent structural study showed that G530, A1492 and A1493 form hydrogen bonds in a manner identical with that of both cognate and near-cognate codon-anticodon helices. To understand how the ribosome discriminates between cognate and near-cognate tRNAs, we made 2'-deoxynucleotide and 2'-fluoro substituted mRNAs, which disrupt the hydrogen bonds between the A site codon and G530, A1492 and A1493. Our results show that multiple 2'-deoxynucleotide substitutions in the mRNA substantially inhibit tRNA selection, whereas multiple 2'-fluoro substitutions in the mRNA have only modest effects on tRNA selection. Furthermore, the miscoding antibiotics paromomycin and streptomycin rescue the defects in tRNA selection with the multiple 2'-deoxynucleotide substituted mRNA. These results suggest that steric complementarity in the decoding center is more important than the hydrogen bonds between the A site codon and G530, A1492 and A1493 for tRNA selection.

Functional replacement of two highly conserved tetraloops in the bacterial ribosome.

Ribosomes are RNA-protein complexes responsible for protein synthesis. A dominant structural motif in the rRNAs is an RNA helix capped with a four-nucleotide loop, called a tetraloop. The sequence of the tetraloop is invariant at some positions in the rRNAs but is highly variable at other positions. The biological reason for the conservation of the tetraloop sequence at specific positions in the rRNAs is not clear. In the 16S rRNA, the GAAA tetraloop in helix 8 and the UACG tetraloop in helix 14 are highly conserved and located near the binding site for EF-Tu and EF-G. To investigate whether the structural stability of the tetraloop or the precise sequence of the tetraloop is important for function, we separately changed the GAAA tetraloop in helix 8 to a UACG tetraloop and the UACG tetraloop in helix 14 to a GAAA tetraloop. The effects of the tetraloop replacements on protein synthesis were analyzed in vivo and in vitro. Replacement of the tetraloops in helices 8 and 14 did not significantly affect the growth rate of the Escherichia coli (Δ7rrn) strain. However, the mutant ribosomes showed a slightly reduced rate of protein synthesis in vitro. In addition, we observed a 2-fold increase in the error rate of translation with the mutant ribosomes, which is consistent with an earlier report. Our results suggest that the tetraloops in helices 8 and 14 are highly conserved mainly for their structural stability and the precise sequences of these tetraloops are not critical for protein synthesis.

Functional role of the sarcin-ricin loop of the 23S rRNA in the elongation cycle of protein synthesis.

The sarcin-ricin loop (SRL) is one of the longest conserved sequences in the 23S ribosomal RNA. The SRL has been accepted as crucial for the activity of the ribosome because it is targeted by cytotoxins such as α-sarcin and ricin that completely abolish translation. Nevertheless, the precise functional role of the SRL in translation is not known. Recent biochemical and structural studies indicate that the SRL is critical for triggering GTP hydrolysis on elongation factor Tu (EF-Tu) and elongation factor G (EF-G). To determine the functional role of the SRL in the elongation stage of protein synthesis, we analyzed mutations in the SRL that are known to abolish protein synthesis and are lethal to cells. Here, we show that the SRL is not critical for GTP hydrolysis on EF-Tu and EF-G. The SRL also is not essential for peptide bond formation. Our results, instead, suggest that the SRL is crucial for anchoring EF-G on the ribosome during mRNA-tRNA translocation.

Messenger RNA interactions in the decoding center control the rate of translocation.

During protein synthesis, mRNA and tRNAs are iteratively translocated by the ribosome. Precisely what molecular event is rate limiting for translocation is not known. Here we show that disruption of the interactions between the A-site codon and the ribosome accelerates translocation, suggesting that the release of the mRNA from the decoding center of the ribosome is the rate-limiting step of translocation. These results provide insight into the molecular mechanism of translocation.

Polyamines accelerate codon recognition by transfer RNAs on the ribosome.

The selection of aminoacyl-tRNAs by the ribosome is a fundamental step in the elongation cycle of protein synthesis. tRNA selection is a multistep process that ensures only correct aminoacyl-tRNAs are accepted while incorrect aminoacyl-tRNAs are rejected. A key step in tRNA selection is the formation of base pairs between the anticodon of the aminoacyl-tRNA and the mRNA codon in the A site, called "codon recognition". Here, we report the development of a new, fluorescence-based, kinetic assay for monitoring codon recognition by the ribosome. Using this assay, we show that codon recognition is a second-order binding step under optimal conditions. Additionally, we show that at low Mg(2+) concentrations, the polyamines spermine and spermidine stimulate codon recognition by the ribosome without a loss of fidelity. Polyamines may accelerate codon recognition by altering the structure and dynamics of the anticodon arm of the aminoacyl-tRNA.

Synthesis and photochemical properties of nitro-naphthyl chromophore and the corresponding immunoglobulin bioconjugate.

Synthesis, photochemistry, and biomolecular caging properties of a new chromophore namely 3-nitro-2-naphthalenemethanol are described. This chromophore is photoexcitable with photons in 350-400 nm range and in several solvents including aqueous medium. On irradiation, it gives the expected nitroso-aldehyde photoproduct with high quantum yield (0.6-0.8). Further, it can be conveniently coupled to the amino residues of immunoglobulin (IgG) using diphosgene. Irradiation of the resulting IgG-nitronaphthyl chromophore bioconjugate at 380 nm causes photorelease of IgG as evidenced by Protein-A affinity binding studies. The bioconjugate showed low level of binding to Protein-A. However, the binding increases after irradiation and, thus, modifies the Fc site of the IgG. Electrophoresis studies of the irradiated bioconjugate show that IgG does not undergo fragmentation or molecular weight change under the irradiation conditions. Thus, 3-nitro-2-naphthalenemethanol can be used as a photocaging agent under physiological conditions at wavelengths, which does not cause significant damage to the biomolecule. The work provides new directions for the development of organic chromophores for biomolecular caging applications.