Natural Ligand-Mimetic and Nonmimetic Inhibitors of the Ceramide Transport Protein CERT

Abstract

Lipid transfer proteins (LTPs) are recognized as key players in the inter-organelle trafficking of lipids and are rapidly gaining attention as a novel molecular target for medicinal products. In mammalian cells, ceramide is newly synthesized in the endoplasmic reticulum (ER) and converted to sphingomyelin in the trans-Golgi regions. The ceramide transport protein CERT, a typical LTP, mediates the ER-to-Golgi transport of ceramide at an ER-distal Golgi membrane contact zone. About 20 years ago, a potent inhibitor of CERT, named (1R,3S)-HPA-12, was found by coincidence among ceramide analogs. Since then, various ceramide-resembling compounds have been found to act as CERT inhibitors. Nevertheless, the inevitable issue remains that natural ligand-mimetic compounds might directly bind both to the desired target and to various undesired targets that share the same natural ligand. To resolve this issue, a ceramide-unrelated compound named E16A, or (1S,2R)-HPCB-5, that potently inhibits the function of CERT has recently been developed, employing a series of in silico docking simulations, efficient chemical synthesis, quantitative affinity analysis, protein–ligand co-crystallography, and various in vivo assays. (1R,3S)-HPA-12 and E16A together provide a robust tool to discriminate on-target effects on CERT from off-target effects. This short review article will describe the history of the development of (1R,3S)-HPA-12 and E16A, summarize other CERT inhibitors, and discuss their possible applications.

Chlamydial Infection-Dependent Synthesis of Sphingomyelin as a Novel Anti-Chlamydial Target of Ceramide Mimetic Compounds

“…We previously developed two types of potent CERT inhibitors: (1 R ,3 S )-HPA-12, a ceramide mimetic compound [ 16 , 17 ], and E16A (also known as (1 S ,2 R )-HPCB-5), a ceramide nonmimetic compound ( Figure 1 ) [ 18 ]. HPA-12 has two chiral carbons comprising four stereoisomers ( Figure 1 ); only the (1 R ,3 S )-configuration exhibits potent CERT inhibition activity [ 16 , 19 , 20 , 21 ]. Notably, the initial assigned configuration of active HPA-12 [ 16 , 22 ] was later revised to (1 R ,3 S ) [ 19 , 20 , 21 ].…”

“…HPA-12 has two chiral carbons comprising four stereoisomers ( Figure 1 ); only the (1 R ,3 S )-configuration exhibits potent CERT inhibition activity [ 16 , 19 , 20 , 21 ]. Notably, the initial assigned configuration of active HPA-12 [ 16 , 22 ] was later revised to (1 R ,3 S ) [ 19 , 20 , 21 ]. A racemic mixture of (1 R ,3 R )/(1 S ,3 S )-HPA-12 (hereafter abbreviated as HPA-12-IR) is a control that lacks CERT inhibitory activity ( Figure 1 and Figure 2 D).…”

“…Despite our extensive searches for chlamydia SMS(es), there is no clear ortholog of mammalian SMS in the genomes from organisms belonging to the Chlamydia and Chlamydophila genera. The stereochemistry of natural ceramide more closely resembles (1 R ,3 S )-HPA-12 rather than (1 R ,3 R )-HPA-12 [ 21 ]. Interestingly, the phosphocholine adduct of (1 R ,3 S )-HPA-12 is produced in uninfected wild-type HeLa cells and infected HeLa∆SMS-1/2 cells to almost the same extent.…”

A Mechanistic Model for the Release of Ceramide from the CERT START Domain

“…POGL structurally resembles ceramide and is produced as a byproduct in the synthesis of sphingomyelin wherein the phosphorylcholine head is transferred from phosphatidylcholine to ceramide, liberating POGL. It has been shown that the CERT START can transfer short-chain diacylglycerol (C10-DAG) in vitro but with a lower efficiency (only 5%) of the ceramide suggesting that CERT might mediate Golgi-to-ER trafficking of DAG in exchange for ER-to-Golgi trafficking of ceramide ( 46 , 47 ). In our simulation the POGL lipid does not remain in the cavity for long.…”

Organelles are miscommunicating: Membrane contact sites getting hijacked by pathogens

“…It also helps differentiate on and off target effects when CERT is pharmacologically inhibited. [ 97 ] …”