Live-Cell Identification of Inhibitors of the Lipid Transfer Protein CERT Using Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET)

Abstract

A BRET system is described, in which Nanoluciferase was fused to the lipid transfer protein CERT for efficient energy transfer to a Nile red-labeled ceramide, which is either directly bound to CERT or transported to the adjacent Golgi membrane. Bulk formation of sphingomyelin, a major plasma membrane component in mammals, is dependent on CERT-mediated transfer of its predecessor ceramide. CERT is considered a promising drug target but no direct cell-based methods exist to efficiently identify inhibitors. The utility of the method was demonstrated by a library of 140 derivatives of the CERT inhibitor HPA-12. These were obtained in a combinatorial synthesis using solid-phase transacylation. Screening of the library led to six compounds that were picked and confirmed to be superior to HPA-12 in a subsequent dose-response study and also in an orthogonal lipidomics analysis.

Figure 1

A Mechanism of CERT mediated ceramide transfer from ER to Golgi. B Reaction catalyzed by sphingomyelin synthase 1 (SMS1) C domain structure of CERT different NLuc constructs developed in this study D structure of Nile Red ceramide (NR‐Cer).

Figure 2

Mechanistic studies. A mBRET increases gradually with increased NR‐Cer concentration and HPA‐12 inhibition is partially rescued by NR‐Cer B pre‐treatment of cells with the acid ceramidase inhibitor SACLAC has no effect on BRET efficiency C mBRET in presence of 0.2 μM NR‐Cer for different constructs with or without HPA‐12 and in HeLa cells with or without natural CERT expression. Error bars depict standard deviation. Significance is assessed by student's t‐test *=p<0.05; ** p<0.01.

Scheme 1

Synthesis of the HPA‐12 analogue library. Reagents and conditions: a 1) Boc₂O, 1 M NaOH, quant.; 2) EDCI, DCM, 69 %; 3) NaBH₄, THF, r.t. b 1) DMP, acetone, BF₃OEt₂ 2) EDCI, DMH, NMM, DCM, 43 % over 3 steps c R₁MgBr or R₁Li. d 1) L‐Selectride 2) AcCl in MeOH. e shaking with 10‐fold excess of resin loaded active ester. DMP: 2,2‐Dimethoxypropane, DMH: N,O‐Dimethylhydroxylamin ‐hydrochlorid, NMM: N‐Methylmorpholine.

Figure 3

Screening and evaluation of the HPA‐12 analogue library. A Screening result of 140 compounds at 0.2 μM concentration in comparison to untreated (100 % BRET) or HPA‐12 at 0.2 μM (blue dashed line) or 20 μM. Concentration of NR‐Cer=0.2 μM. B Structural codes for HPA‐12 and six selected hits. C Dose response curves for selected hits, fitted to Hill's equitation. Error bars are omitted for clarity. D Ratios of cellular SM (16 : 0) and Cer (16 : 0) determined by quantitative HPLC‐MS‐MS evaluation. Error bars depict standard deviation. Four asteriscs: p<1 E⁻⁰⁴; five asteriscs p<1 E⁻⁰⁵ determined by student's t‐test.