Monomethyl Auristatin E Grafted-Liposomes to Target Prostate Tumor Cell Lines.

Novel nanomedicines have been engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or short half-life. Lipid-based carriers such as liposomes represent one of the most advanced classes of drug delivery systems. A Monomethyl Auristatin E (MMAE) warhead was grafted on a lipid derivative and integrated in fusogenic liposomes, following the model of antibody drug conjugates. By modulating the liposome composition, we designed a set of particles characterized by different membrane fluidities as a key parameter to obtain selective uptake from fibroblast or prostate tumor cells. Only the fluid liposomes made of palmitoyl-oleoyl-phosphatidylcholine and dioleoyl-phosphatidylethanolamine, integrating the MMAE-lipid derivative, showed an effect on prostate tumor PC-3 and LNCaP cell viability. On the other hand, they exhibited negligible effects on the fibroblast NIH-3T3 cells, which only interacted with rigid liposomes. Therefore, fluid liposomes grafted with MMAE represent an interesting example of drug carriers, as they can be easily engineered to promote liposome fusion with the target membrane and ensure drug selectivity.

Identifying Potent Nonsense-Mediated mRNA Decay Inhibitors with a Novel Screening System.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that degrades mRNAs carrying a premature termination codon. Its inhibition, alone or in combination with other approaches, could be exploited to develop therapies for genetic diseases caused by a nonsense mutation. This, however, requires molecules capable of inhibiting NMD effectively without inducing toxicity. We have built a new screening system and used it to identify and validate two new molecules that can inhibit NMD at least as effectively as cycloheximide, a reference NMD inhibitor molecule. These new NMD inhibitors show no cellular toxicity at tested concentrations and have a working concentration between 6.2 and 12.5 µM. We have further validated this NMD-inhibiting property in a physiopathological model of lung cancer in which the TP53 gene carries a nonsense mutation. These new molecules may potentially be of interest in the development of therapies for genetic diseases caused by a nonsense mutation.

Targeting low molecular weight cyclin E (LMW-E) in breast cancer.

Low molecular weight cyclin E (LMW-E) plays an important oncogenic role in breast cancer. LMW-E, which is not found in normal tissue, can promote the formation of aggressive tumors and can lead to increased genomic instability and tumorigenesis. Additionally, breast cancer patients whose tumors express LMW-E have a very poor prognosis. Therefore, we investigated LMW-E as a potential specific target for treatment either alone or in combination therapy. We hypothesized that because LMW-E binds to CDK2 more efficiently than full length cyclin E, resulting in increased activity, CDK inhibitors could be used to target tumors with LMW-E bound to CDK2. To test the hypothesis, an inducible full length and LMW-E MCF7-Tet-On system was established. Cyclin E (full length (EL) or LMW-E) is only expressed upon induction of the transgene. The doubling times of cells were unchanged when the transgenes were induced. However, upon induction, the kinase activity associated with LMW-E was much higher than that in the EL induced cells or any of the uninduced cells. Additionally only the LMW-E induced cells underwent chromosome aberrations and increased polyploidy. By examining changes in proliferation and survival in cells with induced full length and LMW-E, CDK inhibitors alone were determined to be insufficient to specifically inhibit LMW-E expressing cells. However, in combination with doxorubicin, the CDK inhibitor, roscovitine (seliciclib, CYC202), synergistically led to increased cell death in LMW-E expressing cells. Clinically, the combination of CDK inhibitors and chemotherapy such as doxorubicin provides a viable personalized treatment strategy for those breast cancer patients whose tumors express the LMW-E.

Unexpected matrix interactions in liquid secondary ion mass spectrometry of two pyranosyl mercaptans.

An unexpected interaction with a thioglycerol matrix appeared in the liquid secondary ion mass spectrometry (LSIMS) spectra of two pyranosyl mercaptans [2,3,4,6-tetra-O-acetyl-1-thio-ß-D-glucopyranose (1a) and 2,3,4,6-tetra-O-acetyl-1,5-dithio-ß-D-glucopyranose (1b)] often used to prepare glucosinolates, important thiosaccharidic metabolites found in all plants of the order Brassicales. The reactions, probably occurring in the solvent cage, seem to involve radical mechanisms.

Exatecan Antibody Drug Conjugates Based on a Hydrophilic Polysarcosine Drug-Linker Platform.

We herein report the development and evaluation of a novel HER2-targeting antibody-drug conjugate (ADC) based on the topoisomerase I inhibitor payload exatecan, using our hydrophilic monodisperse polysarcosine (PSAR) drug-linker platform (PSARlink). In vitro and in vivo experiments were conducted in breast and gastric cancer models to characterize this original ADC and gain insight about the drug-linker structure-activity relationship. The inclusion of the PSAR hydrophobicity masking entity efficiently reduced the overall hydrophobicity of the conjugate and yielded an ADC sharing the same pharmacokinetic profile as the unconjugated antibody despite the high drug-load of the camptothecin-derived payload (drug-antibody ratio of 8). Tra-Exa-PSAR10 demonstrated strong anti-tumor activity at 1 mg/kg in an NCI-N87 xenograft model, outperforming the FDA-approved ADC DS-8201a (Enhertu), while being well tolerated in mice at a dose of 100 mg/kg. In vitro experiments showed that this exatecan-based ADC demonstrated higher bystander killing effect than DS-8201a and overcame resistance to T-DM1 (Kadcyla) in preclinical HER2+ breast and esophageal models, suggesting potential activity in heterogeneous and resistant tumors. In summary, the polysarcosine-based hydrophobicity masking approach allowsfor the generation of highly conjugated exatecan-based ADCs having excellent physicochemical properties, an improved pharmacokinetic profile, and potent in vivo anti-tumor activity.

New insights into homopiperazine-based 5-HT1A/5-HT7R ligands: synthesis and biological evaluation.

The synthesis of new N-homopiperazinyl-based ligands is reported. Various structural modifications along with the corresponding biological activities on 5-HT(1A)/5-HT(7) receptors give further insights into this class of serotoninergic ligands. Among the tested central heterocyles, the 7-azaindole gave the best results on the above-mentioned receptors.

Synthesis and antiproliferative evaluation of pyrazolo[1,5-a]-1,3,5-triazine myoseverin derivatives.

Pyrazolo[1,5-a]-1,3,5-triazine myoseverin derivatives 1a-c were prepared from 4-(N-methyl-N-phenylamino)-2-methylsulfanylpyrazolo[1,5-a]-1,3,5-triazine 2. Their cytotoxic activity, inhibition of tubulin polymerization, and cell cycle effects were evaluated. Compounds 1a and 1c are potent tubulin inhibitors and displayed specific antiproliferative activity in colorectal cancer cell lines at micromolar concentrations.

Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases.

Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.

N-&-N, a new class of cell death-inducing kinase inhibitors derived from the purine roscovitine.

Cyclin-dependent kinases (CDKs) and their regulators show frequent abnormalities in tumors. Ten low molecular weight pharmacologic inhibitors of CDKs are currently in clinical trials against various cancers, including the 2,6,9-trisubstituted purine (R)-roscovitine (CYC202/Seliciclib). We here report the characterization of N-&-N1, a bioisoster of roscovitine displaying improved antitumoral properties. N-&-N1 shows exquisite selectivity for CDKs, with 2- to 3-fold enhanced potency compared with (R)-roscovitine. Inhibition of retinoblastoma protein phosphorylation and RNA polymerase II Ser2 phosphorylation in neuroblastoma SH-SY5Y cells exposed to N-&-N1 indicates that N-&-N1 is able to inhibit CDKs in a cellular context. N-&-N1 also down-regulates the expression of RNA polymerase. Cocrystal structures of N-&-N1 and (R)-roscovitine in complex with CDK2/cyclin A reveal that both inhibitors adopt similar binding modes. A competitive assay shows that, compared with (R)-roscovitine, N-&-N1 has reduced affinity for Erk2 and pyridoxal kinase. N-&-N1 triggers cell death in a panel of diverse cell lines. Cell death is accompanied by events characteristic of apoptosis: cytochrome c release, activation of effector caspases, and poly(ADP-ribose) polymerase cleavage. Induction of p53 and p21CIP1 and down-regulation of the Mcl-1 antiapoptotic factor were also observed. Studies in mice show that N-&-N1 has pharmacokinetics properties similar to those of (R)-roscovitine. Altogether, these results show that analogues of (R)-roscovitine can be designed with improved antitumor potential.

Monodisperse polysarcosine-based highly-loaded antibody-drug conjugates.

Antibody-drug conjugates (ADCs) convey highly potent anticancer drugs to antigen-expressing tumor cells, thereby sparing healthy tissues throughout the body. Pharmacokinetics and tolerability of ADCs are predominantly influenced by the drug-antibody ratio (DAR) of the conjugates, which is to-date limited to a value of 3-4 drugs per antibody in ADCs under clinical investigations. Here, we report the synthesis of monodisperse (i.e. discrete) polysarcosine compounds and their use as a hydrophobicity masking entity for the construction of highly-loaded homogeneous ß-glucuronidase-responsive antibody-drug conjugates (ADCs). The highly hydrophilic drug-linker platform described herein improves drug-loading, physicochemical properties, pharmacokinetics and in vivo antitumor efficacy of the resulting conjugates.

Room-Temperature and Transition-Metal-Free Intramolecular α-Arylation of Ketones: A Mild Access to Tetracyclic Indoles and 7-Azaindoles.

A novel approach for the synthesis of tetracyclic indoles and 7-azaindoles is reported. The strategy involves four steps, with a fast rt intramolecular α-arylation of ketones as key step. The reaction was inspected synthetically to achieve the synthesis of 11 novel tetracyclic structures with moderate to very good yields (39-85%). Theoretical combined with experimental studies led us to propose a probable polar mechanism (concerted SNAr).

Structure-activity relationship of S-trityl-L-cysteine analogues as inhibitors of the human mitotic kinesin Eg5.

The human kinesin Eg5 is a potential drug target for cancer chemotherapy. Eg5 specific inhibitors cause cells to block in mitosis with a characteristic monoastral spindle phenotype. Prolonged metaphase block eventually leads to apoptotic cell death. S-trityl-L-cysteine (STLC) is a tight-binding inhibitor of Eg5 that prevents mitotic progression. It has proven antitumor activity as shown in the NCI 60 tumor cell line screen. It is of considerable interest to define the minimum chemical structure that is essential for Eg5 inhibition and to develop more potent STLC analogues. An initial structure-activity relationship study on a series of STLC analogues reveals the minimal skeleton necessary for Eg5 inhibition as well as indications of how to obtain more potent analogues. The most effective compounds investigated with substitutions at the para-position of one phenyl ring have an estimated K i (app) of 100 nM in vitro and induce mitotic arrest with an EC 50 of 200 nM.

Meriolins (3-(pyrimidin-4-yl)-7-azaindoles): synthesis, kinase inhibitory activity, cellular effects, and structure of a CDK2/cyclin A/meriolin complex.

We report the synthesis and biological characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins), a chemical hybrid between the natural products meridianins and variolins, derived from marine organisms. Meriolins display potent inhibitory activities toward cyclin-dependent kinases (CDKs) and, to a lesser extent, other kinases (GSK-3, DYRK1A). The crystal structures of 1e (meriolin 5) and variolin B (Bettayeb, K.; Tirado, O. M.; Marionneau-Lambert, S.; Ferandin, Y.; Lozach, O.; Morris, J.; Mateo-Lozano, S.; Drückes, P.; Schächtele, C.; Kubbutat, M.; Liger, F.; Marquet, B.; Joseph, B.; Echalier, A.; Endicott, J.; Notario, V.; Meijer, L. Cancer Res. 2007, 67, 8325-8334) in complex with CDK2/cyclin A reveal that the two inhibitors are orientated in very different ways inside the ATP-binding pocket of the kinase. A structure-activity relationship provides further insight into the molecular mechanism of action of this family of kinase inhibitors. Meriolins are also potent antiproliferative and proapoptotic agents in cells cultured either as monolayers or in spheroids. Proapoptotic efficacy of meriolins correlates best with their CDK2 and CDK9 inhibitory activity. Meriolins thus constitute a promising class of pharmacological agents to be further evaluated against the numerous human diseases that imply abnormal regulation of CDKs including cancers, neurodegenerative disorders, and polycystic kidney disease.

Correction: Characterization of Transglutaminase 2 activity inhibitors in monocytes in vitro and their effect in a mouse model for multiple sclerosis.

[This corrects the article DOI: 10.1371/journal.pone.0196433.].

Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes.

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.

Synthesis and biological evaluation in rat and cat of [18F]12ST05 as a potential 5-HT6 PET radioligand.

INTRODUCTION: 5-hydroxytryptamine (5-HT)6 receptors represent one of the more recently discovered serotoninergic receptor family. However, no 5-HT6 positron emission tomography (PET) radiotracer is currently used in clinical imaging studies. The purpose of this study was to propose the first fluorinated PET radiotracer for this brain receptor. METHODS: A new compound presenting in vitro high affinity towards the serotoninergic 5-HT6 receptor, N-[2-(1-[(4-fluorophenyl)sulfonyl]-1H-indol-4-yloxy)ethyl]-N,N-dimethylamine, was labelled with fluorine 18 via a nitro-/fluoronucleophilic substitution. Biological evaluations included (i) in vitro and ex vivo autoradiographies in rat brain and (ii) a PET scan on anaesthetized cat. RESULTS AND CONCLUSION: Although the radioligand showed excellent brain penetration, it did not reveal any specific binding to the 5-HT6 receptors indicating that this radiotracer is not suitable for mapping 5-HT6 receptors using PET.

Targeting Integrin-Dependent Adhesion and Signaling with 3-Arylquinoline and 3-Aryl-2-Quinolone Derivatives: A new Class of Integrin Antagonists.

We previously reported the anti-migratory function of 3-aryl-2-quinolone derivatives, chemically close to flavonoids (Joseph et al., 2002). Herein we show that 3-arylquinoline or 3-aryl-2-quinolone derivatives disrupt cell adhesion in a dose dependent and reversible manner yet antagonized by artificial integrin activation such as manganese. Relying on this anti-adhesive activity, a Structure-Activity Relationship (SAR) study was established on 20 different compounds to throw the bases of future optimization strategies. Active drugs efficiently inhibit platelet spreading, aggregation, and clot retraction, processes that rely on αllbß3 integrin activation and clustering. In vitro these derivatives interfere with ß3 cytoplasmic tail interaction with kindlin-2 in pulldown assays albeit little effect was observed with pure proteins suggesting that the drugs may block an alternative integrin activation process that may not be directly related to kindlin recruitment. Ex vivo, these drugs blunt integrin signaling assayed using focal adhesion kinase auto-phosphorylation as a read-out. Hence, 3-arylquinoline and 3-aryl-2-quinolone series are a novel class of integrin activation and signaling antagonists.

Modulation of protein kinase C (PKC)-mediated contraction and the possible role of PKC epsilon in rat mesenteric arteries.

he involvement of protein kinase C (PKC) in isometric tension development of rat mesenteric arteries was investigated. Non-selective inhibition of PKC and selective inhibition of the epsilon isoform were performed using the PKC inhibitor, chelerythrine, and non-viral gene-transfer of a kinase inactive mutant of PKCepsilon (PKCepsilon-KN), respectively. Chelerythrine (2.5 or 5.0 microM) significantly and equally attenuated phenylephrine-induced but not potassium-induced contractions. Higher concentrations of chelerythrine (10 microM) caused the vessels to lose responsiveness to both phenylephrine and potassium chloride. Transfection of blood vessels with epsilon-KN also resulted in significant attenuation of contractile responses to phenylephrine. Potassium chloride-induced responses were not altered in transfected arteries. In a separate group of vessels, the relationship between [Ca2+]i and isometric tension was evaluated. These studies suggested that calcium sensitivity of the contractile apparatus was decreased in vessels when PKC-epsilon activity was compromised. The results of the study suggest that PKC-epsilon can modulate phenylephrine-induced contraction in mesenteric arteries via calcium-independent pathways.

3-Aryl-2-quinolone derivatives: synthesis and characterization of in vitro and in vivo antitumor effects with emphasis on a new therapeutical target connected with cell migration.

Among 25 3-aryl-2-quinolone derivatives synthesized, the antitumor activity of some of them was characterized both in vitro and in vivo. In this series, no compound appeared to be cytotoxic in vitro, as was known by the colorimetric MTT assay carried out on 12 distinct human cancer cell lines obtained from the American Type Culture Collection. Indeed, the concentration values decreasing the growth of the 12 cell lines by at least 50% (IC(50) index) were always higher than 10(-5) M. We then made use of a computer-assisted phase-contrast videomicroscopy system to quantitatively determine in vitro the level of migration of living MCF-7 human breast cancer cells. For example, at 10(-7) M, compounds 7, 13, 16, and 28 markedly decreased the migration level of these MCF-7 human breast cancer cells. The in vivo determination of the maximum tolerated dose showed that all compounds tested were definitively nontoxic. When the nontoxic, antimigratory compound 16 was combined with either doxorubicin or etoposide, two cytotoxic compounds routinely used in the clinic, this led to additive in vivo benefits from this treatment (as compared to individual administrations of the drugs) when the MXT mouse mammary adenocarcinoma was used. Thus, nontoxic antimigratory compounds, including the 2-quinolone derivatives synthesized here, can actually improve the efficiency of antitumor treatment when combined with conventional cytotoxic agents.

New 1-arylindoles based serotonin 5-HT7 antagonists. Synthesis and binding evaluation studies.

Based on 5-HT1A and 5-HT7 ligand MR25003 scaffold, a new series of 1-aryl indole analogues were prepared and evaluated against 5-HT7 receptors. Modulations of aryl moieties provided a large number of new indolic derivatives. Most of compounds tested have displayed 5-HT7 affinity in the nanomolar range. Among them, 1-(naphthyl)indole derivative 3p (Ki (5-HT7) = 4.5 nM) showed also a good selectivity over 5-HT1A, 5-HT2A and 5-HT6 receptors. This compound was pharmacology characterized as an antagonist.