Chimeric VLPs Bearing VP60 from Two Serotypes of Rabbit Haemorrhagic Disease Virus Are Protective against Both Viruses.

The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.

Design, synthesis, and biological evaluation of hybrid molecules containing alpha-methylene-gamma-butyrolactones and polypyrrole minor groove binders.

We have synthesized and evaluated a series of hybrids of polypyrrole minor groove binders structurally related to the natural antitumor agent distamycin A, and alpha-methylene-gamma-butyrolactones with methyl, phenyl, and 4-substituted phenyl groups at the lactone C(gamma) position, denoted 5-17, for in vitro cytotoxic activity against a variety of cancer cell lines. The apoptotic and cytotoxic activities against several tumor cell lines are reported and discussed in terms of their structural differences in relation to both the number of N-methylpyrrole rings and the type of the alkylating unit tethered to the oligopeptidic frame. It may be noted that in general, and especially for 11, 12, and 17, the cytotoxicity of the hybrids was much greater than that of the alpha-methylene-gamma-butyrolactone units 24a-g alone. Using the human leukemia cell line HL-60, we have tested the effects of a selected series of compounds on programmed cell death (apoptosis). The results clearly indicate that 11, 12, and 17, but not 9, are able to induce apoptosis as demonstrated from (i) identification of nuclear changes associated with apoptosis using fluorescence microscopy and (ii) by DNA laddering on agarose gel electrophoresis. Compound 12 was the most potent, especially after a short incubation period. It induced extensive hydrolysis of poly ADP-ribose polymerase (PARP), considered to be a hallmark of apoptosis, which plays a critical role in chromatin architecture and DNA metabolism.

Synthesis of nitro esters of prednisolone, new compounds combining pharmacological properties of both glucocorticoids and nitric oxide.

Glucocorticoids (GC) are widely used in therapy for their many pharmacological properties including antiinflammatory and immunosuppressive actions. However, their use over long periods is hampered by a number of severe side effects. Given the biological properties of nitric oxide (NO) and previous experience with nonsteroidal antiinflammatory agents, we synthesized new chemical entities combining both NO and GC properties. Here we report the synthesis of nitro esters of prednisolone obtained through the esterification, with different linkers, on the hydroxy group at C-21 position of the corticosteroid structure. The alkyl chain, as of the nitrooxy derivative (2), or aromatic linkers, as of o-, m-, and p-nitrooxymethylbenzoate derivatives (3-5), respectively, furnish stable compounds that release NO and inhibit the GC receptors in biological assays. To improve solubility we introduced a more water-soluble linker such as the nitrooxyalkylpiperidine or -piperazine group (6-9). Also these compounds retained properties of both NO and prednisolone. Compound 5 (NCX 1015) was selected for its better profile: enhanced antiinflammatory properties and reduced side effects compared with prednisolone. NCX 1015 is currently under preclinical development.

Inhibition of choline kinase as a specific cytotoxic strategy in oncogene-transformed cells.

Cancer treatment is in the need of selective drugs that can interfere specifically with signalling pathways affected during the carcinogenic process. Identification of new potential molecular targets is the key event in the design of new anticancer strategies. Once identified, attempts for the generation of specific molecules to regulate their function can be achieved. The relevance of deregulation of choline kinase (ChoK, E.C. 2.7.1.32) in oncogene-driven cell transformation has been previously demonstrated. Here we provide strong evidence that MN58b, a selective inhibitor of ChoK, is rather specific to this enzyme, with no effect on a variety of oncogene-activated signalling pathways involved in the regulation of cell proliferation. MN58b does not affect MAPKs, PI3K, and other enzymes involved in the regulation of phospholipid metabolism such as phospholipases C, D, and A2, CTP:phosphocholine cytidylyltransferase, or diacylglycerol choline-phosphotransferase. Consistent with this specificity, ectopic expression of ChoK resulted in resistance to its inhibitor. Finally, nontransformed cells were able to resume cell proliferation after removal of the drug, while transformed cells were irreversibly affected. These results indicate that inhibition of ChoK is a rather specific strategy for the cytotoxic treatment of transformed cells.

Synthesis and biological activity of N-arylpiperazine-modified analogues of KN-62, a potent antagonist of the purinergic P2X7 receptor.

The P2X(7) receptor is involved in several processes relevant to inflammation (cytokine release, NO generation, killing of intracellular pathogens, cytotoxicity); thus, it may be an appealing target for pharmacological intervention. The characterization of native and recombinant P2X(7) receptor continues to be hindered by the lack of specific and subtype-selective antagonists. However, a tyrosine derivative named KN-62 exhibits selective P2X(7) receptor-blocking properties. The present study was designed to evaluate the functional antagonistic properties of a novel series of KN-62-related compounds characterized by the presence of different phenyl-substituted piperazine moieties. Antagonistic activity of KN-62 derivatives was tested on HEK293 cells transduced with the human P2X(7) receptor and monocyte-derived human macrophages, a cell type well-known for the high level of expression of this receptor. The biological responses investigated were ATP-dependent Ca(2+) influx across the plasma membrane, ethidium bromide uptake, and secretion of the cytokine interleukin-1beta. KN-62 was characterized by the presence of a phenylpiperazine moiety, and the presence of a one-carbon linker between the piperazine nitrogen and the phenyl ring (compound 61) increases the activity, while a two-carbon linker (compound 62) decreases biological activity 10-fold. Also, the nature and the position of substituents on the phenyl ring tethered to the piperazine seemed to exert a fundamental influence on the biological activity. In the series of synthesized compounds, the presence of a fluorine in the para position gives the most potent compound (63), while the same atom in the ortho position reduces potency by 3-fold. When the p-fluorine was replaced in the same position with other halogens, such as chlorine (compound 64) or iodine (compound 65), the activity decreased dramatically. We then tested the activity of the four most potent KN-62 derivatives on ATP-stimulated secretion of IL-1beta from monocyte-derived human macrophages, a key cell type in inflammation and innate immunity. Interestingly, compound 68 and 71 caused a complete inhibition of IL-1beta release, while with KN-62, 63, and 85, there was a small residual cytokine secretion even at concentrations exceeding 100 nM. None of the compounds tested on IL-1beta release had any effect on isolated CaMII kinase activity up to 20 microM (not shown).

Spiro[2.2]pentane as a dissymmetric scaffold for conformationally constrained analogues of glutamic acid: focus on racemic 1-aminospiro[2.2]pentyl-1,4-dicarboxylic acids.

In search for novel conformationally constrained analogues of L-glutamic acid, a diastereodivergent synthesis of the four 1-aminospiro[2.2]pentyl-1,4-dicarboxylic acid racemic pairs is reported along with their stereochemical assignment, conformational analysis, and preliminary biological evaluation as potential glutamate (ionotropic and metabotropic) ligands.

Benzoyl and cinnamoyl nitrogen mustard derivatives of benzoheterocyclic analogues of the tallimustine: synthesis and antitumour activity.

A series of benzoyl and cinnamoyl nitrogen mustards tethered to different benzoheterocycles and to oligopyrroles structurally related to netropsin consisting of two pyrrole-amide units and terminating with an amidine moiety have been synthesised and a structure--activity relationship determined. Derivatives 3--10 have been evaluated for their sequence selective alkylating properties and cytotoxicity against human K562 leukaemia cells. They are 2- to 50-fold less cytotoxic than tallimustine, with compound 8 being the most potent member of this series. Among tallimustine isosters, the compounds with an indole 3 or benzothiophene 6 are 4-fold less cytotoxic than tallimustine, while the compounds with an N-methyl indole or benzofuran showed a 7- and 14-fold reduced cytotoxic potency, respectively. Our preliminary results indicate that these derivatives preferentially bind to AT-rich sequence with a sequence selectivity similar to tallimustine.