Hendrik Luesch

Structure and Activity of Largazole, a Potent Antiproliferative Agent from the Floridian Marine Cyanobacterium Symploca Sp

A novel cytotoxic cyclodepsipeptide, termed largazole (1), has been isolated from the marine cyanobacterium Symploca sp. collected in the Florida Keys. Its planar structure was elucidated by 1D and 2D NMR spectroscopy in conjunction with mass spectrometry. The absolute configuration of 1 was determined by chemical degradation followed by chiral HPLC analysis. Largazole (1) possesses densely assembled unusual structural features, including a rare 4-methylthiazoline linearly fused to a thiazole in its cyclic core and a hitherto undescribed 3-hydroxy-7-mercaptohept-4-enoic acid unit incorporated in an ester, thioester, and amide framework. Largazole (1) exhibits potent antiproliferative activity and preferentially targets cancer cells over nontransformed cells.

Total Synthesis and Molecular Target of Largazole, a Histone Deacetylase Inhibitor

Full details of the concise and convergent synthesis (eight steps, 19% overall yield), its extension to the preparation of a series of key analogues, and the molecular target and pharmacophore of largazole are described. Central to the synthesis of largazole is a macrocyclization reaction for formation of the strained 16-membered depsipeptide core followed by an olefin cross-metathesis reaction for installation of the thioester. The biological evaluation of largazole and its key analogues, including an acetyl analogue, a thiol analogue, and a hydroxyl analogue, suggested that histone deacetylases (HDACs) are molecular targets of largazole and largazole is a class I HDAC inhibitor. In addition, structure-activity relationship (SAR) studies revealed that the thiol group is the pharmacophore of the natural product. Largazoles HDAC inhibitory activity correlates with its antiproliferative activity.

A genomic screen for activators of the antioxidant response element

The antioxidant response element (ARE) is a cis-acting regulatory enhancer element found in the 5′ flanking region of many phase II detoxification enzymes. Up-regulation of ARE-dependent target genes is known to have neuroprotective effects; yet, the mechanism of activation is largely unknown. By screening an arrayed collection of ≈15,000 full-length expression cDNAs in the human neuroblastoma cell line IMR-32 with an ARE-luciferase reporter, we have identified several cDNAs not previously associated with ARE activation. A subset of cDNAs, encoding sequestosome 1 (SQSTM1) and dipeptidylpeptidase 3 (DPP3), activated the ARE in primary mouse-derived cortical neurons. Overexpression of SQSTM1 and DPP3 in IMR-32 cells stimulated NF-E2-related factor 2 (NRF2) nuclear translocation and led to increased levels of NAD(P)H:quinone oxidoreductase 1, a protein which is transcriptionally regulated by the ARE. When transfected into IMR-32 neuroblastoma cells that were depleted of transcription factor NRF2 by RNA interference, SQSTM1 and DPP3 were unable to activate the ARE or induce NAD(P)H:quinone oxidoreductase 1 expression, indicating that the ARE activation upon ectopic expression of these cDNAs is mediated by NRF2. Studies with pharmacological inhibitors indicated that 1-phosphatidylinositol 3-kinase and protein kinase C signaling are essential for activity. Overexpression of these cDNAs conferred partial resistance to hydrogen peroxide or rotenone-induced toxicity, consistent with the induction of antioxidant and phase II detoxification enzymes, which can protect from oxidative stress. This work and other such studies may provide mechanisms for activating the ARE in the absence of general oxidative stress and a yet-unexploited therapeutic approach to degenerative diseases and aging.

Telomere-independent Rap1 is an IKK adaptor and regulates NF-κB-dependent gene expression

We describe a genome-wide gain-of-function screen for regulators of NF-κB, and identify Rap1 (Trf2IP), as an essential modulator of NF-κB-mediated pathways. NF-κB is induced by ectopic expression of Rap1, whereas its activity is inhibited by Rap1 depletion. In addition to localizing on telomeres, mammalian Rap1 forms a complex with IKKs (IκB kinases), and is crucial for the ability of IKKs to be recruited to, and phosphorylate, the p65 subunit of NF-κB to make it transcriptionally competent. Rap1-mutant mice display defective NF-κB activation and are resistant to endotoxic shock. Furthermore, levels of Rap1 are positively regulated by NF-κB, and human breast cancers with NF-κB hyperactivity show elevated levels of cytoplasmic Rap1. Similar to inhibiting NF-κB, knockdown of Rap1 sensitizes breast cancer cells to apoptosis. These results identify the first cytoplasmic role of Rap1 and provide a mechanism through which it regulates an important signalling cascade in mammals, independent of its ability to regulate telomere function.

Apratoxin A reversibly inhibits the secretory pathway by preventing cotranslational translocation

Apratoxin A is a potent cytotoxic marine natural product that rapidly inhibits signal transducer and activator of transcription (STAT) 3 phosphorylation by an undefined mechanism. We have used biochemical and proteomics approaches to illuminate upstream molecular events. Apratoxin A inhibits Janus kinase (JAK)/STAT signaling through rapid down-regulation of interleukin 6 signal transducer (gp130). Apratoxin A also depletes cancer cells of several cancer-associated receptor tyrosine kinases by preventing their N-glycosylation, leading to their rapid proteasomal degradation. A proteomics approach revealed that several proteins in the endoplasmic reticulum, the site of N-glycoprotein synthesis, are down-regulated upon apratoxin A exposure. Using in vitro cell free systems, we demonstrated that apratoxin A prevents cotranslational translocation of proteins destined for the secretory pathway. This process is reversible in living cells. Our study indicates that apratoxins are new tools to study the secretory pathway and raises the possibility that inhibition of cotranslational translocation may be exploited for anticancer drug development.

Apratoxin E, a Cytotoxic Peptolide from a Guamanian Collection of the Marine Cyanobacterium Lyngbya bouillonii

A collection of the marine cyanobacterium Lyngbya bouillonii from Guam afforded apratoxin E (1), a new peptide-polyketide hybrid of the apratoxin class of cytotoxins. The planar structure of 1 was elucidated by NMR spectroscopic analysis and mass spectrometry. Configurational assignments of stereocenters in the peptide portion were made by chiral HPLC analysis of the acid hydrolysate. The relative configuration in the polyketide moiety was assigned by comparison of NMR data including proton-proton coupling constants with those of the known analogues. Apratoxin E (1) displayed strong cytotoxicity against several cancer cell lines derived from colon, cervix, and bone, ranging from 21 to 72 nM, suggesting that the alpha,beta-unsaturation of the modified cysteine residue is not essential for apratoxin activity. The 5- to 15-fold reduced activity compared with apratoxin A (2) is attributed to the dehydration in the long-chain polyketide unit, which could affect the conformation of the molecule.

Largazole: From Discovery to Broad-Spectrum Therapy

The cyclic depsipeptide largazole from a cyanobacterium of the genus Symploca is a marine natural product with a novel chemical scaffold and potently inhibits class I histone deacetylases (HDACs). Largazole possesses highly differential growth-inhibitory activity, preferentially targeting transformed over non-transformed cells. The intriguing structure and biological activity of largazole have attracted strong interest from the synthetic chemistry community to establish synthetic routes to largazole and to investigate its potential as a cancer therapeutic. This Highlight surveys recent advances in this area with a focus on the discovery, synthesis, target identification, structure-activity relationships, HDAC8-largazole thiol crystal structure, and biological studies, including in vivo anticancer and osteogenic activities.

Isolation and biological evaluation of 8-epi-malyngamide C from the Floridian marine cyanobacterium Lyngbya majuscula.

A new stereoisomer of malyngamide C, 8-epi-malyngamide C (1), and the known compound lyngbic acid [(4E,7S)-7-methoxytetradec-4-enoic acid] were isolated from a sample of Lyngbya majuscula collected near Bush Key, Dry Tortugas, Florida. The structure of 1 was determined by NMR and MS experiments. The absolute configuration of 1 was determined by selective Mitsunobu inversion of C-8 to give malyngamide C, as determined by NMR, MS, and comparison of specific rotation. Both 1 and malyngamide C were found to be cytotoxic to HT29 colon cancer cells (IC(50) 15.4 and 5.2 microM, respectively) and to inhibit bacterial quorum sensing in a reporter gene assay.

Anticolon Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor

Histone deacetylases (HDACs) are validated targets for anticancer therapy as attested by the approval of suberoylanilide hydroxamic acid (SAHA) and romidepsin (FK228) for treating cutaneous T cell lymphoma. We recently described the bioassay-guided isolation, structure determination, synthesis, and target identification of largazole, a marine-derived antiproliferative natural product that is a prodrug that releases a potent HDAC inhibitor, largazole thiol. Here, we characterize the anticancer activity of largazole by using in vitro and in vivo cancer models. Screening against the National Cancer Institutes 60 cell lines revealed that largazole is particularly active against several colon cancer cell types. Consequently, we tested largazole, along with several synthetic analogs, for HDAC inhibition in human HCT116 colon cancer cells. Enzyme inhibition strongly correlated with the growth inhibitory effects, and differential activity of largazole analogs was rationalized by molecular docking to an HDAC1 homology model. Comparative genomewide transcript profiling revealed a close overlap of genes that are regulated by largazole, FK228, and SAHA. Several of these genes can be related to largazoles ability to induce cell cycle arrest and apoptosis. Stability studies suggested reasonable bioavailability of the active species, largazole thiol. We established that largazole inhibits HDACs in tumor tissue in vivo by using a human HCT116 xenograft mouse model. Largazole strongly stimulated histone hyperacetylation in the tumor, showed efficacy in inhibiting tumor growth, and induced apoptosis in the tumor. This effect probably is mediated by the modulation of levels of cell cycle regulators, antagonism of the AKT pathway through insulin receptor substrate 1 down-regulation, and reduction of epidermal growth factor receptor levels.

Synthesis and activity of largazole analogues with linker and macrocycle modification.

To characterize largazoles structural requirements for histone deacetylase (HDAC) inhibitory and antiproliferative activities, a series of analogues with modifications to the side chain or 16-membered macrocycle were prepared and biologically evaluated. Structure-activity relationships suggested that the four-atom linker between the macrocycle and octanoyl group in the side chain and the (S)-configuration at the C17 position are critical to repression of HDAC activity. However, the valine residue in the macrocycle can be replaced with alanine without significant loss of activity.