**1. Introduction**

Sphingolipids (SLs) are key modulators of physiological processes including the cell cycle, apoptosis, angiogenesis, stress, and inflammation [1]. Among SLs, ceramides (Cers) and sphingosine-1-phosphate (S1P) are the most studied (Figure 1) and often exert opposing biological functions. Cancer cells show a shift in the balance between proapoptotic ceramide (Cer) and cancer-promoting S1P. This phenomenon is associated with pancreatic cancer progression and poor therapeutic outcomes [2,3]. Biochemical dysregulation of SL metabolism can be used as a biomarker and prognostic factor in pancreatic cancer [4–6]. Further studies suggest that Cer generation and accumulation are critical determinants facilitating apoptosis in pancreatic cancer cells in response to cytotoxic agents, including gemcitabine (GMZ) [7], which highlights the significance of manipulating these pathways to overcome resistance of pancreatic cancer to current therapies. Cer and its biosynthetic

**Citation:** Afrin, F.; Mateen, S.; Oman, J.; Lai, J.C.K.; Barrott, J.J.; Pashikanti, S. Natural Products and Small Molecules Targeting Cellular Ceramide Metabolism to Enhance Apoptosis in Cancer Cells. *Cancers* **2023**, *15*, 4645. https://doi.org/ 10.3390/cancers15184645

Academic Editors: Barbara De Filippis, Marialuigia Fantacuzzi and Alessandra Ammazzalorso

Received: 6 August 2023 Revised: 8 September 2023 Accepted: 11 September 2023 Published: 20 September 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

derivatives are important to rapidly dividing cells such as cancer cells because Cer is a basic unit of stable lipid membranes that supports transmembrane functionality and integrity [8].

**Figure 1.** Ceramide biosynthesis and metabolism pathway.

The polarity in the structure of SLs makes them a basic unit of a membrane or vesicle. These are biosynthesized not only in the mammalian system but also in other eukaryotic and prokaryotic organisms, and in marine plants. Most notable are the ones that are secondary metabolites from fungi. The metabolites from fungi are a survival strategy in the ecosystem of vertebrates [9]. These secondary metabolites are a source of hits for medicinal chemistry approaches towards developing drugs involving sphingolipid biochemistry. This review is focused on ceramide-metabolizing enzymes, which have the capacity to control ceramide flux. Most of these natural products have attracted the attention of research groups with the aim of accomplishing total synthesis, as well as others that perform SAR studies. Some of these have resulted in the identification of small molecule hits. A list of natural products and small molecules for each of the ceramide-metabolizing enzymes, as well as their clinical relevance, is provided in the following sections. The synthesis of small molecules targeting these enzymes strengthened the application of a chiral pool strategy involving diastereoselective and enantioselective syntheses. Fingolimod, an FDA-approved medication, is used for treating relapsing forms of multiple sclerosis. Fingolimod was developed from the fungal metabolite myriocin. Fingolimod has a structural resemblance to sphingosine. Miglustat is another FDA-approved medication targeting glucosylceramide synthase. Miglustat is prescribed to treat Gaucher disease [10]. Miglustat has a structural resemblance to the enzyme substrate. These properties have encouraged academia and the pharma industry to probe these pathways further in recent years.

Given the broad biological significance of SLs, this review has focused on updating the knowledge of medicinal chemistry approaches used to increase cellular Cer levels. The pharmacological goal is to inhibit enzymatic functions that increase cytotoxic Cer, thus inducing cellular apoptosis. Knowledge of the pathway and its associated enzymes paves the way for identifying medicinal chemistry approaches targeting these enzymes, which could help overcome chemotherapy-resistant cancer cells by exploring combination therapies that target the Cer metabolism pathway. A brief discussion about the effect of the natural products, small molecules, on the catalysis of these enzymes is also discussed.

#### **2. The Ceramide Biosynthesis Pathway**

Central to SLs is Cer, which constitutes the hydrophobic backbone of all complex SLs (e.g., glycosphingolipids (GS), sphingomyelin (SM), cerebrosides, and gangliosides) and structurally consists of a fatty acyl of variable chain lengths bound to an amino group of a sphingoid base. The fatty acyl chains are, in general, saturated or monounsaturated and can contain an OH group linked to C-2 or to the terminal carbon atom (α- and ω-hydroxy fatty acids, Figure 1). Among ceramide-containing SLs, those containing long (C16–20) and very long (C22–24) acyl chains are the most abundant in mammalian cells, but Cers with longer acyl chains (C26–36) are also found in epidermal keratinocytes and male germ cells during their differentiation and maturation [8].

Cer is biosynthesized starting from L-serine in the de novo synthetic pathway (Figure 1). Cer biosynthesis involves cellular serine palmitoylation using the cofactor-activated palmitoyl CoA by serine palmitoyl transferase, resulting in 3-keto sphingosine. This is a ratedetermining step in the biosynthesis of SLs. A cellular enantioselective reduction in the ketone catalyzed by ketosphingosine reductase allows the required 1,2 *anti*-amino alcohol system to be formed. N-palmitoylation of ketosphingosine followed by desaturation results in the trans-alkene Cer. The structural and stereochemical core of Cer has the inherent chirality of *L*-serine, N-palmitoylation, and lipophilic alkyl chain modifications. There are different types of Cers based on the side chain substitutions on the polar head group—both N-alkyl and the alkyl side chains.

The Cer metabolic pathway is very dynamic and can result in various SLs being synthesized to accommodate cellular needs and enhance cell signaling pathways. Cermetabolizing enzymes are cell fate specific and expressed based on the physiological role of the cell. Cancer cells tend to upregulate enzymes that promote the production of SLs and cell membrane stability. The remainder of this review will highlight the importance of six different classes of SL-metabolizing enzymes: ceramidase, sphingosine kinase (SK), sphingomyelin synthase (SMS), 3-ketosphinganine reductase, dihydroceramide desaturase, and ceramide synthases.

#### **3. Ceramidase**

Ceramidases (CDases) are a group of ceramide-metabolizing enzymes that hydrolyze Cer to produce sphingosine. Sphingosine is then further metabolized into S1P by SK1 or SK2. In humans, there are five known CDases genes. The CDases expressed by these genes can be divided into three categories depending on the pH required for their optimal catalytic activity: (i) acid ceramidase (encoded by the *ASAH1* gene), (ii) neutral ceramidase (encoded by the *ASAH2* gene), and (iii) alkaline ceramidase (encoded by the *ACER1*, *ACER2*, and *ACER3* genes) [11].

#### *3.1. Acid Ceramidase*

Acid ceramidase (AC)/*ASAH1* is also called N-acylsphingosine amidohydrolase. AC has a molecular weight of 50 kDa and requires a pH of 4.2–4.3 for its optimal activity [12]. Under these acidic conditions, the byproduct, sphingosine amine, exits as an ammonium species providing the active site tolerance for this charged functional group. The 3D crystal structure of AC is generated using EzCADD utilizing PDB file (Figure 2) [13,14]. After performing EZ pocket calculation, one of the binding pockets is shown with Cys143 [13]. AC hydrolyzes the amide bond in unsaturated ceramides with C6–C16 acyl chains [15]. It is mainly localized in lysosomes and maintains intralysosomal Cer homeostasis [16]. AC is expressed ubiquitously and has a higher expression in the heart and kidneys [17]. The KM value of AC was determined to be 389 to 413 μM by using 14C-labeled and BODIPYconjugated C-12 Cer substrate, N-lauroylsphingosine [18]. An SL activator protein, Saposin D, is responsible for the enzymatic activity of AC, as evidenced by the Cer accumulation that occurs in the absence of Saposin D [19]. Further studies narrated the activator protein Saposin D having binding interactions with the polar head group of monomeric SL, embedded in intracellular lysosomal membrane, in close proximity to AC in order to perform catalysis [14,20]. This model shows evidence of a multimeric complex lipid–protein interactions for AC catalysis. The altered function of a mutated AC, the overexpression of normal AC, and the dysregulation of activity of AC have highlighted the importance of its role in SL metabolism. The dysregulation of AC is associated with a wide range of diseases, thereby suggesting that AC could be an attractive therapeutic target for drug discovery.

**Figure 2.** 3D crystal structure of human acid ceramidase (ASAH1, aCDase). PDB ID: 5U7Z. Chain A is red, chain B is teal, and dashed lines indicate hydrogen bonding. The active site is located near Cys143 and a binding pocket near the active site was generated using ezPocket with fconv at 89.4, −3.53, and 203.26 (x, y, z), respectively.
