A First-in-Class, Highly Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 6.

Protein arginine methyltransferase 6 (PRMT6) catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes, and is associated with multiple cancers. To date, a highly selective PRMT6 inhibitor has not been reported. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, (R)-2 (SGC6870). (R)-2 is a potent PRMT6 inhibitor (IC50 = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and nonepigenetic targets. Notably, the crystal structure of the PRMT6-(R)-2 complex and kinetic studies revealed (R)-2 binds a unique, induced allosteric pocket. Additionally, (R)-2 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, (R)-2's enantiomer, (S)-2 (SGC6870N), is inactive against PRMT6 and can be utilized as a negative control. Collectively, (R)-2 is a well-characterized PRMT6 chemical probe and a valuable tool for further investigating PRMT6 functions in health and disease.

Discovery of a series of benzopyrimidodiazepinone TNK2 inhibitors via scaffold morphing.

The protein kinase TNK2 (ACK1) is an emerging drug target for a variety of indications, in particular for cancer where it plays a key role transmitting cell survival, growth and proliferative signals via modification of multiple downstream effectors by unique tyrosine phosphorylation events. Scaffold morphing based on our previous TNK2 inhibitor XMD8-87 identified urea 17 from which we developed the potent and selective compound 32. A co-crystal structure was obtained showing 32 interacting primarily with the main chain atoms of an alanine residue of the hinge region. Additional H-bonds exist between the urea NHs and the Thr205 and Asp270 residues.

[99mTc]Tc-DGA1, a Promising CCK2R-Antagonist-Based Tracer for Tumor Diagnosis with Single-Photon Emission Computed Tomography.

Radiolabeled gastrin analogues have been proposed for theranostics of cholecystokinin subtype 2 receptor (CCK2R)-positive cancer. Peptide radioligands based on other receptor antagonists have displayed superior pharmacokinetics and higher biosafety than agonists. Here, we present DGA1, a derivative of the nonpeptidic CCK2R antagonist Z-360 carrying an acyclic tetraamine, for [99mTc]Tc labeling. Preclinical comparison of [99mTc]Tc-DGA1 with [99mTc]Tc-DG2 (CCK2R-agonist reference) was conducted in HEK293-CCK2R/CCK2i4svR cells and mice models, qualifying [99mTc]Tc-DGA1 for further study in patients with CCK2R-positive tumors and single-photon emission computed tomography/CT.

Klebsiella oxytoca enterotoxins tilimycin and tilivalline have distinct host DNA-damaging and microtubule-stabilizing activities.

Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells-a hallmark feature of AAHC-by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis.

Natural products from thioester reductase containing biosynthetic pathways.

Covering: up to 2018 Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospective highlights various molecular families with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products.

The Anxiolytic Etifoxine Binds to TSPO Ro5-4864 Binding Site with Long Residence Time Showing a High Neurosteroidogenic Activity.

The low binding affinity of the approved anxiolytic drug etifoxine (Stresam) at the steroidogenic 18 kDa translocator protein (TSPO) has questioned the specific contribution of this protein in mediating the etifoxine neurosteroidogenic efficacy. Residence time (RT) at the binding site of the classical TSPO ligand PK11195 is emerging as a relevant neurosteroidogenic efficacy measure rather than the binding affinity. Here etifoxine was evaluated for (i) the in vitro neurosteroidogenic activity in comparison to poorly neurosteroidogenic reference TSPO ligands (PK11195 and Ro5-4864) and (ii) the affinity and RT at [3H]PK11195 and [3H]Ro5-4864 binding sites in rat kidney membranes. Etifoxine shows (i) high neurosteroidogenic efficacy and (ii) low affinity/short RT at the [3H]PK11195 site and low affinity/long RT at the [3H]Ro5-4864 site, at which etifoxine competitively bound. These findings suggest that the long RT of etifoxine at the Ro5-4864 binding site could account for its high neurosteroidogenic efficacy.

Mechanistic Investigation of a Non-Heme Iron Enzyme Catalyzed Epoxidation in (-)-4'-Methoxycyclopenin Biosynthesis.

Mechanisms have been proposed for α-KG-dependent non-heme iron enzyme catalyzed oxygen atom insertion into an olefinic moiety in various natural products, but they have not been examined in detail. Using a combination of methods including transient kinetics, Mössbauer spectroscopy, and mass spectrometry, we demonstrate that AsqJ-catalyzed (-)-4'-methoxycyclopenin formation uses a high-spin Fe(IV)-oxo intermediate to carry out epoxidation. Furthermore, product analysis on (16)O/(18)O isotope incorporation from the reactions using the native substrate, 4'-methoxydehydrocyclopeptin, and a mechanistic probe, dehydrocyclopeptin, reveals evidence supporting oxo↔hydroxo tautomerism of the Fe(IV)-oxo species in the non-heme iron enzyme catalysis.

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.

Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors.

The signal peptide peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (Plasmodium SPP (pSPP)), and B-cell immunomodulation and neoplasia (signal peptide peptidase like 2a, (SPPL2a)). To date, no drug-like, selective inhibitors have been reported. We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid ß 1-25 (Aß1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays. Co-transfection of expression plasmids expressing the FBA substrate with SPP/SPPLs were conducted to evaluate cleavage, which was monitored by ELISA, Western Blot and immunoprecipitation/MALDI-TOF Mass spectrometry (IP/MS). No cleavage is detected in the absence of SPP/SPPL overexpression. Multiple γ-secretase inhibitors (GSIs) and (Z-LL)2 ketone differentially inhibited SPP/SPPL activity; for example, IC50 of LY-411,575 varied from 51±79 nM (on SPPL2a) to 5499±122 nM (on SPPL2b), while Compound E showed inhibition only on hSPP with IC50 of 1465±93 nM. Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line. Thus, it is possible to differentially inhibit SPP family members. These SPP/SPPL cleavage assays will expedite the search for selective inhibitors. The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase.

Evaluation of a nonpeptidic ligand for imaging of cholecystokinin 2 receptor-expressing cancers.

UNLABELLED: Tumor-specific targeting ligands were recently exploited to deliver both imaging and therapeutic agents selectively to cancer tissues in vivo. Because the cholecystokinin 2 receptor (CCK2R) is overexpressed in various human cancers (e.g., lung, medullary thyroid, pancreatic, colon, and gastrointestinal stromal tumors) but displays limited expression in normal tissues, natural ligands of CCK2R were recently explored for use in the imaging of CCK2R-expressing cancers. Unfortunately, the results from these studies revealed not only that the peptidic CCK2R ligands were unstable in vivo but also that the ligands that mediated good uptake by tumor tissues also promoted a high level of retention of the radioimaging agent in the kidneys, probably because of capture of the conjugates by peptide-scavenging receptors. In an effort to reduce the normal organ retention of CCK2R-targeted drugs, we synthesized a nonpeptidic ligand of CCK2R and examined its specificity for CCK2R both in vitro and in vivo. METHODS: Nonpeptidic agonists and antagonists of CCK2R described in the literature were evaluated for their affinities and specificities for CCK2R. Z-360, a benzodiazepine-derived CCK2R antagonist with subnanomolar affinity, was selected for complexation to (99m)Tc via multiple spacers. After synthesis and purification, 4 complexes with different physicochemical properties were evaluated for binding to CCK2R-transfected HEK 293 cells. The best conjugate, termed CRL-3-(99m)Tc, was injected into mice bearing CCK2R tumor xenografts and examined by γ scintigraphy and SPECT/CT. The uptake of the conjugate in various organs was also quantified by tissue resection and γ counting. RESULTS: CRL-3-(99m)Tc was shown to bind with low nanomolar affinity to CCK2R in vitro and was localized to tumor tissues in athymic nu/nu mice implanted with CCK2R-expressing tumors. At 4 h after injection, tumor uptake was measured at 12.0 ± 2.0 percentage injected dose per gram of tissue. CONCLUSION: Because the uptake of CRL-3-(99m)Tc by nonmalignant tissues was negligible and retention in the kidneys was only transient, we suggest that CRL-3-(99m)Tc may be a useful radioimaging agent for the detection, sizing, and monitoring of CCK2R-expressing tumors.

Evaluation of a cholecystokinin 2 receptor-targeted near-infrared dye for fluorescence-guided surgery of cancer.

Surgical resection of malignant disease remains one of the most effective tools for treating cancer. Tumor-targeted near-infrared dyes have the potential to improve contrast between normal and malignant tissues, thereby enabling surgeons to more quantitatively resect malignant disease. Because the cholecystokinin 2 receptor (CCK2R and its tumor-specific splice variant CCK2i4svR) is overexpressed in cancers of the lungs, colon, thyroid, pancreas, and stomach, but absent or inaccessible to parenterally administered drugs in most normal tissues, we have undertaken to design a targeting ligand that can deliver attached near-infrared dyes to CCK2R+ tumors. We report here the synthesis and biological characterization of a CCK2R-targeted conjugate of the near-infrared dye, LS-288 (CRL-LS288). We demonstrate that CRL-LS288 binds selectively to CCK2R+ cancer cells with low nanomolar affinity (Kd = 7 × 10(-9) M). We further show that CRL-LS288 localizes primarily to CCK2R-expressing HEK 293 murine tumor xenografts and that dye uptake in these xenografts is significantly reduced when CCK2R are blocked by preinjection of excess ligand (CRL) or when mice are implanted with CCK2R-negative tumors. Because CRL-LS288 is also found to reveal the locations of distant tumor metastases, we suggest that CRL-LS288 has the potential to facilitate intraoperative identification of malignant disease during a variety of cancer debulking surgeries.

Drug ligand-induced activation of translocator protein (TSPO) stimulates steroid production by aged brown Norway rat Leydig cells.

Translocator protein (TSPO; 18 kDA) is a high-affinity cholesterol-binding protein that is integrally involved in cholesterol transfer from intracellular stores into mitochondria, the rate-determining step in steroid formation. Previous studies have shown that TSPO drug ligands are able to activate steroid production by MA-10 mouse Leydig tumor cells and by mitochondria isolated from steroidogenic cells. We hypothesized herein that the direct, pharmacological activation of TSPO might induce aged Leydig cells, which are characterized by reduced T production, to produce significantly higher levels of T both in vitro and in vivo. To test this, we first examined the in vitro effects of the TSPO selective and structurally distinct drug ligands N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27) and benzodiazepine 4'-chlorodiazepam (Ro5-4864) on steroidogenesis by Leydig cells isolated from aged (21-24 months old) and young adult (3-6 months old) Brown Norway rats. The ligands stimulated Leydig cell T production significantly, and equivalently, in cells of both ages, an effect that was significantly inhibited by the specific TSPO inhibitor 5-androsten-3,17,19-triol (19-Atriol). Additionally, we examined the in vivo effects of administering FGIN-1-27 to young and aged rats. In both cases, serum T levels increased significantly, consistent with the in vitro results. Indeed, serum T levels in aged rats administered FGIN-1-27 were equivalent to T levels in the serum of control young rats. Taken together, these results indicate that although there are reduced amounts of TSPO in aged Leydig cells, its direct activation is able to increase T production. We suggest that this approach might serve as a therapeutic means to increase steroid levels in vivo in cases of primary hypogonadism.

Assembly of asperlicin peptidyl alkaloids from anthranilate and tryptophan: a two-enzyme pathway generates heptacyclic scaffold complexity in asperlicin E.

Members of the asperlicin family of fungal metabolites produced by Aspergillus alliaceus are known potent CCK(A) antagonists. Herein, we report the identification of the gene cluster responsible for directing their biosynthesis. We validate and probe the pathway by genetic manipulation, and provide the first biochemical characterization of the oxidative cyclization en route to the heptacyclic asperlicin E by reconstituting the activity of the FAD depend monooxygenase AspB. This report provides the first genetic characterization of a NRPS assembly line that efficiently activates two anthranilate building blocks and illustrates the remarkably efficient biosynthesis of the complex heptacyclic asperlicin E.

Kinetic, crystallographic, and mechanistic characterization of TomN: elucidation of a function for a 4-oxalocrotonate tautomerase homologue in the tomaymycin biosynthetic pathway.

The biosynthesis of the C ring of the antitumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from l-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from that processed by 4-OT. To establish the function and mechanism of TomN and its relationship with 4-OT, we conducted kinetic, mutagenic, and structural studies. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutants of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high-resolution crystal structure (1.4 Å) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with a root-mean-square deviation of 0.81 Å. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.

Phase I pharmacokinetic and pharmacodynamic study of SJG-136, a novel DNA sequence selective minor groove cross-linking agent, in advanced solid tumors.

PURPOSE: This phase I study assessed the maximum tolerated dose (MTD), safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of SJG-136, a sequence-specific DNA cross-linking agent, in patients with advanced cancer. EXPERIMENTAL DESIGN: In schedule A, seven patients received escalating doses of SJG-136 (6, 12, 24, and 48 µg/m(2)) daily for 5 of 21 days. Blood samples were collected for PK analysis on days 1 and 5 of cycle 1. In schedule B, SJG-136 was given daily for 3 of 21 days (N = 17; doses 20, 25, 30, and 35 µg/m(2)). Blood samples were collected on days 1 and 3 of cycles 1 and 2 for PK and PD analysis. Patients in schedule B received dexamethasone and early diuretic care. RESULTS: Schedule A-dose-limiting toxicities included grade 3 edema, dyspnea, fatigue, and delayed liver toxicity (grade 3-4). PK analysis revealed dose-dependent increases in AUC and C(max). Substantial changes in volume of distribution at steady-state occurred after repeated dosing in some patients prior to the onset of edema. Schedule B-the same toxicities were manageable with steroid premedication and diuretic support. No significant myelosuppression occurred on either schedule. DNA interstrand cross-links correlated with systemic exposure of SJG-136 following the second dose in cycle 1 and were still detectable immediately before cycle 2. CONCLUSIONS: The MTD of SJG-136 in this study was 30 µg/m(2) administered on a daily 3× basis with no myelosuppression effects. Coupled with supportive management, SJG-136 is now advancing to a phase II trial in ovarian cancer.

Pharmacokinetics, pharmacodynamics and metabolism of the dimeric pyrrolobenzodiazepine SJG-136 in rats.

PURPOSE: The dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501, SG2000) has potent in vitro antiproliferative activity and in vivo antitumor activity associated with binding in the minor groove of DNA and formation of covalent interstrand DNA cross-links. The pharmacokinetics and in vitro metabolism of SJG-136 and as well as the feasibility of using the Comet assay to measure in vivo interstrand DNA cross-links, was assessed in the rat. METHODS: SJG-136 pharmacokinetics and pharmacodynamics were characterized in rats following single-dose administration of 15 and 50 µg/kg or multiple-dose administration of 25 µg/kg/day for 5 days. DNA damage was measured in peripheral blood mononuclear cells using the Comet assay. SJG-136 oxidative metabolism was characterized in rat liver microsomes. RESULTS: SJG-136 half-life, clearance and volume of distribution values were 9 min, 190 ml/min/m(2), and 1780 ml/m(2), respectively. SJG-136 did not accumulate in plasma during treatment with 25 µg/kg/day for 5 days. Treatment with SJG-136 produced the anticipated DNA interstrand cross-links, as well as DNA strand breaks, in rat PBMCs. Oxidative metabolism of SJG-136 in rat liver microsomes was catalyzed by CYP3A isoforms and produced a previously unreported monomeric metabolite. CONCLUSIONS: Plasma concentrations of SJG-136 associated with pharmacological activity and in vitro antiproliferative activity were achieved with doses that were tolerated by rats. CYP3A isoforms are the predominant P450s catalyzing SJG-136 metabolism. The comet assay detects DNA damage in PBMCs from rats treated with SJG-136 and is being used in clinical trials to monitor in vivo lesions produced by SJG-136.

Computational determination of binding structures and free energies of phosphodiesterase-2 with benzo[1,4]diazepin-2-one derivatives.

Phosphodiesterase-2 (PDE2) is a key enzyme catalyzing hydrolysis of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) that serve as intracellular second messengers. PDE2 has been recognized as an attractive drug target, and selective inhibitors of PDE2 are expected to be promising candidates for the memory enhancer, antidepressant, and anxiolytic agent. In the present study, we examined the detailed binding structures and free energies for PDE2 interacting with a promising series of inhibitors, i.e., benzo[1,4]diazepin-2-one derivatives, by carrying out molecular docking, molecular dynamics (MD) simulations, binding free energy calculations, and binding energy decompositions. The computational results provide valuable insights into the detailed enzyme-inhibitor binding modes including important intermolecular interactions, e.g., the π-π stacking interactions with the common benzo[1,4]diazepin-2-one scaffold of the inhibitors, hydrogen bonding and hydrophobic interactions with the substituents on the benzo[1,4]diazepin-2-one scaffold. Future rational design of new, more potent inhibitors of PDE2 should carefully account for all of these favorable intermolecular interactions. By use of the MD-simulated binding structures, the calculated binding free energies are in good agreement with the experimental activity data for all of the examined benzo[1,4]diazepin-2-one derivatives. The enzyme-inhibitor binding modes determined and the agreement between the calculated and experimental results are expected to be valuable for future rational design of more potent inhibitors of PDE2.

Structure-based and shape-complemented pharmacophore modeling for the discovery of novel checkpoint kinase 1 inhibitors.

Checkpoint kinase 1 (Chk1), a member of the serine/threonine kinase family, is an attractive therapeutic target for anticancer combination therapy. A structure-based modeling approach complemented with shape components was pursued to develop a reliable pharmacophore model for ATP-competitive Chk1 inhibitors. Common chemical features of the pharmacophore model were derived by clustering multiple structure-based pharmacophore features from different Chk1-ligand complexes in comparable binding modes. The final model consisted of one hydrogen bond acceptor (HBA), one hydrogen bond donor (HBD), two hydrophobic (HY) features, several excluded volumes and shape constraints. In the validation study, this feature-shape query yielded an enrichment factor of 9.196 and performed fairly well at distinguishing active from inactive compounds, suggesting that the pharmacophore model can serve as a reliable tool for virtual screening to facilitate the discovery of novel Chk1 inhibitors. Besides, these pharmacophore features were assumed to be essential for Chk1 inhibitors, which might be useful for the identification of potential Chk1 inhibitors.

The effects of 3-methylclonazepam on Schistosoma mansoni musculature are not mediated by benzodiazepine receptors.

Schistosomiasis is one of the most prevalent infectious diseases worldwide and classified as a neglected disease for which there is an urgent need for searching new drug candidates. According to TDR/WHO, existing leads with proven schistosomicidal activity, like meclonazepam, might be the objects of further exploration. Here, we decided to investigate if the benzodiazepine binding sites that we recently characterized in adult Schistosoma mansoni could represent the molecular target of meclonazepam for its effect on worm motility and morphological appearance. The EC(50) of meclonazepam for its contracturant effect is 10-20 times lower than its IC(50) for binding to the worm benzodiazepine binding sites. On the contrary, benzodiazepines like flunitrazepam and diazepam have affinities at least 50 times higher than meclonazepam for these binding sites but did not induce contraction of the worms. We also confirmed the existence of a great similarity between the appearance, kinetics, Emax and external calcium dependency of the contractile effect of praziquantel and meclonazepam. Based on computer-aided molecular modeling calculations, we verified that a certain structural similarity exists between the active enantiomers of both drugs. We further proposed the hypothesis of common pharmacophoric elements including amide and imine subunits and the asymmetric carbons of S-(+)-meclozepam and R-(-)-praziquantel. As a whole, the present data indicate that the contracturant effect of meclonazepam is not a result of its binding to the worm benzodiazepine binding sites but that it shares some basic transduction pathway with praziquantel, even if not through identical molecular targets or binding sites.

Diazepam, em dose única, inibe a migração celular, a estimulação macrofágica e a atividade de TNF-α na reação inflamatória aguda induzida por LPS em camundongos / Diazepam, in a single dose, inhibits cellular chemotaxis, macrophage stimulation, and TNF-α activity in LPS-induced acute inflammatory responses in mice

Os benzodiazepínicos estão entre as drogas mais freqüentemente prescritas em razão de suas propriedades ansiolíticas. O objetivo deste trabalho foi avaliar a influência do diazepam sobre a resposta inflamatória peritoneal aguda induzida por lipopolissacarídeo. Para tanto, camundongos Swiss foram tratados com diazepam (1 ou 10 mg/kg de peso), em dose única, por via subcutânea, uma hora antes do desafio intraperitoneal com lipopolissacarídeo bacteriano. Após 16 horas do desafio, os animais foram sacrificados, coletando-se os lavados peritoneais para determinação do número total de células e das subpopulações de mononucleares e polimorfonucleares, além da atividade de TNF-α e da porcentagem de macrófagos espraiados. Observou-se que o tratamento com diazepam, nas doses de 1 ou 10 mg/kg, reduziu significativamente a porcentagem de macrófagos estimulados por LPS e a liberação de TNF-α independente de estímulo. Houve também significativa redução da migração de leucócitos nos animais estimulados com LPS e tratados com 10 mg/kg de diazepam em relação aqueles não tratados. Concluímos que a administração do diazepam, em dose única, pode influenciar significativamente o influxo celular, a estimulação de macrófagos e a atividade de TNF-α na resposta inflamatória aguda induzida por LPS em camundongos, com possíveis implicações na eficiência da resposta anti-infecciosa.

Benzodiazepines are one of the most frequently prescribed drugs due to their anxiolytic properties. The aim of this study was to evaluate the effects of diazepam on lipopolysaccharide-induced peritoneal acute inflammatory responses. Swiss mice were treated with diazepam in a single dose of 1 or 10 mg/kg- subcutaneously 1 h before an intraperitoneal injection of lipopolysaccharide or sterile saline solution. The mice were killed 16 h after and the cells were washed from the peritoneal cavity to determine the total number of cells and the mononuclear and polimorfonuclear subpopulations, as well as the TNF-alpha activity and percentage of spread macrophages. Our results showed that the diazepam treatment (1 and 10 mg/kg) induced a significant reduction in the LPS-induced macrophage stimulation and TNF-α activity. Diazepam (10 mg/kg) also reduced the inflammatory cellular migration when compared to the control. It can be concluded that the diazepam treatment in a single dose is able to influence the inflammatory cellular influx, macrophage stimulation and TNF-α activity in the acute inflammatory response in mice, having possible implications on the anti-infectious response efficiency.