Synthesis and Biological Activity of Optimized Belactosin C Congeners

a Institut für Organische und Biomolekulare Chemie der Georg-August-Universität Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germany b Charité Universitätsmedizin Berlin CCM Med. Klinik für Kardiologie und Angiologie Kardiologisches Forschungslabor Ziegelstrasse 5-9, D-10117 Berlin, Germany c Center for Integrated Protein Science at the Department Chemie, Lehrstuhl für Biochemie, Technische Universität München, Garching, D-85747, Germany


Introduction
2][3] Despite this interest, however, only a small number of detailed investigations concerning their biological activities have been carried out so far. 4,5n order to better understand their proteasome inhibitory power and the important structure-activity relationships, the prevailing interactions between such molecules and the proteasome should be known.Towards this end, a protected homobelactosin C was initially cocrystallized with the 20S proteasome from Saccharomyces Cerevisiae, and the structure of this crystal was elucidated by X-ray diffraction. 1 On the basis of this analysis it was decided which groups in the inhibitor should be modified in order to improve potentially favorable interactions with the proteasome.In this paper we report the synthesis and detailed structure-activity investigations of some new belactosin C congeners.that 2-isobutylmalic acid monophenylthio ester 4 (Fig. 1) as a key building block could be easily prepared in enantiomerically pure form.6b Subsequent peptide coupling of 4 with the corresponding dipeptide and ensuing in situ b-lactone ring closure provided the diprotected derivatives of belactosins A and C and homobelactosin C, 1, 2 and 3, respectively, which were eventually completely deprotected.
Fig. 1 The naturally occurring proteasome inhibitors belactosin A and C, its non-natural analogue homobelactosin C and a key building block 4 in our total syntheses of 1-3.6b Computer modelling of possible modifications of the belactosin structure showed that attachment of a 2-naphthylethoxycarbonyl (CNAP) group onto the amino terminus in the alanine residue in belactosin C could be favorable for hydrophobic interactions and thus improve its biological activity.On the other hand, introduction of a 4-carboxybenzyl residue capable of both hydrophilic and hydrophobic interactions, onto the ornithine part of belactosin C could also lead to an improved biological activity.
To test these hypotheses, we prepared the analogues 6, 11 and 15 of belactosin C (2) as well as the known derivative 16 according to our previously published strategy.6b Compound 6 with a 2naphthylmethoxycarbonyl protective group on the amino terminus was obtained starting from the 2-naphthylmethoxycarbonylprotected alanine (CNAP-Ala-OH) 5 by coupling it with the appropriately decorated ornithine and subsequently installing the b-lactone moiety by a one-pot acylation/b-lactonization 6b reaction employing the building block 4 (Scheme 1).The synthesis of the belactosin C analogues 10 and 11 containing a tert-butoxycarbonyl (10) and a free carboxylic acid group, respectively, in the benzyl ester moiety, commenced with partial hydrolysis of di-tert-butyl terephthalate 7 followed by a chemoselective reduction of the free carboxylic acid group with tetrabutylammonium borohydride to furnish the benzyl alcohol 8 (Scheme 2).
The DCC-mediated condensation of 8 with the correspondingly protected ornithine gave rise to the N-terminally Boc-protected intermediate 9.This was converted into the tripeptide 10 by way of Fmoc removal, peptide coupling with Cbz-Ala-OH, selective removal of the N-Boc group, and the one-pot acylation/blactonization reaction as a key step.Cleavage of the tert-butyl ester under acidic conditions (trifluoroacetic acid in dichloromethane) yielded the desired belactosin C congener 11 with a free carboxylic acid functionality in the benzyl ester moiety.
Finally, the stable and storable crystalline hydrotrifluoroacetate salt of belactosin C 15 was prepared along the route outlined in Scheme 3. Thus, H-Orn(Cbz)-OtBu 12 was transformed into The belactosin C derivative 16 was synthesized according to the previously published protocol.6b Thus, the novel derivatives of belactosin C 6, 11 and 15 were prepared in sizable quantities employing our original synthetic protocol.6b

Results and discussion
To investigate the effect of the obtained belactosin C congeners on proteasomal activity in mammalian cells, HeLa cells were treated with varying concentrations of compounds 6, 10, 14, and Fig. 2 Biological activities of belactosin C congeners 6, 14 and 16 in comparison to MG132.A) HeLa cells were treated with the indicated concentrations of MG132 and belactosin C congeners 6, 14 and 16 or solvent for 4 h.Cells were lysed, followed by measurement of chymotrypsin-like (ChTL), trypsin-like (TL) and caspase-like (CaspL) activities in the in vitro activity assay employing fluorogenic substrates.Activities are expressed as percentage of a solvent-treated control.Values are given as the means of three independent experiments ± SEM.B) Ear fibroblasts of Ub G76V -GFP1 transgenic mice were treated with increasing concentrations of MG132 and belactosin C congeners 6, 14, and 16 or solvent (C) for 4 h.Accumulation of degradation-prone proteins was analyzed in cell lysates by western blot, using anti-GFP and anti-ubiquitin antibodies.Amido black staining of western blot membranes served as a control for equal protein loading (LC).
16 for 4 h as well as with the widely used proteasome inhibitor MG132.Subsequently, an in vitro activity assay was performed in cell lysates employing the fluorogenic substrates SSLVY-AMC, BzVGR-AMC and ZLLE-AMC to measure the chymotrypsinlike (ChTL), the trypsin-like (TL) and the caspase-like (CaspL) activities respectively.All compounds dose dependently inhibited proteasomal degradation.The ChTL activity was predominantly affected by all substances.As shown in Fig. 2A, the inhibitory effects of compounds 14 and 16 were similar to those of MG132, albeit MG132 was more effective in concentrations below 0.25 mM.Compound 6 turned out to be the most effective inhibitor.Notably, besides its potent inhibitory effect on the ChTL activity, compound 6 showed the most pronounced effect of all tested substances on TL activity, in particular in concentrations above 0.25 mM.Compound 10 displayed a considerably lower inhibitory potency compared to MG132.
The accumulation of polyubiquitinated proteins or proteins that contain a constitutively active degradation signal is one characteristic effect of proteasome inhibition in cells.To detect the levels of degradation-prone proteins that accumulated upon incubation with belactosin C congeners, fibroblasts obtained from ears of Ub G76V -GFP1 mice were treated with varying concentrations of compounds 6, 14, 16, and MG132 for 4 h.Ub G76V -GFP1 mice contain the green fluorescent protein (GFP) fused to a constitutively active degradation signal (Ub G76V ), thereby allowing us to monitor the accumulation of this specific reporter protein in addition to overall polyubiquitinated protein.
Following treatment, cell lysates were analyzed by western blot, using anti-GFP and anti-ubiquitin antibodies.As shown in Fig. 2 B, a concentration-dependent accumulation of endogenous polyubiquitinated proteins, as well as elevated levels of Ub G76V -GFP were caused by substances 6, 14, and 16, thereby confirming their inhibitory effect on proteasomal activity in living cells.In concentrations below 1mM, compounds 14 and 16 lead to a considerably lower accumulation of polyubiquitinated proteins and Ub G76V -GFP compared to MG132.It is noteworthy that a similar effect was observed for compound 6, which appeared to be a more potent inhibitor compared to MG132 as estimated in the in vitro activity assay.
In order to get insight into the mechanism of biological activities of these compounds, compound 6 was co-crystallized with the yeast 20S proteasome (see Fig. 3).
Yeast 20S proteasome crystals were soaked with compound 6 for 24 h at a final concentration of 5 mM.Data collected from the soaked crystals were evaluated by molecular replacement using the coordinates of the yeast 20S proteasome. 7Subsequent bulk solvent correction and positional refinement performed with CNS yielded an R free of 23.7% (Table S1 †).The 2F o -F c -electron density map calculated after density averaging visualized compound 6 in the primed site of the chymotrypsin-like substrate binding channel, for which it exerts a high selectivity (Fig. 3A).In close analogy to what has been observed for bis-protected homobelactosin C (hBelC), 1 the carbonyl carbon atom of the ligand derived from the beta-lactone ring is covalently bound to the free hydroxyl group of the catalytic N-terminal Thr1O y of subunit b5 (Fig. 3B).Except for the interaction of the isoleucine side chain with the S1 specificity pocket, the molecule targets only the primed site of the substrate binding channel.Structural superposition of 6 and hBelC bound to the subunit b5 reveals that shortening of the linker by a methylene group in 6 compared to hBelC only affects the conformation of the inhibitor backbone but not that of the side chains.The perfect match of the benzyl side chains of both compounds indicates an important proteasomal primed site for substrate binding at this position (Fig. 3C).The restricted size of the primed specificity pockets in the yeast b1-and b2subunits 8 explains the selectivity of 6 and hBelC to only bind by their extended benzyl side chain to the chymotrypsin-like active site.Furthermore, comparison of the primary sequences of mammalian subunits b5 and b5i reveals a conspicuous alteration in this primed specificity channel: Ser115 and Glu116 of the human constitutive subunit b5 are replaced by Glu and His in subunit b5i, respectively.The putative primed substrate binding channels in the b1-and b2-subunits modelled on the basis of subunit b5 show similar architectures and exhibit similar significant differences between constitutive and immune subunits. 9Thus, the novel chemical, biological and structural insights into the binding mechanism of belactosin C derivatives might contribute to the development of new anti-inflammatory or anti-cancer drugs targeting the proteasome.

Conclusion
Four novel belactosin congeners have been synthesized according to an established protocol 6b in order to check the hypothesis concerning their structure-activity relations based on the computer modelling of their complexes with the proteasome.The best substances turned out to have an improved biological activity against HeLa cells at least comparable or even superior to the activity of the known proteasome inhibitor MG132.The most active compound 6 is more potent than MG132, thus becoming a promising candidate for further investigations and possible application in cancer therapy.A detailed investigation of the complex of 6 with the proteasome showed an identical binding mode (and thus mechanism of its activity) as the known b-lactone inhibitor homobelactosin C.

Synthesis of belactosin C congeners
General remarks. 1

((2S)-Naphthylmethoxycarbonylamino))propionic acid (CNAP-Ala) (5).
To a stirred ice-cold solution of L-alanine (1.8 g, 20 mmol) and NaOH (1.6 g, 40 mmol) in H 2 O (10 mL) was added 2-naphthylmethyl chloroformate 11 (CNAP-Cl) (6.3 g, 29 mmol).Then water (10 mL) and THF (10 mL) were added, upon which the solution became clear.The reaction mixture was stirred at r. t. for 2 h, and the reaction was then quenched by adding a saturated solution of NaHCO 3 (20 mL).The mixture was extracted with Et 2 O (1 ¥ 30 mL), the ethereal phases were discarded, and the pH value of the aqueous phase was adjusted to 2-3 with 12 M aqueous HCl.Then it was extracted EtOAc (3 ¥ 30 mL), and the combined organic phases were dried.Evaporation of the solvent gave 4.4 g (80%) of the acid 5 as a colorless solid, m.p. 132-133 General procedure (GP1) for Fmoc-deprotection of the benzyl esters and subsequent peptide condensation.To a solution of the respective benzyl ester (1.70 mmol) in THF (3.3 mL) was added at r.t.Et 2 NH (3.3 mL).The mixture was stirred at r. t. for 1 h, then another 2 mL of Et 2 NH was added, and the mixture was stirred for an additional 2 h.The volatiles were removed under reduced pressure at 35 • C. The oily residue was azeotropically distilled off with toluene (2 ¥ 8 mL) under reduced pressure at 45-50 • C, and the crude amine was taken up in CH 2 Cl 2 (5 mL).
A separate flask was charged with the corresponding protected alanine (2.06 mmol), CH 2 Cl 2 (7.5 mL) and HOAt (0.26 g, 1.92 mmol).EDC (0.32 g, 2.04 mmol) was added dropwise at 0 • C within 10 min, and the resulting mixture was stirred at 0 • C for 20 min.To the solution of the prepared crude amine in CH 2 Cl 2 was added TMP (0.66 g, 0.72 mL, 5.43 mmol), and the resulting suspension was added through a cannula to the stirred reaction mixture.This was left to attain r.t.within 16 h and then concentrated under reduced pressure.The residue was taken up in EtOAc (30 mL).The cloudy solution was washed with 1 N aqueous KHSO 4 solution (2 ¥ 30 mL), and saturated aqueous NaHCO 3 solution (2 ¥ 30 mL), then dried and concentrated under reduced pressure.The oily residue was taken up in EtOAc (5 mL) and purified by column chromatography [SiO 2 (50 g), hexane-EtOAc 1 : 1] to give the desired dipeptide.General procedure (GP2) for Boc-removal and subsequent sequential acylation/b-lactonization. To a solution of the respective dipeptide (1.56 mmol) in EtOAc (3 mL) was added 3 N HCl in EtOAc (12 mL).The mixture was stirred at r. t. for 12 h, then concentrated under reduced pressure at 40 • C. The resulting crude solid product was dried in vacuo (0.05 mbar) at r. t. for 3 h, then dissolved in DMF (6 mL) and the solution cooled to -30 • C. A solution of the thioester 4 (440 mg, 1.57 mmol) in CH 2 Cl 2 (20.6 mL) was added first, then HOAt (255 mg, 1.54 mmol), TMP (377 mg, 3.11 mmol), as well as EDC (561 mg, 3.11 mmol), and the resulting mixture was stirred at -30 • C for 6 h, then at r. t. for 30 h.It was then washed with 1 N aqueous KHSO 4 (80 mL), dried and concentrated under reduced pressure.The oily residue was purified by column chromatography [SiO 2 (60 g), hexane-EtOAc (1 : 2)] to give the desired product.

Investigation of biological activities of the new belactosin C congeners
Methods 1. Cell culture.HeLa cells (epithelial cells derived from human cervix carcinoma) were obtained from the American Type Culture Collection.The cells were maintained in RPMI (Gibco, Karlsruhe, Germany) supplemented with 10% FCS, 100 U mL -1 penicillin, and 100 mg mL -1 streptomycin in a humidified 5% CO 2 atmosphere.
Primary mouse ear fibroblasts were isolated from Ub G76V GFP1 mice. 12Mouse ears were washed in 70% EtOH for 2 min and cut into small pieces in DMEM using a scalpel.Pieces were transferred into a 15 mL tube containing 2 mL of 0.25% trypsin.After incubation for 1 h at 37 • C, trypsin was inactivated by adding 5 mL of DMEM/10%FCS.The suspension was centrifuged at 1000 rpm for 5 min.The pellet was resuspended in 1 mL of DMEM) supplemented with 10% FCS, 100 U mL -1 penicillin, and 100 mg mL -1 streptomycin and transferred into a well of a 6 well-plate.Fibroblasts had grown after 1-2 days and covered the well bottom after 3-5 days.

Assay of proteasomal activity.
Proteasome chymotrypsinlike, trypsin-like and caspase-like activities from HeLa cell lysates were determined fluorometrically in a spectramax GEMINI-EM (MolecularDevices) by using synthetic peptides linked to the fluorophor methylcoumarine.ChTL activity was measured by SLLVY-AMC hydrolysis, TL by BzVGR-AMC and CaspL activity by ZLLE-AMC hydrolysis with 360-nm excitation and 460-nm emission wavelengths.
Cells were treated for indicated times with indicated substances or solvent (DMSO) as a control.Cells were subsequently washed with PBS, and then scraped and lysed under hypotonic conditions with repeated cycles of thawing and freezing in liquid nitrogen.Lysates were centrifuged, and the protein content of the supernatant was estimated by BCA protein assay (Pierce).Lysates were incubated for 30 min at 37 • C in incubation buffer containing an ATP-regenerating system (225 mM Tris-HCl, pH 8.2, 45 mM KCl, 7.5 mM Mg(CH 3 COO) 2 , 7.5 mM MgCl 2 , 1.1 mM dithiothreitol, 6 mM ATP, 5 mM phosphocreatine, 0.2 unit of phosphocreatinekinase) and 0.2 mM of the appropriate fluorogenic substrate.Enzymatic activity was normalized to protein concentration and expressed as a percentage of activity of the solvent-treated control.The values are given as the means of three independent experiments ± S.E.M.

Co-crystallisation.
Crystals of the 20S proteasome from S. cerevisiae were grown in hanging drops at 24 • C as has previously been described 13 and incubated for 48 h with the chemical compound 6 at 10 mM.The protein concentration used for the crystallization was 45 mg mL -1 in Tris-HCl (10 mM, pH 7.5) and EDTA (1 mM).The drops contained 1 mL of protein and 1 xmL of the reservoir solution, containing 20 mM of magnesium acetate, 100 mM of morpholino-ethanesulfonic acid (pH 6.9) and 10% of MPD.
The space group belongs to P2 1 with cell dimensions of a = 134.9A ˚, b = 301.6A ˚, c = 144.2A ˚and b = 112.9• (see Table S1 †).Data to 2.7 A ˚for the proteasome : inhibitor-complex were collected using synchrotron radiation with l = 1.0A ˚at the X06SAbeamline in SLS/Villingen/Switzerland. Crystals were soaked in a cryoprotecting buffer (30% MPD, 20 mM of magnesium acetate, 100 mM of morpholino-ethanesulfonic acid pH 6.9) and frozen in a stream of liquid nitrogen gas at 100 K (Oxford Cryo Systems).X-ray intensities were evaluated employing the XDS program package. 14

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observed and calculated structure amplitudes using the program CNS. 15, 16 Electron density was improved by averaging and back transforming the reflections 10 times over the twofold noncrystallographic symmetry axis using the program package MAIN. 17Conventional crystallographic rigid body, positional and temperature factor refinements were carried out with CNS using the yeast 20S proteasome structure as a starting model. 7Modelling experiments were performed with the program MAIN with current crystallographic values of R cryst = 0.212, R free = 0.237 18 (Table S1 †).
Accession number.The atomic coordinates and structure factors have been deposited in the Protein Data Bank (entry code 3TDD).

Fig. 3
Fig. 3 The belactosin C derivative 6 specifically binds to the chymotryptic-like active site by formation of an ester.(a) Stereorepresentation of the chymotryptic-like active site of the yeast 20S proteasome (colored in bisque) in its complex with 6 (colored in green).The covalent linkage of the inhibitor with b5-Thr1O g is drawn in magenta.The electron density map (colored in grey) is contoured from 1s around Thr-1 (colored in black) with 2F o -F c coefficients after twofold averaging.Temperature factor refinement indicates full occupancies of the inhibitor-binding site except for the 2-naphthylmethoxycarbonyl side chain.6 has been omitted for phasing.Amino acid residues stabilizing 6 are colored in black.(b) Surface representation of the chymotryptic-like active site in its complex with 6 covalently bound to Thr-1 (depicted in white).Surface colors indicate positive and negative electrostatic potentials contoured from 15 kT/e (intense blue) to -15 kT/e (intense red).(c) Structural superposition of 6 and bisbenzyl-protected homobelactosin C (hBelC), including Thr-1 with respect to subunit b5.6 is shown in green, hBelC as well as the active site Thr1 are drawn in black.