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Review
. 2021 Dec 9;26(24):7469.
doi: 10.3390/molecules26247469.

Mipsagargin: The Beginning-Not the End-of Thapsigargin Prodrug-Based Cancer Therapeutics

John T Isaacs  1   2   3 , William Nathaniel Brennen  1   3 , Søren Brøgger Christensen  4 , Samuel R Denmeade  1   2   3
Affiliations
Review

Mipsagargin: The Beginning-Not the End-of Thapsigargin Prodrug-Based Cancer Therapeutics

John T Isaacs et al. Molecules. .

Abstract

Søren Brøgger Christensen isolated and characterized the cell-penetrant sesquiterpene lactone Thapsigargin (TG) from the fruit Thapsia garganica. In the late 1980s/early 1990s, TG was supplied to multiple independent and collaborative groups. Using this TG, studies documented with a large variety of mammalian cell types that TG rapidly (i.e., within seconds to a minute) penetrates cells, resulting in an essentially irreversible binding and inhibiting (IC50~10 nM) of SERCA 2b calcium uptake pumps. If exposure to 50-100 nM TG is sustained for >24-48 h, prostate cancer cells undergo apoptotic death. TG-induced death requires changes in the cytoplasmic Ca2+, initiating a calmodulin/calcineurin/calpain-dependent signaling cascade that involves BAD-dependent opening of the mitochondrial permeability transition pore (MPTP); this releases cytochrome C into the cytoplasm, activating caspases and nucleases. Chemically unmodified TG has no therapeutic index and is poorly water soluble. A TG analog, in which the 8-acyl groups is replaced with the 12-aminododecanoyl group, afforded 12-ADT, retaining an EC50 for killing of <100 nM. Conjugation of 12-ADT to a series of 5-8 amino acid peptides was engineered so that they are efficiently hydrolyzed by only one of a series of proteases [e.g., KLK3 (also known as Prostate Specific Antigen); KLK2 (also known as hK2); Fibroblast Activation Protein Protease (FAP); or Folh1 (also known as Prostate Specific Membrane Antigen)]. The obtained conjugates have increased water solubility for systemic delivery in the blood and prevent cell penetrance and, thus, killing until the TG-prodrug is hydrolyzed by the _targeting protease in the vicinity of the cancer cells. We summarize the preclinical validation of each of these TG-prodrugs with special attention to the PSMA TG-prodrug, Mipsagargin, which is in phase II clinical testing.

Keywords: Mipsagargin; SERCA; Thapsia garganica; Thapsigargin; apoptosis; calcium homeostasis; _targeted prodrugs; tissue-specific proteases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structure for TG, its amine-containing 8-O-(12-aminododecanyl)-8-O-debutanoyl TG analogue [12ADT], and its peptide conjugates. Amino acid sequences used to restrict TG prodrug killing until hydrolyzed by: Prostate-Specific Antigen; hK2. Prostate Specific Membrane; PSMA and Antigen Fibroblast Activation Protein (FAP). Note: for the PSA, hK2, and FAP peptide prodrugs, the N-terminal amino group is capped with a morpholino-protection group. Open arrows denote sites of selective enzymatic hydrolysis.
Figure 2
Figure 2
Overview of compartmental calcium gradients and their function in mammalian cells (Redrawn from reference [26]. Abbreviations: ER (Endoplasmic Reticulum); SERCA (Sarco/endoplasmic reticulum calcium ATPase); PMCA (Plasma membrane calcium ATPase); R (Receptor); PLC (Phospholipase C); IP3 (inositol-1,4,5-trisphosphate); IP3R (Inositol-1,4,5-trisphosphate receptor); BiP (Binding-Immunoglobulin Protein also known as Heat Shock 70 kDa Protein 5 or Glucose-Regulated Protein, 78 kDa); CRT (Calreticulin); CNX (Calnexin); PDIA6 (Protein Disulfide Isomerase Family A Member 6); CSQ (Calsequestrin); ERp57 (ER protein 57 also known as Protein Disulfide Isomerase Family A Member 3); STIM (Stromal Interaction Molecule 1); SARAF (Store-Operated Calcium Entry Associated Regulatory Factor); SOCE (Store-operated Ca2+ entry); Orai1 (ORAI Calcium Release-Activated Calcium Modulator 1); UPR (Unfolded Protein Response); IRE (Inositol-Requiring Enzyme also known as ERN1); PERK (PRKR-Like Endoplasmic Reticulum Kinase also known as EIF2AK3); ATF6 (Activating Transcription Factor 6); Voltage-gated Ca2+ channel (VOC); Transient receptor potential channel (TRPC); Na+/Ca2+ exchanger (NCX); and Mitochondrial Ca2+ uniporter (MCU).
Figure 3
Figure 3
(A) Time course of changes in cytoplasmic Ca2+ vs. indicated biochemical parameter following exposure of prostate cancer cells to 500 nM Thapsigargin from reference [35]. (B) Overview of the apoptotic pathways induced by TG inhibition of SERCA pump involving depletion of ER Ca2+, elevation of cytoplasmic Ca2+, and the activation of calmodulin/calcineurin/calpain cytoplasmic-mitochondrial-dependent caspase/nuclease activation and irreversible DNA fragmentation from reference [35]. See text for details. (C–F) Kinetic changes following treatment of prostate cancer cells with 500 nM TG normalized on a per cell basis in: (C) Gadd153 (also known as BiP) mRNA denoted with the line from reference [37] and the protein level in the insert from reference [15]; (D) Indicated proteins from reference [38]; (E) Protein synthesis and calmodulin protein from reference [32]; and (F) Cell cycle progression based upon flow cytometer determined DNA content vs. cell size (Small arrows indicate position of G0/G1 vs. S vs. G2/Mitosis) from reference [32]; (G) Cytoplasmic Ca2+ in control vs. CAM inhibitory peptide microinjected cells from reference [37].
Figure 4
Figure 4
Kinetic changes following treatment of prostate cancer cells to 500 nM Thapsigargin in: (A) μM rise in cytoplasmic Ca2+ vs. loss of clonogenic survival vs. irreversible nuclear DNA fragmentation from reference [37]; (B) Upregulation of TRPC1 and 3 protein from reference [39]; (C) Percentage of cells expressing activated caspase 3 intracellularly detected based upon a cell penetrant fluorescence substrate and expressing phosphatidyl-serine extracellularly detected based upon plasma membrane binding of fluorescently labeled annexin V from reference [40]; (D) Cytochrome C protein release from mitochondria to cytoplasm from reference [37]; (D) Percentage of cells with mitochondrial fragmentation from reference [41]; (E) Translocation of AIF from mitochondria (black arrows) at time zero vs. to cell nucleus (white arrows) at 48 h post-TG exposure [42]; (F) Cell counting for mitochondrial fragmentation in prolonged treatment TG showed the second phase fragmentation of mitochondria after a 32 h incubation [41]; (G) Irreversible fragmentation of genomic DNA from reference [37]; and (H) Cellular fragmentation into apoptotic bodies with arrow and arrow heads indicating same cell followed longitudinally from reference [32].
Figure 5
Figure 5
Overview of the downstream signaling pathways in the (A) <1 μM Ca2+ initiation phase vs. (B) >1 μM execution phase of prostate cancer cell death induced by Thapsigargin.
Figure 6
Figure 6
Photograph of Soren Christensen and his wife Helle, along with Carston Jakobsen, a postdoctoral fellow from the Christensen laboratory, and Samuel Denmeade and John Isaacs boxing up the >100 kg of Thapsia garganica seeds harvested from Ibiza, Spain in the first week of July.
Figure 7
Figure 7
In vivo response of LNCaP human prostate cancer xenografts in intact male immune-deficient mice to: (A) PSA-TG prodrug given via SQ alzet mini-pump at 25 mg/kg/week (i.e., 2.5 μmoles/kg/day) for 4 weeks from reference [45]; (B) hK2-TG prodrug given IV at 6 mg/kg/injection (i.e., 3.67 μmoles/kg/dose) once a day for 4 consecutive days from reference [60]; (C) FAP-TG prodrug given i.v. at 6.8 mg/kg (4 μmoles/kg/dose) once a day for 3 injections vs. docetaxel given i.v. at 0.39 μmoles on every 3 days for 3 injections from reference [67]; (D) PSMA-TG prodrug (also known as G202) given i.v. at 56 mg/kg (i.e., 40 μmoles/kg/dose) once a day for 3 injections vs. docetaxel given i.v. at 0.39 μmoles on every 3 days for 3 injections from reference [64]. (E) Response of established (0.8 cc) MCF-7 human breast cancers growing in mice given 2 daily intravenous injections at 56 mg/kg of Mipsagargin prodrug (G202) alone and in combination with 10 mg/kg/d oral Tasquinimod (TasQ). Results are presented as relative tumor size normalized to tumor volume at initiation of treatment. p < 0.05 for combination (combo) group vs. monotherapies after day 49 [68]. (F) Structure of prostate-specific antigen (PSA)-activated prodrug of L-Leu-12ADT with 2-fluoro-5-maleimidobenzamide covalently bound to the N-terminal of PSA substrate histidine-serine-serine-lysine-leucine-glutamic acid (HSSKLQ) for coupling to the cysteine 34 of human serum albumin.
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