Discovery of Tricyclic Clerodane Diterpenes as Sarco/Endoplasmic Reticulum Ca²⁺-ATPase Inhibitors and Structure–Activity Relationships

Abstract

Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 μM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented.

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“…In order to visualize the chemical space of the compounds of interest in 3D, and to gather information on its mechanism of action, a PCA with ChemGPS-NP 32 ( http://chemgps.bmc.uu.se ) was carried out using a database of two 28 cancer chemotherapeutics reported in De Ford et al 33 . Subsequent cluster analysis was performed with CheS-Mapper 34 ( http://ches-mapper.org/ ).…”

“…ChemGPS-NP comprises 35 molecular descriptors that are subdivided into eight principal components (PC) with physicochemical properties considered. Data were analyzed as previously described 33 . The SMILES and PC values for the compounds tested are shown in Table 1 .…”

The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling

“…We previously reported potent cytotoxic effects of CJ in CCRF-CEM cells (IC 50 =0.7 μ M). 17 Continuing these studies, we now assessed the viability of other T-ALL cells (CEM-ADR5000 and Jurkat cells) as well as CD3+ cells from healthy human donors after 24 h of CJ treatment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 2a and b , CJ was able to induce cell death in T-ALL cells in the low micromolar concentration range.…”

“…The IC 50 value of CJ in CCRF-CEM cells was previously obtained. 17 The data are presented as mean±S.D. ( n = 3), the IC 50 values were calculated by non-linear regression.…”

“…We have previously reported that representatives of this class of secondary plant metabolites are preferentially cytotoxic to leukemia cells 16 and that they directly inhibit the SERCA pump. 17 However, the exact mechanism of cytotoxicity and the reason for their preference toward leukemia cells remain poorly understood. Here, we addressed these issues in T-ALL cells exhibiting mutations in the HD-domain of Notch1 (CCRF-CEM), a doxorubicin-resistant version of these cells (CEM-ADR5000), and Jurkat cells that possess extracellular juxtamembrane expansion mutations of Notch1.…”