Discovery of a potent and highly specific b2 proteasome inhibitor from a library of copper complexes
Tongliang Zhou a, Yuanbo Cai b, Lei Liang a,⇑, Lingfei Yang a, Fengrong Xu a, Yan Niu a, Chao Wang a, Jun-Long Zhang b,⇑, Ping Xu a,⇑
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
b Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Rare Earth Materials Chemistry and Applications, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
a r t i c l e i n f o a b s t r a c t
Article history: Received 10 June 2016 Revised 29 September 2016 Accepted 14 October 2016 Available online xxxx
Keywords:
Proteasome inhibitors
Schiff base
Copper complex
Drug design
Structure–activity relationships
We reported the synthesis, characterization and biological activity of several copper(II) Schiff base com-plexes, which exhibit high proteasome inhibitory activities with particular selectivity of b2 subunit. Structure–activity relationships information obtained from complex Na2[Cu(a4s1)] demonstrated that distinct bonding modes in b2 and b5 subunits determines its selectivity and potent inhibition for b2 subunit.
2016 Elsevier Ltd. All rights reserved.
Metal complexes have been widely used in clinic application since the great discovery of cisplatin by Rosenberg in the late 1960s.1–5 However, severe side effects like nephrotoxicity, ototox-icity restrict their use as clinic treatment,6–8 thus development of new metal complexes with low cytotoxicity, low drug-resistance, high selectivity as well as potent efficacy is highly demanded. Among them, copper, as an essential intracellular cofactor for many enzymes, is playing vital roles in many physiological pro-cesses. It has gained rapid growing interests and their complexes have been applied as antitumor drug candidates.9–13 Up to date, several copper complexes such as some copper–Phenanthroline complexes have been propelled into pre-clinical research as antitu-mor drug candidates, which demonstrated considerable antitumor potential and relatively lower side effects compared with some platinum drugs. A typically proposed mechanism regarding the antitumor activity is that copper complexes are able to generate reactive oxygen species (ROS) which trigger DNA degradation and apoptosis of cancer cells.14 Recent Letters demonstrated cop-per complexes also interface cell processes via enzyme inhibi-tion.15–17 For example, Dou demonstrated some copper Schiff base complexes which have significant antitumor activity, associ-
⇑ Corresponding authors. Tel.: +86 8280 1505 (L.L.), +86 6276 7034 (J.-L.Z.), +86 8280 2632 (P.X.).
E-mail addresses: [email protected] (L. Liang), [email protected] (J.-L. Zhang), [email protected] (P. Xu).
http://dx.doi.org/10.1016/j.bmcl.2016.10.043
0960-894X/ 2016 Elsevier Ltd. All rights reserved.
ated with proteasome inhibition. The detailed mechanism is prob-ably cytotoxic effect raised by ROS which is induced by these copper Schiff base complexes.15 Nevertheless, in this Letter, we focus on screening copper Schiff base complexes as selective pro-teasome b2 inhibitors.
The Ubiquitin–Proteasome pathway (UPP), as the major path-way of intracellular protein degradation in eukaryotic cells, is essential for some key cellular functions, such as cell cycling, signal
transduction, antigen processing for appropriate immune responses, and apoptosis.18–20 It also plays a pivotal role in the ori-
gin, evolution and transfer of malignant carcinoma.21 It has been reported that tumor cells are more sensitive to proteasome inhibi-tors than normal cells and thus proteasomes are recognized as promising antitumor drug targets. Recently, many proteasome inhibitors have been explored extensively as potential antitumor agents and several successful hits including bortezomib, carfil-zomib, and marizomib, were approved by FDA in 2003, 2012, and 2014, respectively, for the treatment of multiple myeloma.22–24
It is well known 26S proteasome is a large multicatalytic pro-tein complex, consisting of two 19S regulatory particles and one 20S catalytic core. Crystal structure (PDB:3MG6) shows that the active sites of the 20S proteasome are mainly located on the b1, b2 and b5 subunits, which have caspase-like (C-L), trypsin-like (T-L), and chymotrypsin-like (ChT-L) activities, respectively. Recent studies on the function of these subunits demonstrated that
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2 T. Zhou et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
inhibition of b5 subunit could achieve therapeutic effects.25–31 However, co-inhibition of proteasome b1 or b2 subunit have attracted much attention, since inhibition of b1 or b2 subunit is also important in some cancer cell lines.32 So far the effect of inhibition of b1 or b2 subunit has not been fully elucidated mainly due to the lack of specific inhibitor. Therefore, exploration of selective protea-some b1 or b2 inhibitors may provide us more information to study the mechanism and further develop novel drug candidates, as well as avoiding side effects. Herein we report the copper Schiff base complexes as selective proteasome b2 inhibitors with valuable structure–activity relationship.
The Schiff base contains a C@N double bond as a functional group prepared by condensation of an aldehyde or ketone with a primary amine. As ligand, Schiff base coordinates metals in various states by the lone pair of the nitrogen atom of the C@N moiety and other electron-rich functional groups.33 In the previous Letters, various copper Schiff base complexes have been designed with dif-
ferent substituted groups. The synthetic method has also been explored and optimized.34–36 In this work, we hypothesized that
modification of the Schiff base ligand would change the docking mode of the inhibitor in the active site of proteasome. As shown in Figure 1, various diamines (a) and salicylaldehydes (s) were employed to construct tridentate and tetradentate Schiff base ligands by typical methods.
Before assessment of the copper Schiff base complexes, we pre-pared various Schiff base ligands and measured their activity toward proteasome inhibition (Table 1). Different diamines and salicylaldehydes were chosen to construct a library for further study of structure–activity relationships (Scheme 1). It is revealed that the synthesized Schiff base ligands showed no selectivity toward all three b subunits though some have inhibitory activities. To increase the water solubility of the ligands, sulfonate groups were introduced to the salicylaldehyde moiety (s3). However, all the Schiff base ligands containing s3 showed no inhibitory activity except a5s3 which exhibited IC50 10 lM for all b subunits. a2s3, a3s3, a4s3, a6s3, a8s3, a9s3 and the tridentate ligand a2(s3)1/2 showed negligible activities. Moreover, tetradentate ligands including a1s1, a4s1, a6s1, a7s1 showed no activities neither. To identify the diamine moiety, we used the 5-carboxylic acid
Figure 1. Different diamines and salicylaldehydes used in this work.
Table 1
Schiff base ligands as proteasome inhibitorsa
Ligand IC50b (lM) Ligand IC50 (lM)
a1s1 NIc
a4s4 b1 = 1.26 ± 0.02
b2 = 1.19 ± 0.01
b5 = 1.29 ± 0.08
a2(s3)1/2 NI a4s5 b1 = 1.65 ± 0.06
b2 = 1.71 ± 0.08
b5 = 1.82 ± 0.18
a2s3 NI a5s3 b1 = 9.38 ± 0.27
b2 = 12.47 ± 0.29
b5 = 10.24 ± 0.30
a3s2 b1 = 7.35 ± 0.16 a6s1 NI
b2 = 8.20 ± 0.18
b5 = 8.02 ± 0.14
a3s3 NI a6s3 NI
a3s5 b1 = 9.52 ± 0.27 a7s1 NI
b2 = 12.32 ± 0.28
b5 = 10.05 ± 0.29
a4s1 NI a8s3 NI
a4s3 NI a9s3 NI
Bortezomibd 4.2 ± 0.34
a Values represent the mean ± SD of three independent experiments, each based on four biological replicates.
b Percent inhibition at 25 lg mL 1.
c NI: no inhibition.
d Positive control.
substituted diamine a4 to build Schiff base ligands with disubsti-tuted 3,5-dichloro and 3,5-dibromo salicylaldehydes (s4, s5). To our delight, a4s4, a4s5 showed obvious improvement of protea-some inhibitory activity around 1 lM despite a4s3 was incompe-tent. Bulky diamine moiety binaphthyl diamine a9 was used to replace a4, but the activity disappeared. Furthermore, when the salicylaldehyde moiety s5 was fixed, a3s5 was prepared to com-pare with a4s5. Although both of them have activity, a3s5 showed higher IC50 approximately 6 times to a4s5. Therefore, both the dia-mine and the salicylaldehyde are crucial, and precise collaboration of these two moieties is essential for the rational design of metal Schiff base complexes as proteasome inhibitors.
Then we synthesized series of copper(II) and platinum(II) com-plexes in different solvents with modest to good yields (Scheme 1). To confirm the accurate structure of the copper(II) complex, proper diffusion of ethanol into the aqueous solution of Na2[Cu(a2s3)] afforded brownish red crystals. Before this, crystal structure of Na2[Cu(a2s3)] with half-hydrolyzed C@N bond has been reported by Rogez and co-workers.37 In this work, we obtained the intact single crystal of Na2[Cu(a2s3)] which crystallizes in the triclinic P1 space group. The X-ray diffraction reveals a complete Schiff base ligand in the space lattice consists of NNOO tetradentate coordina-tion with a central copper ion structure. It is also clear to see the two deprotonated sulfonate groups on the salicylaldehydes bond-ing sodium ions (Fig. 2).
Although complexation of copper ion with Schiff base ligand might change the structure significantly, the primary results of proteasome inhibition of the Schiff base ligands are still valuable. We considered that complexation of copper ion with Schiff base ligand might change the activity by alteration of the molecular configuration and further the bonding mode in the active site of proteasome. Before the evaluation of copper(II) complexes, we examined the fluorescence spectrum of our compounds including all the Schiff base ligands and copper(II) complexes in the presence of the reaction buffer, all of them showed negligible fluorescence intensity under the test condition of proteasome inhibition exper-iment. Furthermore, we tested the fluorescence of Schiff base ligand with addition of magnesium ion in HEPEs buffer but no obvious change of fluorescence spectrum has been observed which indicated that magnesium ion has no influence to our
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T. Zhou et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
R1 R
1
CHO N NH2 N NH
R1 + R4 OH EtOH R4 OH Cu(OAc)2 Cu
R4 O
H2N reflux MeOH
NH2
R3 R2 R 3 R R R
2 3
a as1/ 2 2
s Cu(as 1/2 )
1/2
R1 R1
CHO N N N N
R1 + 2R4 OH EtOH R OH HO R Cu(OAc)2 R Cu R
4 4 OO
H2N NH2 reflux 4 MeOH 4
R3 R2 R R R 2 R R R R 2 R
a s 3 2 as 3 3 2 3
Cu(as)
Scheme 1. Synthetic routes of tridentate and tetradentate copper(II) Schiff base complexes.
Figure 2. An ORTEP view (50% ellipsoids) of Na2[Cu(a2s3)] (CCDC 847754).
experiments.38 Then we did control experimental to eliminate the possible quenching and we found the copper(II) complexes does not quench the final fluorescent readout. All of these preliminary experiments showed the possible deviation of our results inter-rupted by photoluminescence does not exist thus we initiated our biological evaluation of these copper(II) complexes.
As shown in Table 2, although the Schiff base ligands and their corresponding copper complexes exhibited nonparallel relation-
ship on the proteasome inhibition, we still found encouraging results that Na2[Cu(a3s3)], Na2[Cu(a4s4)], Na2[Cu(a4s5)] and
Table 2
Copper Schiff base complexes as proteasome b2 inhibitorsa
Complex IC50b (lM)
Complex IC50 (lM)
Na2[Cu(a1s1)] NIc
Na2[Cu(a4s5)] 7.65 ± 0.24
Na2{Cu[a2(s3)1/2]} NI Na2[Cu(a5s3)] NI
Na2[Cu(a2s3)] 1.08 ± 0.09 Na2[Cu(a6s1)] NI
Na2[Cu(a3s2)] NI Na2[Cu(a6s3)] NI
Na2[Cu(a3s3)] 7.01 ± 0.14 Na2[Cu(a7s1)] NI
Na2[Cu(a4s1)] 0.89 ± 0.02 Na2[Cu(a8s3)] NI
Na2[Cu(a4s3)] 23.94 ± 0.32 Na2[Cu(a9s3)] 6.78 ± 0.18
Na2[Cu(a4s4)] 3.77 ± 0.21 Bortezomibd 4.2 ± 0.34
a Values represent the mean ± SD of three independent experiments, each based on four biological replicates.
b Percent inhibition at 25 lg mL 1.
c NI: no inhibition.
d Positive control.
Na2[Cu(a9s3)] showed obvious proteasome b2 inhibitory activities with negligible effect on b1 and b5 subunits. It is noteworthy that Na2[Cu(a4s1)] showed submicromolar IC50 toward b2 subunit. Besides, Na2[Cu(a2s3)] also showed b2 inhibitory activity approxi-mate to Na2[Cu(a4s1)]. Parallel to the results of Schiff base ligands, 5-carboxylic acid substituted diamine a4 played positive role in proteasome b2 inhibition. All copper complexes containing a4 moi-ety exhibited moderate to excellent activities. In detail, s3 still played negative role similar to the results obtained from Schiff base ligands. In spite of the negative effect of s3 involved in the specific type of a4 family, it reversed the effect in Na2[Cu(a2s3)] and Na2[Cu(a9s3)]. Structure differentiation affecting the interac-tion between copper complexes and their targets is accountable for the proteasome inhibition though the details are not clear.
To reveal the structure–activity relationships, AutoDock was often used in our previous study as one of the powerful tools.39 Herein, Autodock 4.2 was used to predict of the bonding mode between the potent copper complexes and different proteasome
b subunits by docking Cu(a4s1) into b2 and b5 subunits (PDB: 5cgi) of proteasome respectively. As shown in Figure 3a and b, the 3,4-diaminobenzoic acid moiety stretched into the pocket of b2, generating hydrogen bonds between OH(Thr1)-COOH, C@O (Arg19)-COOH, NH(Lys33)-COOH. Meanwhile, the salicylaldehyde groups are buried in the gap, with the two oxygen atoms as part of the hydrogen bond network together with Gln22. In another case, the binding mode of Cu(a4s1) in b5 subunit is distinguished.
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Figure 3. (a) and (b) Conformation of Cu(a4s1) in proteasome b2 subunit. (c) (d) Cu(a4s1) in proteasome b5 subunit.
As shown in Figure 3c and d, the spatial orientation of Cu(a4s1) in b5 subunit is opposite. The hydrogen bonds formed by nearest resi-dues of amino acids including Thr1, Gly47 with two phenoxy oxy-gen atoms of salicylaldehydes [OH(Thr1)-O, OH(Gly47)-O]. Besides, the phenoxy group of Tyr170 might has interaction with the copper center.
Comparing the bonding mode of Cu(a4s1) in b2 and b5 subunits, it is observed clearly that the buried depth and spatial orientation of inhibitor in each pocket, the amount and pattern of hydrogen bonds are quite different. Due to the carboxylic group, the spatial orientation of Cu(a4s1) in b2 and b5 subunits are opposite while Cu(a4s1) embedded deeper in b2 than in b5. Moreover, the amount of hydrogen bonds between Cu(a4s1) and target in b2 subunit is more than that in b5 subunit. All of these differences might con-tribute to the selective proteasome inhibition of copper(II) complexes.
In summary, we demonstrated the design, synthesis and biolog-ical evaluation of series of copper(II) Schiff base complexes as pro-teasome inhibitors. Consequently we discovered several copper(II) Schiff base complexes which exhibited highly inhibitory activities toward proteasome with selective b2 subunit inhibition. Together, some valuable structure–activity relationships information has been obtained in our present study which may lead further opti-mization for copper complexes as antitumor drug candidates. Mechanistic study and other evaluation of these copper Schiff base complexes as antitumor agents is now underway in our laboratory.
Acknowledgments
This work was supported by the National Natural Science Foun-dation of China (21202003/21571007/21271013), the Beijing Nat-ural Science Foundation (7162110).
Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.10. 043.
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M.; Tokhunts, R. A.; Amir, O.; Goddard, A. L.; Pelphrey, P. M.; Wright, D. L.;Highly Selective Inhibitor Library