Autophagic flux blockage by accumulation of weakly basic tenovins leads to elimination of B-Raf mutant tumour cells that survive vemurafenib

Abstract

Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.

SIRT1 regulates metabolism and leukemogenic potential in CML stem cells

“…Our previous studies used the SIRT1 inhibitor, Tenovin 30 (TV30), which has recently been shown to have additional autophagy inhibitory effects that could affect mitochondrial mass and function independently of SIRT1. Therefore, for the current studies, we used a congener, Tenovin 39OH (TV39OH), which retains SIRT1 inhibitory effects, but does not have autophagy inhibitory properties (35). CML BM CD34 + cells were treated with TV39OH (5 μM), the TKI NIL (1 μM), or a combination.…”

Small molecule activators of the p53 response

“…Other SirT1 inhibitors such as the tenovins and inhauzin have been described to increase p53 levels and function (Lain et al, 2008; Zhang et al, 2012). However, at least in the case of the tenovins, the activation of p53 may be due to additional modes of action of these molecules as it has been shown previously that certain tenovins are capable of demonstrating target engagement with SirT1 without cells responding with a rise in levels or activity of p53 (Ladds et al, 2018a).…”

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology

“…S1, A and B ). This modeling study and its correlation with the results seen in both the enzymatic assay and thermal shift data ( 11 , 14 , 18 ) served as a proof of principle for modeling the interactions of the tenovins with other targets.…”

“…We previously conducted a target confirmation study with the tenovins (see Table 1 for structures) by examining their ability to interact with SirT1 in cells using a cellular thermal shift assay (CETSA) to ensure that the compound series interacted with its purported target ( 14 ). In the present study, we have used molecular modeling to examine the energetics of interactions of these molecules in their interaction with SirT1 ( Fig.…”

“…The ability of tenovins to block autophagic flux has been recently found to be a property of the tertiary aliphatic amine that was added to the structure of tenovin 1 to increase aqueous solubility ( 12 , 13 , 14 ). Through structure-activity relationship studies using our previously published tenovin analogues ( 14 , 15 ), we uncover the ability of certain tenovins to inhibit two novel pathways: the de novo pyrimidine synthesis pathway by inhibiting DHODH, and also nucleoside transport. The blockage of DHODH by another chemical class adds to our previous findings that DHODH is a frequently hit target of small molecules ( 16 ).…”