Jacques Fahy

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.

DNA methyltransferase inhibitors in cancer: a chemical and therapeutic patent overview and selected clinical studies

Introduction: DNA methylation is an epigenetic modification that modulates gene expression without altering the DNA base sequence. It plays a crucial role in cancer by silencing tumor suppressor genes (TSG). The DNA methyltransferases (DNMT) are the enzymes that catalyze DNA methylation and they are interesting therapeutical targets since DNA methylation is reversible such that an aberrant hypermethylation of DNA can be reverted by inhibition of DNMTs. Today, two drugs are on the market for the treatment of myelodysplastic syndrome, azacitidine and decitabine. Areas covered: Here, we present a review of the patents describing the chemistry and biological activities of novel DNMT inhibitors and discuss select clinical studies. Expert opinion: DNMT inhibitors have shown efficacy in clinics. However, highly efficient and specific DNMT inhibitors have not yet been identified. Improving methods will certainly lead to the prediction of novel directly binding inhibitors in the future.

Targeting DNA methylation with small molecules: what's next?

DNA methylation is a mammalian epigenetic mark that is involved in defining where and when genes are expressed, both in normal cells and in the context of diseases. Like other epigenetic marks, it is reversible and can be modulated by chemical agents. Because it plays an important role in cancer by silencing certain genes, such as tumor suppressor genes, and by reactivating other regions, such as repeated elements, it is a promising therapeutic target. Two compounds are already approved to treat hematological cancers. Many efforts have been carried out to discover new molecules that are able to efficiently inhibit DNA methylation in cancer cells. We will briefly overview the foremost of these efforts by focusing on what we have learned to this point on non-nucleoside inhibitors and on what we consider to be the features of an ideal inhibitor.

Novel artificial endonucleases inhibit base excision repair and potentiate the cytotoxicity of DNA-damaging agents on L1210 cells.

A series of molecules with apurinic/apyrimidic (AP) endonuclease activity targeted to abasic sites in DNA, which incorporate an intercalating moiety linked to a purine base by a polyamino chain and recognize and cleave abasic sites in DNA with high efficiency, has been studied. The aim was first to establish whether these compounds were inhibitors of base excision DNA repair, since abasic sites are generated during this process. Using an extension of a recently established methodology, two members of this series have been identified as definite repair inhibitors. Secondly, the potential of using such compounds as sensitizers of alkylating agents has been investigated by determining the cytotoxic activity of these compounds on L1210 cells in culture. A concentration-dependent potentiation of nitrosoureas has been demonstrated, but interpretation is complicated by the inherent cytotoxic properties of the test compounds themselves. Such molecules, however, provide interesting lead compounds for new strategies aimed at enhancing the cytotoxic potential of clinically useful DNA-damaging agents.

Antitumor activity of pyridoisoquinoline derivatives F91873 and F91874, novel multikinase inhibitors with activity against the anaplastic lymphoma kinase.

The anaplastic lymphoma kinase (ALK) is a validated target for the therapy of different malignancies. Aberrant expression of constitutively active ALK chimeric proteins has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL) and has been detected in other cancers such as inflammatory myofibroblastic tumors, diffuse large B-cell lymphomas, certain non-small-cell lung cancers, rhabdomyosarcomas, neuroblastomas and glioblastomas. In the course of a screening program aimed at identifying kinase inhibitors with novel scaffolds, the two pyridoisoquinoline derivatives F91873 and F91874, were identified as multikinase inhibitors with activity against ALK in a biochemical screen. F91873 and F91874 also inhibited nucleophosmin–ALK and signal transducer and activator of transcription 3 phosphorylation in the ALCL cell line COST with the same potency. Both F91873 and F91874 behaved as ATP noncompetitive inhibitors and inhibited cell proliferation of the ALK(+) ALCL cell lines COST, PIO, and Karpas299 ALCL. This growth inhibition effect was associated with a G1-phase cell cycle arrest. Furthermore, administration of F91874 to severe combined immunodeficient mice bearing COST tumor xenografts resulted in a significant antitumor efficacy at 15 mg/kg/day, illustrating the potential utility of such compounds in the treatment of ALK-related pathologies.

Synthesis and Antiproliferative and Metabolic Evaluations of Novel Securinine Derivatives.

New securinine analogues have been prepared by semisynthesis. Two series were developed using either Suzuki or Sonogashira cross coupling reactions. The in vitro cytotoxicity of the compounds was assayed against HCT-116 colon cancer cells. The most potent derivatives showed promising growth inhibition on four tumoral cell lines giving a valuable insight on the structure-activity relationship (SAR) of securinine. Moreover, high antiproliferative effect against A-375 (melanoma) was observed with IC50 up to 60 nM.

Abstract 1925: Trifluoromethylated artemisinin dimers demonstrate a potent anti-cancer activity in vitro and in vivo

Beside their widely recognised efficacy as antimalaric drugs, artemisinin derivatives are also known for their cytotoxic properties since the early nineties. More recently, artemisinin dimers (ADs) were shown to possess an increased potential as anti-cancer agents due to their ability to inhibit cancer cells proliferation in the low nanomolar range. Taking advantage of an original chemistry, we synthesized a new series of trifluoromethylated artemisnin dimers which proved to be particularly potent depending on the size and nature of the linker. Different mode of linkage were performed by either carbon 16 or carbon 10 and were tested in vitro and in vivo. Best combination was found in a hybrid linking position delivering non-symmetrical C10-C16 ADs. One of the lead compound of this series, F98458 demonstrated a moderate (but statiscally significative) anti-tumor activity in mice against a human melanoma xenograft that is, to our knowledge, the first demonstration of in vivo antitumor properties of ADs in preclinical models. Many efforts to understand the molecular origin of the cytotoxic effect of monomeric artemisinin derivatives have been undertaken. One of the aims of this study was to investigate the mechanism of action of ADs in comparison with artemisinin: the antimalaric drug artesunate, a representative monomeric derivative of artemisinin, is known to induce DNA damage and ROS generation while our ADs did not under the same experimental conditions. Moreover ADs did not induce apoptosis (caspase 3/7, annexin V), leading us to investigate cell death related to senescence and autophagy. We have also synthesized partially reduced ADs possessing only one endoperoxide functionality. These compounds did show the same potency than non reduced ADs, underlaying that a tight molecular recognition is involved in its biological response. All these observations suggest that monomeric artemisinin derivatives such as artesunate and ADs possess distinct modes of action. The high in vitro potency associated with the in vivo response led us to synthesize affinity probes in order to identify the molecular target of ADs which could provide fundamental informations for further developpement of these compounds as anti-cancer agents. This work currently underway will also be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1925. doi:1538-7445.AM2012-1925

Abstract 3261: Novel hemisynthetic derivatives of Amaryllidaceae alkaloids exert antitumor activity on xenograft melanoma models

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Alkaloids pancratistatin and narciclasine, isolated from Amaryllidaceae, have been shown to present antitumor activity on preclinical models, including melanoma. Despite extensive investigations, pancratistatin or narciclasine analogues did not succeed to provide sufficient therapeutic benefit, [1] mostly hampered by poor water solubility and systemic toxicity [1]. To overcome those limitations, we developed a chemistry program aimed at improving both their physicochemical and pharmacological properties. A literature survey suggests that any structural modification of this highly oxigenated isocarbostiryl specific scaffold would lead to a loss of activity except on position 1, difficult to achieve but interesting to explore. A series of 40 novel 1-aminopancratistatin derivatives was synthesised starting from large quantities of narciclasine extracted from Narcissus bulbs. These novel compounds exhibit an improved pharmaceutical profile facing standard solubility, plasmatic and microsomal stability assays. Moreover, a subset of narciclasine derivatives demonstrated a significant enhancement of the antiproliferative effect with IC50 reaching the nM range, consistent with a major induction of pro-apoptotic signals, including mitochondrial depolarisation. To explore the mode of action of these alkaloids derivatives, we tested their capacity to inhibit protein synthesis, compared their pharmacological profile with 80 anticancer compounds on 16 tumor cell lines (oncoprofile.16) and explored their impact on major cellular functions such as cellular integrity, cell cycle, topoisomerases inhibition, DNA damage and synthesis. We demonstrated thus that inhibition of DNA synthesis is a key component of their mechanism of action. Based on the pharmacological properties, a structure-activity card can be built to select different categories of pancrastistatin derivatives depending on their ability to inhibit preferentially protein synthesis or/and DNA synthesis. Interestingly, the dimethylaminomethyl benzamide derivative F98604 exhibited a significant antiproliferative activity on the murine B16 and human A375 melanoma xenograft models associated with an increased therapeutic index as compared with the natural compound narciclasine. In conclusion, modulation of position 1 on the narciclasine pharmacophore overcomes the solubility issue and is a key position to modify their pharmacological profiles, this opening new perspectives as anticancer agents active against melanoma. 1. A. Kornienko, A. Evidente Chem. Rev. 2008, 108, 1982 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3261. doi:10.1158/1538-7445.AM2011-3261

Abstract 5461: Novel antitumor amino-pancratistatin derivatives inhibitors of protein and DNA synthesis

The Amaryllidaceous alkaloids pancratistatin and narciclasine are known to exert marginal antitumor properties but associated with toxicities. Despite extensive investigations, pancratistatin or analogues did not succeed to provide sufficient therapeutic benefit although a new pro-drug strategy was applied recently to narciclasine. We decided to undertake a chemistry program aimed at modifying the skeleton of this attractive pharmacophore in order to improve its therapeutic application. At first sight, literature survey suggests that any change in the structure would lead to a loss of activity but a careful examination of published data indicated that position −1 of the molecule may be of potential interest although poorly investigated. We report here the synthesis of a series of 39 novel derivatives of 1-aminopancratistatin by chemical modification of the naturally available narciclasine extracted from Narcissus bulbs. These new compounds present improved pharmaceutical properties such as higher aqueous solubility (from 300 µg/ml for narciclasine up to 2000 µg/ml) without loss of stability tested on human and murine microsomal assays (70-90% stability after 1h incubation). A sub-set of these novel derivatives demonstrated a drastic increase of the cytotoxic activity reaching nM range tested on a panel of 8 different cell lines. The higher antiproliferative activity translates towards the induction of stronger pro-apoptotic signals evidenced with Annexin V, caspase 3/7 and JC-1 assay. Besides the previously described inhibition of protein synthesis, our studies originally demonstrated that part of the mechanism of action involves inhibition of DNA synthesis as well. The contribution of these two pharmacological properties elaborates a SAR (structure - activity relationship) and segregates our new pancrastistatin derivatives into different categories depending on their ability to inhibit protein synthesis, DNA synthesis or both. Based on their differential in vitro activity profile, the compounds were selected for evaluation of their in vivo antitumor properties on murine and xenografted models. As a major result F 98604 (dimethylaminomethyl benzamide derivative of pancratistatin) exhibited significant and reproducible activity on the murine B16 sc and human A375 melanoma models in terms of tumor growth inhibition as well as increased therapeutic index. The present SAR suggests that modification of the natural pancratistatin or narciclasine pharmacophore at the position −1 open new perspectives as potential anticancer agents with novel pharmacological profiles. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5461.