Acidosis, like hypoxia, is a prevalent characteristic of solid tumor microenvironment (Corbet and Feron, 2017b, Pillai et al., 2019). Today it is well acknowledged that acidosis plays a significant role in the inception and advancement of tumors. Additionally, it contributes to drug resistance and the dissemination of metastases. These effects stem from various changes in the phenotype of cancer cells exposed to extracellular acidosis. These alterations encompass evading apoptosis, eluding immune surveillance, initiating (epi)genetic reprogramming, displaying reduced proliferation or entering a quiescent state, undergoing epithelial-to-mesenchymal transition, and experiencing disrupted metabolism (Peppicelli et al., 2017, Vander Linden and Corbet, 2019). Notably, acidic cancer cells become less dependent on glucose to support their metabolic needs but instead preferentially use lipids (Corbet and Feron 2017a). We have previously shown that acidic cancer cells actually become so addicted to fatty acids that their uptake ends up exceeding their needs, and storage of excess fatty acids as triglycerides (TG) is then required to prevent lipotoxicity and ferroptosis (Corbet et al., 2020, Dierge et al., 2021). More generally, TG storage into lipid droplets is increasingly described as a safeguard mechanism to maintain cancer cell viability and proliferation (Du et al., 2017, Hoy et al., 2017). Consequently, targeting the synthesis and accumulation of TGs is emerging as a potential therapeutic avenue to treat cancers.

Diacylglycerol O-acyltransferase 1 (DGAT1) constitutes one of the two enzymes responsible for catalyzing the synthesis of TGs in the human body (Cases et al., 1998). Its function involves the esterification of diacylglycerol (DAG) using acyl-CoA, producing TGs within the endoplasmic reticulum (ER). In human physiology, this enzymatic reaction holds significance for storing metabolic energy, particularly in organs like the intestine, liver, and adipose tissue (Yen et al., 2008). Furthermore, DGAT1 assumes a role in safeguarding the endoplasmic reticulum from the buildup of bioactive lipids stemming from an excess of fatty acids (Chitraju et al., 2017). Clinical trials for diabetes or heart failure conditions have revealed that DGAT1 inhibitors can induce gastrointestinal side effects in a dose-dependent manner, which curtails their use for managing long-term metabolic ailments (Denison et al., 2014). Nevertheless, investigations involving these inhibitors in animal models propose a potential therapeutic role in treating cancers characterized by disrupted TG metabolism, such as glioblastoma (Cheng et al., 2020) and prostate (Nardi et al., 2019) cancers.

pH Low Insertion Peptides (pHLIP) are a unique peptide class that has garnered significant attention in biophysics and biomedical research. These peptides are water-soluble at neutral pH but undergo a conformational change and spontaneously insert into lipid bilayers when exposed to mildly acidic environments, such as those found in tumor tissues (Reshetnyak et al., 2006, Andreev et al., 2007, Reshetnyak et al., 2007, Vavere et al., 2009). The mechanism behind pHLIP insertion involves the transformation of the peptide from a water-soluble state to a transmembrane α-helical structure. At neutral pH, pHLIPs adopt a coil-like conformation, which becomes an α-helix upon insertion into the lipid bilayer under acidic conditions. The binding of peptides of the pHLIP family with the lipid bilayer of the membrane is predominantly driven by hydrophobic interactions since pHLIPs are moderately hydrophobic peptides. In cancer research, pHLIPs have been used to deliver drugs selectively to acidic tumor environments, potentially reducing off-target effects and enhancing therapeutic efficacy (An et al., 2010, Wijesinghe et al., 2011, Moshnikova et al., 2013, Burns et al., 2015, Song et al., 2016, Burns et al., 2017, Gayle et al., 2021).

In this study, we explore the conjugation of two DGAT1 inhibitors to pHLIPs. Our investigation includes assessing the integration of these conjugates into lipid bilayers of varying compositions, using steady-state fluorescence (SSF). Additionally, we utilize circular dichroism spectroscopy (CD) to analyze their structural conformation. Furthermore, we evaluate the conjugates' capacity to influence cancer cell growth within 3D tumor spheroid models mimicking the spontaneous development of a pH gradient.