Targeting squalene epoxidase in the treatment of metabolic-related diseases: current research and future directions

Intended audience and need for this review

SQLE is the second rate-limiting enzyme of cholesterol synthesis and plays an important role in cholesterol synthesis, alteration of the intestinal gut microbiota, and tumor immunity. In addition, SQLE is expressed in many tissues and organs and is involved in the occurrence and development of a variety of metabolic-related diseases. However, current studies on its function, expression, role in metabolic-related diseases and the development of clinical applications are not comprehensive. This article reviews advancements in research into the role of SQLE in metabolic-related diseases in recent years. An in-depth study of SQLE expression regulation is of particular importance for the treatment of metabolic-related diseases with good pharmacological activity.

Structure of SQLE

SQLE was first discovered in rat liver microsomes in 1969 (Yamamoto & Bloch, 1970). Like most cholesterol enzymes, SQLE is located in the endoplasmic reticulum or on lipid droplets, and the SQLE gene is located on human chromosome 8q24.13 (Nagai et al., 1997).

As an essential lipid component of mammalian cell membranes, cholesterol is critical for cell survival and proliferation and coordinates multiple membrane receptor signaling pathways by maintaining the stability of lipid rafts (Dang & Cyster, 2019). Almost all mammals can synthesize cholesterol from acetyl coenzyme (acetyl-CoA) through a series of 20 enzymatic reactions, including the mevalonate (MVA) pathway, SQLE biosynthesis, and subsequent reactions (Fig. 1). 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and SQLE are two key rate-limiting enzymes for cholesterol synthesis (Howe et al., 2017). The HMGCR-mediated mevalonate pathway is a key step in the de novo synthesis of cholesterol in the body. HMGCR-like SQLE activity is precisely regulated by intracellular cholesterol levels via feedback, which results in a second rate-limiting step in cholesterol synthesis (Ryder, 1988, 1991). The two rate-limiting enzymes, HMGCR and SQLE, are important factors in the cholesterol synthesis pathway. SQLE is responsible for the first oxidative step in cholesterol synthesis: it oxidizes squalene to 2,3-epoxysqualene. When the activity of the enzyme lanosterol synthase, which converts 2,3-epoxysqualene to lanosterol, is low, SQLE also converts 2,3-epoxysqualene to dioxidosqualene. The end product of this shunt pathway, 24(S),25-epoxycholesterol, is a ligand for the liver X receptor (LXR), which increases ATP-binding cassette transporter protein A1 (ABCA1) levels to promote cholesterol efflux (Seiki & Frishman, 2009; Gill et al., 2011). Therefore, the catalytic reaction of SQLE is essential for cholesterol synthesis.

Regulation and function of SQLE

SQLE is a direct target of sterol regulatory element-binding proteins (SREBPs). There are three SREBPs: SREBP1a, SREBP1c, and SREBP2, of which SREBP2 is a transcription factor that regulates genes involved in cholesterol synthesis and homeostasis in a cholesterol-dependent manner (Bengoechea-Alonso, Aldaalis & Ericsson, 2022). SQLE proteins contain cholesterol-sensing structural domains that regulate the proteasomal degradation of SQLE. SQLE expression in cells is subject to complex regulatory systems, including transcription, posttranscriptional regulation at the mRNA level, and posttranslational regulation.

Transcriptional regulation

The transcription factor SREBP2 directly regulates the mRNA levels of enzymes involved in cholesterol metabolism, such as SQLE, by binding to sterol regulatory element (SRE) sequences in the promoters of target genes (Brown, Radhakrishnan & Goldstein, 2018). When the cholesterol level in the endoplasmic reticulum (ER) is less than 5% of the total intracellular lipid level, SREBPs are activated, and SREBP cleavage-activating protein (SCAP) undergoes a conformational change to dissociate from the Insig-1 protein. The SCAP–SREBP2 complex is then disassembled and detached from the ER membrane and transported to the Golgi complex via coat protein complex II (COPII) vesicles. This protein is subsequently cleaved by site-1 protease (S1P) and site-2 protease (S2P) protein hydrolase cleavage and converted to the activated nuclear form SREBP2 (nSREBP2). Immediately thereafter, nSREBP2 enters the nucleus as a homodimer and binds to SRE in the promoter of the target gene SQLE to increase SQLE mRNA levels (Griffiths & Wang, 2021). In addition, oxygen sterol-binding protein-like 2 (OSBPL2) deficiency promotes nuclear entry of the specificity protein 1 (SP1) transcription factor and SREBP2 in the SQLE promoter to upregulate SQLE expression and increase cholesterol and cholesteryl ester accumulation through inhibition of the AMP-activated protein kinase (AMPK) pathway (Zhang et al., 2019). In addition, the transcription factors nuclear factor Y (NF-Y) and SP1 act synergistically with nSREBP2 to upregulate SREBP2 gene expression.

Oncogenes/tumor-suppressor genes are also involved in SQLE transcriptional regulation independent of SREBP2. The proto-oncogene MYC upregulates SQLE transcription by binding to the response element 1 (RE1) of the SQLE gene in cancer (Yang et al., 2021). The p53 gene is a common antioncogene in human cancers with a high ability to control cholesterol synthesis. Under low sterol conditions, p53 mediates SREBP2 regulation of SQLE transcription. In castration-resistant prostate cancer (CRPC), PTEN/p53-deficient tumors are dependent on cholesterol metabolism and upregulate SQLE by activating SREBP2 transcription to satisfy the cholesterol demand of tumor cells and promote the growth and progression of CRPC (Shangguan et al., 2022). Under normal sterol conditions, p53 directly represses SQLE expression in an SREBP2-independent manner, and p53 represses SQLE transcription through direct binding to Response Element 2 (RE2) in hepatocellular carcinoma (HCC) cells. p53 deficiency increases SQLE expression even when ER membrane cholesterol levels are normal or elevated (Sun et al., 2021).

Posttranscriptional regulation

A large body of evidence suggests that the expression profiles of long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs) are commonly dysregulated in human cancers. miRNA-133b (Qin et al., 2017; Wang et al., 2022), miRNA-205 (Kalogirou et al., 2021), miRNA-612 (Liu et al., 2020), miRNA-579-3p (Qian et al., 2023), miRNA-1179 (Li, Jiang & Zhao, 2023), miRNA-584-5p (Li et al., 2022b) and miRNA-363-3p (You et al., 2022) can interact with SQLE mRNAs, act as negative regulators of gene expression at the posttranscriptional level, and play important roles in tumor cell differentiation, proliferation, and apoptosis through the miRNA/SQLE axis. Some lncRNAs and circRNAs act as competing endogenous RNAs (ceRNAs) that sponge their corresponding miRNAs, thus decreasing their inhibitory effects on their targets (Tay, Rinn & Pandolfi, 2014; Qian et al., 2023). Qin et al. (2021) reported that in breast cancer, the lncRNA lnc030 is highly expressed in cancer stem cells and is positively correlated with SQLE expression. Poly(rC)-binding protein 2 (PCBP2) consists of 3 K homology (KH) domains (Silvera, Gamarnik & Andino, 1999). Lnc030 interacts with the KH2 of PCBP2, and the 3′ untranslated region (3′UTR) of SQLE mRNA binds to the KH3 of PCBP2. Lnc030 synergistically enhances the stability of SQLE mRNA with PCBP2. This leads to an increase in cholesterol synthesis, which, in turn, activates the PI3K/AKT signaling pathway and is involved in the regulation of the stemness characteristics of breast cancer stem cells (BCSCs). miRNA-133b and miRNA-205 were reported to reduce SQLE mRNA levels more rapidly by binding to the 3′UTR of SQLE mRNA (Qin et al., 2017; Kalogirou et al., 2021; Wang et al., 2022). circ_0000182 was shown to cause SQLE overexpression by sponging miRNA-579-3p. This miRNA then loses the ability to regulate SQLE and thus promotes cholesterol synthesis and proliferation in gastric adenocarcinoma cells (Qian et al., 2023). In summary, miRNAs, lncRNAs, and circRNAs can exert their effects on various cancers by modulating the expression of SQLE (Table 1). Targeting the interactions between miRNAs, lncRNAs, circRNAs, and SQLE holds promise for providing new strategies for the clinical treatment of patients with cancer.

Posttranslational regulation

SQLE is also posttranslationally regulated. The regulation of SQLE protein stability and activity, mainly through the cholesterol membrane-associated ring-CH-type finger 6 (MARCH6)-proteasomal degradation axis, is dependent on specific structural domains in SQLE proteins (Sharpe, Coates & Brown, 2020). These domains include the cholesterol-dependent amphiphilic helical structure formed by the Gln62-Leu73 sequence (Chua et al., 2017), which can be embedded in the hydrophobic interior of membranes while interacting with the hydrophilic environment at the membrane surface. It regulates SQLE by interacting with cholesterol molecules and plays an important role in cholesterol-dependent degradation. The first 100 amino acids of SQLE (SQLE N-100) are attached to the ER membrane in the form of a re-entrant loop (Howe et al., 2015), which senses cholesterol in the cytoplasm. When intracellular cholesterol accumulates, the anchoring of the SQLE protein to the ER membrane tightens. This results in partial exposure of the amphipathic helical structure of the Gln62-Leu73 sequence to the cytoplasmic environment, preventing proteasomal degradation. The ubiquitination process also requires the ubiquitin-conjugating enzyme E2 J2 (UBE2J2). MARCH6 approaches the SQLE protein and recognizes serine residues near the cholesterol-dependent amphiphilic helix in the SQLE protein. UBE2J2 works in concert with MARCH6 to attach the ubiquitin molecule from the E1 enzyme to the SQLE protein to be ubiquitinated to label it as a protein to be degraded so that it becomes a ubiquitin-protease substrate of the degradation system. The valosin-containing protein (VCP) is then recruited to extract the ubiquitinated SQLE substrate, where it dissociates from the ER. VCP then cooperates with other proteins to mediate entry into the proteasomal degradation pathway. Excess cholesterol can stimulate SQLE degradation by inhibiting MARCH6 self-degradation (Sharpe et al., 2019).

The proteasomal degradation pathway of MARCH6-VCP can be regulated independently of cholesterol. The N-terminus of SQLE is partially degraded through a unique ubiquitination pathway, which leads to the conversion of full-length SQLE to a truncated form. The enzyme activity of the truncated SQLE is associated with cholesterol resistance, implying that the function of SQLE is not completely lost under high-cholesterol conditions. Thus, the function of this gene may complement cholesterol metabolism under cancer conditions (Coates, Capell-Hattam & Brown, 2021). In addition to cholesterol, squalene directly binds to the SQLE N-100 structural domain to change its conformation. This results in the inability of MARCH6 to ubiquitinate SQLE or label it for degradation, thus increasing the stability of SQLE (Nathan, 2020). Unsaturated fatty acids (USFAs) can also stabilize SQLE levels via the regulatory blockade of SQLE ubiquitination by MARCH6. Upregulation of the cancer-associated microprotein CASIMO1 increases SQLE levels by interacting with SQLE proteins (Polycarpou-Schwarz et al., 2018).

To better understand the regulatory mechanisms of SQLE, its regulation at the transcriptional, posttranscriptional, and posttranslational levels is illustrated in Fig. 2 (adapted from Zou et al., 2022). Overall, the main mechanism of SQLE regulation is the cholesterol-dependent feedback regulation of SQLE by the SREBP2 transcriptional and ubiquitin proteasomal degradation pathways. SREBP2 activation in tumor tissues leads to high SQLE expression. The activation of oncogenes, deletion of cancer suppressor genes, and deletion of miRNAs and cancer-associated proteins directly or indirectly upregulate SQLE expression. Different tumor cells or specific subpopulations have specific SQLE regulatory mechanisms (Du, Rokavec & Hermeking, 2023).

Because of the crucial role of the 2,3-epoxysqualene derivative lanosterol in fungal membrane synthesis, SQLE has long been investigated as an antifungal target with increasing relevance to human health and disease (Astruc et al., 1977). Many studies have shown that SQLE can meet the high energy requirements for the rapid growth of diseased cells, and the dysregulation of SQLE has been found in a variety of metabolic-related diseases. This has become a hot topic in the field of targeted diagnosis and therapy (Ryder, 1992).

The role and clinical significance of SQLE in metabolism-related diseases

SQLE-targeted therapeutic strategies

SQLE has been reported to act as an oncogene in various cancers. Moreover, the dysregulation of SQLE has been associated with the inhibition of apoptosis and increased cell proliferation and invasiveness, and a high abundance of SQLE in tumors indicates a poorer prognosis. As a novel and attractive therapeutic target for anticancer treatment, SQLE has been increasingly used in preclinical studies to reveal its antitumor effects and related mechanisms. The first SQLE-targeted inhibitors disrupted the synthesis of ergosterol in antifungal bodies, thereby killing or inhibiting the fungus (Barrett-Bee & Dixon, 1995). Currently, SQLE inhibitors are mainly classified as allylamines, natural compounds, or their derivatives (Abe et al., 2000; Zhang et al., 2024). Research on the novel use of established SQLE inhibitors is increasing, and targeting SQLE is considered a new and promising therapy for metabolic diseases (Table 2).

Allylamine

Since the discovery in 1981 that naftifine has high broad-spectrum antifungal activity, it has become the cornerstone for the commercialization of next-generation inhibitor drugs, such as butenafine and tolnaftate (Petranyi, Georgopoulos & Mieth, 1981). Terbinafine is a common antifungal agent that has been proposed as a new therapeutic strategy for human cancers (Chua, Coates & Brown, 2018). The main SQLE inhibitor used in preclinical antitumor studies is terbinafine, which inhibits cell proliferation, induces G0/G1 cell cycle arrest, apoptosis, and autophagy by inhibiting SQLE or SQLE-independent inhibition, and slows tumor growth in vivo in a dose-dependent manner (Xu et al., 2023). In addition to the presence of neurological and dermal toxicity, poor drug resistance were detected for SQLE compared with NB-598 or Cmpd-4″, which had IC50 values in the range of 10–60 nM (Padyana et al., 2019; Brown, Chua & Yan, 2019; Nagaraja et al., 2020). The IC50 was determined to be 7.7 µM, with a maximum inhibition of 65% at an inhibitor concentration of 100 µM, suggesting that terbinafine is not the best SQLE inhibitor (Padyana et al., 2019).

The compound NB598, obtained by modifying the aromatic moiety of terbinafine, is another highly specific inhibitor of mammalian SQLE, with the best responses in neuroblastoma and lung cancer and good drug sensitivity and high efficacy in small-cell lung cancer cell lines (Mahoney et al., 2019; Padyana et al., 2019). Further modification of NB598 yielded silyl derivatives that are also able to inhibit the enzymatic activity of SQLE. However, preclinical studies revealed that gastrointestinal and dermal toxicities were not tolerated by dogs and monkeys treated with a gavage of allylamine inhibitors (NB-598 and Cmpd-4″) for small cell lung cancer treatment (Nagaraja et al., 2020). Cmpd-4″ has been reported to share the same high potency as NB-598 for the time-dependent inhibition of SQLE but suffers from the same gastrointestinal and dermal toxicities (Padyana et al., 2019). Naftifine and terbinafine, which are used as antifungal agents, cause similar adverse effects, and this toxicity may limit the potential therapeutic benefit of metabolic disease treatment. This toxicity is attributed to the fact that the site of action of both terbinafine and NB-598 is Y195, and the tertiary amine group in the inhibitor structure forms a hydrogen bond with Y195. This prevents Y195 from interacting with glutamine (Q168) at position 168, inhibiting the conversion of SQLE to the active state. Thus, all the catalytic reactions of SQLE are inhibited, resulting in greater neurological and dermal toxicity (Padyana et al., 2019; Nagaraja et al., 2020). The IC50 values of allylamine inhibitors in mammalian cells are several orders of magnitude greater than those in fungi, and large doses are often required to achieve therapeutic efficacy, such as antitumor effects (Ryder, 1988). Hence, there is a need for careful assessments of the tolerance to adverse effects. NB-598 is a preclinical drug without much safety or pharmacological data. FR194738 is derived from NB-598 and has an IC50 of only 2.1 nM, making it one of the most potent inhibitors. Compared with NB-598, FR194738 has a similar potency to NB-598 in terms of its ability to inhibit cholesterol synthesis, but it has improved lipophilic and pharmacokinetic properties (Sawada, Washizuka & Okumura, 2004; Zhang et al., 2024). As a specific SQLE inhibitor, FR194738 has little effect on cholesterol upstream or downstream (Sawada et al., 2001). The SQLE-specific inhibitor FR194738 attenuates the growth of desmoplasia-resistant prostate cancer in vitro in PC3 cells and in vivo in a mouse xenograft model harboring PC3 cells (Shangguan et al., 2022). Like its parent compound, FR194738 is a preclinical drug with limited safety and pharmacologic data that require additional basic and clinical studies.

Natural compounds and their derivatives

Many natural compounds and their derivatives may be clinically safe SQLE inhibitors that can effectively and selectively inhibit SQLE activity. For example, Abe et al. (2000) reported that green tea polyphenols, the main component of which is galloyl-containing epigallocatechin gallate (EGCG), noncompetitively inhibit SQLE by scavenging reactive oxygen species from the active site of the enzyme. The team synthesized galloyl groups, such as dodecyl ester and gallate dodecyl ester, as SQLE inhibitors, which are widely used in food additives for antioxidant purposes. The metabolites of EGCG are also inhibitory, and other plant extracts, such as beta-carotene, anthocyanins, tannins, fo-ti, and rhubarb, are also rich in galloyl groups. Grape skins and red wine are rich in the galloyl polyphenolic compound resveratrol, which reversibly and noncompetitively inhibits SQLE activity, with cholesterol-lowering and cardiovascular disease-preventive effects. Although EGCG still has few side effects when consumed at high doses, it has low bioavailability and a short half-life to reach effective therapeutic concentrations.

Ellagitannin analogs of pedunculin and eugenol also showed significant inhibitory efficacy. Gupta & Porter (2001) reported that garlic and five of its compounds—S-allylcysteine, alliin, diallyl disulfide, selenocystine, and diallyl trisulfide—were effective in inhibiting SQLE. Unlike the mechanism of inhibition by tea polyphenols, the inhibitory effect of garlic extract on SQLE is irreversible. This is because the arylcysteine in garlic binds to the active site of the SQLE, leading to its inactivation. Additionally, telluride, which is present at high levels in garlic, is not highly selective for SQLE. Instead, it can interact with other proteins and inhibit SQLE activity in Schwann cells via the blood‒brain barrier. This interaction leads to the inhibition of cholesterol synthesis and the accumulation of squalene, which can adversely affect myelin sheath formation and severely impede neurotransmission. This ultimately results in peripheral ganglionic neural degeneration, segmental demyelination, and paralysis of peripheral nerves (Wagner, Toews & Morell, 1995; Laden & Porter, 2001). Moreover, different types of SQLE inhibitors offer diverse frameworks for creating novel compounds that mitigate side effects and enhance affinity. However, further investigations are needed to determine their therapeutic potential in treating metabolic diseases.