FRET-Based Method for Direct, Real-Time Measurement of DNA Methyltransferase Activity.

DNA methyltransferase activity is associated with a host of diseases, including cancers, where global hypomethylation of the genome, as well as marked changes in local DNA methylation patterns, can be both diagnostic and prognostic for the disease. Despite this, we currently lack a method for directly measuring the activity of the DNA methyltransferases, which would support the development of DNA methyltransferase-targeted therapies. Here, we demonstrate an assay for the direct measurement of methyltransferase activity, in real time. We employ a fluorescent methyltransferase cofactor analogue, which when bound by the enzyme to a labeled target DNA sequence results in fluorescence resonance energy transfer (FRET) between the donor dye (DNA) and the acceptor dye (cofactor). We demonstrate that the method can be used to monitor the activity of DNA MTases in real time and can be applied to screen inhibitors of the DNA methyltransferases. We show this in both bulk phase and single molecule imaging experiments, highlighting the potential application of the assay in screening and biophysical studies of methyltransferase function.

Site-Selective and Rewritable Labeling of DNA through Enzymatic, Reversible, and Click Chemistries.

Current methods for bioconjugation rely on the introduction of stable linkers that lack the required versatility to perform sequential functionalizations. However, sequential manipulations are an increasing requirement in chemical biology because they can underpin multiple analyses of the same sample to provide a wider understanding of cell behavior. Here, we present a new method to site-selectively write, remove, and rewrite chemical functionality to a biomolecule, DNA in this case. Our method combines the precision and robustness of methyltransferase-directed labeling with the reversibility of acyl hydrazones and the efficiency of click chemistry. Underpinning the method is a new S-adenosyl-l-methionine derivative to site-selectively label DNA with a bifunctional chemical handle containing an acyl hydrazone-linker and a terminal azide. Functional tags are conjugated via the azide and can be removed (i.e., untagged) when needed at the acyl hydrazone via exchange with hydroxyl amine. The formed hydrazide-labeled DNA is a versatile intermediate that can be either rewritten to reset the original chemical handle or covalently reacted with a permanent tag. This ability to write, tag, untag, and permanently tag DNA is exploited to sequentially introduce two fluorescent dyes on DNA. Finally, we demonstrate the potential of the method by developing a protocol to sort labeled DNA using magnetic beads, with subsequent amplification of the sorted DNA sample for further analysis. The presented method opens new avenues for site-selective bioconjugation and should underpin integrative approaches in chemical biology where sequential functionalizations of the same sample are required.

Spermine-NBD as fluorescent probe for studies of the polyamine transport system in Leishmania donovani.

This study describes the synthesis of fluorescent probes as potential substrates for the polyamine transport system (PTS) of Leishmania donovani. A competitive radioassay was used to determine the most efficient probe. We observed that the conjugate spermine-nitrobenzofurazan (Spm-NBD) was able to compete with [3H]-spermidine in L. donovani at a potent IC50 of 60 µM.

Synthesis and antikinetoplastid evaluation of bis(benzyl)spermidine derivatives.

This study describes the synthesis and the biological evaluation of twenty-four original bis(benzyl)spermidines. Structural modifications of the polyamine scaffold were performed in order to avoid easily metabolized bonds. Some bis(benzyl)polyamine derivatives have demonstrated promising activity in vitro against Trypanosoma brucei gambiense and Leishmania donovani. From the enzymatic experiments on trypanothione reductase, we observed that this enzyme was not targeted by our compounds. In vivo evaluation on Swiss mice model infected by T. b. gambiense or L. donovani was done with the most interesting compound of the series.

Polyamine-based analogs and conjugates as antikinetoplastid agents.

Naturally occurring polyamines: putrescine, spermidine and spermine are crucial for Kinetoplastid growth and persistence. These aliphatic polycations are either biosynthesized or internalized into Kinetoplastid by active transport. Impairing the polyamine metabolism using polyamine derivatives is an interesting path in the search of new antikinetoplastid chemotherapy. In the past 30 years, research interest in this field has been constantly expanding and recent results demonstrated that the discovery of a polyamine-based antikinetoplastid drug is undoubtedly possible. In this paper, all the polyamine derivatives previously described to present an antikinetoplastid activity are reported. This review is organized around three main parts which are diamine, triamine and tetramine derivatives. Each part includes the description of the series of molecules and, their in vitro and in vivo activity when available. Structure-activity relationships of these derivatives are discussed and the most promising structures for a positive outcome are eventually highlighted.

Synthesis and in vitro antikinetoplastid activity of polyamine-hydroxybenzotriazole conjugates.

Thirteen new polyamine derivatives coupled to hydroxybenzotriazole have been synthesized and evaluated for their in vitro antikinetoplastid activity. Trypanosoma Trypanothione reductase (TryR) was envisioned as a potential target. Among all tested molecules, only one compound, a N3-spermidine-benzotriazole derivative, displayed relevant inhibitory activity on this enzyme but was not active on parasites. The corresponding Boc-protected spermidine-benzotriazole was however trypanocidal against Trypanosoma brucei gambiense with an IC50 value of 1µM and was completely devoid of cytotoxicity. On the intramacrophage amastigotes of Leishmania donovani, a N2-spermidine conjugate of this series, exhibited an interesting IC50 value of 3µM associated with both low cytotoxicity against axenic Leishmania donovani. These new compounds are promising leads for the development of antikinetoplastid agents and their targets have to be deciphered.

Design, synthesis and in vitro antikinetoplastid evaluation of N-acylated putrescine, spermidine and spermine derivatives.

A structure-activity relationship study on polyamine derivatives led to the synthesis and the determination of antikinetoplastid activity of 17 compounds. Among them, a spermidine derivative (compound 13) was specifically active in vitro against Leishmania donovani axenic amastigotes (IC50 at 5.4µM; Selectivity Index >18.5) and a spermine derivative (compound 28) specifically active against Trypanosoma brucei gambiense (IC50 at 1.9µM; Selectivity Index >52).