Design, synthesis and preliminary evaluation of a series of histone deacetylase inhibitors carrying a benzodiazepine ring.

A series of new histone deacetylase inhibitors were designed and synthesized based on hybridization between SAHA or oxamflatin and 5-phenyl-1,4-benzodiazepines. The compounds were tested for their enzyme inhibitory activity on HeLa nuclear extracts, and on human recombinant HDAC1 and HDAC6. Antiproliferative activity was tested on different cancer cells types, while proapoptotic activity was primarily tested on NB4 cells. The compounds showed IC50 values similar to those of SAHA. Compound (S)-8 displayed interesting activity against hematological and solid malignancies.

Influence of ring size on the cognition-enhancing activity of DM235 and MN19, two potent nootropic drugs.

A series of analogs of DM235 and MN19, characterized by rings with different size, have been prepared and evaluated for their nootropic activity in the mouse passive-avoidance test. It was found that the optimal ring size for the analogs of DM235, showing endocyclic both amidic groups, is 6 or 7 atoms. For the compounds structurally related to MN19, carrying an exocyclic amide group, the piperidine ring is the moiety which gives the most interesting compounds.

The new low-toxic histone deacetylase inhibitor S-(2) induces apoptosis in various acute myeloid leukaemia cells.

Histone deacetylase inhibitors (HDACi) induce tumour cell cycle arrest and/or apoptosis, and some of them are currently used in cancer therapy. Recently, we described a series of powerful HDACi characterized by a 1,4-benzodiazepine (BDZ) ring hybridized with a linear alkyl chain bearing a hydroxamate function as Zn(++)--chelating group. Here, we explored the anti-leukaemic properties of three novel hybrids, namely the chiral compounds (S)-2 and (R)-2, and their non-chiral analogue 4, which were first comparatively tested in promyelocytic NB4 cells. (S)-2 and partially 4--but not (R)-2--caused G0/G1 cell-cycle arrest by up-regulating cyclin G2 and p21 expression and down-regulating cyclin D2 expression, and also apoptosis as assessed by cell morphology and cytofluorimetric assay, histone H2AX phosphorylation and PARP cleavage. Notably, these events were partly prevented by an anti-oxidant. Moreover, novel HDACi prompted p53 and α-tubulin acetylation and, consistently, inhibited HDAC1 and 6 activity. The rank order of potency was (S)-2 > 4 > (R)-2, reflecting that of other biological assays and addressing (S)-2 as the most effective compound capable of triggering apoptosis in various acute myeloid leukaemia (AML) cell lines and blasts from patients with different AML subtypes. Importantly, (S)-2 was safe in mice (up to 150 mg/kg/week) as determined by liver, spleen, kidney and bone marrow histopathology; and displayed negligible affinity for peripheral/central BDZ-receptors. Overall, the BDZ-hydroxamate (S)-2 showed to be a low-toxic HDACi with powerful anti-proliferative and pro-apototic activities towards different cultured and primary AML cells, and therefore of clinical interest to support conventional anti-leukaemic therapy.

Design, synthesis and preliminary biological evaluation of new hydroxamate histone deacetylase inhibitors as potential antileukemic agents.

This study concerns the synthesis of new histone deacetylase inhibitors (HDACi) characterized by a 1,4-benzodiazepine ring used as the cap, joined through an amide function or a triple bond as connection units, to a linear alkyl chain bearing the hydroxamate function as Zn2+-chelating group. Biological tests performed in human acute promyelocytic leukemia NB4 cells showed that new hybrids can induce histone H3/H4 acetylation, growth arrest, and also apoptosis. Notably, chiral compounds exhibit stereoselective activity.

Radiopacity of tantalum-loaded acrylic bone cement.

Radiopacifying agents are commonly added to bone cements to enhance the visibility of the cement in radiography. The radiopacifiers usually employed may, however, have undesired effects on the mechanical properties of the cement. A potentially new radiopacifier is tantalum, which in the present work was evaluated in terms of radiopacity. Bone cements containing different percentages of tantalum were compared with plain bone cement as well as with formulations containing different percentages of the commonly used radiopacifier barium sulphate. The radiopacity was assessed quantitatively and qualitatively, by measuring with a digital densitometer the optical density of the cement on X-ray films, and consulting the expertise of ten orthopaedic surgeons. It was found that tantalum does present radiopacity, but not as high as barium sulphate under the specific conditions applied to this study.

A procedure and criterion for bone cement fracture toughness tests.

Nowadays, two procedures, based on the recommendation of two American standards (ASTM E399 and ASTM D5045), are used to determine the fracture toughness, KIc, of bone cement. However, there is a lack of knowledge about the equivalence of the two testing methods applied to bone cement. Additionally, in spite of the recommendation of several authors to introduce a rejection criterion for specimens based on the size of defects found in the fracture surface, no data are available about the effect of porosity within the material on the KIc of bone cement. The aims of this study were to verify whether the KIc values calculated for bone cement using the two procedures are comparable and whether macroporosity within the tested samples affects the KIc value of bone cement, and, if so, to establish a rejection criterion for specimen selection. Samples of pure polymethyl methacrylate (PMMA) were tested by both procedures. Additionally, samples showing defects (macroporosity) of different sizes and located in different positions within the specimen were tested. The KIc value determined following the ASTM E399 procedure was 13 per cent lower than that calculated following the ASTM D5045 procedure. In the first series a lower data scatter was observed. Also, the presence of macroporosity on the fracture surface of the specimen affected the KIc value of bone cement. Therefore, the mechanical behaviour of samples was affected by defects within the material. Since it is possible to mould specimens without macroporosity, it seems recommendable to reject specimens with macroporosity on the fracture surface before calculating the KIc value of bone cement.

Structure-affinity relationships of a unique nicotinic ligand: N(1)-dimethyl-N(4)-phenylpiperazinium iodide (DMPP).

DMPP is a well-known nicotinic agonist that does not fit any proposed pharmacophore for nicotinic binding and represents a unique ligand among the hundreds of nicotinic agonists studied in the past decades. A systematic modulation of the chemical structure of DMPP, aimed to establish its structure-affinity relationships, is reported. The research has allowed to identify molecules such as 11c, 13c, 14c, and 28c, with affinities for alpha(4)beta(2) receptors in the low nanomolar range, some 2 orders of magnitude lower than the lead compound. The agonistic properties of the most interesting compounds have been assessed by measuring their analgesic activity on mice (hot-plate test). Another result of the research was the identification of DMPP analogues, such as 3a (K(i) = 90 nM) and 14b (K(i) = 180 nM), that maintain affinity for the central nicotinic receptor when the ammonium function is changed into an aminic one and are therefore possible leads for drug development in neurodegenerative diseases.

Molecular cloning and expression of an interferon-inducible protein encoded by gene 203 from the gene 200 cluster.

We report here the complete coding sequence of a 203 cDNA, a member of the interferon-inducible Ifi 200 gene family. By combining reverse-transcriptase PCR and rapid amplification of cDNA ends (RACE) techniques we have obtained a 3.8-kb cDNA corresponding to a 203 mRNA. When used as a probe in northern analysis, its 3' segment hybridized to a 3.8-kb interferon-inducible mRNA, whereas the 5'-end additionally hybridized to a less abundant interferon-inducible 1.8-kb mRNA. Nucleotide sequence analysis revealed that the two mRNAs share the 5'-untranslated region and the same open reading frame, which encodes a hydrophilic protein composed of 408 amino acids. The difference between them is due to a 3'-untranslated region extended by alternative polyadenylation site selection. Furthermore, 203 mRNA was found to be inducible by interferon-alpha in various murine cell lines. Using polyclonal antibodies raised against a segment specific for the 203 protein, we established that p203 protein levels increase on treatment with interferon-alpha in murine fibroblasts and that p203 is located in the nucleus.

The murine cytomegalovirus immediate-early 1 protein stimulates NF-kappa B activity by transactivating the NF-kappa B p105/p50 promoter.

The transcription of murine cytomegalovirus (MCMV) immediate-early (IE) genes is regulated by a large and complex enhancer containing several consensus binding sites for the ubiquitous transcription factor NF-kappa B. To verify whether MCMV, like the human CMV, can activate NF-kappa B-dependent transcription, we transfected murine embryo fibroblasts cells with a construct containing three copies of the NF-kappa B element in front of the homologous minimal MCMV IE1-3 promoter. Upon MCMV infection the reporter gene activity was transactivated to about three-fold above the basal level. The specificity of this transactivation was demonstrated by the lack of any significant effect on the activity of DNA constructs containing either a mutated NF-kappa B trimer or an ATF/CRE trimer. Gel shift assays with a NF-kappa B probe revealed that MCMV infection activated DNA binding proteins showing NF-kappa B characteristics. The DNA-binding activity remained elevated during the course of infection and was associated to an increase in the steady-state mRNA levels for the NF-kappa B subunit p105/p50. Since the promoter of the p105/p50 gene was transactivated by MCMV infection during the period in which the IE proteins are expressed, the role of the two major IE transcriptional regulatory proteins was examined. In cotransfection experiments, the IE1 protein transactivated the p105/p50 promoter, whereas the IE3 was ineffective in increasing the transcription of the reporter gene. Taken as a whole, these results demonstrate that MCMV, like its human counterpart, regulates the cellular NF-kappa B activity needed for the initial induction of the IE genes and the progression of the viral replicative cycle.