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. 2017 Jul 17;22(7):1198.
doi: 10.3390/molecules22071198.

Application of An Improved HPLC-FL Method to Screen Serine Palmitoyl Transferase Inhibitors

Simone Bertini  1 , Giuseppe Saccomanni  2 , Sara Del Carlo  3 , Maria Digiacomo  4 , Claudia Gargini  5 , Ilaria Piano  6 , Giuseppe Matteo Campisi  7 , Riccardo Ghidoni  8 , Marco Macchia  9 , Clementina Manera  10
Affiliations

Application of An Improved HPLC-FL Method to Screen Serine Palmitoyl Transferase Inhibitors

Simone Bertini et al. Molecules. .

Abstract

In this work, we reported the application and validation of an improved high-performance liquid chromatography method coupled with a fluorimetric detector (HPLC-FL) to screen the activity of two heterocyclic derivatives reported as serine palmitoyl transferase (SPT) inhibitors. The analytical conditions were optimized in terms of the derivatization procedure, chromatographic condition, extraction procedure, and method validation according to EMEA guidelines. Once fully optimized, the method was applied to assess the SPT-inhibitory activity of the above-mentioned derivatives and of the reference inhibitor myriocin. The obtained results, expressed as a percentage of residual SPT activity, were compared to those obtained with the reference radio immune assay (RIA). The good correlation between the two types of assay demonstrated that the improved HPLC-FL method is suitable for a preliminary and rapid screening of potential SPT-inhibitors.

Keywords: HPLC; SPT; enzymatic assay; screening; serine palmitoyl transferase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structures of the 1,3-dihydro-2H-benzimidazol-2-oxo-derivatives which were screened using the radio immune assay (RIA) and HPLC-FL method.
Scheme 1
Scheme 1
Reagents and conditions: (i) anhydr. DMF, DIPEA, microwave, 15 min., 130 °C, 5 bar; (ii) anhydr. THF, absol. EtOH, Pd-C, RT, 4 h; (iii) CDI, anhydr. THF, DMAP, microwave, 15 min., 130 °C, 7 bar; (iv) CF3COOH, CH2Cl2, RT, 2 h; (v) anhydr. DMF, Et3N, 2-Br-4’-chloroacetophenone, 0 °C → RT, 2 h; (vi) TMSN3, TBAF, 125 °C, 24 h; (vii) CF3COOH, CH2Cl2, RT, 2 h; (viii) anhydr. DMF, Et3N, 2-Br-4’-chloroacetophenone, 2 h, 0 °C → RT.
Figure 2
Figure 2
Different derivatization times of 200 nM sphinganine samples: area values obtained by analysis of triplicates. Values are expressed as mean ± SD, n = 6, from two independent experiments.
Figure 3
Figure 3
HPLC-FL chromatograms of a blank sample (black), a standard sphinganine sample (blue) and cell lysate sample after incubation (red).
Figure 4
Figure 4
Calibration curve of sphinganine.
Figure 5
Figure 5
Human serine palmitoyl transferase (SPT) initial velocity evaluation.
Figure 6
Figure 6
Dose-dependent inhibition of human SPT by myriocin (HPLC-FL assay).
See this image and copyright information in PMC

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