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Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B 1 , manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC 50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1α protein accumulation without affecting HIF-1α mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors.

The degree of tumor hypoxia correlates with advanced disease stages and treatment resistance. The transcription factor hypoxia-inducible factor-1 (HIF-1) promotes tumor cell adaptation and survival under hypoxic conditions. Therefore, specific HIF-1 inhibitors represent an important new class of potential tumor-selective therapeutic agents. A T47D human breast tumor cell-based reporter assay was used to examine extracts of plants and marine organisms for inhibitors of HIF-1 activation. Bioassay-guided fractionation of the lipid extract of the red alga Laurencia intricata yielded a structurally novel diterpene laurenditerpenol (1). The structure of 1 was determined spectroscopically. The relative configurations of the substituents of each ring system were assigned based on NOESY correlations. The absolute configurations of positions C-1 was determined by the modified Mosher ester procedure (directly in NMR tubes). Compound 1 potently inhibited hypoxia-activated HIF-1 (IC 50 : 0.4 μM) and hypoxia-induced VEGF (a potent angiogenic factor) in T47D cells. Compound 1 selectively inhibits HIF-1 activation by hypoxia but not iron chelator induced activation. Further, 1 suppresses tumor cell survival under hypoxic conditions without affecting normoxic cell growth. Compound 1 inhibits HIF-1 by blocking the induction of the oxygen-regulated HIF-1α protein. Mitochondrial respiration studies revealed that 1 suppresses oxygen consumption.Rapid tumor growth outstrips the capability of existing blood vessels to supply oxygen and nutrients. As a result, a common feature of solid tumors is the existence of hypoxic regions. Tumor hypoxia directly correlates with advanced disease stages and poor prognosis. 1 Unlike normal cells from the same tissue, tumor cells are often chronically hypoxic. Hypoxic tumor cells are more resistant than normoxic tumor cells to radiation treatment and chemotherapy and these hypoxic tumor cells are considered an important contributor to disease relapse. 1 Current approaches to overcome tumor hypoxia target the direct effects of hypoxia -lack of cellular oxygen. 1 Presently, there is no drug that specifically targets hypoxic tumor cells. Recent results from clinical studies on tirapazamine (a bioreductive drug that selectively kill hypoxic tumor cells) have indicated the significant potential of drugs that target tumor hypoxia. 2,3 Our focus is to discover new drug leads that target the important indirect effect * Joint Corresponding Authors to whom correspondence should be addressed. In order to discover HIF-1 functional antagonists, we have established a T47D human breast tumor cell-based reporter assay in a 96-well plate format. The activity of HIF-1 is monitored using a luciferase reporter gene under the control of HRE from the erythropoietin gene (pTK-HRE3-luc). 9 Natural product-rich extracts of terrestrial and marine organisms were examined for inhibitors of hypoxia-induced HIF-1 activation. The lipid extract of a Jamaican sample of the red alga Laurencia intricata Lamouroux (Rhodomelac...

Theopapuamide (1), a new cytotoxic peptide has been isolated from the lithistid sponge Theonella swinhoei from Papua New Guinea. The structure was established by analysis of NMR, mass spectrometry and chemical methods. The undecapeptide (1) contains several unusual amino acid residues, of which the occurrence of β-methoxyasparagine and 4-amino-5-methyl-2,3,5-trihydroxyhexanoic acid (Amtha) is unprecedented in natural peptides. Compound 1 also contains a amide linked fatty acid moiety, 3-hydroxy-2,4,6-trimethyl-octanoic acid (Htoa). Theopapuamide (1) was cytotoxic against CEM-TART and HCT-116 cell lines with EC 50 values of 0.5 μM and 0.9 μM respectively.Marine sponges have proven to be a significant source of biologically active cyclic peptides and depsipeptides. 1 Among these sponge depsipeptides, callipeltin A (from New Caledonian sponge Callipelta sp.), 2 neamphamide A (from a Papua New Guinea sponge Neamphius huxleyi) 3 and Papuamides A-B (from Papua New Guinea sponges Theonella mirabilis and Theonella swinhoei) 4 are well known for their potent HIV-inhibitory activity and their structurally unique features incorporating several modified amino acid residues. For instance, the atypical amino acid residues 3,4-dimethyl-L-glutamine and β-methoxytyrosine are common to all of the above mentioned marine depsipeptides but to date have not been described elsewhere. The rarity of these atypical amino acid residues has inspired their chemical synthesis. 5 The structural diversity found among lithistid sponge metabolites (genus Theonella and Callipelta) has been attributed to symbiotic microorganisms. 1 As part of our continuing studies on Theonella swinhoei from Papua New Guinea, 6 the aqueous CH 3 CN extract of the sponge was analyzed and proved active in an in vitro anti-HIV assay. Fractionation of the active extract resulted in the isolation of a new cyclic depsipeptide, theopapuamide (1). This paper describes the isolation, structure elucidation, and stereochemical analysis of theopapuamide (1).The crude aqueous CH 3 CN extract of T. swinhoei (family Theonellidae) was concentrated under vacuum and fractionated by C18 flash column chromatography. Further purification on Diaion HP-20 resin followed by CN-HPLC afforded the new cyclic undecapeptide, *To whom correspondence should be addressed. Tel: (801) In conjunction with NMR analysis, the gross structure elucidation of 1 was guided by standard amino acid analysis, 9 which revealed molar concentrations of ~1:2:1 for Asx, Thr and Leu respectively. The presence of two N-methylated amino acid residues were suggested based on the characteristic 1 H and 13 C chemical shifts of the N-methyl groups at δ H 2.81 (δ C 30.8) and δ H 2.88 (δ C 31.8). HMBC correlations were used to identify these N-methylated amino acids as NMeLeu and NMeGln, respectively. Additionally, the presence of a methoxy-bearing amino acid residue was suggested by the characteristic 1 H and 13 C chemical shifts of the O-methyl group at δ H 3.34 (δ C 60.3). Based on HMBC correlations, the methoxy...