Frédéric Ausseil

DNA methylation inhibitors in cancer: Recent and future approaches

This review presents the different human DNA methyltransferases (DNMTs), their biological roles, their mechanisms of action and their role in cancer. The description of assays for detecting DNMT inhibitors (DNMTi) follows. The different known DNMTi are reported along with their advantages, drawbacks and clinical trials. A discussion on the features of the future DNMT inhibitors will conclude this review.

Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer.

BackgroundThe multicellular tumor spheroid (MCTS) is an in vitro model associating malignant-cell microenvironment and 3D organization as currently observed in avascular tumors.MethodsIn order to evaluate the relevance of this model for pre-clinical studies of drug combinations, we analyzed the effect of gemcitabine alone and in combination with the CHIR-124 CHK1 inhibitor in a Capan-2 pancreatic cell MCTS model.ResultsCompared to monolayer cultures, Capan-2 MCTS exhibited resistance to gemcitabine cytotoxic effect. This resistance was amplified in EGF-deprived quiescent spheroid suggesting that quiescent cells are playing a role in gemcitabine multicellular resistance. After a prolonged incubation with gemcitabine, DNA damages and massive apoptosis were observed throughout the spheroid while cell cycle arrest was restricted to the outer cell layer, indicating that gemcitabine-induced apoptosis is directly correlated to DNA damages. The combination of gemcitabine and CHIR-124 in this MCTS model, enhanced the sensitivity to the gemcitabine antiproliferative effect in correlation with an increase in DNA damage and apoptosis.ConclusionsThese results demonstrate that our pancreatic MCTS model, suitable for both screening and imaging analysis, is a valuable advanced tool for evaluating the spatio-temporal effect of drugs and drug combinations in a chemoresistant and microenvironment-depending tumor model.

Parthenolide Inhibits Tubulin Carboxypeptidase Activity

Microtubules are centrally involved in cell division, being the principal components of mitotic spindle. Tubulin, the constituent of microtubules, can be cyclically modified on its alpha-subunit by enzymatic removal of the COOH-terminal tyrosine residue by an ill-defined tubulin carboxypeptidase (TCP) and its readdition by tubulin tyrosine ligase (TTL). We and others have previously shown that suppression of TTL and resulting accumulation of detyrosinated tubulin are frequent in human cancers of poor prognosis. Explanations for the involvement of TTL and detyrosinated tubulin in tumor progression arise from the recent discovery that tubulin detyrosination leads to CAP-Gly protein mislocalization, which correlates with defects in spindle positioning during mitosis. Impaired control of spindle positioning is one factor favoring tumor invasiveness. Thus, TCP could be a target for developing novel therapeutic strategies against advanced stages of cancers. Inhibitors of TCP, by reversing abnormal detyrosinated tubulin accumulation in tumor cells, could impair tumor progression. TCP has never been isolated and this has hampered search of specific inhibitors. In this article, we describe a cell-based assay of TCP activity and its use to screen a library of natural extracts for their inhibitory potency. This led to the isolation of two sesquiterpene lactones. We subsequently found that parthenolide, a structurally related compound, can efficiently inhibit TCP. This inhibitory activity is a new specific property of parthenolide independent of its action on the nuclear factor-kappaB pathway. Parthenolide is also known for its anticancer properties. Thus, TCP inhibition could be one of the underlying mechanisms of these anticancer properties.

Bioactive flavonoids of Tanacetum parthenium revisited

Bio-guided fractionation of an extract from Tanacetum parthenium showing activity as mitotic blocker allowed the isolation and identification of santin 3, jaceidin 2 and centaureidin 1. The latter two closely related flavonols, which, to the best of our knowledge, are isolated here together for the first time, form a mixture difficult to resolve and which is probably the reason for the confusion in the literature regarding their occurrence. Centaureidin 1 had an IC50 of 1 microM while jaceidin 2 and santin 3 were 200 times less active.

Alisiaquinones and alisiaquinol, dual inhibitors of Plasmodium falciparum enzyme targets from a New Caledonian deep water sponge.

Four new meroterpenes, alisiaquinones A-C (1-3) and alisiaquinol (4), were isolated from a New Caledonian deep water sponge. Their structures and relative stereochemistry were elucidated by spectroscopic data analysis. They are related to xestoquinone, but showed unusual substitution on a tetrahydrofuran junction. They displayed micromolar range activity on two enzymatic targets of importance for the control of malaria, the plasmodial kinase Pfnek-1 and a protein farnesyl transferase, as well as on different chloroquine-sensitive and -resistant strains of Plasmodium falciparum. Alisiaquinone C displayed a submicromolar activity on P. falciparum and a competitive selectivity index on the different plasmodial strains.

Physalin B, a novel inhibitor of the ubiquitin-proteasome pathway, triggers NOXA-associated apoptosis

The ubiquitin-proteasome pathway plays a critical role in the degradation of proteins involved in tumor growth and has therefore become a target for cancer therapy. In order to discover novel inhibitors of this pathway, a cellular assay reporter of proteasome activity was established. Human DLD-1 colon cancer cells were engineered to express a 4 ubiquitin-luciferase (DLD-1 4Ub-Luc) reporter protein, rapidly degraded via the ubiquitin-proteasome pathway and designed DLD-1 4Ub-Luc cells. Following treatment with reference proteasome inhibitors, the 4Ub-Luc protein accumulated in DLD-1 4Ub-Luc cells and a 80-fold increase in luciferase-produced bioluminescence signal was measured, as compared to untreated cells. The screening of over 30,000 compounds using this DLD-1 4Ub-Luc assay led to the identification of physalin B as a novel inhibitor of the ubiquitin-proteasome pathway. Indeed, physalin B induced an increase in bioluminescence from DLD-1 4Ub-Luc cells, at concentrations also producing an accumulation of ubiquitinated proteins and inhibiting TNFalpha-induced NF-kappaB activation. Physalin B did not inhibit catalytic activities of purified proteasome and interfered with cellular proteasomal catalytic activities at 4- to 8-fold higher concentrations than that required to induce significant increase in bioluminescence and accumulation of ubiquitinated proteins in DLD-1 4Ub-Luc cells. Furthermore, physalin B proved to be cytotoxic, triggered apoptosis in DLD-1 4Ub-Luc cells and induced the proapoptotic protein NOXA, characteristic of the proteasome signaling pathway. Therefore, the use of the DLD-1 4Ub-Luc assay allowed the identification of a novel inhibitor of the ubiquitin-proteasome pathway that might interfere with proteasome functions in a different way from reference proteasome inhibitors.

Design, Synthesis and Biological Evaluation of 4-Amino-N-(4-aminophenyl)benzamide Analogues of Quinoline-Based SGI-1027 as Inhibitors of DNA Methylation

Quinoline derivative SGI‐1027 (N‐(4‐(2‐amino‐6‐methylpyrimidin‐4‐ylamino)phenyl)‐4‐(quinolin‐4‐ylamino)benzamide) was first described in 2009 as a potent inhibitor of DNA methyltransferase (DNMT) 1, 3A and 3B. Based on molecular modeling studies, performed using the crystal structure of Haemophilus haemolyticus cytosine‐5 DNA methyltransferase (MHhaI C5 DNMT), which suggested that the quinoline and the aminopyridimine moieties of SGI‐1027 are important for interaction with the substrates and protein, we designed and synthesized 25 derivatives. Among them, four compounds—namely the derivatives 12, 16, 31 and 32—exhibited activities comparable to that of the parent compound. Further evaluation revealed that these compounds were more potent against human DNMT3A than against human DNMT1 and induced the re‐expression of a reporter gene, controlled by a methylated cytomegalovirus (CMV) promoter, in leukemia KG‐1 cells. These compounds possessed cytotoxicity against leukemia KG‐1 cells in the micromolar range, comparable with the cytotoxicity of the reference compound, SGI‐1027. Structure–activity relationships were elucidated from the results. First, the presence of a methylene or carbonyl group to conjugate the quinoline moiety decreased the activity. Second, the size and nature of the aromatic or heterocycle subsitutents effects inhibition activity: tricyclic moieties, such as acridine, were found to decrease activity, while bicyclic substituents, such as quinoline, were well tolerated. The best combination was found to be a bicyclic substituent on one side of the compound, and a one‐ring moiety on the other side. Finally, the orientation of the central amide bond was found to have little effect on the biological activity. This study provides new insights in to the structure–activity relationships of SGI‐1027 and its derivative.

Development of a universal radioactive DNA methyltransferase inhibition test for high-throughput screening and mechanistic studies

DNA methylation is an important epigenetic mark in eukaryotes, and aberrant pattern of this modification is involved in numerous diseases such as cancers. Interestingly, DNA methylation is reversible and thus is considered a promising therapeutic target. Therefore, there is a need for identifying new small inhibitors of C5 DNA methyltransferases (DNMTs). Despite the development of numerous in vitro DNMT assays, there is a lack of reliable tests suitable for high-throughput screening, which can also give insights into inhibitor mechanisms of action. We developed a new test based on scintillation proximity assay meeting these requirements. After optimizing our assay on human DNMT1 and calibrating it with two known inhibitors, we carried out S-Adenosyl-l-Methionine and DNA competition studies on three inhibitors and were able to determine each mechanism of action. Finally, we showed that our test was applicable to 3 other methyltransferases sources: human DNMT3A, bacterial M.SssI and cellular extracts as well.

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitinproteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Ź factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

New insights on the mechanism of quinoline-based DNA methyltransferase inhibitors

Background: 4-Aminoquinoline SGI-1027 and analogs inhibit DNA methylation, which is deregulated in cancers. Results: These compounds induce deviations from Michaelis-Menten equations in DNA competition experiments and interact with DNA. Conclusion: They are competitive inhibitors for the DNA substrate of the DNA methyltransferase and non-competitive for the methyl group donor, S-adenosyl-l-methionine. Significance: These findings suggest a mechanism of inhibition for these 4-aminoquinoline-based DNMT inhibitors. Among the epigenetic marks, DNA methylation is one of the most studied. It is highly deregulated in numerous diseases, including cancer. Indeed, it has been shown that hypermethylation of tumor suppressor genes promoters is a common feature of cancer cells. Because DNA methylation is reversible, the DNA methyltransferases (DNMTs), responsible for this epigenetic mark, are considered promising therapeutic targets. Several molecules have been identified as DNMT inhibitors and, among the non-nucleoside inhibitors, 4-aminoquinoline-based inhibitors, such as SGI-1027 and its analogs, showed potent inhibitory activity. Here we characterized the in vitro mechanism of action of SGI-1027 and two analogs. Enzymatic competition studies with the DNA substrate and the methyl donor cofactor, S-adenosyl-l-methionine (AdoMet), displayed AdoMet non-competitive and DNA competitive behavior. In addition, deviations from the Michaelis-Menten model in DNA competition experiments suggested an interaction with DNA. Thus their ability to interact with DNA was established; although SGI-1027 was a weak DNA ligand, analog 5, the most potent inhibitor, strongly interacted with DNA. Finally, as 5 interacted with DNMT only when the DNA duplex was present, we hypothesize that this class of chemical compounds inhibit DNMTs by interacting with the DNA substrate.