Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

Nucleic acids analogues, i.e., oligonucleotide N3′→P5′ phosphoramidates and N3′→P5′ thio‐phosphoramidates, containing 3′‐amino‐3′‐deoxy nucleosides with various 2′‐substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, ΔT_m, relative to their phosphodiester counterparts, reaches 2.2–4.0° per modified nucleoside. 2′‐OH‐ (RNA‐like), 2′‐O‐Me‐, and 2′‐ribo‐F‐nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2′‐deoxy‐ and 2′‐fluoro‐phosphoramidate compounds form extremely stable triple‐stranded complexes with either single‐ or double‐stranded phosphodiester DNA oligonucleotides. Melting temperature, T_m, of these triplexes exceeds T_m values for the isosequential phosphodiester counterparts by up to 35°. 2′‐Deoxy‐N3′→P5′ phosphoramidates adopt RNA‐like C3′‐endo or N‐type nucleoside sugar‐ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2′‐deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H‐mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio‐phosphoramidates conjugated with lipid groups are cell‐permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3′→P5′ thio‐phosphoramidate conjugated to 5′‐palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase‐I and Phase‐I/II clinical trials as potential broad‐spectrum anticancer agent.     

TatD is a central component of a Tat translocon‐initiated quality control system for exported FeS proteins in Escherichia coli

Bacterial Tat systems export folded proteins, including FeS proteins such as NrfC and NapG, which acquire their cofactors before translocation. NrfC and NapG are proofread by the Tat pathway, and misfolded examples are degraded after interaction with the translocon. Here, we identify TatD as a crucial component of this quality control system in Escherichia coli. NrfC/NapG variants lacking FeS centres are rapidly degraded in wild‐type cells but stable in a ΔtatD strain. The precursor of another substrate, FhuD, is also transiently detected in wild‐type cells but stable in the ΔtatD strain. Surprisingly, these substrates are stable in ΔtatD cells that overexpress TatD, and export of the non‐mutated precursors is inhibited. We propose that TatD is part of a quality control system that is intimately linked to the Tat export pathway, and that the overexpression of TatD leads to an imbalance between the two systems such that both Tat‐initiated turnover and export are prevented.     

Bioinformatic Comparison of Bacterial Secretomes

The rapid increasing number of completed bacterial genomes provides a good op-portunity to compare their proteomes. This study was undertaken to specifically compare and contrast their secretomes-the fraction of the proteome with pre-dicted N-terminal signal sequences, both type Ⅰ and type Ⅱ. A total of 176 theoreti-cal bacterial proteomes were examined using the ExProt program. Compared with the Gram-positives, the Gram-negative bacteria were found, on average, to con-tain a larger number of potential Sec-dependent sequences. In the Gram-negative bacteria but not in the others, there was a positive correlation between proteome size and secretome size, while there was no correlation between secretome size and pathogenicity. Within the Gram-negative bacteria, intracellular pathogens were found to have the smallest secretomes. However, the secretomes of certain bacte-ria did not fit into the observed pattern. Specifically, the secretome of Borrelia burgdoferi has an unusually large number of putative lipoproteins, and the signal peptides of mycoplasmas show closer sequence similarity to those of the Gram-negative bacteria. Our analysis also suggests that even for a theoretical minimal genome of 300 open reading frames, a fraction of this gene pool (up to a maximum of 20%) may code for proteins with Sec-dependent signal sequences.     

The importance of valency in enhancing the import and cell routing potential of protein transduction domain-containing molecules

Protein transduction domains (PTDs) are peptides that afford the internalization of cargo macromolecules (including plasmid DNA, proteins, liposomes, and nanoparticles). In the case of polycationic peptides, the efficiency of PTDs to promote cellular uptake is directly related to their molecular mass or their polyvalent presentation. Similarly, the efficiency of routing to the nucleus increases with the number of nuclear localization signals (NLS) associated with a cargo. The quantitative enhancement, however, depends on the identity of the PTD sequence as well as the targeted cell type. Thus the choice and multivalent presentation of PTD and NLS sequences are important criteria guiding the design of macromolecules intended for specific intracellular localization. This review outlines synthetic and recombinant strategies whereby PTDs and signal sequences can be assembled into multivalent peptide dendrimers and promote the uptake and routing of their cargoes. In particular, the tetramerization domain of the tumour suppressor p53 (p53^tet) is emerging as a useful scaffold to present multiple routing and targeting moieties. Short cationic peptides fused to the 31-residue long p53^tet sequence resulted in tetramers displaying a significant enhancement (up to 1000 fold) in terms of their ability to be imported into cells and delivered to the cell nucleus in relation to their monomeric analogues. The design of future polycationic peptide dendrimers as effective delivering vehicles will need to incorporate selective cell targeting functions and provide solutions to the issue of endosomal entrapment.     

HIV-1 Clade B Tat, but Not Clade C Tat, Increases X4 HIV-1 Entry into Resting but Not Activated CD4+ T Cells

CXCR4-using human immunodeficiency virus, type 1 (HIV-1) variants emerge late in the course of infection in >40% of individuals infected with clade B HIV-1 but are described less commonly with clade C isolates. Tat is secreted by HIV-1-infected cells where it acts on both uninfected bystander cells and infected cells. In this study, we show that clade B Tat, but not clade C Tat, increases CXCR4 surface expression on resting CD4⁺ T cells through a CCR2b-dependent mechanism that does not involve de novo protein synthesis. The expression of plectin, a cytolinker protein that plays an important role as a scaffolding platform for proteins involved in cellular signaling including CXCR4 signaling and trafficking, was found to be significantly increased following B Tat but not C Tat treatment. Knockdown of plectin using RNA interference showed that plectin is essential for the B Tat-induced translocation of CXCR4 to the surface of resting CD4⁺ T cells. The increased surface CXCR4 expression following B Tat treatment led to increased function of CXCR4 including increased chemoattraction toward CXCR4-using-gp120. Moreover, increased CXCR4 surface expression rendered resting CD4⁺ T cells more permissive to X4 but not R5 HIV-1 infection. However, neither B Tat nor C Tat was able to up-regulate surface expression of CXCR4 on activated CD4⁺ T cells, and both proteins inhibited the infection of activated CD4⁺ T cells with X4 but not R5 HIV-1. Thus, B Tat, but not C Tat, has the capacity to render resting, but not activated, CD4⁺ T cells more susceptible to X4 HIV-1 infection.     

HIV-1 Tat protein enhances RANKL/M-CSF-mediated osteoclast differentiation

Impaired osteoblast/osteoclast cross-talk and bone structure homeostasis resulting in osteopenia/osteoporosis are often observed in HIV seropositive patients but the causal mechanisms remain unsettled. This study analyzed the biological effects of Tat on peripheral blood monocyte-derived osteoclast differentiation. Tat enhances osteoclast differentiation and activity induced by RANKL plus M-CSF treatment increasing both the mRNA expression of specific osteoclast differentiation markers, such as cathepsin K and calcitonin receptor, and TRAP expression and activity. These Tat-related biological effects may be related, at least in part, to the induction of c-fos expression and AP-1 activity. c-fos up-regulation was triggered by Tat when cell cultures were co-treated with RANKL/M-CSF and an analysis of c-fos promoter with c-fos deletion mutant constructs disclosed specific c-fos promoter domains targeted by Tat. Together, these results show that Tat may be considered a viral factor positively modulating the osteoclastogenesis and then bone resorption activity suggesting a pathogenetic role of this viral protein in the HIV-related osteopenia/osteoporosis.     

Cytoplasmic transduction peptide (CTP): new approach for the delivery of biomolecules into cytoplasm in vitro and in vivo

The protein transduction domain (PTD) of HIV-1 TAT has been extensively documented with regard to its membrane transduction potential, as well as its efficient delivery of biomolecules in vivo. However, the majority of PTD and PTD-conjugated molecules translocate to the nucleus rather than to the cytoplasm after transduction, due to the functional nuclear localization sequence (NLS). Here, we report a cytoplasmic transduction peptide (CTP), which was deliberately designed to ensure the efficient cytoplasmic delivery of the CTP-fused biomolecules. In comparison with PTD, CTP and its fusion partners exhibited a clear preference for cytoplasmic localization, and also markedly enhanced membrane transduction potential. Unlike the mechanism underlying PTD-mediated transduction, CTP-mediated transduction occurs independently of the lipid raft-dependent macropinocytosis pathway. The CTP-conjugated Smac/DIABLO peptide (Smac-CTP) was also shown to be much more efficient than Smac-PTD in the blockage of the antiapoptotic properties of XIAP, suggesting that cytoplasmic functional molecules can be more efficiently targeted by CTP-mediated delivery. In in vivo trafficking studies, CTP-fused beta-gal exhibited unique organ tropisms to the liver and lymph nodes when systemically injected into mice, whereas PTD-beta-gal exhibited no such tropisms. Taken together, our findings implicate CTP as a novel delivery peptide appropriate for (i) molecular targeting to cytoplasmic compartments in vitro, (ii) the development of class I-associated CTL vaccines, and (iii) special drug delivery in vivo, without causing any untoward effects on nuclear genetic material.     

The immunophilin ligand GPI1046 protects neurons from the lethal effects of the HIV-1 proteins gp120 and Tat by modulating endoplasmic reticulum calcium load

The dysfunction and death of neuronal cells is thought to underlie the cognitive manifestations of human immunodeficiency virus (HIV)-associated neurological disorders. Although HIV-infected patients are living longer owing to the effectiveness of anti-retroviral therapies, the number of patients developing neurological disorders is on the rise. Thus, there is an escalating need for effective therapies to preserve cognitive function in HIV-infected patients. Using HIV-protein-induced neurotoxicity as a model system, we tested the effectiveness of a non-immunosuppressive immunophilin ligand to attenuate gp120 and Tat-induced modification of neuronal function. The immunophilin ligand GPI1046 attenuated endoplasmic reticulum (ER) calcium release induced by gp120 and Tat and protected neurons from the lethal effect of these neurotoxic HIV proteins. Both inositol 1,4,5 trisphosphate (IP(3)) and ryanodine-sensitive ER calcium release was attenuated by pre-incubation with GPI1046. Using the sarco/endoplasmic reticulum calcium pump inhibitor thapsigargin to release ER calcium, we determined that GPI1046 reduced the total ER calcium load. These findings suggest that non-immunosuppressive immunophilin ligands may be useful neuroprotective drugs in HIV dementia.     

MMP-9 antisense oligodeoxynucleotide exerts an inhibitory effect on osteoclastic bone resorption by suppressing cell migration

We have previously shown that matrix metalloproteinases (MMPs) play a role in osteoclastic bone resorption by facilitating migration of osteoclastic cells toward bone surface through matrices. Of MMPs identified so far, MMP-9 is likely the most important proteinase for the action, since osteoclasts express this enzyme at a tremendously high level. However, no direct evidence has been provided to demonstrate its contribution to bone resorption. In this study, to address this point, we used an MMP-9 antisense phosphothiorate oligodeoxynucleotide (S-ODN), which was shown to inhibit the protein synthesis of MMP-9 efficiently. We demonstrated that the antisense S-ODN inhibited osteoclastic pit formation on matrigel-coated dentine slices in a concentration-dependent manner with a maximum reduction of total pit volume by 53% at 10 microM. These results, taken together, suggest that MMP-9 is involved in osteoclastic bone resorption process possibly by facilitating migration of osteoclasts through proteoglican-rich matrices.