ENGLERINS: Englerin A was isolated based on the activity of the organic extract of the bark of Phyllanthus engleri Pax in the NCI 60 cell screen. The extract was identified in a retrospective bioinformatic analysis of testing data for 68,000 extracts, which sought to identify samples with the most selectivity against renal cancer cell lines. Bioassay guided fractionation of the extract led to isolation of the novel sesquiterpene diester, englerin A. Englerin A was isolated in high yield (1-4 g/kg dry wt.) from stem bark and root bark of the Tanzanian tree, Phyllanthus engleri Pax (Euphorbiaceae). Isolation required three purification steps. Other plant parts from the initial collection did not contain appreciable amounts of englerins and were devoid of anticancer activity. Three collections of bark collected from the original location in Iringa Province, Tanzania have all yielded similar amounts of englerin A, showing that natural collections are a viable source for preclinical development. The NCI currently possesses 6 g of pure englerin A which is available for development activities, isolated from Tanzanian bark. I recently reported a series of chlorinated analogues derived from englerin A, one of which has activity only 2.5-fold weaker than the natural product. Structure-activity studies have established several important points: a) Cell growth inhibition is not simply due to release of glycolate, a well-known but low-potency renal toxin, since a reverse ester analogue which cannot generate glycolate is active. b) The cinnamate moiety tolerates substantial variation without loss of activity. The cinnamate double bond plays a rigidifying role but its electronic contributions are not important. c) The isopropyl group plays an important role in activity, since its simplification to ethyl and methyl groups rapidly decreases potency. d) The cinnamate benzene ring is not required to be aromatic. Our current hypothesis is that the net effect of englerin A is to simultaneously starve the cells of glucose while creating an addiction to glucose. We believe that the selectivity depends on cells expressing both PKC-theta and HSF1 and/or being highly glucose dependent. Sensitivity to englerin A also correlates directly with sensitivity to 2-deoxyglucose, further highlighting the link between englerin A sensitivity, glucose dependence and PKC-theta activation. Englerin A was shown to be active in two different xenograft models. Pharmaceutical formulation and other preclinical development is ongoing. Collaborations with three synthetic chemistry groups have resulted in a number of active analogues which are being evaluated in tandem with the natural product. In addition, we have discovered that englerin A has excellent in vitro activity in a large panel of Ewing's sarcoma cell lines. SCHWEINFURTHINS: I isolated schweinfurthins A and B from the African plant Macaranga schweinfurthii Pax. The compounds displayed potent and selective activity against central nervous system, renal, and breast cancer cell lines in the NCI 60 cell assay, with GI50 values for four sensitive CNS tumor cell lines in the 10-25 nM range. The spectrum of anticancer activity did not match that of any currently used agent, indicating that these compounds might be acting at a previously unrecognized target or through a novel mechanism. Thus far, a total of 11 schweinfurthins have been isolated from nature. Synthetic strategies have been developed by the Wiemer lab (University of Iowa) to provide a reliable source of natural schweinfurthins and synthetic analogues for further biological testing. In the case of schweinfurthin F, total synthesis of the (R,R,R) and (S,S,S) enantiomers and comparisons of spectral data, optical rotations, and bioassay data with those reported for the natural product have resulted in assignment of the natural compounds as the (R,R,R) isomers. These synthetic efforts have continued, and most of the naturally occurring schweinfurthins have now been obtained by total synthesis. Investigations into the mechanism of action of schweinfurthins have yet to identify a proximate molecular target; however, in glioblastoma cell lines, it appears that a defective neurofibromatosis type 1 (NF1) pathway confers sensitivity. With a grant from the Children's Tumor Foundation, we are conducting a massive siRNA screen for potential targets at a CRO. My collaboration with the Lockett lab (FNLCR) focused on the clear effect of natural schweinfurthins on the cellular actin cytoskeleton in sensitive cell lines. Development of pattern recognition algorithms for cellular images has enabled quantitation of changes in F-actin distribution with drug treatment. A synthetic analogue of schweinfurthin A has shown activity in an allograft model of peripheral nerve sheath tumor driven by an NF1 defect. Pharmaceutical development is ongoing.
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