Evaluation of an NPE library by qHTS
In an effort to improve the process of identifying biological activity from NPE libraries we tested extracts in qHTS format across 35 diverse assays that were optimized for 1536-well format (Table S1
). To reduce the ionic, optical and aggregation-based interference from high salt concentration, pigments, polymeric organics and other resinous materials, we prepared the extracts using XAD-resin to enrich the NPs from these genetically diverse and pure culture actinomycete and fungal strains while reducing the content of biomass at the extremes of polarity (e.g. non-organic or highly lipophilic).
The NPE library we subsequently prepared for qHTS contained 15,704 samples that comprised 13 separate 7-concentration inter-plate titration series, such that overall 91 individual 1536-well plates were maintained as a titration archive for screening (Figure S1b
). As a dynamic library that increases in size over time, a subset of the archived library (~5,300 samples) was tested in the diverse assay panel to estimate the relative activity of the extracts. By examining data from a single tested concentration, qHTS data can also be examined as a traditional single-point high throughput screen. A retrospective analysis of the activity from the preliminary testing of the NPE library across the assays in this manner resulted in a broad range of activity from none to several thousand actives () at either 3 or 6 standard deviations (SD) as a cut-off for biological activity. From this subset, ‘hit’ rates spanning 0–80% for 3SD or an average of 11%, and 0–50% for 6SD or average of 4.5% were observed. The highest hit rates, those above 5% (3SD) were all limited to biochemical assays except for one that measured cellular viability (assay 26, ). Using qHTS analysis among the assays tested, 11 gave high quality class 1a CRCs (see ). These assays were comprised of two cell-based assays and nine biochemical assays that employed detection formats of fluorescence, absorbance and bioluminescence, with five of the 11 based on firefly luciferase (FLuc) outputs. Initially we decided to follow-up actives from screens employing purified molecular targets, based primarily on a high quality pharmacological response in a single assay. A particular extract (Papua New Guinea 05441) with high apparent potency 1a curves in all three solvent extracts was identified for Calmodulin Kinase IIa (assay #25, ), however a re-test with a new extract from the same strain did not yield the original activity (Figure S2a
), illustrating the challenges of working with uncharacterized mixtures and the importance of initial selection criteria limiting the number of follow-ups. However, several assays (#19, #20, #26) based on a common FLuc output showed CRCs from a number of NPEs suggesting to us that these NPEs potentially contained FLuc inhibitors ( and S2b
). More restricted in prevalence, however, were class 1a CRCs (e.g., Costa Rica strain 05545 and 06085). Using this observation to strengthen the commitment to proceed with de-replication, we re-cultured strain 05545 and initiated the isolation of the active constituents of this NPE ().
Activity analysis of NPEs in various assays
Bioactivity guided de-replication of NPE 05545
Structural characterization of a series of aspulvinones
The strain 05545, a marine fungus from the genus Aspergillus
(determined by 18S rRNA gene sequence analysis, data not shown) was isolated from Costa Rica marine sediments, collected in Isla Despensa, Guanacaste Conservation Area in December 2005. The terrestrial Aspergillus
sp. is known by the production of pulvinone derivatives, unsaturated tetronic acids with an aryl substituent at C2, an arylmethylene substituent at C4 and isoprene residues in the aryl rings (Nobutoshi Ojima, 1975
The marine fungus was scaled-up to 5L, and the active extracts were subjected to chromatographic separation using a combination of MPLC and HPLC (). The fractionation of active extracts was followed by secondary bioassay to guide isolation of six new derivatives, aspulvinones I-CR to M-CR (4
), in addition to known aspulvinones E, F and H (1
), as well as (Hiroshi Sugiyama, 1979
; Nobutoshi Ojima, 1975
) butyrolactone I and III (10
) and (K. V. Rao, 2000
; Rajesh R. Parvatkar, 2009
; Xuemei Niu, 2008
) benzofuran (12
)(Dervilla M. X. Donnelly, 1988
; Hung-Yi Huang, 2008
) (See ). Aspulvinone F (2
) was reported in 1975 with an incorrect structure (Nobutoshi Ojima, 1975
), and in 1979 it was reconsidered by Begley et al. (1979)
, who suggested that aspulvinone F likely bears a dihydrofuran ring instead of an epoxide. Our NMR data and X-ray crystallographic analysis reveals that the proposed revised structure is formally confirmed, and the absolute configuration of the chiral center was established as R
Structures of isolated metabolites
NMR and X-ray analysis of aspulvinone F (2)
Aspulvinone I-CR (4) was isolated as a pale yellow solid. The HREIMS gave an [M]− ion at m/z 479.1659, consistent with the molecular formula C27H28O8, requiring 14 sites of unsaturation and 16 amu more than compound 2 (C27H28O7). The 1H NMR and 13C NMR data indicated that the structure of 4 is very similar to compound 2. The most significant differences in the NMR data reside in the high-field shift effect of the signal at C-23 (δH 3.19, δC 31.8) and the presence of a sp3 methine instead of a sp3 methylene at C-24, (δH 4.61, δC 91.4). These data indicated that compound 4 bears a dihydrofuran ring fused to the benzene ring, as opposed to the dihydropyran ring present in compound 2. This structure was further supported by COSY, HSQC and HMBC spectra. COSY correlations between H2-23 and H2-24, as well as between H2-18 and H2-19 supported the presence of two dihydrofuran ring systems. HMBC correlation from H2-18 to C-9 and C-10; H-8 to C-10; H-7 and H-11 to C-9; H2-23 to C-15 and C-17; and H-17 to C-23 confirmed the presence of a dihydrofuran fused to each of the benzene rings.
Aspulvinone J-CR (5) was obtained as a pale yellow solid. Its molecular formula C27H28O7 was established by HREIMS [M+Na]+ ion at 487.1733, indicating 14 degrees of unsaturation. The NMR features of 5 were similar to those of 4 except that the right hand benzene ring is trisubstituted, showing a sp2 methine at C-13 in 5 (δH 7.81, δC 127.8). A COSY cross-peak between H-13 and H-14 as well as HMBC correlations between H-13 and C-2, C-11 and C-15 confirmed these assignments. These assignments were re-confirmed by co-crystal structure of 5 with the FLuc (see below).
Aspulvinone K-CR (6) was isolated as a brown-yellow optically active oil. The [M+Na]+ ion at m/z 521.1788 in the HREIMS suggested C27H30O9 as the molecular formula, which indicated 18 additional amu, with one less unsaturation site than compound 4. A significant change in the 1H NMR and 13C NMR was also observed in the methine C-19, which shifted from δH 4.63 and δC 90.7 in 4 to δH 3.64 and δC 79.8 in 6. These modifications together with the shift of C-9, from δC 161.0 in 4 to δC 157.2 in 6, suggested that there are a hydroxyl and 2,3-dihydroxy-3-methylbutyl group upon C-9 and C-10, respectively, instead of the dihydrofuran ring.
Aspulvinone L-CR (7) isolated as a yellow oil, gave a [M+Na]+ ion at m/z 505.1838 in the positive ion HREIMS, consistent with the molecular formula of C27H30O8, and requiring one less unsaturation site than compound 2. NMR data of compound 7 were similar to those of compound 2 except for C-18 and C-19. The shifts of C-18 from δH 3.18 and δC 30.0 in 2 to δH 2.96, 2.66 and δC 34.8 in 7, as well as the change in C-19 from δH 4.60 and δC 89.9 in 2 to δH 3.63 and δC 81.1 in 7, suggests that in this compound the formation of the dihydrofuran ring did not occur. Instead, there is the open form where C-10 bears a 2,3-dihydroxy-3-methylbutyl group, as in compound 6.
Aspulvinone M-CR (8) was isolated as a yellow solid. The HREIMS gave an [M+Na]+ ion at m/z 503.1682 consistent with the molecular formula C27H28O8 and the same degrees of unsaturation as compound 2. The only difference compared with compound 2 was the shift of C-24 from δH 1.77 and δC 33.3 in 2 to δH 3.72 and δC 71.0 in 8 due to a secondary hydroxyl group at C-24.
Aspulvinone N-CR (9) was obtained as a yellow crystalline oil with the molecular formula C27H28O7 on the basis of HREIMS data, [M+H]+ ion at m/z 465.1913. The level of unsaturation is the same as in compound 2, but the difference in the shifts of methylene C-18 and methine C-19 indicated the presence of a dihydropyran with a hydroxyl group at C-19 instead of the dihydrofuran ring present in 2. Compared with 2, compound 9 showed shifts in the signal H2-18 from δH 3.18 to 3.03, 2.72, and H-19 signal shifted from δH 4.60 to 3.75. Also, the 13C signals of C-18 and C-19 shifted from δC 89.9 to 68.7 and from δC 71.1 to 77.2, respectively. COSY correlations between H2-18 and H-19 and HMBC correlations between H2-18 and C-9, C-10, C-11, C-19 and C-20, as well as between H-19 with C-10, C-18, C-21 and C-22 were consistent with a hydroxyl group at C-19.
The aspulvinone family of natural products has a more complex carbon framework than the related pulvinones due to the incorporation of two isoprene units. The ortho
position to the hydroxyl group serves as the site for alkylation by a dimethylallyl diphosphate mediated by a presumed prenyltransferase. Based on isolation of aspulvinones K-CR and L-CR (compounds 6
), we surmise that prenylation is followed by epoxidation or di-hydroxylation of the double bond with subsequent cyclization, resulting in the five or six membered ring heterocycles. Whether this represents a common biosynthetic theme that includes pyran ring formation in the notoamide/stephacidin/paraherquamide class of fungal derived alkaloid natural products remains to be explored in detail (Ding et al., 2010
X-ray co-crystal of aspulvinone J-CR bound to FLuc
To further characterize the physical interaction of the aspulvinone series with Fluc, apo crystals of the Photinus pyralis luciferase protein were soaked in the presence of aspulvinone J-CR (5) one of the more potent aspulvinone congeners (). Following structural determination by molecular replacement and subsequent refinement to a resolution of 1.7 Å, the resulting electron density maps were examined for ligand binding. Aspulvinone J-CR (5) was clearly bound as the observed difference electron density map (Fo-Fc), which displayed prominent peaks greater than 3σ that were consistent with this compound ().
Structure of FLuc containing bound aspulvinone J-CR (5)
Aspulvinone J-CR (5) fully occupies the D-luciferin binding site with the isopropyl alcohol at one terminus of the structure positioning deeply into the D-luciferin binding pocket and the other isopropyl alcohol extending into the ATP binding site (). The aspulvinone molecule adopts a mostly planar arrangement across the three-ring system. However, the plane of the benzofuran ring that is positioned in the D-luciferin binding region is angled 18.9° relative to the mean plane defined by the hydroxyl-butyrolactone core (). The benzofuran ring in the ATP binding region is only 5.8° relative to the hydroxyl-butyrolactone mean plane. The isopropyl termini are oriented on the same side of the three-ring system. In general, the driving force of aspulvinone binding to luciferase is highly enriched by hydrogen bond interactions (). Two oxygen atoms of the hydroxyl-butyrolactone core are able to form hydrogen bonds to the backbone –NH of Gly316 and the hydroxyl group of Ser347, with the optimal H-bonding distance of 2.73Å and 2.85Å, respectively. Notably, the isopropyl alcohol on both ends of the aspulvinone J-CR (5) structure could form tight binding with the surrounding residues through a hydrogen bond matrix. The one fitting deeply into the D-luciferin binding pocket forms a direct hydrogen bond to Arg218 and also a few water-mediated hydrogen bonds to Arg218, Ala222, Phe227 and Asn229 through two structural water molecules. In contrast, the other isopropyl alcohol extending into the AMP binding site is involved in hydrogen bonding with His245 and the backbone carbonyl oxygen of Gly316. Additionally, the oxygen of the dihydrobenzofuran ring could further help the molecule lock on the correct binding mode through the hydrogen bond interaction to Thr343.
Based on the co-crystal structure of Fluc with aspulvinone J-CR (5
), we further examined aspulvinone F (2
) which is ~30-fold less potent than 5
as an FLuc inhibitor (). The docking model of 2
indicated that it could adopt the same binding orientation and maintain nearly all key interactions as 5
. However, the isopropyl alcohol extending into the AMP binding site was missing in 2
. As this isopropyl alcohol forms hydrogen bonds with His245 and the backbone carbonyl oxygen of Gly316 in 5
, the absence of such a moiety in 2
could explain this significant potency shift (Figure S32
In a previous study, we elucidated inhibitor-based protein stabilization as the mechanism by which the small molecule PTC124 increases the activity of a FLuc reporter protein used in an assay designed to discover nonsense codon suppressor compounds (Auld et al., 2009a
; Auld et al., 2008b
). We found that PTC124 stabilized the half-life of the enzyme by reacting at the FLuc active site with ATP to form a multi-substrate adduct inhibitor (MAI), which we determined from crystallographic studies (Auld et al., 2010
; Thorne et al., 2010b
). This potent inhibitor can be displaced from the enzyme through the use of typical luciferase detection reagents that contain high concentrations of substrates including CoASH which presumably thiolytically cleaves the MAI (Auld et al., 2010
), thus allowing detection of FLuc enzyme activity in the assay. The PTC124-AMP adduct effectively fills the active site of FLuc. Alignment of the aspulvinone co-crystal structure and the structure of PTC124-AMP adduct in the luciferase active site revealed a similar binding orientation in the D-luciferin pocket (). The hydroxyl-butyrolactone core of aspulvinone occupies a similar position to the oxadiazole of PTC124. However it was found that the central ring system of aspulvinone J-CR forms an angle of 50.8 degrees between the mean planes defined by the butyrolactone ring (5-membered) and the aryl ring of Phe247. This is too large for a π-π (face-to-face, quadrupole-quadrupole) interaction or the aromatic stacking observed in the PTC124-AMP-FLuc structure, and more closely approximates a herringbone (edge-to-face, dipole-quadrupole) interaction (Burley and Petsko, 1985
). The planar nature of both ligands makes the molecules well accommodated in the unusually long and linear binding pocket of D-luciferin.
Superposition of aspulvinone J-CR (5) and PTC124-AMP adduct within FLuc binding pocket