S. Brøgger Christensen

Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy

A prostate-specific membrane antigen–activated prodrug selectively kills cancer cells and is being tested in patients with advanced cancer. An Old Approach Is New Again In the 1995 film The Last Supper, a group of graduate students invite a diverse cast of characters for a series of Sunday dinners. After one guest threatens the lives of several of the students, subsequent dinners turn deadly. If the guest holds views that the group considers toxic to society, then the house wine is made poisonous and served only to the unwanted houseguest, who promptly dies. In a related scenario, Denmeade et al. use a prodrug to seek out and selectively poison unsavory guests that are toxic to the body—namely, cancer cells. The new work describes the development of a thapsigargin (TG) prodrug that is activated in the vasculature of solid tumors by tumor endothelial cells. The carboxypeptidase prostate-specific membrane antigen (PSMA)—which is selectively expressed on the surface of prostate cancer cells, including metastatic ones, and tumor, but not normal, endothelial cells—cleaves and activates the prodrug extracellularly in the tumor microenvironment. The activated cytotoxic moiety then poisons neighboring cancer cells within sites of metastases by entering the cells and inhibiting the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump, which is essential to the function of all normal and tumor cell types. The authors showed that treatment with the prodrug caused significant tumor regression in two mouse xenograft models of human prostate cancer and one model of human breast cancer with relatively little toxicity—less than that of the maximally tolerated dose of the widely used cancer drug docetaxel. Although the targeted prodrug concept is not new, the current approach has several features that make it superior to many previous ones. First, unlike most cytotoxic cancer drugs, TG is not cell cycle–dependent and thus can kill nondividing cancer cells. Furthermore, drug toxicity is expected to be low, because the PSMA substrate in the prodrug is cleaved primarily by prostate cancer cells and in the vicinity of tumor endothelial cells. In fact, the authors report that studies in cynomolgus monkeys showed minimal toxic effects except in the kidney, and even that renal toxicity was minimal to mild and reversible at the low drug dose. As with all cancer drugs, the new findings will require clinical validation in ongoing studies. However, this unusual therapeutic approach has the potential to be an effective and selective ouster of unwanted invaders that threaten their hosts. Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer.

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 Ca2+-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 Ca2+ 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.

Isolation of an Angiotensin Converting Enzyme (ACE) inhibitor from Olea europaea and Olea lancea

The aqueous extract of the leaves of Olea europaea and Olea lancea both inhibited Angiotensin Converting Enzyme (ACE) in vitro. A bioassay-directed fractionation resulted in the isolation of a strong ACE-inhibitor namely the secoiridoid 2-(3,4-dihydroxyphenyl)ethyl 4-formyl-3-(2-oxoethyl)-4 E-hexenoate (oleacein) (IC(50) = 26 μM). Five secoiridoid glycosides (oleuropein, ligstroside, excelcioside, oleoside 11-methyl ester, oleoside) isolated from Oleaceous plants showed no significant ACE-inhibition whereas, after enzymatic hydrolysis, the ACE-inhibition at 0.33 mg/ml was between 64% to 95%. Secoiridoids have not been described previously in the literature as inhibitors of ACE. Oleacein showed a low toxicity in the brine shrimp (Artemia satina) lethality test (LC(50) (24 h) = 969 ppm).

A Trojan Horse in Drug Development: Targeting of Thapsigargins Towards Prostate Cancer Cells

Available chemotherapeutics take advantage of the fast proliferation of cancer cells. Consequently slow growth makes androgen refractory prostate cancer resistant towards available drugs. No treatment is available at the present, when the cancer has developed metastases outside the prostate (T4 stage). Cytotoxins killing cells irrespective of the phase of the cell cycle will be able to kill slowly proliferating prostate cancer cells. Lack of selectivity, however, prevents their use as systemic drugs. Prostate cancer cells secrete characteristic proteolytic enzymes, e.g. PSA and hK2, with unusual substrate specificity. Conjugation of cytotoxins with peptides, which are selective substrates for PSA or hK2, will afford prodrugs, from which the active drug only will be released in close vicinity of the cancer cells. Based on this strategy prodrugs targeted at prostate cancer cells have been constructed and evaluated as potential drugs for prostate cancer. The potency of the thapsigargins as apoptotic agents make these naturally occurring sesquiterpene lactones attractive lead compounds. Intensive studies on structure-activity relationships and chemistry of the thapsigargins have enabled construction of potent derivatives enabling conjugation with peptides. Studies on the mechanism of action of the thapsigargins have revealed that the cytoxicity is based on their ability to inhibit the intracellular sarco-/endoplasmtic calcium pump.

Thapsigargin analogues for targeting programmed death of androgen-Independent prostate cancer cells

A number of analogues of thapsigargin, a selective inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPases have been synthesized. In all of the prepared analogues the butanoyl residue at O-8 has been replaced with a residue containing an aromatic amine. The amine can be used as an anchoring point for attaching a peptide group sensitive to the proteolytic enzyme, prostate specific antigen, secreted by prostate cancer cells. Like thapsigargin, the analogues are capable of elevating the cytoplasmic Ca2+ concentration approximately sevenfold when tested at effective cytotoxic doses. The analogues in which the 8-O-butanoyl group has been replaced with 3-(4-aminophenyl)propanoyl or 4-aminocinnamoyl were found potently to induce programmed cell death of the prostate cancer cells.

Limonoids from Khaya senegalensis

A new limonoid was isolated from the bark of Khaya senegalensis and identified as 2,6-dihydroxyfissinolide. The assignments of some signals in the (13)C NMR spectrum of fissinolide have been reversed and the signals of the (1)H NMR spectrum of methyl 3beta-acetoxy-6-hydroxy-1-oxomeliac-14-enoate have been assigned. The limonoids only showed moderate antiprotozoal activities.

Synthesis of antiparasitic licorice chalcones

Abstract A simple, inexpensive high yield method for preparation of echinatin and licochalcone C is described. Echinatin inhibits the growth of Leishmania parasites in concentrations, in which the proliferation of lymphocytes is only slightly affected, indicating that the compound might be used for treatment of leishmaniasis.

Novel inhibitory activity of the Staphylococcus aureus NorA efflux pump by a kaempferol rhamnoside isolated from Persea lingue Nees

OBJECTIVES To isolate a plant-derived compound with efflux inhibitory activity towards the NorA transporter of Staphylococcus aureus. METHODS Bioassay-guided isolation was used, with inhibition of ethidium bromide efflux via NorA as a guide. Characterization of activity was carried out using MIC determination and potentiation studies of a fluoroquinolone antibiotic in combination with the isolated compound. Everted membrane vesicles of Escherichia coli cells enriched with NorA were prepared to study efflux inhibitory activity in an isolated manner. RESULTS The ethanolic extract of Persea lingue was subjected to bioassay-guided fractionation and led to the isolation of the known compound kaempferol-3-O-α-L-(2,4-bis-E-p-coumaroyl)rhamnoside (compound 1). Evaluation of the dose-response relationship of compound 1 showed that ethidium bromide efflux was inhibited, with an IC(50) value of 2 μM. The positive control, reserpine, was found to have an IC(50) value of 9 μM. Compound 1 also inhibited NorA in enriched everted membrane vesicles of E. coli. Potentiation studies revealed that compound 1 at 1.56 mg/L synergistically increased the antimicrobial activity of ciprofloxacin 8-fold against a NorA overexpresser, and the synergistic activity was exerted at a fourth of the concentration necessary for reserpine. Compound 1 was not found to exert a synergistic effect on ciprofloxacin against a norA deletion mutant. The 2,3-coumaroyl isomer of compound 1 has been shown previously not to cause acute toxicity in mice at 20 mg/kg/day. CONCLUSIONS Our results show that compound 1 acts through inhibition of the NorA efflux pump. Combination of compound 1 with subinhibitory concentrations of ciprofloxacin renders a wild-type more susceptible and a NorA overexpresser S. aureus susceptible.

Absolute configurations of the histamine liberating sesquiterpene lactones thapsigargin and trilobolide

Abstract The absolute configuration of trilobolide and the revised one of thapsigargin classify the compounds as a group of guaianolides previously not found in higher plants.

Modifications of the α,β-Double bond in chalcones only Marginally affect the antiprotozoal activities

Abstract Methods for selective alkylation of chalcones in the α- or β-position and for selective reduction of the α,β-double bond have been developed. The antiparasitic potencies of the α,β-double bond modified chalcones only differ marginally from the potencies of the parent chalcones indicating that the propenone residue only functions as a spacer between the two benzene rings, which are the true pharmacophore.