Kirk Gustafson

Two new HIV-inhibitory depsipeptides, stellettapeptins A (1) and B (2), were isolated from an extract of the marine sponge Stelletta sp., collected from northwestern Australia. Structures of these cyclic nonribosomal peptides were elucidated on the basis of extensive NMR data analysis, and chemical degradation and derivatization studies. Stellettapeptins contain numerous nonproteinogenic amino acid residues and they are the first peptides reported to contain a 3-hydroxy-6,8-dimethylnon-4-(Z)-enoic acid moiety. Compounds 1 and 2 potently inhibit infection of human T-lymphoblastoid cells by HIV-1RF with EC50 values of 23 and 27 nM, respectively.

The molecular formula of monocillinol A (1), established as C 11 H 13 NO 5 by HR-FABMS measurements, required six double bond equivalents in the molecule. IR absorption bands at ν 1724 and 1667 cm −1 suggested the presence of α,β-unsaturated ester and amide ...

... TERPENOIC ACID GLYCERIDES FROM THE DORID NIJDIBRANCH ARCHIDORIS MONTEREYENSIS Kirk Gustafson and Raymond J. Andersen* Marie HM Chen and Jon Clardy ... The authors would also like to acknowledge financial assistance to Dr. DJ Faulkner. ...

Page 1. J. Am. Chem. SOC. 1994,116, 9337-9338 9337 Table 1. Amino Acid Analysis of Circulins A and B Circulins A and B Novel HIV-Inhibitory Macrocyclic Peptides from the Tropical Tree Chassalia parvifofial Kirk R. Gustafson, Raymond C. Sowder II,t Louis E. Henderson? ...

Larvae of the marine bryozoan Bugula neritina are defended against potential predators by high concentrations of bryostatins, which are produced by a bacterial symbiont of the bryozoan. From the larvae of B. neritina, three bryostatins, bryostatin 10 (1), the novel bryostatin 20 (2), and an as yet uncharacterized bryostatin, were isolated that were unpalatable to fish. These deterrent bryostatins represent the first example from the marine environment of a microbial symbiont producing an antipredator defense for its host. The structure of bryostatin 20 (2) was determined by spectral comparison with previously described bryostatins.

Four new macrocyclic polypeptides were isolated and identified from an extract of the tropical tree Chassalia parvifolia. Circulins C-F are 29-30 amino acid cyclic peptides in which the entire primary amino acid chain is covalently cyclized via peptide bonds. Their structures were deduced from a combination of FABMS analyses, N-terminal Edman degradation, endoproteinase digestion, and amino acid analyses. All the peptides share a high degree of sequence homology and contain six cysteine residues forming three intramolecular disulfide bridges. Circulins C-F inhibited the cytopathic effects of in vitro HIV-1 infection with EC(50) values of 50-275 nM.

... (4) Gustafson, KR; Cardellina, JH, 11 ... J. Nat. Prod. 1992,55, 207-213. (7) Gustafson, KR; Cardellina, JH, 11; McMahon, JB; Pan-nell, LK; Cragg, GM; Boyd, MR The peltatoh, novel HIV inhibitory catechol derivatives from Pothomorphe peltata. J. Org. Chem. 1992,57,2809-2811. ...

... 3. Ochtodene p=.013 0 t_) SQ-g^ e|? ~~ ^ tO Q 1 0 8 l 0 0 0: Q: 0 8 Fig. ... Ochtodes does not grow on Guam and is not reported from any of the islands of Micronesia (Tsuda & Wray, 1977). ... Ecol. Monogr., Vol. 56, pp. 343-363. COLEY, PD, JP BRYANT & FS CHAPIN III, 1985. ...

Cyclotides are disulfide rich macrocyclic plant peptides that are defined by their unique topology in which a head-to-tail cyclized backbone is knotted by the interlocking arrangement of three disulfide bonds. This cyclic cystine knot motif gives the cyclotides exceptional resistance to thermal, chemical, or enzymatic degradation. Over 100 cyclotides have been reported and display a variety of biological activities, including a cytoprotective effect against HIV infected cells. It has been hypothesized that cyclotides from one subfamily, the Möbius subfamily, may be more appropriate than bracelet cyclotides as drug candidates given their lower toxicity to uninfected cells. Here, we report the anti-HIV and cytotoxic effects of three cyclotides, including two from the Möbius subfamily. We show that Möbius cyclotides have comparable inhibitory activity against HIV infection to bracelet cyclotides and that they are generally less cytotoxic to the target cells. To explore the structure activity relationships (SARs) of the 29 cyclotides tested so far for anti-HIV activity, we modeled the structures of the 21 cyclotides whose structures have not been previously solved. We show that within cyclotide subfamilies there is a correlation between hydrophobicity of certain loop regions and HIV inhibition. We also show that charged residues in these loops impact on the activity of the cyclotides, presumably by modulating membrane binding. In addition to providing new SAR data, this report is a mini-review that collates all cyclotide anti-HIV information reported so far and provides a resource for future studies on the therapeutic potential of cyclotides as natural anti-HIV agents.

Deregulation of the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR)-70kDa ribosomal protein S6 kinase 1 (p70(S6K)) pathway is commonly observed in many tumors. This pathway controls proliferation, survival, and translation, and its overactivation is associated with poor prognosis for tumor-associated survival. Current efforts focus on the development of novel inhibitors of this pathway. In a cell-based high-throughput screening assay of 15,272 pure natural compounds, we identified pomiferin triacetate as a potent stabilizer of the tumor suppressor programmed cell death 4 (Pdcd4). Mechanistically, pomiferin triacetate appeared as a general inhibitor of the PI3K-Akt-mTOR-p70(S6K) cascade. Interference with this pathway occurred downstream of Akt but upstream of p70(S6K). Specifically, mTOR kinase emerged as the molecular target of pomiferin triacetate, with similar activities against mTOR complexes 1 and 2. In an in vitro mTOR kinase assay pomiferin triacetate dose-dependently inhibited mTOR with an IC50 of 6.2 μM. Molecular docking studies supported the interaction of the inhibitor with the catalytic site of mTOR. Importantly, pomiferin triacetate appeared to be highly selective for mTOR compared to a panel of 17 lipid and 50 protein kinases tested. As a consequence of the mTOR inhibition, pomiferin triacetate efficiently attenuated translation. In summary, pomiferin triacetate emerged as a novel and highly specific mTOR inhibitor with strong translation inhibitory effects. Thus, it might be an interesting lead structure for the development of mTOR- and translation-targeted anti-tumor therapies.

A novel anti-HIV protein, cyanovirin-N (CV-N), was isolated from an aqueous cellular extract of the cultured cyanobacterium (blue-green alga) Nostoc ellipsosporum, purified by reverse-phase HPLC, and sequenced by N-terminal Edman degradation of the intact protein and peptide fragments produced by endoproteinase digestions. CV-N consists of a single 101 amino acid chain which exhibits significant internal sequence duplication, but no significant homology to previously described proteins or to the transcription products of known nucleotide sequences. Alignment of residues 1-50 with residues 51-101 reveals 13 conservative amino acid changes as well as direct homology between 16 amino acid residues. CV-N contains four cysteines which form two intrachain disulfide bonds. The positions of the disulfide linkages were established by fast atom bombardment mass spectral studies of peptide fragments generated by a tryptic digestion of the native protein. Reductive cleavage of these crosslinks resulted in loss of anti-HIV activity.

Site-directed mutagenesis of DNA constructs coding for the novel, HIV-inactivating proteins cyanovirin-N (CV-N) and FLAG-cyanovirin-N (F-CV-N) was performed using mutagenic oligonucleotide primers in the polymerase chain reaction or by a restriction site elimination maneuver. The mutant constructs were expressed in Escherichia coli and the recombinant protein products were tested for binding to the HIV surface envelope glycoprotein gp 120 and for antiviral activity against infectious HIV. Results showed an overall very high correlation (r2 > 0.9) between the relative gp120 binding affinities and the anti-HIV activities of CV-N, F-CV-N, and the various mutants. An outlier, however, was a mutant which lacked one of the internal disulfide linkages normally present in CV-N and which showed modest gp120 binding but no antiviral activity against HIV. These findings are consistent with the view that gp120 binding is a necessary but not sufficient requirement for the HIV-inactivating activity of CV-N and related proteins; the sequence specificities for gp120 binding and anti-HIV activity are not identical.

In previous work, botryllamides discovered from the marine ascidian Botryllus tyreus were characterized as selective inhibitors of the ABCG2 multidrug transporter. However, the structural basis for this activity could not be established. In this study, botryllamide F, the core botryllamide structure, and botryllamide G, the most potent botryllamide ABCG2 inhibitor, were synthesized along with a series of structural variants for

Cyanovirin-N (CV-N), an 11-kDa cyanobacterial protein, potently inactivates diverse strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and also prevents virus-to-cell fusion, virus entry, and infection of cells in vitro. These properties make CV-N an attractive candidate for use as a topical microbicide to prevent the sexual transmission of HIV. We evaluated the efficacy of gel-formulated, recombinant CV-N gel asa topical microbicide in male macaques (Macaca fascicularis) that were rectally challenged with a chimeric SIV/HIV-1 virus known as SHIV89.6P. All of the untreated macaques were infected and experienced CD4+T cell depletion. In contrast, none of the macaques that received either 1% or 2% CV-N gel showed evidence of SHIV89.6P infection. Neither CV-N nor placebo gels produced any adverse effects in any macaque following the rectal application. These results indicate that CV-N gel as a topical microbicide can prevent rectal transmission of SHIV in macaques. These studies encourage clinical evaluation of CV-N as a topical microbicide to prevent sexual transmission of HIV in humans.