Potent Cytotoxic Peptides from the Australian Marine Sponge Pipestela candelabra

Two consecutive prefractionated fractions of the Australian marine sponge extract, Pipestela candelabra, were identified to be selectively active on the human prostate cancer cells (PC3) compared to the human neonatal foreskin fibroblast non-cancer cells (NFF). Twelve secondary metabolites were isolated in which four compounds are new small peptides. Their structures were characterized by spectroscopic and chemical analysis. These compounds inhibited selectively the growth of prostate cancer cells with IC50 values in the picomolar to sub-micromolar range. Structure-activity relationship of these compounds is discussed.


Introduction
Pipestela has been characterized as a new sponge genus sharing some shape features and molecular DNA sequences with ten other genera in the family Axinellidae: Auletta, Axinella, Cymbastela, Dragmacidon, Dragmaxia, Pararhaphoxya, Phakellia, Phycopsis, Ptilocaulis, and Reniochalina [1]. The genus Pipestela is distributed only across the north-eastern region of Australia, and is very common from the Great Barrier Reef, Coral Sea, Papua New Guinea, Solomon Islands, and Vanuatu [2]. Chemical investigation of the sponge P. candelabra was first reported in 2012 with the identification OPEN ACCESS of four cyclodepsipeptides, including jaspamide and pipestelides A-C, from a sample collected in the Solomon Islands [3]. However, a different structure class, hemiasterlin and milnamide analogues, was found in this chemical study on the Australian sponge P. candelabra.
Hemiasterlins and milnamides are two related small cytotoxic peptide families from marine sponges. Their structural features contain a dipeptide side chain of two unnatural amino acid residues, tert-leucine and N-methylvinylogous valine. This dipeptide fragment is incorporated with another atypical amino acid residue tri-or tetra-methylated tryptophan to produce the hemiasterlins or connected to a tetrahydro-β-carboline residue to form the milnamides. So far, six hemiasterlin derivatives (hemiasterlin or milnamide B [4], hemiasterlin A [5,6], hemiasterlin B [5], hemiasterlin C [6], crinamide A [5] and crinamide B [5]), and three milnamide analogues (milnamide A [7], milnamide C [8] and milnamide D [8,9]) have been identified from four sponge genera including Auletta [6,7], Cymbastela [5,9], Hemiasterella [4], and Siphonochalina [6]. These related natural products show potent cytotoxicity against many cancer cell lines, such as murine leukemia P388, human breast cancer MCF7, human glioblastoma/astrocytoma U373, human ovarian cancer HEY, human colon cancers LOVO and HT-29, human lung cancer A549, and murine melanoma B16-F10 [5,7]. In addition, hemiasterlin was a more potent in vitro cytotoxin and antimitotic agent than either of the anticancer drugs taxol or vincristine [10]. This made it stand out as an extremely exciting natural product lead structure for an anticancer drug development program. Many synthetic methodologies have been developed to produce a large number of hemiasterlin analogues [10][11][12][13]. From structure-activity relationship studies, the tert-leucine and N-methylvinylogous valine residues were found to be essential for bioactivity [10]. In 2003, the first hemiasterlin analogue HTI-286 (13) was selected for evaluation in a phase I clinical trial in patients with advanced solid tumors [10,14]. A phase II trial of HTI-286 has been halted since there were no objective responses and common toxicities observed included neutropaenia, nausea, alopecia, and pain [15]. However, there remains a particular interest to develop and examine the therapeutic effect of novel hemiasterlin derivatives. In 2009, another hemiasterlin analogue E7974 (14) with the N-isopropyl-D-pipecolic acid substituent replacing the N-methyltryptophan residue showed strong in vivo antitumor efficacy in many human xenograft cancer models and overcame a second mechanism of drug resistance present in cancer cells [16]. Results from phase I clinical trial have recently revealed that the compound E7974 remains a promising candidate for the treatment of several forms of advanced solid tumors in colorectal, pancreatic and liposarcoma cancer patients [17]. Human clinical testing of E7974 is currently ongoing [17].

Results and Discussion
Chemical degradation and subsequent Marfey's amino acid analysis [25][26][27] revealed that all new compounds 1-3 shared the same L configuration of the tert-leucine residue with other known milnamides and hemiasterlines (Supplementary Figure S23). Structurally, milnamides E-G (1-3) differ from milnamides A, C, and D (5-7) only by the lack of the methyl substituent at N-9. Absolute configurations of milnamides A, C, and D were previously determined by synthesis or X-ray diffraction analysis [8,24]. Therefore, it has been well established that absolute configurations of the new milnamides E-G (1-3) can be assigned by comparing the signs of Cotton effects (CEs) and specific optical rotations [5,8]. This is the first report for the milnamides in which there is an absence of a methyl group on the indolic part. These new milnamides E-G (1-3) were only identified from the new genus Pipestela candelabra while other milnamides A (5), C (6), and D (7) with a methyl group on the indolic part have been reported from other genera Auletta sp. [7,8], Cymbastela sp. [9], and also from the P. candelabra in this study.
Compound 4 was obtained as a white amorphous solid. The (+)-HRESIMS showed a signal [M + H] + ([C 46 H 73 N 6 O 9 ] + ) at m/z 853.5470 indicating the molecular formula C 46 H 72 N 6 O 9 . NMR analysis revealed that this compound contained six residues including one trimethylated tryptophan (tt), two tert-leucines (tert-Leu), two N-methylvinylogous valines (mvv) and one 2-hydroxyacetic acid (haa). HMBC correlations and ROESY correlations allowed establishing a tripeptide substructure A (tt-tert-Leu-mvv) ( Figure 5), which was identical to the tripeptide structure of hemiasterlin (8) and hemiasterlin A (9). The remaining atoms in 4 were in a side chain B whose sequence (haa-tert-Leu-mvv) was confirmed by HMBC correlations from H-30 (δ H 4.73 ppm) to C-28 (δ C 167.6 ppm), H-38 (δ H 2.94 ppm) to C-31 (δ C 170.3 ppm) ( Figure 5). The observation of HMBC correlations from H-27 (δ H 6.34 ppm) to C-2 (δ C 125.2 ppm) and C-9 (δ C 136.5 ppm) supported the linkage between A and B at an indole nitrogen N-1 and C-27 ( Figure 5). This connection was supported by ROESY correlations from H-27 to H-2 (δ H 7.20 ppm) and H-8 (δ H 7.54 ppm). A planar structure of a new hemiasterlin, hemiasterlin D (4), was determined as in Figure 5. The presence of two L-tert-Leu residues in 4 was established since only a single peak corresponding to L-tert-Leu was observed in the ion mass extraction chromatogram of its Marfey's derivatives (Supplementary Figure S23). Compound 4 showed the same sign of optical rotation with our isolated hemiasterlin (8) [6]) (literature did not report the optical value of hemiasterlin B [5]). On the basis of biogenetic considerations, the two N-methylvinylogous valines and the trimethylated tryptophan residue of 4 were proposed to have an S-configuration as those in the other hemiasterlin analogues. Hemiasterlin D is the first hemiasterlin with a peptide side chain containing 2-hydroxyacetic acid, tert-leucine and N-methylvinylogous valine residues at N-1.
Antiproliferative effect of isolated compounds was evaluated against the human prostate cancer cell line (PC3) and the human neonatal foreskin fibroblast non-cancer cell line (NFF). All compounds showed potent inhibition against prostate cancer cells with IC 50 values from the picomolar to sub-micromolar range ( Table 2). Selective indexes between PC3 cells and NFF cells were from 2.6 to 8.3 indicating these compounds had some selective activity towards prostate cancer cells. Cytotoxic results also indicated that breaking the β-carboline system in milnamides to form the tryptophan system in hemiasterlins increased cytotoxicity.
In the milnamide family, milnamide A (5) exhibited the strongest activity (IC 50 = 11.0 nM) and was 3-fold more potent than milnamide C (6) and 35-fold more potent than milnamide C (7). A similar trend was observed in the activity of milnamide E to G (1-3). The results indicated that replacing the methylene at C-1 with a carbonyl or an imine led to a decrease in cytotoxicity. Losing the N-methyl at N-9 in the β-carboline ring converting 5 to 1, 6 to 2, and 7 to 3 also reduced the activity. These data suggested that the N-methyl at N-9 might contribute to the milnamide cytotoxicity. Compared with milnamides (1-3 and 5-7), hemiasterlins (4, 8, and 9) demonstrated much more potent cytotoxicity and showed higher selectivity towards noncancer cells. Their activity against the PC3 cells was in the picomolar to sub-nanomolar range. In the examination of hemiasterlins, Andersen et al. [10] found that the N-methyl indole had better activity than the NH indole. Here a methyl at the indole nitrogen N-1 in hemiasterlin (8) was found six-fold more cytotoxic than an NH indole in hemiastermin A (9). This cytotoxicity result was comparable with those reported for other cancer cell lines [5,6]. In our study, the new compound, hemiasterlin D (4) showed inhibition with IC 50 values of 2.20 and 8.16 nM for PC3 and NFF cells, respectively. Although the new hemiasterlin D (4) here was less potent than the known analogues, the results showed that cytotoxicity was maintained when replacing the methyl of the N-methylindole in the tetramethyltryptophan residue with a long side chain. This result may offer a new position at N-1 for hemiasterlin to be developed for more selective activity and better pharmaceutical properties.
The three depsipeptides, geodiamolides D-F (10-12), which were found co-occurring with the milnamides and hemiasterlins, also displayed strong cytotoxic activity against the PC3 cells and showed 3-to 5-fold selectivity with the NFF cells. Geodiamolide D (10) showed the strongest activity with an IC 50 value of 33.1 nM, which was 4-fold more potent than geodiamolide E (11) and 5-fold more active than geodiamolide F (12). This data indicated that there was a difference in cytotoxicity due to the halogen atoms attached to the geodiamolide skeleton. The cytotoxic trend of geodiamolides D-F (10-12) against PC3 cells decreased from iodine to bromine and chlorine. This trend in the human PC3 cell line was totally different from the previous cytotoxic trend reported for these compounds in the murine leukemia L1210 cell line (IC 50 values of 62.2, 24.2, and 11.2 nM for geodiamolides D, E and F, respectively) [19]. How these halogen atoms influence cytotoxicity is something that warrants further investigation.

General Experimental Procedures
Optical rotations were measured on JASCO P-1020 polarimeter (10 cm cell). Circular dichroism spectra were measured on JASCO J-715 Spectropolarimeter Circular Dichroism/Optical Rotatory Dispersion. UV spectra were recorded on a CAMSPEC M501 UV/VIS spectrophotometer. NMR spectra were recorded at 30 °C on a Varian Inova 600 MHz spectrometer. The 1 H and 13 C chemical shift were referenced to the DMSO-d 6 solvent peak at δ H 2.50 and δ C 39.52 ppm. Standard parameters were used for the 2D NMR spectra obtained, which included gCOSY, gHSQCAD ( 1 J CH = 140 Hz), gHMBCAD ( n J CH = 8 Hz) and ROESY. Mass spectra were acquired using a Mariner TOF mass spectrometer (Applied Biosystems Pty Ltd.

Animal Material
A first sample of P. candelabra was collected at the depth of 36 m, Wilson Reef, Coral Sea, Queensland, Australia. It was identified as Pipestela candelabra (phylum Porifera, class Demospongiae, order Halichondrida, family Axinellidae). A voucher specimen QMG320579 has been deposited at the Queensland Museum, South Brisbane, Queensland, Australia.
A second sample characterised as the same taxonomy with the first specimen Pipestela candelabra was collected at the depth of 20 m, Houghton Reef, Howick Group, Queensland, Australia. A voucher specimen QMG320790 has been deposited at the Queensland Museum, South Brisbane, Queensland, Australia.
A second freeze-dried sample of P. candelabra (30 g) collected at Houghton Reef, Howick Group, was also extracted exhaustively with hexane (750 mL), DCM (4 × 250 mL) and MeOH (4 × 250 mL), respectively. The DCM and MeOH extracts were combined and then evaporated solvents to yield a yellow residue (7.5 g). This crude extract was pre-adsorbed onto C 18 (10 g) and packed dry into a cartridge (25 × 50 mm), which was connected to a C 18

Peptide Hydrolysis and LC/MS Analysis of Marfey's Derivatives
Peptide samples (150 μg) were dissolved in degassed 6 N HCl (500 μL) and heated at 120 °C for 8 h. The solvent was removed under dry nitrogen and the resulting material was subjected to further derivatization for a stereochemical assignment.
The hydrolysate mixture or the amino acid standard was added a solution of L-FDAA 1% (w/w) in acetone and 100 μL of a 1 N NaHCO 3 solution. The vial was heated at 50 °C for 3 h and the contents were neutralized with 2 N HCl (50 μL) after cooling to room temperature. An aliquot of the L-FDAA derivative was dried under dry nitrogen, diluted in MeOH and loaded on a Phenomenex Luna column (C 18 , 3 μm, 4.6 mm × 50 mm) using a linear gradient from 5% MeOH (0.1% FA)-95% H 2 O (0.1% FA) to 100% MeOH (0.1% FA) in 12 min. FDAA derivatives were detected by absorption at 340 nm and assignment was secured by ion mass extraction.

Cytotoxicity Assay
Human prostate adenocarcinoma cells (PC3) and human neonatal foreskin fibroblast (noncancer cells, NFF) were grown in media RPMI-1640 (Life Technologies, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS). Cells were grown under 5% CO 2 in a humidified environment at 37°C. Fifty microlitres of media containing 500 cells were added to a 384 well microtitre plate (Perkin Elmer, Waltham, MA, USA, part number: 6007660) containing 0.2 μL of a compound. Final compound concentration range tested was 10 μM to 1 pM (final DMSO concentration of 0.4%). Each concentration in media was tested in triplicate and in two independent experiments. Cells and compounds were then incubated in 72 h at 37 °C, 5% CO 2 and 80% humidity. Cell proliferation was measured with the addition of 10 μL of a 60% Alamar blue (Invitrogen, Carlsbad, CA, USA) solution in media to each well of the microtitre plate to give a final concentration of 10% Alamar blue. The plates were incubated at 37 °C, 5% CO 2 and 80% humidity within 24 h. The fluorescence of each well was read at excitation 535 nm and emission 590 nm on the Perkin Elmer EnVision Multilabel Reader 2104. Eight-point concentration response curves were then analysed using non-linear regression and IC 50 values determined by using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). Paclitaxel and doxorubicin were used during each screening as positive control compounds.

Conclusions
Contributing to the chemical investigation of the new sponge genus Pipestela candelabra, twelve secondary metabolites belonging to three structure classes including milnamide, hemiasterlin and geodiamolide were isolated. Three new members of the milnamide family, milnamides E-G (1-3), and one new member of the hemiasterlin family, hemiasterlin D (4), were identified. Milnamides F (2) and G (3) were identified in the sponge collected at Wilson Reef while hemiasterlin D was only discovered in the sample collected at Houghton Reef. However, all three structure classes were found in samples of the P. candelabra collected at two locations which are 124 km apart.
All new and known natural products showed potent and selective inhibition against the human prostate cancer cells (PC3). Hemiasterlin D (4) is the first example of a hemiasterlin substituted at N-1 by a peptide side chain, which is identical to the peptide side chain at N-13. The compound has less cytotoxicity (IC 50 of 2.20 nM) compared to hemiasterlin (8) and hemiasterlin A (9) (IC 50 of 0.0484 nM and 0.269 nM, respectively) but it opens the possibility for further structural variation at N-1. This is a first report of the preliminary structure-activity relationships of the milnamide family. The C-1 and N-9 in milnamides are important positions affecting their cytotoxicity in PC3 cells.