A thiourea-bridged 99mTc(CO)3-dipicolylamine-2-nitroimidazole complex for targeting tumor hypoxia: Utilizing metabolizable thiourea-bridge to improve pharmacokinetics.

The 2-nitroimidazole based 99mTc-radiopharmaceuticals are widely explored for imaging tumor hypoxia. Radiopharmaceuticals for targeting hypoxia are often lipophilic and therefore, show significant uptake in liver and other vital organs. In this context, lipophilic radiopharmaceuticals with design features enabling faster clearance from liver may be more desirable. A dipicolylamine-NCS bifunctional chelator that could generate a thiourea-bridge up on conjugation to primary amine bearing molecule was used to synthesize a 2-nitroimidazole-dipicolyl amine ligand for radiolabeling with 99mTc(CO)3 core. Corresponding Re(CO)3-analogue was prepared to establish the structure of 2-nitroimidazole-99mTc(CO)3 complex prepared in trace level. The 2-nitroimidazole-99mTc(CO)3 complex showed a hypoxic to normoxic ratio of ~2.5 in CHO cells at 3 h. In vivo, the complex showed accumulation and retention in tumor with high tumor to blood and tumor to muscle ratio. The study demonstrated the utility of metabolizable thiourea-bridge in 2-nitroimidazole-99mTc(CO)3 complex in inducing faster clearance of the radiotracer from liver. The dipicolylamine-NCS bifunctional chelator reported herein can also be used for radiolabeling other class of target specific molecules with 99mTc(CO)3 core.

Impact of aliphatic acyl and aromatic thioamide substituents on the anticancer activity of Ru(II)-p-cymene complexes with acylthiourea ligands-in vitro and in vivo studies.

Six different acylthiourea ligands (L1-L6) and their corresponding Ru(II)-p-cymene complexes (P1-P6) were designed to explore the structure-activity relationship of the complexes upon aliphatic chain and aromatic conjugation on the C- and N-terminals, respectively. The compounds were synthesized and adequately characterized using various analytical and spectroscopic techniques. The structures of P2-P6, solved using single crystal X-ray diffraction (XRD), confirmed the neutral monodentate coordination of the S atoms of the acylthiourea ligands to Ru(II) ions. In silico studies showed an increase of lipophilicity for the ligands with an increase in alkyl chain length or aromatic conjugation at the C- or N-terminal, respectively. Subsequently, mitogen-activated protein kinases (MAPK) were predicted as one of the primary targets for the complexes, which showed good binding affinity towards extracellular signal-regulated kinases (ERK1, ERK2 and ERK5), c-Jun N-terminal kinase (JNK) and p38 of the MAPK pathway. Henceforth, the complexes were tested for their anticancer activity in lung carcinoma (A549) and cisplatin-resistant lung carcinoma (cisA549R) cells and human umbilical vein epithelial normal cells (HUVEC). Interestingly, an increase in chain length or aromatic conjugation led to an increase in the activity of the complexes, with P5 (7.73 and 13.04 µM) and P6 (6.52 and 14.45 µM) showing the highest activity in A549 and cisA549R cells, which is better than the positive control, cisplatin (8.72 and 44.28 µM). Remarkably, we report the highest activity yet observed for complexes of the type [(η6-p-cymene)RuIICl2(S-acylthiourea)] in the tested cell lines. Aqueous solution studies showed that complexes P5 and P6 are rapidly hydrolyzed to produce solely aquated species that remained stable for 24 h. Staining assays and flow cytometric analyses of P5 and P6 in A549 cells revealed that the complexes induced apoptosis and arrested the cell cycle predominantly in the S phase. In vivo studies demonstrated the higher toxicity of cisplatin and a comparatively higher survival rate of mice injected with the most active complex P6. Histological analyses revealed that treatment with P6 at high doses of up to 8 mg kg-1 did not cause any palpable damage to the tested organs.

Synthesis, Antifungal Activity, and 3D-QSAR Study of Novel Nopol-Derived 1,3,4-Thiadiazole-Thiourea Compounds.

A series of novel nopol derivatives bearing the 1,3,4-thiadiazole-thiourea moiety were designed and synthesized by multi-step reactions in search of potent natural product-based antifungal agents. Their structures were confirmed by FT-IR, NMR, ESI-MS, and elemental analysis. Antifungal activity of the target compounds was preliminarily evaluated by in vitro methods against Fusarium oxysporum f. sp. cucumerinum, Cercospora arachidicola, Physalospora piricola, Alternaria solani, Gibberella zeae, Rhizoeotnia solani, Bipolaris maydis, and Colleterichum orbicalare at 50 µg/mL. All the target compounds exhibited better antifungal activity against P. piricola, C. arachidicola, and A. solani. Compound 6j (R = m, p-Cl Ph) showed the best broad-spectrum antifungal activity against all the tested fungi. Compounds 6c (R = m-Me Ph), 6q (R = i-Pr), and 6i (R = p-Cl Ph) had inhibition rates of 86.1%, 86.1%, and 80.2%, respectively, against P. piricola, much better than that of the positive control chlorothalonil. Moreover, compounds 6h (R = m-Cl Ph) and 6n (R = o-CF3 Ph) held inhibition rates of 80.6% and 79.0% against C. arachidicola and G. zeae, respectively, much better than that of the commercial fungicide chlorothalonil. In order to design more effective antifungal compounds against A. solani, analysis of the three-dimensional quantitative structure-activity relationship (3D-QSAR) was carried out using the CoMFA method, and a reasonable and effective 3D-QSAR model (r2 = 0.992, q2 = 0.753) has been established. Furthermore, some intriguing structure-activity relationships were found and are discussed by theoretical calculation.

Molecular structure, NBO analysis of the hydrogen-bonded interactions, spectroscopic (FT-IR, FT-Raman), drug likeness and molecular docking of the novel anti COVID-2 molecule (2E)-N-methyl-2-[(4-oxo-4H-chromen-3-yl)methylidene]-hydrazinecarbothioamide (Dimer) - quantum chemical approach.

Prospective antiviral molecule (2E)-N-methyl-2-[(4-oxo-4H-chromen-3-yl)methylidene]-hydrazinecarbothioamide has been probed using Fourier transform infrared (FTIR), FT-Raman and quantum chemical computations. The geometry equilibrium and natural bond orbital analysis have been carried out with density functional theory employing Becke, 3-parameter, Lee-Yang-Parr method with the 6-311G++(d,p) basis set. The vibrational assignments pertaining to different modes of vibrations have been augmented by normal coordinate analysis, force constant and potential energy distributions. Drug likeness and oral activity have been carried out based on Lipinski's rule of five. The inhibiting potency of 2(2E)-methyl-2-[(4-oxo-4H-chromen-3-yl)methylidene]-hydrazinecarbothioamide has been investigated by docking simulation against SARS-CoV-2 protein. The optimized geometry shows a planar structure between the chromone and the side chain. Differences in the geometries due to the substitution of the electronegative atom and intermolecular contacts due to the chromone and hydrazinecarbothioamide were analyzed. NBO analysis confirms the presence of two strong stable hydrogen bonded NH⋯O intermolecular interactions and two weak hydrogen bonded CH⋯O interactions. The red shift in NH stretching frequency exposed from IR substantiates the formation of NH⋯O intermolecular hydrogen bond and the blue shift in CH stretching frequency substantiates the formation of CH⋯O intermolecular hydrogen bond. Drug likeness, absorption, distribution, metabolism, excretion and toxicity property gives an idea about the pharmacokinetic properties of the title molecule. The binding energy of the nonbonding interaction with Histidine 41 and Cysteine 145, present a clear view that 2(2E)-methyl-2-[(4-oxo-4H-chromen-3-yl)methylidene]-hydrazinecarbothioamide can irreversibly interact with SARS-CoV-2 protease.

Nanoparticles Loaded with a New Thiourea Derivative: Development and In vitro Evaluation Against Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by protozoa of the genus Leishmania. Current treatments are restricted to a small number of drugs that display both severe side effects and a potential for parasites to develop resistance. A new N-(3,4-methylenedioxyphenyl)-N'- (2-phenethyl) thiourea compound (thiourea 1) has shown promising in vitro activity against Leishmania amazonensis with an IC50 of 54.14 µM for promastigotes and an IC50 of 70 µM for amastigotes. OBJECTIVE: To develop a formulation of thiourea 1 as an oral treatment for leishmaniasis, it was incorporated into Nanoparticles (NPs), a proven approach to provide long-acting drug delivery systems. METHODS: Poly (D,L-Lactic-co-Glycolic Acid) (PLGA) polymeric NPs containing thiourea 1 were obtained through a nanoprecipitation methodology associated with solvent evaporation. The NPs containing thiourea 1 were characterized for Encapsulation Efficiency (EE%), reaction yield (% w/w), surface charge, particle size and morphology by Transmission Electron Microscopy (TEM). RESULTS: NPs with thiourea 1 showed an improved in vitro leishmanicidal activity with a reduction in its cytotoxicity against macrophages (CC50>100 µg/mL) while preserving its IC50 against intracellular amastigotes (1.46 ± 0.09 µg/mL). This represents a parasite Selectivity Index (SI) of 68.49, which is a marked advancement from the reference drug pentamidine (SI = 30.14). CONCLUSION: The results suggest that the incorporation into NPs potentiated the therapeutic effect of thiourea 1, most likely by improving the selective delivery of the drug to the phagocytic cells that are targeted for infection by L. amazonensis. This work reinforces the importance of nanotechnology in the acquisition of new therapeutic alternatives for oral treatments.

Benzothiourea Derivatives Target the Secretory Pathway of the Human Fungal Pathogen Cryptococcus neoformans.

Cryptococcus neoformans is one of the most important human fungal pathogens and causes life-threatening meningoencephalitis in immunocompromised patients. The current gold standard therapy for C. neoformans meningoencephalitis is based on medications that are over 50 years old and is not readily available in regions with high disease burden. Here, we report the mycologic, mechanistic, and pharmacologic characterization of a set of benzothioureas with highly selective fungicidal activity against C. neoformans. In addition, to direct antifungal activity, benzothioureas inhibit C. neoformans virulence traits. On the basis of a set of phenotypic, biochemical, and biophysical assays, the benzothioureas (BTUs) inhibit the late secretory pathway (post-Golgi), possibly through a direct interaction with Sav1, an orthologue of the Sec4-class small GTPase. Importantly, pharmacological characterization of the BTUs indicates it readily penetrates the blood-brain barrier. Together, our data support the further development of this scaffold as an antifungal agent with a novel mechanism of action against C. neoformans.

Phase I study of TAS-115, a novel oral multi-kinase inhibitor, in patients with advanced solid tumors.

TAS-115 is a novel MET, VEGFR, FMS and PDGFR inhibitor, developed to improve the continuity of drug administration with a relatively short half-life. We assessed its tolerability, safety, pharmacokinetics, efficacy, and pharmacodynamics in patients with solid tumors. This open-label, dose-escalation phase I study of TAS-115 consisted of three parts: part 1 (TAS-115 was administered orally once daily [SID]); part 2 and an expansion part (SID in a 5 days on/2 days off [5-on/2-off] schedule for 21 days per cycle). In part 1 (200-800 mg SID administered to 21 patients), systemic exposure after single administration increased almost dose-proportionally. Three dose-limiting toxicities (DLTs) were observed in three patients: grade 3 rash (650 mg), thrombocytopenia with bleeding, and rash (800 mg). The maximum tolerated dose (MTD) was determined as 650 mg SID. In part 2, the 5-on/2-off schedule was evaluated at the MTD to improve treatment exposure. No DLTs were observed and no patients required treatment interruption in cycle 1. During part 2 and the expansion part (N = 61), grade ≥3 treatment-related adverse events were reported in 47 patients, with neutropenia (24.6%), hypophosphatemia (21.3%), anemia, and thrombocytopenia (14.8% each), and leukocytopenia (11.5%) occurring in ≥10% of patients. The best overall response was stable disease in 31 of 82 patients (37.8%). An apparent reduction in fluorodesoxyglucose-uptake and bone scan index was observed in some patients. TAS-115 was generally well tolerated, with manageable toxicities and recommended phase II dose was estimated as 650 mg SID, 5-on/2-off. Furthermore, promising antitumor activity was observed.

Synthesis, antitumor testing and molecular modeling study of some new 6-substituted amido, azo or thioureido-quinazolin-4(3H)-ones.

A new series of 6-substituted amido, azo or thioureido-quinazolin-4(3H)-one was synthesized and tested for their in-vitro antitumor activity. Compounds 21, 53 and 60 showed broad spectrum antitumor activity with average IC50 values of 6.7, 7.6 and 9.1 µM, respectively compared with methotrexate (1, IC50 19.26 µM). As an attempt to reveal the mechanism of the antitumor potency, cell cycle analysis and DHFR inhibition were performed. Compounds 59 and 61 induced their cytotoxicity in Hela (IC50 10.6 µM) and HCT-116 (IC50 15.5 µM) cell lines, respectively through Pre-G1 apoptosis, inhibiting cell growth at G2-M phase. Compounds 29, 33, 59 and 61 showed DHFR inhibitory potency at IC50 0.2, 0.2, 0.3 and 0.3 µM, respectively. The active DHFR inhibitors showed high affinity binding toward the amino acid residues Thr56, Ser59 and Ser118. The active compounds obeyed Lipinski's rule of five and could be used as template model for further optimization.

Synthesis of sulfadiazinyl acyl/aryl thiourea derivatives as calf intestinal alkaline phosphatase inhibitors, pharmacokinetic properties, lead optimization, Lineweaver-Burk plot evaluation and binding analysis.

To seek the new medicinal potential of sulfadiazine drug, the free amino group of sulfadiazine was exploited to obtain acyl/aryl thioureas using simple and straightforward protocol. Acyl/aryl thioureas are well recognized bioactive pharmacophore containing moieties. A new series (4a-4j) of sulfadiazine derived acyl/aryl thioureas was synthesized and characterized through spectroscopic and elemental analysis. The synthesized derivatives 4a-4j were subjected to calf intestinal alkaline phosphatase (CIAP) activity. The derivative 4a-4j showed better inhibition potential compared to standard monopotassium phosphate (MKP). The compound 4c exhibited higher potential in the series with IC50 0.251 ±â€¯0.012 µM (standard KH2PO4 4.317 ±â€¯0.201 µM). Lineweaver-Burk plots revealed that most potent derivative 4c inhibition CIAP via mixed type pathway. Pharmacological investigations showed that synthesized compounds 4a-4j obey Lipinsk's rule. ADMET parameters evaluation predicted that these molecule show significant lead like properties with minimum possible toxicity and can serve as templates in drug designing. The synthetic compounds show none mutagenic and irritant behavior. Molecular docking analysis showed that compound 4c interacts with Asp273, His317 and Arg166 amino acid residues.

First Bispecific Inhibitors of the Epidermal Growth Factor Receptor Kinase and the NF-κB Activity As Novel Anticancer Agents.

The activation of the NF-κB transcription factor is a major adaptive response induced upon treatment with EGFR kinase inhibitors, leading to the emergence of resistance in nonsmall cell lung cancer and other tumor types. To suppress this survival mechanism, we developed new thiourea quinazoline derivatives that are dual inhibitors of both EGFR kinase and the NF-κB activity. Optimization of the hit compound, identified in a NF-κB reporter gene assay, led to compound 9b, exhibiting a cellular IC50 for NF-κB inhibition of 0.3 µM while retaining a potent EGFR kinase inhibition (IC50 = 60 nM). The dual inhibitors showed a higher potency than gefitinib to inhibit cell growth of EGFR-overexpressing tumor cell lines in vitro and in a xenograft model in vivo, while no signs of toxicity were observed. An investigation of the molecular mechanism of NF-κB suppression revealed that the dual inhibitors depleted the transcriptional coactivator CREB-binding protein from the NF-κB complex in the nucleus.

Development of Multifunctional Pyrimidinylthiourea Derivatives as Potential Anti-Alzheimer Agents.

Starting from a screening-hit compound, via structure modifications and optimizations, a series of nonfused and nonassembly pyrimidinylthiourea derivatives (2-5) was designed, synthesized, and evaluated as novel multifunctional agents against Alzheimer's disease. Biological activity results demonstrated that compounds 5r and 5t exhibited potent inhibition and excellent selectivity toward acetylcholinesterase (AChE, 5r, IC50 = 0.204 µM, SI > 196; 5t, IC50 = 0.067 µM, SI > 597), specific metal-chelating ability, significant antioxidant effects, modulation of metal-induced Aß aggregation, inhibition of ROS production by copper redox cycle, low cytotoxicity, and moderate neuroprotection to human neuroblastoma SH-SY5Y cells. Moreover, compound 5r displayed appropriate blood-brain barrier (BBB) permeability both in vitro and in vivo and could improve memory and cognitive function of scopolamine-induced amnesia mice. The multifunctional profiles of 5r and its effectivity in AD mice highlight these structurally distinct pyrimidinylthiourea derivatives as prospective prototypes in the research of innovative multifunctional drugs for Alzheimer's disease.

Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction.

Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a transcription factor-binding site in Mediator as a novel therapeutic strategy in fungal infectious disease.

Blood-brain transfer and antinociception of linear and cyclic N-methyl-guanidine and thiourea-enkephalins.

Enkephalins are active in regulation of nociception in the body and are key in development of new synthetic peptide analogs that target centrally located opioid receptors. In this study, we investigated the in vivo blood-brain barrier (BBB) penetration behavior and antinociceptive activity of two cyclic enkephalin analogs with a thiourea (CycS) or a N-methyl-guanidine bridge (CycNMe), and their linear counterparts (LinS and LinNMe) in mice, as well as their in vitro metabolic stability. (125)I-LinS had the highest blood-brain clearance (K1=3.46µL/gmin), followed by (125)I-LinNMe, (125)I-CycNMe, and (125)I-CycS (K1=1.64, 0.31, and 0.11µL/gmin, respectively). Also, these peptides had a high metabolic stability (t1/2>1h) in mouse serum and brain homogenate, and half-inhibition constant (Ki) values in the nanomolar range with predominantly µ-opioid receptor selectivity. The positively charged NMe-enkephalins showed a higher antinociceptive activity (LinNMe: 298% and CycNMe: 205%), expressed as molar-dose normalized area under the curve (AUC) relative to morphine, than the neutral S-enkephalins (CycS: 122% and LinS: 130%).

Synthesis and in vitro urease inhibitory activity of N,N'-disubstituted thioureas.

Thiourea derivatives (1-38) were synthesized and evaluated for their urease inhibition potential. The synthetic compounds showed a varying degree of in vitro urease inhibition with IC50 values 5.53 ± 0.02-91.50 ± 0.08 µM, most of which are superior to the standard thiourea (IC50 = 21.00 ± 0.11 µM). In order to ensure the mode of inhibition of these compounds, the kinetic study of the most active compounds has been carried out. Most of these inhibitors were found to be mixed-type of inhibitors, except compounds 13 and 30 which were competitive, while compound 19 was identified as non-competitive inhibitor with Ki values between 8.6 and 19.29 µM.

4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), a potent inhibitor of bacterial phosphopantetheinyl transferase that attenuates secondary metabolism and thwarts bacterial growth.

4'-Phosphopantetheinyl transferases (PPTases) catalyze a post-translational modification essential to bacterial cell viability and virulence. We present the discovery and medicinal chemistry optimization of 2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides, which exhibit submicromolar inhibition of bacterial Sfp-PPTase with no activity toward the human orthologue. Moreover, compounds within this class possess antibacterial activity in the absence of a rapid cytotoxic response in human cells. An advanced analogue of this series, ML267 (55), was found to attenuate production of an Sfp-PPTase-dependent metabolite when applied to Bacillus subtilis at sublethal doses. Additional testing revealed antibacterial activity against methicillin-resistant Staphylococcus aureus , and chemical genetic studies implicated efflux as a mechanism for resistance in Escherichia coli . Additionally, we highlight the in vitro absorption, distribution, metabolism, and excretion and in vivo pharmacokinetic profiles of compound 55 to further demonstrate the potential utility of this small-molecule inhibitor.

Bacterial ß-glucuronidase inhibition protects mice against enteropathy induced by indomethacin, ketoprofen or diclofenac: mode of action and pharmacokinetics.

1. We have previously demonstrated that a small molecule inhibitor of bacterial ß-glucuronidase (Inh-1; [1-((6,8-dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea]) protected mice against diclofenac (DCF)-induced enteropathy. Here we report that Inh-1 was equally protective against small intestinal injury induced by other carboxylic acid-containing non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin (10 mg/kg, ip) and ketoprofen (100 mg/kg, ip). 2. Inh-1 provided complete protection if given prior to DCF (60 mg/kg, ip), and partial protection if administered 3-h post-DCF, suggesting that the temporal window of mucosal protection can be extended for drugs undergoing extensive enterohepatic circulation. 3. Pharmacokinetic analysis of Inh-1 revealed an absolute bioavailability (F) of 21% and a short t1/2 of <1 h. This low F was shown to be due to hepatic first-pass metabolism, as confirmed with the pan-CYP inhibitor, 1-aminobenzotriazole. 4. Using the fluorescent probe 5 (and 6)-carboxy-2',7'-dichlorofluorescein, we demonstrated that Inh-1 did not interfere with hepatobiliary export of glucuronides in gall bladder-cannulated mice. 5. These data are compatible with the hypothesis that pharmacological inhibition of bacterial ß-glucuronidase-mediated cleavage of NSAID glucuronides in the small intestinal lumen can protect against NSAID-induced enteropathy caused by locally high concentrations of NSAID aglycones.

Degradation of MAC13243 and studies of the interaction of resulting thiourea compounds with the lipoprotein targeting chaperone LolA.

The discovery of novel small molecules that function as antibacterial agents or cellular probes of biology is hindered by our limited understanding of bacterial physiology and our ability to assign mechanism of action. We previously employed a chemical genomic strategy to identify a novel small molecule, MAC13243, as a likely inhibitor of the bacterial lipoprotein targeting chaperone, LolA. Here, we report on the degradation of MAC13243 into the active species, S-(4-chlorobenzyl)isothiourea. Analogs of this compound (e.g., A22) have previously been characterized as inhibitors of the bacterial actin-like protein, MreB. Herein, we demonstrate that the antibacterial activity of MAC13243 and the thiourea compounds are similar; these activities are suppressed or sensitized in response to increases or decreases of LolA copy number, respectively. We provide STD NMR data which confirms a physical interaction between LolA and the thiourea degradation product of MAC13243, with a Kd of ~150 µM. Taken together, we conclude that the thiourea series of compounds share a similar cellular mechanism that includes interaction with LolA in addition to the well-characterized target MreB.

Calcium dependence of damage to mouse motor nerve terminals following oxygen/glucose deprivation.

Motor nerve terminals are especially sensitive to an ischemia/reperfusion stress. We applied an in vitro model of this stress, oxygen/glucose deprivation (OGD), to mouse neuromuscular preparations to investigate how Ca(2+) contributes to stress-induced motor terminal damage. Measurements using an ionophoretically-injected fluorescent [Ca(2+)] indicator demonstrated an increase in intra-terminal [Ca(2+)] following OGD onset. When OGD was terminated within 20-30min of the increase in resting [Ca(2+)], these changes were sometimes reversible; in other cases [Ca(2+)] remained high and the terminal degenerated. Endplate innervation was assessed morphometrically following 22min OGD and 120min reoxygenation (32.5°C). Stress-induced motor terminal degeneration was Ca(2+)-dependent. Median post-stress endplate occupancy was only 26% when the bath contained the normal 1.8mM Ca(2+), but increased to 81% when Ca(2+) was absent. Removal of Ca(2+) only during OGD was more protective than removal of Ca(2+) only during reoxygenation. Post-stress endplate occupancy was partially preserved by pharmacological inhibition of various routes of Ca(2+) entry into motor terminals, including voltage-dependent Ca(2+) channels (ω-agatoxin-IVA, nimodipine) and the plasma membrane Na(+)/Ca(2+) exchanger (KB-R7943). Inhibition of a Ca(2+)-dependent protease with calpain inhibitor VI was also protective. These results suggest that most of the OGD-induced motor terminal damage is Ca(2+)-dependent, and that inhibition of Ca(2+) entry or Ca(2+)-dependent proteolysis can reduce this damage. There was no significant difference between the response of wild-type and presymptomatic superoxide dismutase 1 G93A mutant terminals to OGD, or in their response to the protective effect of the tested drugs.

Chronic peripheral administration of somatostatin receptor subtype-4 agonist NNC 26-9100 enhances learning and memory in SAMP8 mice.

Selective somatostatin receptor subtype agonists have been proposed as a means to mitigate learning and memory loss associated with Alzheimer's disease. The first aim of this study evaluated blood-to-brain transport and regional brain distribution of NNC 26-9100, a selective somatostatin subtype-4 (sst4) receptor agonist. The entry rate of (131)I-NNC 26-9100 was K(i)=0.25 µl/g min, with an ~93% association with the parenchymal component. The second goal of this study was to evaluate the effect of chronic NNC 26-9100 administration (i.p.) on learning and memory, brain Aß(x-42) levels, and protein expression of sst4 receptor and amyloid precursor protein (APP) in the senescence-accelerated mouse p8 (SAMP8) model of Alzheimer's disease. Mice chronically treated with NNC 26-9100 showed improved learning (day 21) and memory (day 28) using the T-maze paradigm (20 and 200 µg). Ex vivo tissue analyses showed a decline in Aß(x-42) levels at the 20 µg dose, while no alterations were observed in sst4 receptor or APP protein expression compared to vehicle controls. These findings indicate NNC 26-9100 is taken up into key brain regions associated with learning and memory. Furthermore, chronic administration of NNC 26-9100 improved learning and memory and decreased Aß(x-42) brain levels. These results suggest sst4 receptor agonists may provide a viable therapy in the treatment of Alzheimer's disease and other forms of cognitive impairment.

Bioactivation of antituberculosis thioamide and thiourea prodrugs by bacterial and mammalian flavin monooxygenases.

The thioamide and thiourea class of antituberculosis agents encompasses prodrugs that are oxidatively converted to their active forms by the flavin monooxygenase EtaA of Mycobacterium tuberculosis. Reactive intermediates produced in the EtaA-catalyzed transformations of ethionamide and prothionamide result in NAD(+)/NADH adducts that inhibit the enoyl CoA reductase InhA, the ultimate target of these drugs. In the case of thiacetazone and isoxyl, EtaA produces electrophilic metabolites that mediate the antibacterial activity of these agents. The oxidation of the thioamide/thiourea drugs by the human flavin monooxygenases yields similar reactive metabolites that contribute to the toxicities associated with these second line antituberculosis drugs.