Translational T-box riboswitches bind tRNA by modulating conformational flexibility.

T-box riboswitches are noncoding RNA elements involved in genetic regulation of most Gram-positive bacteria. They regulate amino acid metabolism by assessing the aminoacylation status of tRNA, subsequently affecting the transcription or translation of downstream amino acid metabolism-related genes. Here we present single-molecule FRET studies of the Mycobacterium tuberculosis IleS T-box riboswitch, a paradigmatic translational T-box. Results support a two-step binding model, where the tRNA anticodon is recognized first, followed by interactions with the NCCA sequence. Furthermore, after anticodon recognition, tRNA can transiently dock into the discriminator domain even in the absence of the tRNA NCCA-discriminator interactions. Establishment of the NCCA-discriminator interactions significantly stabilizes the fully bound state. Collectively, the data suggest high conformational flexibility in translational T-box riboswitches; and supports a conformational selection model for NCCA recognition. These findings provide a kinetic framework to understand how specific RNA elements underpin the binding affinity and specificity required for gene regulation.

Mechanisms of catalytic RNA molecules.

Ribozymes are folded catalytic RNA molecules that perform important biological functions. Since the discovery of the first RNA with catalytic activity in 1982, a large number of ribozymes have been reported. While most catalytic RNA molecules act alone, some RNA-based catalysts, such as RNase P, the ribosome, and the spliceosome, need protein components to perform their functions in the cell. In the last decades, the structure and mechanism of several ribozymes have been studied in detail. Aside from the ribosome, which catalyzes peptide bond formation during protein synthesis, the majority of known ribozymes carry out mostly phosphoryl transfer reactions, notably trans-esterification or hydrolysis reactions. In this review, we describe the main features of the mechanisms of various types of ribozymes that can function with or without the help of proteins to perform their biological functions.

Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand.

T-box riboswitches are cis-regulatory RNA elements that regulate the expression of proteins involved in amino acid biosynthesis and transport by binding to specific tRNAs and sensing their aminoacylation state. While the T-box modular structural elements that recognize different parts of a tRNA have been identified, the kinetic trajectory describing how these interactions are established temporally remains unclear. Using smFRET, we demonstrate that tRNA binds to the riboswitch in two steps, first anticodon recognition followed by the sensing of the 3' NCCA end, with the second step accompanied by a T-box riboswitch conformational change. Studies on site-specific mutants highlight that specific T-box structural elements drive the two-step binding process in a modular fashion. Our results set up a kinetic framework describing tRNA binding by T-box riboswitches, and suggest such binding mechanism is kinetically beneficial for efficient, co-transcriptional recognition of the cognate tRNA ligand.

Chemoproteomic Approach to Explore the Target Profile of GPCR ligands: Application to 5-HT1A and 5-HT6 Receptors.

Determination of the targets of a compound remains an essential aspect in drug discovery. A complete understanding of all binding interactions is critical to recognize in advance both therapeutic effects and undesired consequences. However, the complete polypharmacology of many drugs currently in clinical development is still unknown, especially in the case of G protein-coupled receptor (GPCR) ligands. In this work we have developed a chemoproteomic platform based on the use of chemical probes to explore the target profile of a compound in biological systems. As proof of concept, this methodology has been applied to selected ligands of the therapeutically relevant serotonin 5-HT1A and 5-HT6 receptors, and we have identified and validated some of their off-targets. This approach could be extended to other drugs of interest to study the targeted proteome in disease-relevant systems.

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum.

Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome.

New inhibitors of angiogenesis with antitumor activity in vivo.

Angiogenesis is a requirement for the sustained growth and proliferation of solid tumors, and the development of new compounds that induce a sustained inhibition of the proangiogenic signaling generated by tumor hypoxia still remains as an important unmet need. In this work, we describe a new antiangiogenic compound (22) that inhibits proangiogenic signaling under hypoxic conditions in breast cancer cells. Compound 22 blocks the MAPK pathway, impairs cellular migration under hypoxic conditions, and regulates a set of genes related to angiogenesis. These responses are mediated by HIF-1α, since the effects of compound 22 mostly disappear when its expression is knocked-down. Furthermore, administration of compound 22 in a xenograft model of breast cancer produced tumor growth reductions ranging from 46 to 55% in 38% of the treated animals without causing any toxic side effects. Importantly, in the responding tumors, a significant reduction in the number of blood vessels was observed, further supporting the mechanism of action of the compound. These findings provide a rationale for the development of new antiangiogenic compounds that could eventually lead to new drugs suitable for the treatment of some types of tumors either alone or in combination with other agents.

Tb(3+)-tRNA for LRET studies of protein synthesis.

When suitably labeled bulk tRNAs are transfected into cells they give rise to FRET (fluorescence resonance energy transfer) signals via binding to ribosomes that provide a measure of total protein synthesis. Application of this approach to monitoring rates of specific protein synthesis requires achieving a very high signal-to-noise ratio. Such high ratios may be attainable using LRET (luminescence resonance energy transfer) in place of FRET. Lanthanide complexes containing an antenna chromophore are excellent LRET donors. Here we describe the synthesis of a Phe-tRNA(Phe) labeled with a Tb(3+) complex, denoted Tb(3+)-Phe-tRNA(Phe) that, notwithstanding the bulkiness of the Tb(3+) complex, is active in protein synthesis.

Combined effect of a peptide-morpholino oligonucleotide conjugate and a cell-penetrating peptide as an antibiotic.

A cell-penetrating peptide (CPP)-morpholino oligonucleotide (MO) conjugate (PMO) that has an antibiotic effect in culture had some contaminating CPPs in earlier preparations. The mixed conjugate had gene-specific and gene-nonspecific effects. An improved purification procedure separates the PMO from the free CPP and MO. The gene-specific effects are a result of the PMO, and the nonspecific effects are a result of the unlinked, unreacted CPP. The PMO and the CPP can be mixed together, as has been shown previously in earlier experiments, and have a combined effect as an antibiotic. Kinetic analysis of these effects confirm this observation. The effect of the CPP is bacteriostatic. The effect of the PMO appears to be bacteriocidal. An assay for mutations that would alter the ability of these agents to affect bacterial viability is negative.

The antibacterial cell division inhibitor PC190723 is an FtsZ polymer-stabilizing agent that induces filament assembly and condensation.

Cell division protein FtsZ can form single-stranded filaments with a cooperative behavior by self-switching assembly. Subsequent condensation and bending of FtsZ filaments are important for the formation and constriction of the cytokinetic ring. PC190723 is an effective bactericidal cell division inhibitor that targets FtsZ in the pathogen Staphylococcus aureus and Bacillus subtilis and does not affect Escherichia coli cells, which apparently binds to a zone equivalent to the binding site of the antitumor drug taxol in tubulin (Haydon, D. J., Stokes, N. R., Ure, R., Galbraith, G., Bennett, J. M., Brown, D. R., Baker, P. J., Barynin, V. V., Rice, D. W., Sedelnikova, S. E., Heal, J. R., Sheridan, J. M., Aiwale, S. T., Chauhan, P. K., Srivastava, A., Taneja, A., Collins, I., Errington, J., and Czaplewski, L. G. (2008) Science 312, 1673-1675). We have found that the benzamide derivative PC190723 is an FtsZ polymer-stabilizing agent. PC190723 induced nucleated assembly of Bs-FtsZ into single-stranded coiled protofilaments and polymorphic condensates, including bundles, coils, and toroids, whose formation could be modulated with different solution conditions. Under conditions for reversible assembly of Bs-FtsZ, PC190723 binding reduced the GTPase activity and induced the formation of straight bundles and ribbons, which was also observed with Sa-FtsZ but not with nonsusceptible Ec-FtsZ. The fragment 2,6-difluoro-3-methoxybenzamide also induced Bs-FtsZ bundling. We propose that polymer stabilization by PC190723 suppresses in vivo FtsZ polymer dynamics and bacterial division. The biochemical action of PC190723 on FtsZ parallels that of the microtubule-stabilizing agent taxol on the eukaryotic structural homologue tubulin. Both taxol and PC190723 stabilize polymers against disassembly by preferential binding to each assembled protein. It is yet to be investigated whether both ligands target structurally related assembly switches.

Development of Fluorescent Ligands for the Human 5-HT1A Receptor.

In this work, we report the design and synthesis of a set of fluorescent probes targeting the human 5-HT1A receptor (h5-HT1AR). Among the synthesized compounds, derivative 4 deserves special attention as being a high-affinity ligand (K i = 2 nM) with good fluorescent properties (I em > 1000 au and a fluorescence quantum yield, Φf, of 0.26), which enables direct observation of the h5-HT1AR in cells. Thus, it represents the first efficacious fluorescent probe for the specific labeling of h5-HT1AR in cells. Our results provide the basis for the introduction of a variety of tags in scaffolds of G protein-coupled receptor (GPCR) ligands that enable visualization, covalent binding, or affinity pull-down of receptors. These strategies should contribute to the optimization of the therapeutic exploitation of known or new members of the GPCR superfamily by providing valuable information about their location or level of expression.

Further naphthylcombretastatins. An investigation on the role of the naphthalene moiety.

By synthesis and biological studies of new naphthalene analogues of combretastatins, we have found that the naphthalene is a good surrogate for the isovanillin moiety (3-hydroxy-4-methoxyphenyl) of combretastatin A-4, always generating highly cytotoxic analogues when combined with the 3,4,5-trimethoxyphenyl or related systems. On the other hand, when the naphthalene replaces the 3,4,5-trimethoxyphenyl moiety, the cytotoxic activity is largely decreased. The most cytotoxic naphthalene analogues of combretastatins, which also produce inhibition of tubulin polymerization, exerted their antimitotic effects through microtubule network disruption and subsequent G(2)/M arrest of the cell cycle in human cancer cells.