Rh(I)-Catalyzed Allenic Pauson-Khand Reaction to Access the Thapsigargin Core: Influence of Furan and Allenyl Chloroacetate Groups on Enantioselectivity.

Thapsigargin (Tg) is a potent SERCA pump inhibitor with the potential to treat cancer and COVID-19. We have extended the scope of the asymmetric allenic Pauson-Khand reaction to furan-tethered allene-ynes, a stereoconvergent transformation affording the 5,7,5-ring system of Tg in good yields and high enantioselectivity. Computational studies of the oxidative cyclization step show that the furan and chloroacetate groups contribute to this high selectivity.

Targeting oncogenic Notch signaling with SERCA inhibitors.

P-type ATPase inhibitors are among the most successful and widely prescribed therapeutics in modern pharmacology. Clinical transition has been safely achieved for H+/K+ ATPase inhibitors such as omeprazole and Na+/K+-ATPase inhibitors like digoxin. However, this is more challenging for Ca2+-ATPase modulators due to the physiological role of Ca2+ in cardiac dynamics. Over the past two decades, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) modulators have been studied as potential chemotherapy agents because of their Ca2+-mediated pan-cancer lethal effects. Instead, recent evidence suggests that SERCA inhibition suppresses oncogenic Notch1 signaling emerging as an alternative to γ-secretase modulators that showed limited clinical activity due to severe side effects. In this review, we focus on how SERCA inhibitors alter Notch1 signaling and show that Notch on-target-mediated antileukemia properties of these molecules can be achieved without causing overt Ca2+ cellular overload.

Large Scale Conversion of Trilobolide into the Payload of Mipsagargin: 8-O-(12-Aminododecanoyl)-8-O-Debutanoylthapsigargin.

In spite of the impressing cytotoxicity of thapsigargin (Tg), this compound cannot be used as a chemotherapeutic drug because of general toxicity, causing unacceptable side effects. Instead, a prodrug targeted towards tumors, mipsagargin, was brought into clinical trials. What substantially reduces the clinical potential is the limited access to Tg and its derivatives and cost-inefficient syntheses with unacceptably low yields. Laser trilobum, which contains a structurally related sesquiterpene lactone, trilobolide (Tb), is successfully cultivated. Here, we report scalable isolation of Tb from L. trilobum and a transformation of Tb to 8-O-(12-aminododecanoyl)-8-O-debutanoylthapsigargin in seven steps. The use of cultivated L. trilobum offers an unlimited source of the active principle in mipsagargin.

IRE1α and IGF signaling predict resistance to an endoplasmic reticulum stress-inducing drug in glioblastoma cells.

To date current therapies of glioblastoma multiforme (GBM) are largely ineffective. The induction of apoptosis by an unresolvable unfolded protein response (UPR) represents a potential new therapeutic strategy. Here we tested 12ADT, a sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, on a panel of unselected patient-derived neurosphere-forming cells and found that GBM cells can be distinguished into "responder" and "non-responder". By RNASeq analysis we found that the non-responder phenotype is significantly linked with the expression of UPR genes, and in particular ERN1 (IRE1) and ATF4. We also identified two additional genes selectively overexpressed among non-responders, IGFBP3 and IGFBP5. CRISPR-mediated deletion of the ERN1, IGFBP3, IGFBP5 signature genes in the U251 human GBM cell line increased responsiveness to 12ADT. Remarkably, >65% of GBM cases in The Cancer Genome Atlas express the non-responder (ERN1, IGFBP3, IGFBP5) gene signature. Thus, elevated levels of IRE1α and IGFBPs predict a poor response to drugs inducing unresolvable UPR and possibly other forms of chemotherapy helping in a better stratification GBM patients.

Fragment Molecular Orbital Study of the Interaction between Sarco/Endoplasmic Reticulum Ca2+-ATPase and its Inhibitor Thapsigargin toward Anti-Malarial Development.

Plasmodium falciparum, the causative agent of malignant malaria, is insensitive to thapsigargin (TG), a well-known inhibitor of the human sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). To understand the key factor causing the difference of the sensitivity, the molecular interaction of TG and each SERCA was analyzed by the fragment molecular orbital (FMO) method. While the major component of the interaction energy was the nonpolar interaction, the major difference in the molecular interaction arose from the polar interaction, namely, the hydrogen bonding interaction with a hydroxyl group of TG. Additionally, we successfully confirmed these FMO calculation results by measuring the inhibitory activity of a synthesized TG derivative. Our calculations and experiments indicated that, by replacing the hydroxyl group of TG with another functional group, the sensitivities of TG to human and P. falciparum SERCAs can be reversed. This study provides important information to develop antimalarial compounds targeting P. falciparum SERCA.

Divergent synthesis of thapsigargin analogs.

Thapsigargin (3) is a potent inhibitor of the SERCA-pump protein, with potential for application in a variety of medicinal areas. The efficient and scalable syntheses of thapsigargin (3) and nortrilobolide (2) have been disclosed previously. To demonstrate the modularity of the previous routes, three natural products (compounds 6, 13, 15) and four analogs (compounds 17-20) have been divergently prepared from a common building block featuring varied acyl chains at the C2, C3, and C8 positions. Biological tests revealed that all of the compounds prepared displayed promising activity profiles.

Inhibition of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase by thapsigargin analogs induces cell death via ER Ca2+ depletion and the unfolded protein response.

Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA inhibition induces cell death are incompletely understood. Here, we report that low (0.1 µm) concentrations of Tg and Tg analogs with various long-chain substitutions at the O-8 position extensively inhibit SERCA1a-mediated Ca2+ transport. We also found that, in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2-4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover, knockdown of two key store-operated Ca2+ entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca2+ entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca2+ stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response. We conclude that ER Ca2+ drainage and sustained unfolded protein response activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca2+ levels and SOCE are not required.

Differential effects of thapsigargin analogues on apoptosis of prostate cancer cells: complex regulation by intracellular calcium.

The inhibition of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) by thapsigargin (Tg) and Tg-type analogues is considered to trigger cell death by activation of apoptotic pathways. Some of these analogues may be useful as antineoplastic agents after appropriate targeting as peptide conjugated prodrugs to cancer cells. With this in mind, this study evaluates the effect on LNCaP androgen-sensitive cancer cells of thapsigargin substituted with 12-aminododecanoyl linkers and Leu (Leu-8ADT), aspartate (Asp-8ADT) or Boc-8ADT. Our results show that both Leu-8ADT and Asp-8ADT result in rapid ER calcium depletion and an influx of calcium across the plasma membrane by activation of store-operated calcium entry. By contrast, ER Ca(2+) depletion by Boc-8ADT is a very slow process that does not perceptibly increase cytosolic Ca(2+) and activate store-operated calcium entry, because the inhibition of SERCA with this compound is very slow. Nevertheless, we find that Boc-8ADT is a more efficient inducer of apoptosis than both Tg and Leu-8ADT. Compared with Tg and the other analogues, apoptosis induced by Asp-8ADT is very modest, although this compound also activates store-operated calcium entry and at high concentrations (1 µm) causes severe morphological changes, reflecting decreased cell viability. We conclude that many factors need to be considered for optimization of these compounds in antineoplastic drug design. Among these ER stress induced by Ca(2+) endoplasmic reticulum mobilization seems particularly important, whereas the early cytosolic increase of Ca(2+) concentration preceding the executive phase of apoptosis appears to be of no, or little, consequence for a subsequent apoptotic effect.

Targeting carcinoma-associated fibroblasts within the tumor stroma with a fibroblast activation protein-activated prodrug.

BACKGROUND: Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG. METHODS: The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9-11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided. RESULTS: FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC(50) = 3.5 nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure. Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206 mm(3), 95% CI = 0.068 to 0.344 mm(3); control: mean = 0.580 mm(3), 95% CI = 0.267 to 0.893 mm(3)) and 0.24 (treated: mean = 0.131 mm(3), 95% CI = 0.09 to 0.180 mm(3); control: mean = 0.543 mm(3), 95% CI = 0.173 to 0.913 mm(3)), respectively, on day 21 after therapy. CONCLUSIONS: This study validates the proteolytic activity of FAP as a target for the activation of a systemically delivered cytotoxic prodrug and demonstrates that targeted killing of cells within the stromal compartment of the tumor microenvironment can produce a therapeutic response.

Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca2+-ATPase with thapsigargin and thapsigargin analogs.

Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca(2+)-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.

Progress toward the synthesis of the basiliolides and transtaganolides: an intramolecular pyrone Diels-Alder entry into a novel class of natural products.

Efforts directed toward the synthesis of a basiliolide/transtaganolide model system are disclosed. A highly endo-selective intramolecular pyrone Diels-Alder (IMPDA) cycloaddition rapidly constructs the tricyclic core of the basiliolides and transtaganolides.

A prostate-specific antigen activated N-(2-hydroxypropyl) methacrylamide copolymer prodrug as dual-targeted therapy for prostate cancer.

Prostate cancer targeted peptide prodrugs that are activated by the serine protease activity of prostate-specific antigen (PSA) are under development in our laboratory. To enhance delivery and solubility of these prodrugs, macromolecular carriers consisting of N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers were covalently coupled to a PSA-activated peptide prodrug. HPMA copolymers are water-soluble, nonimmunogenic synthetic carriers that exhibit promise for drug delivery applications. These macromolecular copolymers enter the interstitium of solid tumors by the enhanced permeability and retention effect. The PSA-activated peptide substrate imparts selectivity because it is specifically hydrolyzed to release a cytotoxin at the site of prostate tumor. Enzymatically active PSA is present in high amounts in the extracellular fluid of a tumor, but PSA is inactivated in blood by binding to serum protease inhibitors. As an initial proof of concept, the HPMA copolymer was synthesized with a peptide substrate (HSSKLQ) bound to a fluorophore, 7-amino-4-methylcoumarin (AMC). PSA cleavage of the HPMA-HSSKLQ-AMC copolymer was observed, which led to the synthesis of an HPMA-based copolymer with the prodrug SSKYQ-L12ADT [HPMA-morpholinocarbonyl-Ser-Ser-Lys-Tyr-Gln-Leu-12-aminododecanoyl thapsigargin (JHPD)]. L12ADT is a potent analogue of the highly cytotoxic natural product thapsigargin. HPMA-JHPD was hydrolyzed by PSA in vitro and was toxic to prostate cancer cells in the presence of active PSA. The HPMA-JHPD produced no systemic toxicity when given at a 500 micromol/L L12ADT equivalent dose. Analysis of tumor tissue from mice treated with a single or multiple dose of the HPMA-JHPD copolymer showed release and accumulation of the L12ADT toxin within the tumor tissue.

Total synthesis of five thapsigargins: guaianolide natural products exhibiting sub-nanomolar SERCA inhibition.

Herein we describe the total synthesis of five guaianolide natural products: thapsigargin, thapsivillosin C, thapsivillosin F, trilobolide and nortrilobolide. Prodrug derivatives of thapsigargin have shown selective in vivo cytotoxicity against prostate tumours and the need for further investigation of this phenomenon highlights the importance of these total syntheses. The first absolute stereochemical assignment of thapsivillosin C is also delineated.

Design and total synthesis of unnatural analogues of the sub-nanomolar SERCA inhibitor thapsigargin.

Thapsigargin is a densely oxygenated guaianolide which displays potent sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) binding affinities. The total syntheses of designed unnatural analogues of this important natural product are described. This article constitutes the chemical synthesis behind an ongoing project. Rational modifications have been made to the lactone region of thapsigargin in order to obtain derivatives for future structure-activity relationship studies.

Pharmacokinetics, biodistribution, and antitumor efficacy of a human glandular kallikrein 2 (hK2)-activated thapsigargin prodrug.

BACKGROUND: Prostate cancer cells secrete unique proteases such as prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) that represent targets for the activation of prodrugs as systemic treatment of metastatic prostate cancer. Previously, a combinatorial peptide library was screened to identify a highly active peptide substrate for hK2. The peptide was coupled to an analog of the potent cytotoxin thapsigargin, L12ADT, to generate an hK2-activated prodrug that was efficiently hydrolyzed by purified hK2, stable to hydrolysis in human and mouse plasma in vitro and selectively toxic to hK2 producing prostate cancer cells in vitro. METHODS: In the current study, toxicology, pharmacokinetics, prodrug biodistribution, and antitumor efficacy studies were performed to evaluate the hK2-activated prodrug in vivo. RESULTS: The single intravenous maximally tolerated dose of prodrug was 6 mg/kg (i.e., 3.67 micromole/kg) which produced peak serum concentration of approximately 36 microM and had a half-life of approximately 40 min. In addition, over a 24 hr period <0.5% of free L12ADT analog was observed in plasma. The prodrug demonstrated significant antitumor effect in vivo while it was being administered, but prolonged intravenous administration was not possible due to local toxicity to tail veins. Subcutaneous administration of equimolar doses produced lower plasma AUC compared to intravenous dosing but equivalent intratumoral levels of prodrug following multiple doses. CONCLUSIONS: The hK2-activated prodrug was stable in vivo. The prodrug, however, was rapidly cleared and difficult to administer over prolonged dosing interval. Additional studies are underway to assess antitumor efficacy with prolonged administration of higher subcutaneous doses of prodrug. Second-generation hK2-activated thapsigargin prodrugs with increased half-lives and improved formulations are also under development.

SERCA-inhibiting activity of C-19 terpenolides from Thapsia garganica and their possible biogenesis.

An investigation of Thapsia garganica afforded a series of tetracyclic C-19 dilactones, whose production was dependent on the time and location of the collection. These unusual tetrahomosesquiterpenoids are presumably biosynthesized via a carbon dioxide-triggered electrophilic polyolefin cyclization. Despite the structural differences with thapsigargin, these compounds showed SERCA-inhibiting properties.

Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer.

OBJECTIVE: Prostate cancer cells secrete the unique protease human glandular kallikrein 2 (hK2) that represents a target for proteolytic activation of cytotoxic prodrugs. The objective of this study was to identify hK2-selective peptide substrates that could be coupled to a cytotoxic analogue of thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump that induces cell proliferation-independent apoptosis through dysregulation of intracellular calcium levels. METHODS: To identify peptide sequence requirements for hK2, a combination of membrane-bound peptides (SPOT analysis) and combinatorial chemistry using fluorescence-quenched peptide substrates was used. Peptide substrates were then coupled to 8-O-(12[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), a potent analogue of thapsigargin, to produce a prodrug that was then characterized for hK2 hydrolysis, plasma stability, and in vitro cytotoxicity. RESULTS: Both techniques indicated that a peptide with two arginines NH2-terminal of the scissile bond produced the highest rates of hydrolysis. A lead peptide substrate with the sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR) was hydrolyzed by hK2 with a Km of 26.5 micromol/L, kcat of 1.09 s(-1), and a kcat/Km ratio of 41,132 s(-1) mol/L(-1). The GKAFRR-L12ADT prodrug was rapidly hydrolyzed by hK2 and was stable in plasma, whereas the GKAFRR-L peptide substrate was unstable in human plasma. The hK2-activated thapsigargin prodrug was not activated by cathepsin B, cathepsin D, and urokinase but was an excellent substrate for plasmin. The GKAFRR-L12ADT was selectively cytotoxic in vitro to cancer cells in the presence of enzymatically active hK2. CONCLUSION: The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.

Synthesis of the thapsigargins.

The thapsigargins are a family of complex guaianolides with potent and selective Ca(2+)-modulating properties. This article documents the evolution of a synthetic route through several iterations to a final practical and scaleable synthetic route capable of generating both unnatural and natural products based around the guaianolide skeleton.