Validation and standardization of the ETP-based activated protein C resistance test for the clinical investigation of steroid contraceptives in women: an unmet clinical and regulatory need.

Background Regulatory bodies recommend the use of an assay based on the assessment of the endogenous thrombin potential (ETP) for the investigation of the activated protein C resistance (APCr) in the development of steroid contraceptives in women. However, the assays described in the literature are home-made and not standardized regarding the method, the reagents, the reference plasma and the quality controls. In the absence of any commercially available method, we aimed at validating the ETP-based APCr assay. Methods The validation was performed according to regulatory standards. The method targets a 90% inhibition of the ETP in healthy donors in the presence of APC compared to the same condition in the absence of APC. As a large-scale production of a pool of plasma from well-selected healthy donors is impossible, algorithms were applied to a commercial reference plasma to correlate with the selected pool. Results Repeatability and intermediate precision passed the acceptance criteria. The assay demonstrated a curvilinear dose response to protein S and APC concentrations (R2 > 0.99). Analysis of plasma samples from 47 healthy individuals (22 women not taking combined hormonal contraceptives [CHC], and 25 men not Factor V Leiden carriers) confirmed the validity of the test, with a mean inhibition percentage of 90%. Investigations in 15 women taking different contraceptives and in two subjects with Factor V Leiden confirmed the good sensitivity and performance of the assay. Conclusions This validation provides the pharmaceutical industry, the regulatory bodies and physicians with a reproducible, sensitive and validated gold-standard ETP-based APCr assay.

A new potential anti-cancer beta-carboline derivative decreases the expression levels of key proteins involved in glioma aggressiveness: A proteomic investigation.

Gliomas remain highly fatal due to their high resistance to current therapies. Deregulation of protein synthesis contributes to cancer onset and progression and is a source of rising interest for new drugs. CM16, a harmine derivative with predicted high blood-brain barrier penetration, exerts antiproliferative effects partly through translation inhibition. We evaluated herein how CM16 alters the proteome of glioma cells. The analysis of the gel-free LC/MS and auto-MS/MS data showed that CM16 induces time- and concentration-dependent significant changes in the total ion current chromatograms. In addition, we observed spontaneous clustering of the samples according to their treatment condition and their proper classification by unsupervised and supervised analyses, respectively. A two-dimensional gel-based approach analysis allowed us to identify that treatment with CM16 may downregulate four key proteins involved in glioma aggressiveness and associated with poor patient survival (HspB1, BTF3, PGAM1, and cofilin), while it may upregulate galectin-1 and Ebp1. Consistently with the protein synthesis inhibition properties of CM16, HspB1, Ebp1, and BTF3 exert known roles in protein synthesis. In conclusion, the downregulation of HspB1, BTF3, PGAM1 and cofilin bring new insights in CM16 antiproliferative effects, further supporting CM16 as an interesting protein synthesis inhibitor to combat glioma.

Reversal of tumoral immune resistance by inhibition of tryptophan 2,3-dioxygenase.

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO1) is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance, and IDO1 inhibition is an active area of drug development. Tryptophan 2,3-dioxygenase (TDO) is an unrelated hepatic enzyme that also degrades tryptophan along the kynurenine pathway. Here, we show that enzymatically active TDO is expressed in a significant proportion of human tumors. In a preclinical model, TDO expression by tumors prevented their rejection by immunized mice. We developed a TDO inhibitor, which, upon systemic treatment, restored the ability of mice to reject TDO-expressing tumors. Our results describe a mechanism of tumoral immune resistance based on TDO expression and establish proof-of-concept for the use of TDO inhibitors in cancer therapy.

Indoleamine 2,3-dioxygenase inhibitory activity of derivatives of marine alkaloid tsitsikammamine A.

Tsitsikammamines are marine alkaloids whose structure is based on the pyrroloiminoquinone scaffold. These and related compounds have attracted attention due to various interesting biological properties, including cytotoxicity, topoisomerase inhibition, antimicrobial, antifungal and antimalarial activity. Indoleamine 2,3-dioxygenase (IDO1) is a well-established therapeutic target as an important factor in the tumor immune evasion mechanism. In this preliminary communication, we report the inhibitory activity of tsitsikammamine derivatives against IDO1. Tsitsikammamine A analogue 11b displays submicromolar potency in an enzymatic assay. A number of derivatives are also active in a cellular assay while showing little or no activity towards tryptophan 2,3-dioxygenase (TDO), a functionally related enzyme. This IDO1 inhibitory activity is rationalized by molecular modeling studies. An interest is thus established in this class of compounds as a potential source of lead compounds for the development of new pharmaceutically useful IDO1 inhibitors.

New selective carbonic anhydrase IX inhibitors: synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides.

Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC50 value, is restored at 20% level with 1µM CA IX inhibitor.

Effects of SiC nanoparticles orally administered in a rat model: biodistribution, toxicity and elemental composition changes in feces and organs.

BACKGROUND: Silicon carbide (SiC) presents noteworthy properties as a material such as high hardness, thermal stability, and photoluminescent properties as a nanocrystal. However, there are very few studies in regard to the toxicological potential of SiC NPs. OBJECTIVES: To study the toxicity and biodistribution of silicon carbide (SiC) nanoparticles in an in vivo rat model after acute (24h) and subacute (28days) oral administrations. The acute doses were 0.5, 5, 50, 300 and 600mg·kg(-1), while the subacute doses were 0.5 and 50mg·kg(-1). RESULTS: SiC biodistribution and elemental composition of feces and organs (liver, kidneys, and spleen) have been studied by Particle-Induced X-ray Emission (PIXE). SiC and other elements in feces excretion increased by the end of the subacute assessment. SiC did not accumulate in organs but some elemental composition modifications were observed after the acute assessment. Histopathological sections from organs (stomach, intestines, liver, and kidneys) indicate the absence of damage at all applied doses, in both assessments. A decrease in the concentration of urea in blood was found in the 50mg·kg(-1) group from the subacute assessment. No alterations in the urine parameters (sodium, potassium, osmolarity) were found. CONCLUSION: This is the first study that assesses the toxicity, biodistribution, and composition changes in feces and organs of SiC nanoparticles in an in vivo rat model. SiC was excreted mostly in feces and low traces were retrieved in urine, indicating that SiC can cross the intestinal barrier. No sign of toxicity was however found after oral administration.

Quantification of nanoparticles in aqueous food matrices using Particle-Induced X-ray Emission.

Nanoparticles (NPs) of SiO(2) (15 nm) or Ag (20 - 40 nm) were dispersed in water, coffee and milk at several aqueous dilutions. The NPs dispersions concentrations were quantified with an ion beam technique: Particle-Induced X-ray Emission. Additional measurements in relation to the state of the NPs dispersions were done: particle size distribution by centrifuge liquid sedimentation and the extreme surface composition by X-ray photoelectron spectroscopy. The particle size distribution of SiO(2) and Ag NPs dispersions in water and Ag NPs in coffee remained mostly as primary particles with hydrodynamic diameters close to the reported pristine NPs diameter. SiO(2) NPs agglomerated in coffee. In milk, both NPs presented an adsorption with milk lipids. Extreme surface composition corroborated adsorption in milk and showed that SiO(2) agglomerates adsorb some coffee components. A linear tendency in the measurement of the concentration dilutions of all dispersions was measured, and a lack of media influence in the slope of each curve was found. Limits of detection with the current setup were estimated at 0.5 and 0.3 mg/ml for SiO(2) and Ag NPs, respectively.

BM-573 inhibits the development of early atherosclerotic lesions in Apo E deficient mice by blocking TP receptors and thromboxane synthase.

Atherosclerosis is the principal cause of mortality in industrialized countries. Its development is influenced by several mediators of which thromboxane A(2) (TXA(2)) and 8-iso-PGF(2α) have recently received a lot of attention. This study aimed to investigate the effect of a dual thromboxane synthase inhibitor and thromboxane receptor antagonist (BM-573) and ASA on lesion formation in apolipoprotein E-deficient mice. The combination of ASA and BM-573 was also studied. Plasma measurements demonstrated that the treatments did not affect body weight or plasma cholesterol levels. BM-573, but not ASA, significantly decreased atherogenic lesions as demonstrated by macroscopic analysis. Both treatments alone inhibited TXB(2) synthesis but only BM-573 and the combination therapy were able to decrease firstly, plasma levels of soluble intracellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) and secondly, the expression of these proteins in the aortic root of Apo E. These results were confirmed in endothelial cell cultures derived from human saphenous vein endothelial cells (HSVECs). In these cells, BM-573 also prevented the increased mRNA expression of ICAM-1 and VCAM-1 induced by U-46619 and 8-iso-PGF(2α). Our results show that a molecule combining receptor antagonism and thromboxane synthase inhibition is more efficient in delaying atherosclerosis in Apo E(-/-) mice than sole inhibition of TXA(2) formation.

Anti-virulence strategy against Brucella suis: synthesis, biological evaluation and molecular modeling of selective histidinol dehydrogenase inhibitors.

In the facultative intracellular pathogen Brucella suis, histidinol dehydrogenase (HDH) activity, catalyzing the last step in histidine biosynthesis, is essential for intramacrophagic replication. The inhibition of this virulence factor by substituted benzylic ketones was a proof of concept that disarming bacteria leads to inhibition of intracellular bacterial growth in macrophage infection. This work describes the design, synthesis and evaluation of 19 new potential HDH inhibitors, using a combination of classical approaches and docking studies. The IC(50)-values of these inhibitors on HDH activity were in the nanomolar range, and several of them showed a 70-100% inhibition of Brucella growth in minimal medium. One selected compound yielded a strong inhibitory effect on intracellular replication of B. suis in human macrophages at concentrations as low as 5 µM, with an overall survival of intramacrophagic bacteria reduced by a factor 10(3). Docking studies with two inhibitors showed a good fitting in the catalytic pocket and also interaction with the second lipophilic pocket binding the cofactor NAD(+). Experimental data confirmed competition between inhibitors and NAD(+) at this site. Hence, these inhibitors can be considered as promising tools in the development of novel anti-virulence drugs.

Indol-2-yl ethanones as novel indoleamine 2,3-dioxygenase (IDO) inhibitors.

Indoleamine 2,3-dioxygenase (IDO) is a heme dioxygenase which has been shown to be involved in the pathological immune escape of diseases such as cancer. The synthesis and structure-activity relationships (SAR) of a novel series of IDO inhibitors based on the indol-2-yl ethanone scaffold is described. In vitro and in vivo biological activities have been evaluated, leading to compounds with IC(50) values in the micromolar range in both tests. Introduction of small substituents in the 5- and 6-positions of the indole ring, indole N-methylation and variations of the aromatic side chain are all well tolerated. An iron coordinating group on the linker is a prerequisite for biological activity, thus corroborating the virtual screening results.

Surface properties and cell adhesion onto allylamine-plasma and amine-plasma coated glass coverslips.

Surface properties of nanoparticles to be used for radioimmunotherapy need to be optimized to allow antibody conjugation while ensuring biocompatibility. We aimed to investigate cell adhesion and proliferation onto different coatings to be used for nanoparticles. C, CH(x) or SiO(x) coatings deposited onto glass coverslips by magnetron deposition as well as nitrogen functionalized materials synthetized using different reactive sputtering conditions and PPAA (plasma polymerized allylamine) coating, were compared. Amine functionalization did increase hydrophilicity in all the materials tested. Biocompatibility was assessed by measuring cell viability, morphology, attachment, spreading, and pro-inflammatory cytokine secretion. The results show that C and CN(x) were the most biocompatible substrates while SiO(x) and SiO(x)N(y) were the most toxic materials. PPAA coatings displayed unexpectedly an intermediate biocompatibility. A correlation could be observed between wettability and cell proliferation except for C coated surface, indicating that more complex processes than hydrophilicity alone are taking place that affect cell functions.

NF-kappaB inducing kinase (NIK) inhibitors: identification of new scaffolds using virtual screening.

As a wide variety of pro-inflammatory cytokines are involved in the development of rheumatoid arthritis (RA), there is an urgent need for the discovery of novel therapeutic strategies. Among these, the inhibition of the NF-kappaB inducing kinase (NIK), a key enzyme of the NF-kappaB alternative pathway activation, represents a potential interesting approach. In fact, NIK is involved downstream of many tumor necrosis factor receptors (TNFR) like CD40, RANK or LTbetaR, implicated in the pathogenesis of RA. But, up to now, the number of reported putative NIK inhibitors is extremely limited. In this work, we report a virtual screening (VS) study combining various filters including high-throughput docking using a 3D-homology model and ranking by using different scoring functions. This work led to the identification of two molecular fragments, 4H-isoquinoline-1,3-dione (5) and 2,7-naphthydrine-1,3,6,8-tetrone (6) which inhibit NIK with an IC(50) value of 51 and 90 microM, respectively. This study opens new perspectives in the field of the NF-kappaB alternative pathway inhibition.

Design, solid-phase synthesis, and biological evaluation of novel 1,5-diarylpyrrole-3-carboxamides as carbonic anhydrase IX inhibitors.

Following previous studies we herein report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 1,5-diarylpyrrole-3-carboxamides prepared by a solid-phase strategy involving a PS(HOBt) resin. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. This study revealed that the 3-position of the pyrrole was opened to the solvent, so we introduced an amino side-chain, protonated at physiological pH both to enhance the aqueous solubility and to decrease the cell membrane penetration. This strategy consisted of preparing membrane-impermeant inhibitors that may selectively target the tumor-associated hCA IX. Physico-chemical characterizations including aqueous solubility and lipophilic parameters are described. Pharmacological studies revealed high hCA IX inhibitory potency in the nanomolar range. Some compounds are selective for hCA IX displaying hCA I/hCA IX and hCA II/hCA IX ratios higher than 20 and 5, respectively.

Ligand-based and structure-based virtual screening to identify carbonic anhydrase IX inhibitors.

A three-dimensional pharmacophore model of CA IX inhibitors was generated and used to screen the ZINC database of commercially available compounds. The hits were docked in a CA IX homology model. By visualizing the binding mode and score of these compounds, six derivatives were selected and evaluated for their inhibitory potency against CA IX. A highly active CA IX inhibitor was identified which may be used as a lead to design novel such derivatives.

Novel 3-carboxamide-coumarins as potent and selective FXIIa inhibitors.

Recently, FXIIa was highlighted as an original attractive target for the development of new anticoagulant drugs with low rates of therapy-related hemorrhages. In this work, we describe the development of a new series of 3-carboxamide-coumarins that are the first potent and selective nonpeptidic inhibitors of FXIIa.

Carbonic anhydrase inhibitors: bioreductive nitro-containing sulfonamides with selectivity for targeting the tumor associated isoforms IX and XII.

2-Substituted-5-nitro-benzenesulfonamides incorporating a large variety of secondary/tertiary amines were explored as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), with the aim of designing bioreductive inhibitors targeting the hypoxia overexpressed, tumor-associated isozymes. The compounds were ineffective inhibitors of the cytosolic isoform I, showed a better inhibition of the physiologically relevant CA II (KIs of 8.8-4975 nM), and strongly inhibited the tumor-associated CA IX and XII (KIs of 5.4-653 nM). Some of these compounds showed excellent selectivity ratios for the inhibition of the tumor-associated isozymes over the cytosolic ones (in the range of 10-1395). The X-ray crystal structure of the adduct of hCA II with the lead molecule 2-chloro-5-nitro-benzenesulfonamide as well as molecular modeling studies for interaction with hCA IX afforded a better understanding of factors governing the discrimination of the two isoforms for this type of bioreductive compound targeting specifically hypoxic tumors.

Synthesis and pharmacological evaluation of a new targeted drug carrier system: beta-cyclodextrin coupled to oxytocin.

Beta-cyclodextrin (beta-CD) was monofunctionalized into its carboxylic derivative and then conjugated to the N-side of oxytocin (OT), a nonapeptide involved in human behavior and myometrium contraction. On isolated rat myometrium, this conjugate (beta-CD-OT) partly preserves the contracting activity of OT (EC(50) = 0.40 microM vs 1.7 nM). Moreover, the contraction induced frequency is also lowered by beta-CD-OT. This novel hydrophilic targeted carrier could form a host-guest complex with prostaglandins and their derivatives used as labor inducers or with anticancer drugs used in cervix and endometrial cancer. This strategy can improve the solubility, the stability, and/or the biological activity of these drugs as well as reducing their side-effects.

Targeting tumor-associated carbonic anhydrase IX in cancer therapy.

Carbonic anhydrase isoform IX (CA IX) is highly overexpressed in many types of cancer. Its expression, which is regulated by the HIF-1 transcription factor, is strongly induced by hypoxia and correlates with a poor response to classical chemo- and radiotherapies. CA IX contributes to acidification of the tumor environment by efficiently catalyzing the hydration of carbon dioxide to bicarbonate and protons, thereby leading to acquisition of metastatic phenotypes and chemoresistance to weakly basic anticancer drugs. Inhibition of this enzymatic activity by specific inhibitors, such as the sulfonamide indisulam, reverts these processes, establishing a clear-cut role for CA IX in tumorigenesis. Thus, selective CA IX inhibitors could prove useful for elucidating the role of CA IX in hypoxic cancers, for controlling the pH imbalance in tumor cells and for developing diagnostic or therapeutic applications for tumor management. Indeed, fluorescent inhibitors and membrane-impermeant sulfonamides have recently been used as proof-of-concept tools, demonstrating that CA IX is an interesting target for anticancer drug development.

Mechanism-based thrombin inhibitors: design, synthesis, and molecular docking of a new selective 2-oxo-2H-1-benzopyran derivative.

New 2-oxo-2H-1-benzopyran derivatives were prepared to optimize 2a,b, initially developed as mechanism-based alpha-chymotrypsin (alpha-CT) inhibitors, into potent and selective thrombin (THR) inhibitors. From this study, 22, characterized by a 2-(N-ethyl-2'-oxoacetamide)-5'-chlorophenyl ester side chain, was shown to be a good THR inhibitor (ki/KI = 3455 M(-1) x s(-1)), displaying an excellent selectivity profile against other serine proteases such as factor Xa, trypsin, and alpha-CT. Docking analysis of this compound into the different protein structures revealed the molecular basis responsible for its potency and selectivity.

Data in support of a harmine-derived beta-carboline in vitro effects in cancer cells through protein synthesis.

A harmine-derived beta-carboline, CM16, inhibits cancer cells growth through its effects on protein synthesis, as described in "A harmine-derived beta-carboline displays anti-cancer effects in vitro by targeting protein synthesis" (Carvalho et al., 2017)[1]. This data article provides accompanying data on CM16 cytostatic evaluation in cancer cells as well as data related to its effects on transcription and translation. After confirming the cytostatic effect of CM16, we investigated its ability to arrest the cell cycle in the glioma Hs683 and SKMEL-28 melanoma cell lines but no modification was evidenced. According to the global protein synthesis inhibition induced by CM16 [1], transcription phase, a step prior to mRNA translation, evaluated by labelled nucleotide incorporation assay was not shown to be affected under CM16 treatment in the two cell lines. By contrast, mRNA translation and particularly the initiation step were shown to be targeted by CM16 in [1]. To further decipher those effects, we established herein a list of main actors in the protein synthesis process according to literature survey for comparative analysis of cell lines displaying different sensitivity levels to CM16. Finally, one of these proteins, PERK, a kinase regulating eIF2-α phosphorylation and thereby activity, was evaluated under treatment with CM16 in a cell-free system.