Raymond J. Andersen

Regression of Castrate-Recurrent Prostate Cancer by a Small-Molecule Inhibitor of the Amino-Terminus Domain of the Androgen Receptor

Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.

A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds

We present a yeast chemical-genomics approach designed to identify genes that when mutated confer drug resistance, thereby providing insight about the modes of action of compounds. We developed a molecular barcoded yeast open reading frame (MoBY-ORF) library in which each gene, controlled by its native promoter and terminator, is cloned into a centromere-based vector along with two unique oligonucleotide barcodes. The MoBY-ORF resource has numerous genetic and chemical-genetic applications, but here we focus on cloning wild-type versions of mutant drug-resistance genes using a complementation strategy and on simultaneously assaying the fitness of all transformants with barcode microarrays. The complementation cloning was validated by mutation detection using whole-genome yeast tiling microarrays, which identified unique polymorphisms associated with a drug-resistant mutant. We used the MoBY-ORF library to identify the genetic basis of several drug-resistant mutants and in this analysis discovered a new class of sterol-binding compounds.

Effects of nutrient limitation on toxin production and composition in the marine dinoflagellate Protogonyaulax tamarensis

Toxin production was measured by high pressure liquid chromatography (HPLC) when the marine dinoflagellate Protogonyaulax tamarensis (NEPCC 255) was grown under nitrogen or phosphorus limitation. The major toxins found in P. tamarensis (255) consisted of (N21-SO3-)STX (11%), (N21-SO3-)NeoSTX (44%), and [(N21-SO3-)GTX2 plus (N21-SO3-)GTX3] (20%). Total toxin content on a per cell basis was high for cultures in log phase (30 to 40 fmol cell-1) and then decreased to ca 20 fmol cell-1 as the cultures entered stationary phase. There was a gradual decrease in the toxin content per cell during nitrogen-limited stationary phase to ca 3 fmol cell-1 or less. Phosphorus-limited cultures showed a markedly different response than nitrogen-limited cultures. Toxin content in P-limited cells dramatically increased at the start of stationary phase, reaching levels 3 to 4 times that observed in control and nitrogen-limited cultures. These results cannot be explained by changes in the average cell volume. Eventhough dramatic effects on the total toxin concentration were observed in response to nutrient limitation (N or P), the toxin composition (on a percent basis) remained constant. This suggests that the individual toxin composition of a given isolate is a fixed genetic trait and not a transient response to changing environmental factors.

Variation in paralytic shellfish toxin composition within the Protogonyaulax tamaronsis/catenella species complex; red tide dinoflagellates

Abstract Unialgal isolates of the Protogonyaulax (—Gonyaulax) tamarensis/catenella species complex, a group of dinoflagellates which causes paralytic shellfish poisoning (PSP), were subjected to toxin analysis by HPLC. Protogonyaulax isolates from widely separated geographical locations were compared, including the northeastern Pacific (British Columbia and Washington State), eastern Canada, Portugal, the United Kingdom and New Zealand. Two distantly related gonyaulacoid species were also analyzed, but the presence of PSP toxins was not detected. Although Protogonyaulax isolates varied markedly in total toxin concentration and toxicity, even through the culture cycle, the toxin ratios of individual isolates were distinctive and relatively constant. No toxins were detected in the Plymouth (U.K.) isolate of P. tamarensis, from the species type locality. Two isolates from Vancouver Island (British Columbia), which were previously considered to be non-toxic according to the mouse bioassay, revealed weak toxin spectra by HPLC. Within populations from English Bay (British Columbia) the toxin profiles of tamarensoid isolates tended to be conservative. However, this was not the case for the catenelloid forms from Washington State, which displayed a greater degree of toxin heterogeneity. Significantly, there was no identifiable relationship between toxicity or toxin profiles and the morphological characteristics conventionally used to separate the two dominant morphotypes into species within this species complex.

Motuporin, A Potent Protein Phosphatase Inhibitor Isolated from the Papua New Guinea Sponge Theonella swinhoei Gray

Abstract Motuporin ( 1 ), a cyclic pentapeptide that is a potent protein phosphatase-1 inhibitor and cytotoxin, has been isolated from the marine sponge Theonella swinhoei collected in Papua New Guinea. The structure of motuporin was elucidated by spectroscopic analysis and chemical degradation.

Chemical and biological evidence links microcystins to salmon ‘netpen liver disease’

Evidence is presented that links microcystins to a severe liver disease that occurs in Atlantic salmon that are netpen-reared in coastal British Columbia. Liquid chromatography-linked protein phosphatase bioassay analysis of extracts of liver tissue taken from Atlantic salmon afflicted with netpen liver disease showed the presence of an inhibitor of protein phosphatase that was chromatographically indistinguishable from microcystin-LR. Analysis of liver tissue from healthy control fish showed a complete absence of microcystin-LR. Intraperitoneal injection of microcystin-LR into healthy Atlantic salmon re-created the pathologic changes of netpen liver disease, including diffuse necrosis and hepatic megalocytosis.

An androgen receptor N-terminal domain antagonist for treating prostate cancer

Hormone therapies for advanced prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD), but these ultimately fail and the disease progresses to lethal castration-resistant prostate cancer (CRPC). The mechanisms that drive CRPC are incompletely understood, but may involve constitutively active AR splice variants that lack the LBD. The AR N-terminal domain (NTD) is essential for AR activity, but targeting this domain with small-molecule inhibitors is complicated by its intrinsic disorder. Here we investigated EPI-001, a small-molecule antagonist of AR NTD that inhibits protein-protein interactions necessary for AR transcriptional activity. We found that EPI analogs covalently bound the NTD to block transcriptional activity of AR and its splice variants and reduced the growth of CRPC xenografts. These findings suggest that the development of small-molecule inhibitors that bind covalently to intrinsically disordered proteins is a promising strategy for development of specific and effective anticancer agents.

Bioaccumulation and clearance of microcystins from salt water mussels, mytilus edulis, and in vivo evidence for covalently bound microcystins in mussel tissues

Over a period of 3 days saltwater mussels, Mytilus edulis, were fed a cyanobacteria, Microcystis aeruginosa, that contained a high concentration of microcystins. The mussels were killed on a periodic basis over the course of 2 months. Mussels were also collected at two sites were high levels of microcystins in tissues had been noted. A strategy based on the chemically unique nature of the C20 beta-amino acid, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6- dienoic acid (Adda), portion of the microcystins was used in conjunction with a protein phosphatase (PPase) assay to analyse for both covalently bound microcystins and free microcystins in the mussel tissues. The mussel PPase assay results were compared with the Lemieux oxidation gas chromatography-mass spectrometry (GCMS) analysis. Less than 0.1% of the total microcystin burden in the mussel tissue was found to be extractable with MeOH. Thus, direct evidence was provided for the existence of covalently bound microcystins in mussel tissues in vivo. The mussels rapidly cleared the covalently bound microcystins when transferred to untreated seawater. Within 4 days the total microcystin burden dropped from a high of 336.9 (+/- 45.8) micrograms/g wet tissue to 11.3 (+/- 2.6) micrograms/g. After 4 days postexposure until completion of the experiment the total levels remained below the detection limits of the GCMS method. The levels of free microcystins, extracted with MeOH and detected by the PPase assay, fell from 204 ng/g wet tissue to a residual 14 ng/g over a 53 day postexposure period. Presumably the bound microcystin present in the mussel tissue exists as a covalent complex with the PP-1 and PP-2A enzymes. We conclude that in any shellfish monitoring program it is the total tissue microcystin burden that needs to be considered.

Cytotoxic peptides hemiasterlin, hemiasterlin A and hemiasterlin B induce mitotic arrest and abnormal spindle formation

Abstract Purpose: Hemiasterlin, hemiasterlin A and hemiasterlin B are newly isolated cytotoxic tripeptides with potential as antitumor drugs. We wished to determine their mechanism of cytotoxicity. Methods: We studied their effect on cell survival, cell cycle progression, and microtubule morphology in MCF-7 human mammary carcinoma cells. Results: At the nanomolar concentrations at which they were cytotoxic, the peptides induced arrest in mitotic metaphase. Hemiasterlin A produced abnormal mitotic spindles like those produced by the microtubule inhibitors taxol, nocodazole and vinblastine at low concentrations. At high concentrations hemiasterlin A did not cause microtubule bundling like taxol, but caused microtubule depolymerization like nocodazole and vinblastine. Conclusions: The hemiasterlins probably exert their cytotoxic effect by inhibiting spindle microtubule dynamics.

The Search for Novel Human Pancreatic α‐Amylase Inhibitors: High‐Throughput Screening of Terrestrial and Marine Natural Product Extracts

Specific inhibitors of human pancreatic α‐amylase (HPA) have potential as oral agents for the control of blood glucose levels in the treatment of diabetes and obesity. In a search for novel inhibitors, a library of 30 000 crude biological extracts of terrestrial and marine origin has been screened. A number of inhibitory extracts were identified, of which the most potent was subjected to bioassay‐guided purification. A family of three glycosylated acyl flavonols, montbretins A–C, was thereby identified and characterized as competitive amylase inhibitors, with Ki values ranging from 8.1–6100 nM. Competitive inhibition by myricetin, which corresponds to the flavone core, and noncompetitive inhibition by a second fragment, ethyl caffeiate, suggest a binding mode for these inhibitors.