Natural product analogs

Gossypol is a natural product derived from cottonseed extracts that causes male infertility and has been studied extensively in humans as a male contraceptive. This compound is cytotoxic to a variety of cancer cell lines and was advanced to human clinical trial on this basis even though its mechanism of action was not clear. More recently, gossypol was discovered in a natural product library screens to bind Bcl-XL [25]. Given its clinical history and this possible mechanistic rational, gossypol has been the focus of many recent investigations [25-30]. The (-)-enantiomer, 1, is responsible for the majority of both the cytotoxic and spermicidal activities. It binds both Bcl-XL (K; = 0.57 mM) and Bcl-2 (K = 0.46 mM) and induces cytochrome c release, caspase activation and apoptotic death in numerous cell lines. (-)-Gossypol, 1, reverses the protection afforded by both Bcl-2 and Bcl-XL overexpression in Jurkat T leukemia cells with an IC50 of 18.1 mM and 22.9 mM, respectively, and dose dependently induces cytochrome c release from isolated mitochondria in these cell lines [31]. In the in vivo setting, 1 significantly enhanced the antitumor activity of X-ray irradiation leading to tumor regression in a PC-3 murine xenograft model of human prostate cancer [32] and potentiated the effect of CHOP therapy

(cyclophosphamide-adriamycin-vincristine-prednisone) in mouse xenograft models of diffuse large cell lymphoma [33].

Guided by NMR structural analysis and molecular modeling, several analogs have been designed that lack the two aldehyde groups found in gossypol [34]. One of these, apogossypol 2, retains moderate binding affinity for Bcl-XL (K; = 2.3 mM). Time lapsed confocal microscopy experiments show the ability of 2 to displace a fluorescently labelled BH3-only protein (GFP-Bcl-Gs) from the mitochondria of cells containing wild-type Bcl-XL but not those transfected with an inactive Bcl-XL mutant (R139M). This compound has also been evaluated against 12 primary patient-derived samples of chronic lymphocytic leukemia to show a response in only half the samples with a composite LD50 of approximately 16 mM. Most recently, a synthetic analog 3, designed based on the 3D structure of gossypol in complex with Bcl-XL has been reported to bind both Bcl-2 (K = 0.088 mM) and Bcl-XL (K; = 1.49 mM) and to inhibit growth of human breast (MDA-MB-231, IC50 = 1.54 mM) and prostate (PC-3, IC50 = 1.82 mM) cancer cell lines [35].

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High throughput screens have identified three additional classes of polyphenols that bind Bcl-XL [36]. NMR structural studies have confirmed binding of 4, 5, and 6 to the BH3 binding groove of Bcl-XL. Purpurogallin, 4, is an antioxidant found in edible oils and has moderate affinity for Bcl-XL (K = 2.2 mM). Theaflavanin, 5, and (-)-catechin-3 gallate, 6, are black and green tea extracts, respectively, with submicromolar affinities for both Bcl-XL (0.25 mM, 012 mM, respectively) and Bcl-2 (0.28 mM, 0.40 mM, respectively). Although the gallate group of 6 is required for binding, a detailed structure activity relationship has not been reported. A mechanistic link between these effects and modulation of Bcl-2 family members has not been established.

Tetrocarcin A (TC-A), 7, is a Gram-positive antibacterial agent isoloated from Actinomycete indentified in a cell-based high throughput screen of natural product libraries to reverse the protection afforded by Bcl-2 and Bcl-XL overexpression against pro-apoptotic stimuli [37]. Modification of the C-9 sugar subunit produced compounds that maintain the ability to reverse Bcl-2 protection, but lack the parent antibacterial activity. Removal of the C-9 sugar moiety altogether or replacement with non-sugar substituents abolished both activities [38]. Although TC-A induces apoptosis in a concentration dependent fashion, this effect is independent of the expression level of Bcl-2 family member (Bcl-2, Bax or Bid). Direct binding of 7 to Bcl-2 family proteins has not been demonstrated. The mechanism of action of 7 remains controversial and has recently been postulated to involve activation of the ER-stress pathway [39,40].

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Antimcycin A, a Streptomyces derived antibiotic was identified from a screen of compounds with known effects on mitochondrial function for their ability to selectively kill cells with high versus low Bcl-XL expression in isogenic cell lines [41,42]. High Bcl-XL expression levels not only did not protect against antimycin A, but actually markedly enhanced antimycin A induced apoptotic cell death. The intrinsic fluorescence of antimycin A3, 8a, increased proportionally in the presence of increasing concentrations of Bcl-2 protein with maximal effect at a 1:1 stoic-hiometry, suggesting a direct binding interaction. This effect was competitively reversed by addition of increasing concentrations of Bak BH3 peptide suggesting specific binding to the BH3 hydrophobic binding groove. Compound 8a has an apparent Kd of 2.5 mM for Bcl-2. Although 2-methoxy antimycin A3, 8b, has no effect on mitochondrial respiration, it retains binding affinity for Bcl-2 and the ability to selectively kill Bcl-XL overexpressing cell lines. 2-Benzoyl antimycin A3, 8c, has no detectable affinity for Bcl-2 and no effect on Bcl-XL overexpressing cells.

Chelerythrine, 9, was identified in an FPA-based high throughput screen of a natural product library consisting of 107,423 extracts from a variety of sources [43]. It exhibits moderate Bcl-XL binding affinity (FPA IC50 = 1.5 mM) and disrupts Bak/Bcl-XL interactions in an in vitro pulldown assay. Chelerythrine concentration dependently induces cytochrome c release from isolated mitochondria and shows specific killing at concentrations greater than 2 mM in the Bcl-XL overexpressing tumor cell line SH-SY5Y.

The GX15 series of Bcl-2 inhibitors is derived from the family of prodigiosin tripyrrole natural products that are produced by microorganisms such as Streptomyces and Serratia and contain the common 4-methoxy-2,2!-bipyrrole ring system found in 10. The antibiotic, cytotoxic and more recently immunosuppressive activities of this family of natural products have been ascribed to a number of mechanisms of action and have recently been reviewed [44]. GX15 analogs have been shown by NMR studies and molecular modeling to competitively bind Bcl-2 [45] and 10 binds Bcl-w with three-fold higher affinity than Bik BH3 peptide [46]. An advanced lead in this series, GX15-070, reportedly binds Bcl-w (Kd = 0.44 mM) and Mcl-1 (Kd = 0.49 mM), disrupts Mcl-1/Bak interactions in SK-Mel melanoma cells and is cytotoxic (EC50 = 1.7 mM) to primary patient derived B-cell chronic lymphocytic leukemia (CLL) cells. GX15-070 also exhibited in vivo antitumor efficacy in murine models of cervical and prostate carcinomas [47]. Phase I clinical trials for the treatment of CLL were initiated with GX15-070 in January 2005 [48].

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