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F. H. Al-Awadhi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
evaluation of tasiamide F (1), a novel inhibitor of cathepsins D and
E from a marine cyanobacterium Lyngbya sp.
standards. The configuration of lactic acid was examined by chiral
HPLC revealing the presence of L-lactic acid. To determine the
configuration of the statine unit, modified Marfey’s analysis20,21
was carried out and tasiamide B (2) was used as a standard to
2. Results and discussion
liberate the (4S)
potential dehydration and rehydration). Portions of the hydroly-
sates of 1 and 2 were derivatized with both -FDLA and DL-FDLA
c-amino acid (mixture of 3S,4S and 3R,4S due to
2.1. Isolation and structure determination
L
and the retention times were compared. The analysis revealed S
configuration at C-28. The relative configuration of the Phe-derived
statine unit was established based on NMR analysis of the coupling
constants of the methylene protons at C-26 to the hydroxy
methine at C-27.22 The downfield methylene proton H-26
(dH 2.30) showed a larger coupling (J 9.5 Hz) to H-27 compared
to the upfield methylene proton (dH 2.16) (J 3.8 Hz), suggesting
3S,4S configuration (recorded in DMSO-d6). Similar coupling
constants were observed when 1H NMR data were recorded in
CDCl3 (H-26; dH 2.50, J 8.2 Hz) and (H-26; dH 2.35, J 4.8 Hz), further
supporting 3S,4S configuration.
Samples of the cyanobacterium Lyngbya sp. were collected from
patch reefs in Cocos Lagoon, Guam. The freeze dried sample was
subjected to solvent extraction with 1:1 EtOAc–MeOH. The non-
polar extract was then partitioned between EtOAc and H2O. The
EtOAc soluble fraction was further fractionated by diol column
chromatography eluting with a gradient of increasing polarity
starting with DCM–hexanes. The fraction eluted with 1:1 EtOAc–
MeOH was further purified by reversed-phase HPLC using MeCN–
H2O mixtures of increasing polarity to yield compound 1 (Fig. 1).
The HRESIMS of 1 in the positive mode exhibited a molecular
ion peak at m/z 1001.5325 [M+Na]+ suggesting a molecular
formula of C50H74N8O12 with eighteen degrees of unsaturation.
The structure of 1 (Fig. 1) was determined using a combination
of 1D and 2D NMR techniques. The 1H and 13C NMR spectra sug-
gested the presence of several characteristic signals corresponding
2.2. Biological evaluation
The similarity of tasiamide F (1), in terms of its structure and in
particular the presence of a statine unit, to the natural aspartic pro-
tease inhibitor pepstatin A (6) and other related marine cyanobac-
terial natural products (Fig. 2), suggested that 1 might also have
aspartic protease inhibitory activity. To evaluate the antiprote-
olytic activity, tasiamides F and B, 1 and 2, were tested in vitro side
by side against cathepsins D and E, and BACE1 (Fig. 3). Both com-
pounds exhibited a low nanomolar inhibitory activity against
cathepsins D and E (Table 2); indicating that tasiamides 1 and 2
are ꢀ30-fold more potent against cathepsin D compared with
to
a
-protons (ꢀdH 4–5 ppm), exchangeable protons of amides (ꢀdH
7–8 ppm), two N-methyl signals (ꢀdH 2.7–2.9 ppm; ꢀdC 29–30 Hz),
and one O-methyl (dH 3.62 ppm) suggesting a peptide structure.
Examination of the 2D spectra (HSQC, COSY, HMBC, and ROESY)
of 1 in DMSO-d6 (Table 1 and Supporting information) revealed
the presence of Gly and Ile as proteinogenic amino acids as well
as O-CH3-Pro, N-CH3-Phe, N-CH3-Gln, lactic acid and a statine unit
[4-amino-3-hydroxy-5-phenylpentanoic
acid,
Ahppa].
The
sequence of these units was established by the analysis of HMBC
and ROESY spectra and was further confirmed by ESIMS fragmen-
tation (Fig. 1). Analysis of the minor set of signals in the proton
NMR spectra in three different solvents (DMSO-d6, CDCl3, and
CD3OD; Figs. S3, S9, S10) showed the presence of changing
resonance-doubling ratios (6:1 in DMSO-d6 and CDCl3 and 2:1 in
CD3OD) from aprotic to protic solvents, indicating the presence
of conformers in solution.
Compound 1 is a tasiamide B analogue, with key differences
between the structures being the replacement of amino acid
residues in tasiamide B (2) to Ala ? Gly; Leu ? Ile; Val ? Ile, and
was named tasiamide F (1) in light of existence of other tasiamides
(Fig. 2). Other structurally related cyanobacterial compounds
include grassystatin C (3),9 bearing a Leu-derived statine core,
and tasiamide E (4) and tasiamide (5) which lack the statine unit
(Fig. 2).10,11,19
grassystatin C (3; 1.62 lM), while the cathepsin E inhibitory activ-
ity is similar (grassystatin C: 42.9 nM).9 Grassystatins A–C, bearing
the Leu-derived statine unit, are more selective towards cathepsin
E inhibition (ꢀ20- to 38-fold).9 Likewise, several designed tasi-
amide B analogues bearing the Phe-derived statine unit are more
selective towards inhibiting cathepsin D,12 thus demonstrating
that the selectivity can be tuned and these structural scaffolds
can serve as a starting point for further development of selective
aspartic protease inhibitors. When tested against BACE1, an
enzyme involved in the pathogenesis of Alzheimer’s disease,13 tasi-
amide F (1) was found to be 12- to 30-fold less potent in inhibiting
this enzyme when compared to cathepsins D and E (Table 2). Inter-
estingly, despite the minor structural differences, 2 was ꢀ8 fold
more potent in inhibiting BACE1 compared to 1. Based on previous
SAR and molecular docking studies, it has been shown that Phe,
Ala, Leu, and Val in tasiamide B (2) are critical for BACE1 activity
and significantly affect the inhibition through hydrophobic interac-
tions with the receptor’s pocket.17 Therefore, minor modifications
in these residues appear to be responsible for the altered activity
against BACE1 compared with 1.
To establish the absolute configuration, a portion of 1 (100 lg)
was hydrolyzed using 6 N HCl (110 °C, 24 h) and analyzed by chiral
HPLC–MS. The analyses revealed retention times corresponding to
L-Pro, N-Me-D-Phe, L-Ile, N-Me-L-Gln by comparison with authentic
2.3. Molecular docking
In order to provide some insight to the structural basis of 1 and
2, molecular docking experiments were carried out using Auto-
Dock Vina.23 The crystal structure of pepstatin A bound to cathep-
sin D (PDB: 1LYB)24 was used for docking. Pepstatin A was
successfully redocked into cathepsins D and E (Fig. S1) before
attempting to dock tasiamides B (2) and F (1). Compounds 1 and
2
were then docked into cathepsin
D
(PDB: 1LYB)24
(Fig. 4A and B) and homology model of cathepsin
a
E
(Fig. 4C and D), since the only published crystal structure that is
available is for an early activation intermediate (PDB: 4PEP),25
and the interactions between each of the compounds and the
receptors’ binding pockets were examined. Despite the minor
Figure 1. Tasiamide F (1) with ESIMS fragmentation pattern.