Parasites account for huge economic losses by infecting agriculturally important plants and animals. Furthermore, morbidity and death caused by parasites affect a large part of the world population, especially in economically weak regions. Anthelmintic drugs to tackle this challenge remain scarce and their efficiency becomes increasingly endangered by the advent of drug resistance development. In the present study, we assessed the anthelmintic potential of bis-3-chloropiperidines, a family of compounds which have already demonstrated antiproliferative activity against various cell lines. We synthesized and tested the activity of 21 bis-3-chloropiperidine derivatives against two strains of the free-living nematode Caenorhabditis elegans (N2 and DC19) and the parasitic flatworm Schistosoma mansoni. Overall, bifunctional chloropiperidines featuring an aromatic linker performed best against the tested indicator organisms and could be considered for future optimization efforts. Ultimately, out of the 21 analyzed bis-3-chloropiperidines, four derivatives (2, 5, 9 and 11) reduced vitality parameters against S. mansoni and five the motility of C. elegans (2, 4, 5, 13, 21) while exhibiting no or low cytotoxicity.

We report the synthesis and characterization of two novel cisplatin- alkylating agents conjugates. Combining a platinum based cytostatic agent with a sterically demanding alkylating agent could potentially induce further DNA damage, block cell repair mechanisms and keep the substrate active against resistant tumor cell lines. The 3-chloropiperidines utilized as ligands in this work are cyclic representatives of the N-mustard family and were not able to coordinate platinum on their own. The introduction of a second coordination site, in form of a pyridine moiety, led to the isolation of the desired conjugates. They were characterized with HRMS, CHN-analyses and XRD. We concluded this work by examining the cytotoxicity of the ligands and the obtained complexes with MTT assays in human cancer cell lines. While the ligands showed hardly any activity, the novel conjugates both displayed a high antiproliferative and cytotoxic potency in a panel of three cell lines. Moreover, both complexes were able to largely circumvent the acquired cisplatin resistance of A2780cisR ovarian cancer cells, both in the MTT assay and a flow-cytometric apoptosis assay.

Omics-based technologies are driving major advances in precision medicine, but efforts are still required to consolidate their use in drug discovery. In this work, we exemplify the use of multi-omics to support the development of 3-chloropiperidines, a new class of candidate anticancer agents. Combined analyses of transcriptome and chromatin accessibility elucidated the mechanisms underlying sensitivity to test agents. Furthermore, we implemented a new versatile strategy for the integration of RNA- and ATAC-seq (Assay for Transposase-Accessible Chromatin) data, able to accelerate and extend the standalone analyses of distinct omic layers. This platform guided the construction of a perturbation-informed basal signature predicting cancer cell lines' sensitivity and to further direct compound development against specific tumor types. Overall, this approach offers a scalable pipeline to support the early phases of drug discovery, understanding of mechanisms, and potentially inform the positioning of therapeutics in the clinic.

Elucidating the structure of RNA and RNA ensembles is essential to understand biological functions. In this work, we explored the previously uncharted reactivity of bis-chloropiperidines (B-CePs) towards RNA. We characterized at the molecular level the different adducts induced by the fast reacting compound B-CeP 1 with RNA. Following an approach based on solution thermal melting coupled with ESI mass spectrometry (STHEM-ESI), we proved the ability of B-CePs to induce inter-molecular cross-links between guanines in double stranded RNA. These results open the possibility of using B-CePs as structural probes for investigating higher-order structures, such as the kissing loop complex established by the dimerization initiation site (DIS) of the HIV-1 genome. We confirmed the potential of B-CePs to reveal the identity of RNA structures involved in long-range interactions, expecting to benefit the characterization of samples that are not readily amenable to traditional high-resolution techniques, and thus promoting the elucidation of pertinent RNA systems associated with old and new diseases.

Specific RNA sequences regulate functions essential to life. The Trans-Activation Response element (TAR) is an RNA stem-bulge-loop structure involved in several steps of HIV-1 replication. In this work, we show how RNA targeting can inhibit HIV-1 nucleocapsid (NC), a highly conserved protein known to catalyze nucleic acid melting and strand transfers during reverse transcription. Our RNA targeting strategy consists of the employment of bis-3-chloropiperidines (B-CePs) to impair RNA melting through bifunctional alkylation. Specific interactions between B-CePs and TAR RNA were analytically investigated by gel electrophoresis and mass spectrometry, allowing the elucidation of B-CePs' recognition of TAR, and highlighting an RNA-directed mechanism of protein inhibition. We propose that B-CePs can freeze TAR tridimensional conformation, impairing NC-induced dynamics and finally inhibiting its functions in vitro.

Nitrogen mustards (NMs) are an old but still largely diffused class of anticancer drugs. However, spreading mechanisms of resistance undermine their efficacy and therapeutic applicability. To expand their antitumour value, we developed bis-3-chloropiperidines (B-CePs), a new class of mustard-based alkylating agent, and we recently reported the striking selectivity for BxPC-3 pancreatic tumour cells of B-CePs bearing aromatic moieties embedded in the linker. In this study, we demonstrate that such tropism is shared by bis-3-chloropiperidines bearing appended aromatic groups in flexible linkers, whereas esters substituted by aliphatic groups or by efficient DNA-interacting groups are potent but nonselective cytotoxic agents. Besides, we describe how the critical balance between water stability and DNA reactivity can affect the properties of bis-3-chloropiperidines. Together, these findings support the exploitation of B-CePs as potential antitumour clinical candidates.