It is yet to be investigated whether both ligands target structurally related assembly switches.

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.

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.

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.EGS technology | gram-negative bacteria | gram-positive bacteria | RNase P M orpholino oligonucleotides (MOs) covalently linked with a cell-penetrating peptide (CPP) can be used as antibiotics (1). Such compounds are potent in the variety of cells they attack (both Gram-positive and Gram-negative bacteria) but are somewhat less efficient than conventional antibiotics today in terms of the concentrations used to kill cells. These compounds work well in curing internal infections in mice (2-4) and are also potent in preventing the growth and development of the malarial parasite, Plasmodium falciparum (5).A very efficient CPP-MO combination (PMO) has been synthesized from a basic peptide from human T cells (6), which has no biological activity on human cells, and an MO. One significant asset of this conjugate is its relative resistance to mutation that will alter its properties, as more than three noncontiguous mutations are needed to inactivate it. Another feature, as with most nucleic acid-based potential drugs, is its universal application against any target gene through the rational design of the base sequence of its MO (1).Many peptides from a variety of organisms are natural antibiotics (7,8). Several derivatives could be used in humans as antibiotics, but this form of therapy has not become popular. Some peptides that resemble signal peptides in their content of basic amino acid residues, such as arginine and lysine, are also useful as CPPs (2-4).The external guide sequence (EGS) technology relies on an RNA introduced in vivo that is complementary to a pathogenic RNA. The resident and ubiquitous RNase P then cleaves the complex, thereby rendering the pathogenic RNA inactive (9). In an investigation of the PMO that relies on EGS technology for its use, both gene-specific and non-gene-specific effects had been ascribed to this and similar compounds (1). Gel filtration purification yielded the PMO and, separately, unreacted CPP and MO. Using larger bed volume columns in scaling up preparations revealed that earlier preparations did have some contamination by chemically unreacted CPP in the PMO fractions. Reassaying of all these purified components indicated that significant anti...

In this work, we report the design and synthesis of a set of fluorescent probes targeting the human 5-HT 1A receptor (h5-HT 1A R). 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, Φ f , of 0.26), which enables direct observation of the h5-HT 1A R in cells. Thus, it represents the first efficacious fluorescent probe for the specific labeling of h5-HT 1A R 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.

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. malaria | intraerythrocytic development | peptide conjugated morpholino oligomer | vivo morpholino oligomer | gene expression

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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.