Christopher D. Pond

Thiazoline Peptides and a Tris-Phenethyl Urea from Didemnum molle with Anti-HIV Activity

As part of our screening for anti-HIV agents from marine invertebrates, the MeOH extract of Didemnum molle was tested and showed moderate in vitro anti-HIV activity. Bioassay-guided fractionation of a large-scale extract allowed the identification of two new cyclopeptides, mollamides E and F (1 and 2), and one new tris-phenethyl urea, molleurea A (3). The absolute configurations were established using the advanced Marfeys method. The three compounds were evaluated for anti-HIV activity in both an HIV integrase inhibition assay and a cytoprotective cell-based assay. Compound 2 was active in both assays with IC(50) values of 39 and 78 μM, respectively. Compound 3 was active only in the cytoprotective cell-based assay, with an IC(50) value of 60 μM.

Isolation of pyrrolocins A-C: cis- and trans-decalin tetramic acid antibiotics from an endophytic fungal-derived pathway.

Three new decalin-type tetramic acid analogues, pyrrolocins A (1), B (2), and C (3), were defined as products of a metabolic pathway from a fern endophyte, NRRL 50135, from Papua New Guinea. NRRL 50135 initially produced 1 but ceased its production before chemical or biological evaluation could be completed. Upon transfer of the biosynthetic pathway to a model host, 1–3 were produced. All three compounds are structurally related to equisetin-type compounds, with 1 and 3 having a trans-decalin ring system, while 2 has a cis-fused decalin. All were active against Mycobacterium tuberculosis, with the trans-decalin analogues 1 and 3 exhibiting lower MICs than the cis-decalin analogue 2. Here we report the isolation, structure elucidation, and antimycobacterial activities of 1–3 from the recombinant expression as well as the isolation of 1 from the wild-type fungus NRRL 50135.

4-quinolone alkaloids from melochia odorata

The methanol extract of Melochia odorata yielded three 4-quinolone alkaloids including waltherione A (1) and two new alkaloids, waltherione C (2) and waltherione D (3). Waltheriones A and C showed significant activities in an in vitro anti-HIV cytoprotection assay at concentrations of 56.2 and 0.84 μM and inhibition of HIV P24 formation of more than 50% at 1.7 and 0.95 μM, respectively. The structures of the alkaloids were established by spectroscopic data interpretation.

Structure and activity of lobophorins from a turrid mollusk-associated Streptomyces sp.

A novel lumun-lumun sampling methodology was used to obtain a large diversity of micromollusks, including the new species Lienardia totopotens. In turn, from L. totopotens we cultivated a Streptomyces sp. strain that contained new and known spirotetronate polyketides, lobophorins (1–5). The structures were elucidated using spectroscopy, and the compounds were evaluated for cytotoxicity to human cells and activity against Mycobacterium tuberculosis, Bacillus subtilis, Pseudomonas aeruginosa and Burkholderia cepacia. Compounds 2–5 showed varying degrees of activity against human cells, M. tuberculosis and B. subtilis in the low μM to mid nM range but were inactive against the other strains, while 1 lacking digitoxose was inactive. Very slight structural changes in 2–5 led to varying antibacterial:cytotoxicity ratios, providing a possible basis to synthesize more selective derivatives.

Tetrahdroxysqualene from Rhus taitensis Shows Antimycobacterial Activity against Mycobacterium tuberculosis

Tuberculosis has become a major health problem, in particular with the emergence of extremely drug resistant tuberculosis (XDRTB). In our search for new therapeutic leads against TB, we isolated a new triterpene (1) from the plant Rhus taitensis collected in Papua New Guinea. Tetrahydroxysqualene (1) was isolated using bioassay-guided fractionation of the methanolic extract of R. taitensis leaves and twigs. The structure of tetrahydroxysqualene (1) was elucidated on the basis of HRESIMS and 1D and 2D NMR spectra. Tetrahydroxysqualene (1) exhibited antituberculosis activity with an MIC of 10.0 microg/mL, while showing only modest cytotoxicity.

Effects of mutations in the F361 to R364 region of topoisomerase I (Topo I), in the presence and absence of 9-aminocamptothecin, on the Topo I-DNA interaction

Steady-state levels and rates of DNA binding and release of wild-type and mutant topoisomerase I (Topo I) proteins were quantified by surface plasmon resonance analysis. The proteins were constructed and expressed as GST fusion proteins. The Topo I mutations analyzed were F361S, R362L and R364G, all altering a highly conserved region of wild-type eukaryotic Topo I. The R362L and R364G mutations resulted in much lower steady-state levels of DNA binding than wild-type. This was due to a large increase in the k(d). The F361S mutation increased the steady-state levels of the protein-DNA interaction by increasing the k(a) 2-fold, while having little effect on the k(d). The F361S mutation has been shown to confer resistance to camptothecin and its analogs. The camptothecin analog 9-aminocamptothecin decreased greatly the overall k(d) of the wild-type Topo I, but had little effect on the F361S mutant. Both the wild-type and the F361S mutant exhibited decreased steady-state levels in the presence of the drug, and this was attributable to decreased association.

Accessing chemical diversity from the uncultivated symbionts of small marine animals

Chemistry drives many biological interactions between the microbiota and host animals, yet it is often challenging to identify the chemicals involved. This poses a problem, as such small molecules are excellent sources of potential pharmaceuticals, pretested by nature for animal compatibility. We discovered anti-HIV compounds from small, marine tunicates from the Eastern Fields of Papua New Guinea. Tunicates are a reservoir for novel bioactive chemicals, yet their small size often impedes identification or even detection of the chemicals within. We solved this problem by combining chemistry, metagenomics, and synthetic biology to directly identify and synthesize the natural products. We show that these anti-HIV compounds, the divamides, are a novel family of lanthipeptides produced by symbiotic bacteria living in the tunicate. Neighboring animal colonies contain structurally related divamides that differ starkly in their biological properties, suggesting a role for biosynthetic plasticity in a native context where biological interactions take place.

Identification of a small topoisomerase I–binding peptide that has synergistic antitumor activity with 9-aminocamptothecin

The topoisomerase I (top1)–targeted camptothecin class of anticancer drugs is important in the treatment of several types of cancers. This class of drug inhibits the top1 enzyme during its catalytic DNA relaxation cycle, stabilizing the transient covalent top1-DNA complex by simultaneous noncovalent interactions with DNA and top1. We examined top1 using phage display because of the significance of this known top1-directed drug action. Several peptides that bind top1 were discovered and these were examined for top1 affinity, top1 catalytic and cleavage complex effects, and cytotoxic effects in cultured cell lines and in an in vivo tumor model. Although several peptides exhibited nanomolar and low-micromolar affinity for top1, none had cytotoxic effects when administered alone. However, in combination with 9-aminocamptothecin, one 15-mer peptide (SAYAATVRGPLSSAS) had synergistic cytotoxic effects with 9-aminocamptothecin both in the cytotoxicity assay and in nude mouse xenograft human tumor models. This report details the investigation of this peptide. [Mol Cancer Ther 2006;5(3):739–45]

Making the most of Papua New Guinea’s biodiversity: Establishment of an integrated set of programs that link botanical survey with pharmacological assessment in “the land of the unexpected”

An integrated and coordinated set of programs has been established to meet International Cooperative Biodiversity Group (ICBG) goals in Papua New Guinea (PNG). Here we give an overview of the PNG ICBG and focus on the key elements and major steps taken to establish a program necessary for the pharmacological assessment of botanicals and traditional medicines in PNG and, by extrapolation, in other developing countries.

Accumulation, Activation and Interindividual Variation of the Epidermal TP53 Protein in Response to Ionizing Radiation in Organ Cultured Human Skin

Abstract Pond, C. D., Leachman, S. A. and Warters, R. L. Accumulation, Activation and Interindividual Variation of the Epidermal TP53 Protein in Response to Ionizing Radiation in Organ Cultured Human Skin. Radiat. Res. 161, 739–745 (2004). In this study, we examined effects of low-dose ionizing radiation on organ cultured human foreskin and, in particular, on the epidermis. Diagnostic, therapeutic, natural environmental and incidental exposures to moderate to low doses of radiation are inevitable and, although information on cultured cells continues to accumulate, little is known about the effects of low-dose radiation on human tissues. Our hypothesis is that ex vivo organ cultured foreskin is a simple and reliable model to study the biochemical effects of low-dose radiation exposure on skin. A model such as this will aid in the identification and quantification of low-dose radiation-induced changes in proteins in human skin and may be useful in the development of a precise, non-invasive, and reliable assay of exposure. In this work, several aspects of skin responses to culture conditions and radiation were examined. The responses of epidermal TP53 from organ cultured skin irradiated in medium with and without serum were found to be similar. TP53 levels in organ cultured neonatal foreskin epidermis were then examined for baseline TP53 expression. After an initial increase at 4 h, the TP53 D01 signal returned to low steady-state levels for at least 72 h. Irradiated skin samples from different individuals revealed variations in the TP53 D01 signal. The dose and temporal response of dermis and epidermis to radiation were examined by Western blotting from 0 to 24 h after exposure. After irradiation and incubation, the epidermis was removed and assayed by Western blotting and was found to have increases in the TP53 D01 epitope and the TP53 phosphoserine 15 (TP53-S15p) epitope that reached a maximum at about 3 h. In the epidermis, doses of 1–5 cGy of radiation were detectable with the TP53 D01, and CDKN1A antibodies and doses greater than 10 cGy were detectable with the TP53-S15p antibody. When the dermis was compared to epidermis, it was found that dermis had a smaller response to radiation and more phosphorylated TP53.