*2.5. Molecular Modeling*

Ceramides have been considered a group of endogenous sphingolipids with the ability to interfere with the signaling pathways both upstream and downstream of p53 [85]. Under normal non-stressed conditions, the p53 transcription factor undergoes ubiquitination by the E3-ligase mouse double minute 2 (MDM2). This process prepares p53 for the proteasomal degradation. Disruption of the complex between p53 and MDM2 leads to p53 accumulation, nuclear translocation, and activation of the downstream apoptotic pathways [86]. The role of ceramides in the regulation of p53 has remained controversial for a long time, with studies suggesting direct and indirect cross-talk. Recently, in 2018, Fekry et al. from the University of North Carolina showed that C16-ceramides could directly interact with p53 [87]. They provided experimental evidence that ceramides bind in close proximity to the BOX V motif of p53, which is a part of the p53–MDM2 interface. Consequently, this binding disrupts the p53-MDM2 binding, reducing the ubiquitination and proteasomal degradation of p53. To gain insights into the molecular determinants of the binding of our ceramides **A** (**1**) and **B** (**2**) into the putative binding site of p53, we decided to perform a molecular docking simulation into the crystal structure of the human p53 (2MEJ). Both molecules showed very similar docked poses to the one proposed by Fekry et al. (Figure 9). In both cases, the central polar part of the compounds was oriented close to the polar residues Arg280 and Asp281 at the base of the H2 helix, probably stabilized by forming water-mediated H bonds. The two long hydrophobic tails of both compounds fit in a complementary fashion into the deep groove between the H2 helix and the Ser240-P250 loop. To our delight, the C10 atom of the acyl chain of both ceramides was docked in close proximity to Ser240 and Ser241. These residues have been proven by the aforementioned research group, using MS-proteomics experiments, to participate in C16-ceramide binding to p53 [87]. Moreover, they used modified C16-ceramide with a diaziridine group to show that the ceramide C10 atom binds proximal to Ser240 and Ser241. Interestingly, a very similar orientation of C10 was noticed in the top-ranked docking poses of both ceramides **A** (**1**) and **B** (**2**).

**Figure 9.** Proposed binding mode of ceramides **1** and **2** into the putative binding site of p53. The surface of the protein is colored by atom type, with carbons in green. Important residues of the binding site are labeled. Docked ligands are shown as cyan sticks.

#### **3. Material and Methods**

## *3.1. Plant Material*

The red algae *Hypnea musciformis* was collected from Safaga at the Egyptian Red Sea in August 2017, air-dried, and stored at low temperature (−24 ◦C) until further processing. The plant was identified by Dr. Tarek Temraz of the Marine Science Department in the Faculty of Science at Suez Canal University in Ismailia, Egypt. A voucher specimen was deposited in the herbarium section of the Pharmacognosy Department of the Faculty of Pharmacy at Suez Canal University in Ismailia, Egypt under registration number SAA-130.
