Pharmacologic Activation of STING in the Bladder Induces Potent Antitumor Immunity in Non-Muscle Invasive Murine Bladder Cancer.

Stimulator of interferon genes (STING) is an innate immune receptor activated by natural or synthetic agonists to elicit antitumoral immune response via type I IFNs and other inflammatory cytokines. Bacillus Calmette-Guerin (BCG) is the standard of care as intravesical therapy for patients with high-risk non-muscle invasive bladder cancer (NMIBC). There are limited options available for patients with NMIBC who developed BCG unresponsiveness. In this study, we characterized in vitro and in vivo antitumor effects of E7766, a macrocyle-bridged STING agonist, via intravesical instillation in two syngeneic orthotopic murine NMIBC tumor models resistant to therapeutic doses of BCG and anti-PD-1 agents. E7766 bound to recombinant STING protein with a Kd value of 40 nmol/L and induced IFNß expression in primary human peripheral blood mononuclear cells harboring any of seven major STING genotypes with EC50 values of 0.15 to 0.79 µmol/L. Intravesical E7766 was efficacious in both NMIBC models with induction of effective immunologic memory in the treated animals. Pharmacologic activation of the STING pathway in the bladder resulted in IFN pathway activation, infiltration of T cells and natural killer (NK) cells, dendritic cell activation, and antigen presentation in bladder epithelium, leading to the antitumor activity and immunity. In addition, measurements of the pharmacodynamic markers, Ifnß1 and CXCL10, in bladder, urine, and plasma, and of STING pathway intactness in cancer cells, supported this mode of action. Taken together, our studies reveal an antitumor immune effect of pharmacologic activation of the STING pathway in bladder epithelium and thus provide a rationale for subsequent clinical studies in patients with NMIBC.

E7766, a Macrocycle-Bridged Stimulator of Interferon Genes (STING) Agonist with Potent Pan-Genotypic Activity.

A strategy for creating potent and pan-genotypic stimulator of interferon genes (STING) agonists is described. Locking a bioactive U-shaped conformation of cyclic dinucleotides by introducing a transannular macrocyclic bridge between the nucleic acid bases leads to a topologically novel macrocycle-bridged STING agonist (MBSA). In addition to substantially enhanced potency, the newly designed MBSAs, exemplified by clinical candidate E7766, exhibit broad pan-genotypic activity in all major human STING variants. E7766 is shown to have potent antitumor activity with long lasting immune memory response in a mouse liver metastatic tumor model. Two complementary stereoselective synthetic routes to E7766 are also described.

Neuropilin-1 drives tumor-specific uptake of chlorotoxin.

BACKGROUND: Chlorotoxin (Cltx) isolated from scorpion venom is an established tumor targeting and antiangiogenic peptide. Radiolabeled Cltx therapeutic (131I-TM601) yielded promising results in human glioma clinical studies, and the imaging agent tozuleristide, is under investigation in CNS cancer studies. Several binding targets have previously been proposed for Cltx but none effectively explain its pleiotropic effects; its true target remains ambiguous and is the focus of this study. METHODS: A peptide-drug conjugate (ER-472) composed of Cltx linked to cryptophycin as warhead was developed as a tool to probe the molecular target and mechanism of action of Cltx, using multiple xenograft models. RESULTS: Neuropilin-1 (NRP1), an endocytic receptor on tumor and endothelial cells, was identified as a novel Cltx target, and NRP1 binding by Cltx increased drug uptake into tumor. Metabolism of Cltx to peptide bearing free C-terminal arginine, a prerequisite for NRP1 binding, took place in the tumor microenvironment, while native scorpion Cltx with amidated C-terminal arginine did not bind NRP1, and instead acts as a cryptic peptide. Antitumor activity of ER-472 in xenografts correlated to tumor NRP1 expression. Potency was significantly reduced by treatment with NRP1 blocking antibodies or knockout in tumor cells, confirming a role for NRP1-binding in ER-472 activity. Higher cryptophycin metabolite levels were measured in NRP1-expressing tumors, evidence of NRP1-mediated enhanced drug uptake and presumably responsible for the superior antitumor efficacy. CONCLUSIONS: NRP1 was identified as a novel Cltx target which enhances tumor drug uptake. This finding should facilitate tumor selection for chlorotoxin-based therapeutics and diagnostics.

Leiodermatolide, a novel marine natural product, has potent cytotoxic and antimitotic activity against cancer cells, appears to affect microtubule dynamics, and exhibits antitumor activity.

Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP-tagged plus end binding protein EB-1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The antitumor activities of leiodermatolide, as well as the proven utility of antimitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research.

Apratoxin A Shows Novel Pancreas-Targeting Activity through the Binding of Sec 61.

Apratoxin A is a natural product with potent antiproliferative activity against many human cancer cell lines. However, we and other investigators observed that it has a narrow therapeutic window in vivo Previous mechanistic studies have suggested its involvement in the secretory pathway as well as the process of chaperone-mediated autophagy. Still the link between the biologic activities of apratoxin A and its in vivo toxicity has remained largely unknown. A better understanding of this relationship is critically important for any further development of apratoxin A as an anticancer drug. Here, we describe a detailed pathologic analysis that revealed a specific pancreas-targeting activity of apratoxin A, such that severe pancreatic atrophy was observed in apratoxin A-treated animals. Follow-up tissue distribution studies further uncovered a unique drug distribution profile for apratoxin A, showing high drug exposure in pancreas and salivary gland. It has been shown previously that apratoxin A inhibits the protein secretory pathway by preventing cotranslational translocation. However, the molecule targeted by apratoxin A in this pathway has not been well defined. By using a (3)H-labeled apratoxin A probe and specific Sec 61α/ß antibodies, we identified that the Sec 61 complex is the molecular target of apratoxin A. We conclude that apratoxin A in vivo toxicity is likely caused by pancreas atrophy due to high apratoxin A exposure. Mol Cancer Ther; 15(6); 1208-16. ©2016 AACR.

Design, Synthesis, and Evaluation of Irciniastatin Analogues: Simplification of the Tetrahydropyran Core and the C(11) Substituents.

The design, synthesis, and biological evaluation of irciniastatin A (1) analogues, achieved by removal of three synthetically challenging structural units, as well as by functional group manipulation of the C(11) substituent of both irciniastatins A and B (1 and 2), has been achieved. To this end, we first designed a convergent synthetic route toward the diminutive analogue (+)-C(8)-desmethoxy-C(11)-deoxy-C(12)-didesmethylirciniastatin (6). Key transformations include an acid-catalyzed 6-exo-tet pyran cyclization, a chiral Lewis acid mediated aldol reaction, and a facile amide union. The absolute configuration of 6 was confirmed via spectroscopic analysis (CD spectrum, HSQC, COSY, and ROESY NMR experiments). Structure-activity relationship (SAR) studies of 6 demonstrate that the absence of the three native structural units permits access to analogues possessing cytotoxic activity in the nanomolar range. Second, manipulation of the C(11) position, employing late-stage synthetic intermediates from our irciniastatin syntheses, provides an additional five analogues (7-11). Biological evaluation of these analogues indicates a high functional group tolerance at position C(11).

Antiproliferative and antiplasmodial compounds from selected Streptomyces species.

In continuation of our ongoing search for bioactive compounds from microbial extracts, we performed antiproliferative and/or antimalarial assays on extracts of 806 microbial species isolated from Madagascan marine organisms, on 1317 species isolated from Madagascan soil samples and on a Streptomyces species (S.4) from a marine sponge collected from the Florida Keys. This work identified active extracts from four Streptomyces isolates (S.1, S.2, S.3 and S.4). The extracts of Streptomyces S.1 and S.2 showed antiproliferative activity against the A2780 ovarian cancer cell line, while those of S.3 and S.4 displayed both antiproliferative and antimalarial activity. Bioassay-guided fractionation coupled with dereplication of the active extracts led to the identification and isolation of nonactin (1), monactin (2), dinactin (3), ±-nonactic acid (4), toyocamycin (5), piperafizine A (6) and a new dipeptide named xestostreptin (7). The structures of all isolated compounds 1-7 were elucidated by analyses of their NMR spectroscopic and mass spectrometric data, and were confirmed by comparison with the data reported in the literature. Compound 6 was crystallized and subjected to X-ray diffraction analysis to confirm its structure as piperafizine A (6). Compounds 1-3 displayed strong antiproliferative activity against A2780 ovarian cancer cells (IC50 values of 0.1, 0.13 and 0.2 µM, respectively), A2058 melanoma cells (IC50 values of 0.2, 0.02 and 0.02 µM, respectively), and H522-T1 non small-cell cancer lung cells (IC50 values of 0.1, 0.01 and 0.01 µM, respectively), while compounds 4 and 7 exhibited weak antiplasmodial activity against the Dd2 strain of Plasmodium falciparum, with IC50 values of 6.5 and 50 µM, respectively.

High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc-) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells.

The cystine-glutamate antiporter (system xc-) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of cystine uptake and glutamate release previously reported in normal human fibroblast cells.

Antiproliferative Compounds from Cleistanthus boivinianus from the Madagascar Dry Forest.

The two new lignans 3α-O-(ß-D-glucopyranosyl)desoxypodophyllotoxin (1) and 4-O-(ß-D-glucopyranosyl)dehydropodophyllotoxin (2) were isolated from Cleistanthus boivinianus, together with the known lignans deoxypicropodophyllotoxin (3), (±)-ß-apopicropodophyllin (4), (-)-desoxypodophyllotoxin (5), (-)-yatein (6), and ß-peltatin-5-O-ß-D-glucopyranoside (7). The structures of all compounds were characterized by spectroscopic techniques. Compounds 1, 4, and 5 showed potent antiproliferative activities against the A2780 ovarian cancer cell line, with IC50 values of 33.0 ± 3.6, 63.1 ± 6.7, and 230 ± 1 nM, respectively. Compounds 2 and 7 showed only modest A2780 activities, with IC50 values of 2.1 ± 0.3 and 4.9 ± 0.1 µM, respectively, while compounds 3 and 6 had IC50 values of >10 µM. Compound 1 also had potent antiproliferative activity against the HCT-116 human colon carcinoma cell line, with an IC50 value of 20.5 nM, and compound 4 exhibited modest antiproliferative activity against the A2058 human caucasian metastatic melanoma and MES-SA human uterine sarcoma cell lines, with IC50 values of 4.6 and 4.0 µM, respectively.

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking.

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment.

Nitrogen-containing dimeric nor-multiflorane triterpene from a Turraea sp.

The new triterpene turranoic acid (1) and the new N-containing nor-triterpene turraenine (2), along with triptocallic acid B (3) and esculentoic acid (4) were isolated from leaves of a Turraea sp. Compounds 1-3 showed weak to moderate in vitro antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum strain FCM29. Compound 1 also displayed weak cytotoxic activity against the nonsmall lung cancer cell line H522-T1 with an IC50 value of 16.4 µM.

Structure elucidation of antiproliferative bisbenzylisoquinoline alkaloids from Anisocycla grandidieri from the Madagascar dry forest.

Antiproliferative bioassay-guided fractionation of the ethanol extract of the stems of Anisocycla grandidieri led to the isolation of the known alkaloids stebisimine (1), (+)-1,2-dehydrotelobine (2), (+)-2'-norcocsuline (3) and puetogaline B (4). Herein, we report the full NMR assignments of all compounds and the X-ray crystallography of single crystals of compounds 1 and 3. Compounds 2 and 3 showed moderate antiproliferative activity against the A2780 human ovarian cancer cell line with IC50 values of 4.1 ± 0.3 and 2.7 ± 0.3 µM, respectively, and they also displayed selective activity toward the H460 (large cell lung cancer), MCF-7 (breast ductal carcinoma), and UACC-257 (melanoma) cell lines.

Antiproliferative Homoisoflavonoids and Bufatrienolides from Urginea depressa.

Investigation of the South African plant Urginea depressa Baker (Asparagaceae Juss.) for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the six new homoisoflavonoids urgineanins A-F (1-6), the two known bufatrienolides 7 and 9, and the new bufatrienolides urginins B and C (8 and 10). Structures were elucidated based on analysis of their 1D and 2D NMR spectra, electronic circular dichroism, and mass spectrometric data. Five of the six new homoisoflavonoids had good antiproliferative activity against the A2780 ovarian cancer, A2058 melanoma, and H522-T1 human non-small-cell lung cancer cells, and urgineanin A (1) had submicromolar activity against all three cell lines. The four bufatrienolides 7-10 had strong antiproliferative activity against the same cell line, with IC50 values of 24.1, 11.2, 111, and 40.6 nM, respectively.

Mycoleptodiscins A and B, cytotoxic alkaloids from the endophytic fungus Mycoleptodiscus sp. F0194.

Two novel reddish-orange alkaloids, mycoleptodiscin A (1) and mycoleptodiscin B (2), were isolated from liquid cultures of the endophytic fungus Mycoleptodiscus sp. that had been isolated from Desmotes incomparabilis in Panama. Elucidation of their structures was accomplished using 1D and 2D NMR spectroscopy in combination with IR spectroscopic and MS data. These compounds are indole-terpenes with a new skeleton uncommon in nature. Mycoleptodiscin B (2) was active in inhibiting the growth of cancer cell lines with IC50 values in the range 0.60-0.78 µM.

Antiproliferative homoscalarane sesterterpenes from two Madagascan sponges.

Dereplication of the antiproliferative ethyl acetate fraction of the Madagascan sponge Carteriospongia sp. led to the detection and isolation of the two known homoscalarane-type sesterterpenes 1 and 2. Investigation of a similar sponge containing closely related compounds afforded the four new antiproliferative homoscalarane sesterterpenes (3 and 5-7). The structures of all isolated compounds were elucidated by spectroscopic methods, including UV, IR and 1D and 2D NMR. Compounds 1, 3 and 5 displayed submicromolar antiproliferative activity against the A2780 ovarian cell line with IC50 values of 0.65, 0.26 and 0.28 µM, respectively, while compounds 6 and 7 showed moderate activity (4.5 and 8.7 µM, respectively). Compounds 3 and 5 also displayed anti-proliferative activity against the H522-T1 non-small cell lung and A2058 human melanoma cancer cell lines.

Structures and cytotoxic evaluation of new and known acyclic Ene-Ynes from an American Samoa Petrosia sp. Sponge.

Four new compounds, (-)-petrosynoic acids A-D (1-4), and five known congeners, pellynols A (5), C (6), D (7), F (8), and I (9), were isolated from a Petrosia sp. marine sponge collected in American Samoa. Isolation work was guided by cytotoxicity against human lung cancer cells (H460). The structures of the C31-C33 polyacetylenes (1-9) were determined on the basis of 1D- and 2D-NMR analysis, mass spectrometry, and comparison of specific rotation values. Compounds 1-9 were found to be broadly cytotoxic with limited selectivity for cancer cells, as they were all moderately active against the A2058 (melanoma), H522-T1 (lung), and H460 (lung) human cancer cell lines as well as IMR-90 quiescent human fibroblast cells.

Two antiproliferative triterpene saponins from Nematostylis anthophylla from the highlands of Central Madagascar.

Investigation of the endemic Madagascan plant Nematostylis anthophylla (Rubiaceae) for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the known triterpene saponin randianin (1), and the two new bioactive triterpene saponins 2"-O-acetylrandianin (2) and 6"-O-acetylrandianin (3). The structures of the two new compounds were elucidated based on analysis of their 1D- and 2D-NMR spectra, and mass spectrometric data. The three isolated triterpene saponins displayed moderate but selective antiproliferative activities, with IC(50) values of 1.2, 1.7, and 2.2 µM, respectively, against the A2780 ovarian cancer, but only weak inhibitions of the proliferation of A2058 melanoma and the H522 lung cancer cell lines.

Atom-based enumeration: new eribulin analogues with low susceptibility to P-glycoprotein-mediated drug efflux.

A series of eribulin analogues was evolved in silico through iterative atom-based enumeration employing a genetic algorithm-derived survival function to minimize predicted PgP-mediated drug efflux. Representatives of the virtual series were subsequently synthesized in the laboratory and tested in vitro for PgP-susceptibility. These new computer-inspired derivatives were found to exhibit high cell growth inhibitory activity and to be among the least sensitive to P-glycoprotein-mediated drug efflux in the eribulin series, thereby validating this approach to in silico molecular design.

Antiproliferative acetogenins from a Uvaria sp. from the Madagascar dry forest.

Investigation of the endemic Madagascan plant Uvaria sp. for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of two new acetogenins. The structures of these two compounds were elucidated on the basis of analysis of their 1D and 2D NMR spectra, circular dichroism, and mass spectrometric data, together with chemical modification. The two acetogenins display weak antiproliferative activity against the A2780 ovarian cancer, the A2058 melanoma, and the H522 lung cancer cell lines.