Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study

Abstract

thiosemicarbazones (tSc) and their metal complexes display diverse biological activities and are active against multiple pathological conditions ranging from microbial infections to abnormal cell proliferation. Ribonucleotide reductase (RNR) is considered one of the main targets of TSCs, yet, the existence of additional targets, differently responsible for the multifaceted activities of TSCs and their metal complexes has been proposed. to set the basis for a more comprehensive delineation of their mode of action, we chemogenomically profiled the cellular effects of bis(citronellalthiosemicar bazonato)nickel(ii) [ni(S-tcitr) 2 ] using the unicellular eukaryote Saccharomyces cerevisiae as a model organism. Two complementary genomic phenotyping screens led to the identification of 269 sensitive and 56 tolerant deletion mutant strains and of 14 genes that when overexpressed make yeast cells resistant to an otherwise lethal concentration of Ni(S-tcitr) 2. Chromatin remodeling, cytoskeleton organization, mitochondrial function and iron metabolism were identified as lead cellular processes responsible for ni(S-tcitr) 2 toxicity. The latter process, and particularly glutaredoxin-mediated iron loading of RNR, was found to be affected by Ni(S-tcitr) 2. Given the multiple pathways regulated by glutaredoxins, targeting of these proteins by Ni(S-tcitr) 2 can negatively affect various core cellular processes that may critically contribute to Ni(S-tcitr) 2 cytotoxicity. Originally discovered as antivirals active against smallpox and other viruses 1 , thiosemicarbazones (TSC) are still attracting significant interest as anticancer agents 2-4. The antiproliferative activity of TSCs has been initially ascribed to their metal (e.g., Fe 2+) sequestration capacity and to the inactivation of ribonucleotide reductase (RNR), the enzyme that converts ribonucleotides into deoxyribonucleotides and whose activity correlates with cell proliferation 5,6. However, additional targets have emerged from more recent studies, including topoisomerase II, the metalloenzymes xanthine oxidase and tyrosinase, and the mitochondria signalling pathway 7-11. One of the first TSC tested in phase I clinical trials was 5-hydroxy-2-formylpyridine thiosemicarbazone (5-HP) 12. However, these trials revealed severe side effects (mainly gastrointestinal toxicity) and fast inactivation via metabolic conversion (glucuronidation) of 5-HP 4. Further investigation has led to the development of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine) 13 , whose efficacy is currently undergoing phase II clinical testing 14-17. This compound shows promising activity against hematologic disorders but not solid tumours 4. The reasons may be an inappropriate drug delivery due to a short plasma half-life, Triapine metabolic conversion, and/or rapid development of drug resistance 4. Recently, phase I clinical trials are being performed to test Triapine in combination therapy with other anticancer drugs 18. Since 2015 4,19,20 , phase I clinical trials were also starte...

Gene-dependent yeast cell death pathway requires AP-3 vesicle trafficking leading to vacuole membrane permeabilization

“…Resistance to cell death was not limited to thermal stress as disruption of AP-3 also conferred resistance to acetic acid, a biproduct of alcohol fermentation known to trigger yeast cell death, unlike other acids (Sousa et al, 2013;Vilela-Moura et al, 2011) (Figure 3H), and to hydrogen peroxide, a mimic of oxidative bursts produced by phagocytic host immune cells (Figure . 3I and 3J). Consistent with our findings, several AP-3 deletion strains were scored as death-resistant in supplementary tables of yeast cell death screens using acetic acid (Sousa et al, 2013), ER-stress (Kim et al, 2012), or thiosemicarbazone Ni(S-tcitr)2 to trigger cell death (Baruffini et al, 2020). Thus, AP-3 appears to contribute to cell death induced by multiple stimuli.…”

Hydrazones and Thiosemicarbazones Targeting Protein-Protein-Interactions of SARS-CoV-2 Papain-like Protease

“…The compound interaction hotspots, including the interactions of the polar thiocarbonyl moiety, resemble the modular composition of other previously described thiocarbazone lead compounds ( Osmaniye et al, 2021 ). While thiosemicarbazones currently attract significant interest as anticancer agents ( Baruffini et al, 2020 ), they also show antiviral activity against smallpox and other viruses ( Kune, 1964 ; Rogolino et al, 2015 ). Hydrazones have shown biological activity for treatment of Alzheimer’s disease, cancer and inflammation with properties rendering them beneficial for medicinal applications ( Wahbeh and Milkowski, 2019 ; de Oliveira Carneiro Brum et al, 2020 ).…”

A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool

“…The plasmids were introduced in the W303-1Arnr2∆/pFL38RNR2 strain, and the wild-type (wt) copy of RNR2 on pFL38 was removed through plasmid shuffling on SC medium supplemented with 2% glucose and 1 g/L 5-FOA (Formedium™, Hunstanton, UK) as previously reported [78], thus obtaining the W303-1Arnr2∆/pFL39rnr2 L362V strain. Mutant RNR2 strains overexpressing RNR1 or RNR4 were obtained by transforming the W303-1Arnr2∆/pFL39rnr2 L362V strain with the empty episomal vector YEplac181 and with the vector-borne RNR1 or RNR4, cloned as previously reported [79].…”