Paul G. Grothaus

An enzyme immunoassay for the determination of taxol and taxanes in Taxus sp. tissues and human plasma

A rapid and sensitive indirect competitive inhibition enzyme immunoassay (CIEIA) method has been developed for quantitating taxanes in extracts of Taxus brevifolia Nutt. tissue and in human plasma. High titer rabbit polyclonal antibodies (pAb) were raised against a conjugate of 7-succinyltaxol and keyhole limpet hemocyanin. The presence of taxane analyte competitively inhibited the binding of the rabbit anti-taxane pAbs to a 7-succinyltaxol-bovine serum albumin solid phase coating antigen. The CIEIA detected taxol and cephalomannine in concentrations as low as 0.3 ng/ml (3.5 x 10(-4) microM), but did not detect baccatin III or 10-deacetylbaccatin III at concentrations below 1000 ng/ml (1.7 microM and 1.8 microM, respectively). This method is useful for estimating the taxane content of T. brevifolia extracts and may be useful for monitoring the taxol plasma level of taxol-treated patients.

Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples

Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palythoa tuberculosa. The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enzyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosa extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection.

Biological activity of 26-succinylbryostatin 1

Bryostatin 1, a macrocyclic lactone, has undergone phase I trials as an anticancer agent. Because of the lipid solubility of this compound it must be delivered either in ethanol or in a PET formulation. During the trial, these vehicles caused a large number of treatment-related side effects. We have synthesized the triethanolamine salt of 26-succinylbryostatin 1 and find that this compound is approx. 100-fold more water soluble than bryostatin 1. Because of the potential for clinical use, we have evaluated the biologic activity of this compound. We find that in a concentration-dependent manner 26-succinylbryostatin 1 is capable of activating protein kinase C (PKC) in vitro and displacing [3H]PDBu from PKC. However, at all concentrations tested the activity was less than the parent compound bryostatin 1. Addition of bryostatin 1 but not 26-succinylbryostatin 1 to U937 leukemic cells in culture stimulated a drop in cytosolic PKC, secondary to translocation of PKC to the membrane. Although 26-succinylbryostatin 1 did not stimulate a drop in the cytosolic levels of PKC, addition to U937 cells activated transcription from an AP-1 enhancer construct and c-Jun protein phosphorylation in a similar fashion to bryostatin 1 and differentiation of U937 cells. Unlike bryostatin 1, 26-succinylbryostatin 1 was unable to cause aggregation of human platelets. Although injection of bryostatin-1 into mice carrying B16 melanoma inhibits tumor growth, there was no significant inhibition of melanoma growth when identical doses of 26-succinylbryostatin 1 were injected. Therefore, 26-succinylbryostatin 1 shares some but not all of the pharmacologic properities of bryostatin 1. This compound can activate protein phosphorylation without lowering cytosolic levels of PKC.

NCI Program for Natural Product Discovery: A Publicly-Accessible Library of Natural Product Fractions for High-Throughput Screening

The US National Cancer Institutes (NCI) Natural Product Repository is one of the worlds largest, most diverse collections of natural products containing over 230,000 unique extracts derived from plant, marine, and microbial organisms that have been collected from biodiverse regions throughout the world. Importantly, this national resource is available to the research community for the screening of extracts and the isolation of bioactive natural products. However, despite the success of natural products in drug discovery, compatibility issues that make extracts challenging for liquid handling systems, extended timelines that complicate natural product-based drug discovery efforts and the presence of pan-assay interfering compounds have reduced enthusiasm for the high-throughput screening (HTS) of crude natural product extract libraries in targeted assay systems. To address these limitations, the NCI Program for Natural Product Discovery (NPNPD), a newly launched, national program to advance natural product discovery technologies and facilitate the discovery of structurally defined, validated lead molecules ready for translation will create a prefractionated library from over 125,000 natural product extracts with the aim of producing a publicly-accessible, HTS-amenable library of >1,000,000 fractions. This library, representing perhaps the largest accumulation of natural-product based fractions in the world, will be made available free of charge in 384-well plates for screening against all disease states in an effort to reinvigorate natural product-based drug discovery.