ITH14001, a CGP37157-Nimodipine Hybrid Designed to Regulate Calcium Homeostasis and Oxidative Stress, Exerts Neuroprotection in Cerebral Ischemia.

During brain ischemia, oxygen and glucose deprivation induces calcium overload, extensive oxidative stress, neuroinflammation, and, finally, massive neuronal loss. In the search of a neuroprotective compound to mitigate this neuronal loss, we have designed and synthesized a new multitarget hybrid (ITH14001) directed at the reduction of calcium overload by acting on two regulators of calcium homeostasis; the mitochondrial Na+/Ca2+ exchanger (mNCX) and L-type voltage dependent calcium channels (VDCCs). This compound is a hybrid of CGP37157 (mNCX inhibitor) and nimodipine (L-type VDCCs blocker), and its pharmacological evaluation revealed a moderate ability to selectively inhibit both targets. These activities conferred concentration-dependent neuroprotection in two models of Ca2+ overload, such as toxicity induced by high K+ in the SH-SY5Y cell line (60% protection at 30 µM) and veratridine in hippocampal slices (26% protection at 10 µM). It also showed neuroprotective effect against oxidative stress, an activity related to its nitrogen radical scavenger effect and moderate induction of the Nrf2-ARE pathway. Its Nrf2 induction capability was confirmed by the increase of the expression of the antioxidant and anti-inflammatory enzyme heme-oxygenase I (3-fold increase). In addition, the multitarget profile of ITH14001 led to anti-inflammatory properties, shown by the reduction of nitrites production induced by lipopolysaccharide in glial cultures. Finally, it showed protective effect in two acute models of cerebral ischemia in hippocampal slices, excitotoxicity induced by glutamate (31% protection at 10 µM) and oxygen and glucose deprivation (76% protection at 10 µM), reducing oxidative stress and iNOS deleterious induction. In conclusion, our hybrid derivative showed improved neuroprotective properties when compared to its parent compounds CGP37157 and nimodipine.

Development of 4,5-dihydro-benzodiazepinone derivatives as a new chemical series of BRD4 inhibitors.

Bromodomains (BRDs) are protein interaction modules that selectively recognize ε -N-lysine residues, serving as key epigenetic readers and play a key role in epigenetic regulation of gene transcription. Bromodomain-containing protein 4 (BRD4), a protein containing two BRDs termed BD1 and BD2, has emerged as an attractive candidate for the development of inhibitors targeting gene transcription in several types of cancers. In this study, we made structural modifications of previously reported BRD4 inhibitors, to develop new chemical scaffold 3,4-dihydroquinoxalin-2(1H)-one. Four series of compounds (compounds 7-10) were synthesized, and the BRD4-inhibitory activity and anti-proliferative effect of these compounds were evaluated. We found compound 10d has remarkable anti-proliferative activities toward leukemia cells and could induce apoptosis by mitochondrial pathways. Notably, the analysis of molecular docking suggested that hydrophobic interaction was essential for compound 10d to bind to BD1. In conclusion, these results demonstrate the potential of compound 10d to be utilized as a BRD4 inhibitor with apoptosis inducing effect in future leukemia therapy.

Cytotoxic and pathogenic properties of Klebsiella oxytoca isolated from laboratory animals.

Klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. Studies suggest that in humans K. oxytoca exerts its pathogenicity in part through a cytotoxin. However, cytotoxin production in animal isolates of K. oxytoca and its pathogenic properties have not been characterized. Furthermore, neither the identity of the toxin nor a complete repertoire of genes involved in K. oxytoca pathogenesis have been fully elucidated. Here, we showed that several animal isolates of K. oxytoca, including the clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on HEp-2 and HeLa cells, indicating the ability to produce cytotoxin. Cytotoxin production appears to be regulated by the environment, and soy based product was found to have a strong toxin induction property. The toxin was identified, by liquid chromatography-mass spectrometry and NMR spectroscopy, as low molecular weight heat labile benzodiazepine, tilivalline, previously shown to cause cytotoxicity in several cell lines, including mouse L1210 leukemic cells. Genome sequencing and analyses of a cytotoxin positive K. oxytoca strain isolated from an abscess of a mouse, identified genes previously shown to promote pathogenesis in other enteric bacterial pathogens including ecotin, several genes encoding for type IV and type VI secretion systems, and proteins that show sequence similarity to known bacterial toxins including cholera toxin. To our knowledge, these results demonstrate for the first time, that animal isolates of K. oxytoca, produces a cytotoxin, and that cytotoxin production is under strict environmental regulation. We also confirmed tilivalline as the cytotoxin present in animal K. oxytoca strains. These findings, along with the discovery of a repertoire of genes with virulence potential, provide important insights into the pathogenesis of K. oxytoca. As a novel diagnostic tool, tilivalline may serve as a biomarker for K oxytoca-induced cytotoxicity in humans and animals through detection in various samples from food to diseased samples using LC-MS/MS. Induction of K. oxytoca cytotoxin by consumption of soy may be in part involved in the pathogenesis of gastrointestinal disease.

Developments of polo-like kinase 1 (Plk1) inhibitors as anti-cancer agents.

Polo-like kinases (Plks) are a family of serine/threonine kinases with a highly conserved N-terminal Ser/Thr kinase catalytic domain and a C-terminal region that play crucial roles in cell cycle progression. Plk1, playing a key role in multiple steps of mitotic progression, is the most studied member of the family. It is overexpressed in a wide spectrum of cancer types and is a promising target in oncology. Most of Plk1 inhibitors competitively bind to the ATP-binding site, which is characterized with unique features. Other inhibitors target regions outside the ATP pocket. In this review some pre-clinical or clinical Plk1 inhibitors are reported, focusing on SAR studies and biological activities, including the kinase activity, in vitro and in vivo anti-tumor efficacy. Those studies exhibited the inhibitors' significant therapeutic effects. Moreover, combination therapies of these Plk1 inhibitors with other anticancer drugs resulted with synergistic effects.

Investigation of the inhibitory effects of the benzodiazepine derivative, 5-BDBD on P2X4 purinergic receptors by two complementary methods.

BACKGROUND/AIMS: ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs. METHODS: We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs. RESULTS: Our data show that ATP (< 1 µM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs. CONCLUSIONS: Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different.

RING-finger type E3 ubiquitin ligase inhibitors as novel candidates for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) significantly affects quality of life. We recently cloned synoviolin, a RING-type E3 ubiquitin ligase implicated in the endoplasmic reticulum-associated degradation (ERAD) pathway. Synoviolin is highly expressed in rheumatoid synovial cells and may be involved in the pathogenesis of RA. Inhibition of synoviolin activity is a potentially useful therapeutic approach for the treatment of RA. We conducted a high-throughput screen of small molecules to find inhibitors of synoviolin autoubiquitination activity. We identified two classes of small molecules, named LS-101 and LS-102, which inhibited synoviolin activity. LS-102 selectively inhibited synoviolin enzymatic activity, while LS-101 inhibited a broad array of RING-type E3 ligases. Moreover, these inhibitors suppressed the proliferation of rheumatoid synovial cells, and significantly reduced the severity of disease in a mouse model of RA. Our results suggest that inhibition of synoviolin is a potentially useful approach in the treatment of RA.

Discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly.

The discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly is described. Synthesis of analogs of the 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione hit established structure-activity relationships. Replacement of the enamine functionality of the hit series with either an imidazole or a pyrazole ring led to compounds that inhibited both capsid assembly and reverse transcriptase. Optimization of the bicyclic benzodiazepine scaffold to include a 3-phenyl substituent led to lead compound 48, a pure capsid assembly inhibitor with improved antiviral activity.

Pre-clinical evaluation of a new orally-active CCK-2R antagonist, Z-360, in gastrointestinal cancer models.

BACKGROUND: Gastrin has a role in gastrointestinal (GI) malignancy. This study provides pre-clinical evaluation of a novel, orally-active gastrin/cholecystokinin-2 receptor (CCK-2R) antagonist, Z-360. METHODS: (125)I gastrin-17 (G17) displacement and G17-stimulated calcium assays were used in classical CCK-2R-transfected cell lines. Akt phosphorylation was assessed by Western blotting. Z-360 efficacy in vivo was evaluated in three human xenograft models, and microvessel density and apoptosis in these models were investigated by immunohistochemistry. RESULTS: Z-360 inhibited (125)I G17 binding to cells expressing CCK-2R, and G17-stimulated signalling. Reduced Akt phosphorylation in an oesophageal cell-line treated with Z-360 was reversed by co-treatment with G17. Z-360 increased survival in a gastric ascites model (p=0.011) and decreased tumour growth in a hepatic metastasis model (81%, p=0.02). In an orthotopic pancreatic model, Z-360 combined with gemcitabine decreased final tumour weight compared to single agents (84%, p=0.002) and there was increased apoptosis and decreased microvessel density in ex vivo tumour tissue. CONCLUSIONS: These results show that the orally-active CCK-2R antagonist, Z-360 has high sub-nM affinity for classical CCK-2R, is well tolerated in vivo and exerts an anti-tumour effect.

Peripheral benzodiazepine receptors in potatoes (Solanum tuberosum).

The peripheral benzodiazepine receptor (PBR), an internal protein of the mammalian mitochondrial membrane, is involved in several metabolic functions such as steroidogenesis, oxidative phosphorylation, and regulation of cell proliferation. Here we report the presence of PBRs in parenchymal and meristematic tissues of potato (Solanum tuberosum). PBRs are heterogeneously distributed in potato and are highly expressed in meristematic cells. In particular the receptor protein is mainly localised in the meristematic nuclear subcellular preparation. This 30-36 kDa protein, which corresponds to PBR, is increased, indeed, in meristematic compared to the parenchymal tissue. This suggests an involvement of this receptor in the regulation of cell plant growth. In addition, the demonstration that PBRs are also present in vegetables supports the hypothesis of a highly conserved receptor system during phylogenesis.

Non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors: past, present, and future perspectives.

Along with nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) have gained a definitive and important place in the treatment of HIV-1 infections, and are in rapid development. These compounds can be grouped into two classes: the first generation NNRTIs, mainly discovered by random screening, and the second generation NNRTIs, developed as a result of comprehensive strategies involving molecular modelling, rationale-based drug synthesis, biological and pharmacokinetic evaluations. The recent boom of NNRTIs is mainly due to their antiviral potency, high specificity and low toxicity. The rapid emergence of drug-resistant HIV-1 strains induced by the first generation drugs is a disadvantage bypassed, in part, by the broad spectrum second generation NNRTIs. Starting from the first generation, this review will focus on the second generation NNRTIs dealing with the recent and most interesting published results, highlighting the guidelines for the development of a third generation of NNRTIs.

Effects of JL13, a pyridobenzoxazepine with potential atypical antipsychotic activity, in animal models for schizophrenia.

JL13 [5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5] benzoxazepine fumarate] is a substance with a close structural resemblance to clozapine. However, it is less sensitive to oxidation and may therefore have less hematological side effects. In the present study, JL13 was compared with clozapine and haloperidol in several animal models for schizophrenia. The paw test represents a screening model for antipsychotic drugs that can discriminate between drugs with extrapyramidal side effects and drugs without. Haloperidol increased both forelimb retraction time and hindlimb retraction time (HRT), whereas both clozapine and JL13 increased only HRT. In the prepulse inhibition paradigm, all three drugs reversed the apomorphine- and the amphetamine-induced disruption of prepulse inhibition. However, whereas haloperidol was equally effective against both dopaminergic drugs, JL13 and clozapine were more effective against amphetamine. Finally, only JL13 was able to increase prepulse inhibition in normal rats, whereas only clozapine reduced basal startle amplitude. Taken together, these data suggest that JL13 may be an effective antipsychotic drug, with a profile similar to clozapine.

7,8-Methylenedioxy-4H-2,3-benzodiazepin-4-ones as novel AMPA receptor antagonists.

The synthesis and anticonvulsant activity of novel 7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones 3a-e, structurally-related to GYKI 52466 1, a well-known noncompetitive AMPA-receptor antagonist, are reported. The new compounds possess marked anticonvulsant properties and, in analogy to 1, antagonize seizures induced by AMPA. In addition, when compared to the model compound 1, compounds 3 show a longer-lasting anticonvulsant activity and a lower toxicity.

Comparison of a DSB-120 DNA interstrand cross-linked adduct with the corresponding bis-tomaymycin adduct: an example of a successful template-directed approach to drug design based upon the monoalkylating compound tomaymycin.

The interstrand cross-linked DSB-120-d(CICG*ATCICG)2 DNA adduct (*indicates covalently modified guanine) was examined by two-dimensional NMR and compared to the bis-tomaymycin adduct on the same oligomer. Tomaymycin and DSB-120 form self-complementary adducts with the d(CICGATCICG)2 duplex sequence in which the covalent linkage sites occur between C11 of either drug and the exocyclic 2-amino group of the single reactive guanine on each strand of d(CICGATCICG)2. In the case of DSB-120, this is evidence for the formation of a guanine--guanine DNA interstrand cross-link. Both drugs show formation of an S stereo-chemistry at the covalent linkage site with an associated 3' orientation. While the majority of DNA in these adducts appears to be B-form, DSB-120 interstrand cross-linking induces atypical properties in the 8I nucleotide, indicated by broadening of the 8IH2 proton resonance, non-C2' endo sugar geometry, and unusually weak internucleotide NOE connectivity to the 7C nucleotide. Tomaymycin does not produce this regional dislocation. For tomaymycin, while there are strong NOE connectivities from protons on the five-membered ring to the 8IH2 proton on the floor of the minor groove, the equivalent internucleotide connectivities in DSB-120 are weaker. This indicates that the tomaymycin tail is close to the floor of the minor groove, while the five-membered ring of DSB-120 is more shallowly immersed, perhaps due to strain from cross-linking with a very short linker unit. Last, the conformational stresses induced on the duplex by DSB-120 appear to make the region of covalent attachment more accessible to solvent than is the case for tomaymycin. The 4GN2Hb resonance appears in 100% D2O on the tomaymycin adduct but is only observed in 90% H2O/10% D2O for the DSB-120 adduct. On the basis of these results, the strategies for template-directed DNA cross-linker design are assessed.