Bioorganic & Medicinal Chemistry Letters 24 (2014) 2098–2104
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Molecular Dynamics simulations of Inhibitor of Apoptosis Proteins and identification of potential small molecule inhibitors Jayanthi Jayakumar, Sharmila Anishetty ⇑ Centre for Biotechnology, Anna University, Chennai 600 025, India
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Article history: Received 20 December 2013 Revised 13 March 2014 Accepted 17 March 2014 Available online 26 March 2014 Keywords: Inhibitor of Apoptosis Proteins (IAPs) Cancer SMAC MD simulations Small molecule inhibitors
a b s t r a c t Chemotherapeutic resistance due to over expression of Inhibitor of Apoptosis Proteins (IAPs) XIAP, survivin and livin has been observed in various cancers. In the current study, Molecular Dynamics (MD) simulations were carried out for all three IAPs and a common ligand binding scaffold was identified. Further, a novel sequence based motif specific to these IAPs was designed. SMAC is an endogenous inhibitor of IAPs. Screening of ChemBank for compounds similar to lead SMAC-non-peptidomimetics yielded a cemadotin related compound NCIMech_000654. Cemadotin is a derivative of natural anti-tumor peptide dolastatin-15; hence these compounds were docked against all three IAPs. Based on our analysis, we propose that NCIMech_000654/dolastatin-15/cemadotin derivatives may be investigated for their potential in inhibiting XIAP, survivin and livin. Ó 2014 Elsevier Ltd. All rights reserved.
One of the main players in apoptosis is a family of cysteine dependent aspartate specific proteases called caspases.1 Caspase activity can be abolished either by its inhibition or through ubiquitinilation and subsequent degradation. Studies indicate that Inhibitor of Apoptosis Proteins (IAPs) are capable of fulfilling both these functions.2 IAPs are anti apoptotic and are endogenous inhibitors of caspases. Under cancerous conditions, cells escape apoptosis and one of the crucial factors contributing to this is a higher level of expression of IAPs. Currently, there are eight known human IAPs: X-linked inhibitor of apoptosis (XIAP), cIAP1, cIAP2, NAIP (NLR family, apoptosis inhibitory protein), livin, ILP2 (IAP-like protein 2), BRUCE and survivin.3 They are characterized by the presence of 1 to 3 copies of a zinc binding Baculoviral IAP Repeat (BIR) domain at their N terminus, each of which is approximately 70 amino acids in length. Some IAPs have a RING finger domain at the C terminus.3 XIAP, survivin and livin are highly expressed in several types of cancer. Of these, XIAP is the most well characterized IAP. It inhibits caspases-3, -7 and -9, and is the only IAP capable of blocking caspase activation both at the initiation and the execution phases.2 Survivin (BIRC5) is the smallest known IAP.4 It is said to inhibit caspases-3 and -95 and is associated with higher tumor grade, advanced stage of cancers and chemo-resistance. It is expressed only in cancer cells or during embryonic development, but not in ⇑ Corresponding author. Tel.: +91 44 22350772; fax: +91 44 22350299. E-mail addresses: (S. Anishetty).
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http://dx.doi.org/10.1016/j.bmcl.2014.03.046 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
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normal cells.5,6 Livin also called as KIAP or ML-IAP (Melanoma IAP) is not detected in most adult tissues but is highly expressed in melanoma cancer cells.7 It has a single BIR domain (through which it interacts with caspases-3, -7 and -9) and a carboxy terminal RING finger domain. SMAC/DIABLO (Second mitochondrial derived activator of caspase, direct inhibitor of apoptosis binding protein with low pI) is a mitochondrial protein which is released into the cytosol along with cytochrome c when the cell undergoes apoptosis. SMAC is an endogenous IAP inhibitor.8 Structural studies indicate that SMAC binds to BIR2 and BIR3 domains of XIAP and inhibits its activity.9 It competes with caspase-9 in binding to BIR3 domain of XIAP.10 SMAC binds to survivin and indirectly inhibits apoptosis.5 Livin can also bind to SMAC.11 XIAP, survivin and livin are amongst the therapeutic targets for cancer. Any molecular level insights into these proteins will be valuable for development of drugs against them. MD simulation studies of XIAP and survivin in complex with SMAC and various inhibitors have provided insights into protein interaction and drug binding modes.4,12,13 In this study, we have performed two sets of Molecular Dynamics (MD) simulations (in the presence and absence of zinc ions) of the three IAPs: XIAP-BIR3, survivin and livin, in order to gain insights into the dynamics of BIR domains and identify a scaffold for rational drug design. We chose BIR3 domain of XIAP since it has been shown to be the minimal region of XIAP that can inhibit caspase-9. To our knowledge this is the first report on MD simulations of livin. Additionally, a comparative MD study of IAPs in the presence and absence of zinc has not been
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reported in literature. A sequence based novel motif, specific to XIAP-BIR3, survivin, livin has been designed. Further, compounds similar to lead SMAC non peptidomimetics were screened from ChemBank database14 and their affinity towards the three IAPs was assessed using molecular docking tools. The crystal structure of XIAP-BIR3 (PDB ID|A:3HL5), survivin (PDB ID|A:2RAW) and livin (PDB ID|A:1OXN) were retrieved from PDB database.15 Since IAPs are zinc metallo-proteins, two sets of simulations were performed for each of these proteins: one in the presence and the other in the absence of zinc ion. The function genrestr from GROMACS version 4 was used to impose distance restraints on the zinc ion such that the protein remains zinc bound. In the other set of simulations, zinc ion was removed before subjecting the proteins to MD simulation. Apart from these two steps all other parameters and setup was similar for both sets of simulations. All atom MD simulations were performed using GROMACS.16 Proteins were solvated with explicit solvent SPC water in a cubic box, which left 0.3 nm space around the solute for XIAP-BIR3, survivin and livin for simulations performed with and without zinc. To neutralize the simulation system, counter ions in the form of Na+ and Cl ions were added as required. Energy minimization was performed using steepest descent method. The system was weakly coupled to an external bath using Berendson’s method with the reference temperature fixed at 300 K. All bonds were constrained with LINCS.17,18 Energy calculations were performed using G43a1 force field in GROMACS. During the simulation, grid type neighbor searching was done and long-range electrostatics was handled using PME.19 Position restrained MD simulation was performed for 50 ps for all the energy minimized structures for each of the six simulations. This was followed by full scale all atom MD simulation. A time step of 2 fs was used. Coordinates were written every 0.5 ps to the trajectory. Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) were computed using built in tools found in GROMACS. Xmgrace was used to plot the graphs [http://plasma-gate.weizmann.ac.il/Grace/]. Residues with RMSF of 0.2 nm and above were considered mobile. Inter-atomic contacts of PDB complexes of XIAP-BIR3 and caspase-9 (PDB ID: 1NW9), XIAP-BIR3 and SMAC (PDB ID: 1G3F), livin-SMAC mimetic (PDB ID:1OXN) were computed using WHAT IF server.20 Representative sequences of XIAP, survivin and livin with UniProt IDs P98170, Q60989, Q9R0I6, Q8JGN5, Q4R1J6, Q50L39, Q804H7,Q28ER3, Q6J1J1, Q8I009, Q6I6F4, O15392, O70201, Q9GLN5, Q5RAH9, Q9JHY7, Q96CA5 and A2AWP0 were retrieved from UniProtKB.21 Multiple sequence alignment was performed using ClustalX22 and a conserved motif was designed. This was scanned against the UniProtKB/SwissProt using ScanProsite tool.23 The precision and recall of the motif was computed taking into consideration the reviewed sequences from UniprotKB. Lead SMAC-non-peptidomimetics were retrieved from literature and compounds similar to these were obtained by searching ChemBank database based on tanimoto coefficient > 0.8 (Supplementary Table S1). Docking of small molecules with the three IAPs was performed using AutoDock-4.24 The standard docking protocol was used for rigid protein and flexible ligand where default grid spacing was 0.375 Angstroms. The IAP family proteins XIAP, survivin and livin have conserved BIR domains. They are zinc-binding proteins with three conserved cysteines and one histidine coordinated to a zinc ion. IAPs contain IAP binding motif (IBM) surface groove that can bind to IBM containing proteins. The IBM is located at the N-terminal region of caspases and pro-apoptotic IAP-antagonists like SMAC. We performed two sets of MD simulations for these three proteins (one in the presence and the other in the absence of zinc ions). Simulation of XIAP-BIR3 was performed for 25 ns in the presence of zinc. The RMSD initially increased to 0.4 nm and remained
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the same till 10 ns. After 10 ns it gradually increased to 0.82 nm and then became stabile around 20 ns and maintained its stability till the end of the simulation (Figure 1a). The protein on the whole was mobile. Residue wise fluctuations are shown in Figure 2a. XIAP binds to caspase-9 and inhibits its activity, while SMAC can bind to XIAP and relieve this inhibition. The BIR3 domain utilizes a two site binding mechanism for caspase binding, one of which is an exosite, the IBM interacting groove. Additionally, a helix distal to the BIR3 domain which packs into the dimer interface of caspase-9, prevents it from going into an active dimeric conformation.2 Caspase-9 and SMAC bind to XIAP-BIR3 in a mutually exclusive way.10 The residues important for interaction with caspase-9, SMAC and IBM groove are tabulated in Table 1. We find that some of the residues (308, 310, 314, and 323) that bind to caspase-9 and SMAC overlap and these were mobile in the simulation. Stable region includes Asn280–Glu282, Phe289–Tyr290, Val298–Phe301, Glu318–His320 and Leu330–Leu331. This includes part of ARTs (antagonist of XIAP) binding interface (Phe272– Leu292) and the zinc coordinated residues His320 and Cys300 of sheet-2. Lys299 of hydrophobic pocket (Val297–Lys299) with a high affinity for SMAC/SMAC mimetics binding27 was also in the stable region. This region binds exclusively to SMAC. Survivin is an important drug target in cancer as it is a nodal protein orchestrating the integration of several pathways related to tumor cell viability and maintenance. It has a single BIR domain, spanning residues 18–88 and a long carboxy terminal alpha helix. It is associated with regulators of cytokinesis, Aurora B kinase, INCENP and Borealin.28 All functional sites of interest are tabulated in Table 1. SMAC inhibits survivin and this results in an indirect method of caspase inhibition wherein binding of SMAC to other IAPs is prevented.5 Leu54, Leu64, Glu65, Trp67, Glu76, and His80 of survivin are said to be important for binding to SMAC.29 MD simulation of survivin was performed for 10 ns in the presence of zinc. The RMSD initially increased to 1.4 nm and then stabilized. The protein became stable around 3.5 ns and remained so till the end of the simulation (Figure 1b). Most part of the protein showed high mobility. The high deviation in RMSD is due to the mobility of its C-terminal helix. This region encompassing residues 97–141 is involved in dimerization and interaction with borealin, a key regulator of chromosome segregation and cytokinesis.28 The other flexible regions include Thr5–Cys33, Pro47–Asp53 and Cys59–Leu96. We observed a similar pattern of certain exclusively SMAC interacting residues (Leu54, Lys62) showing low mobility, compared to residues common to IBM interaction groove and SMAC interaction. Except Cys57 of strand-2 other zinc co-ordinated residues are in the mobile region. Stable region includes Thr34–Cys46 and Leu54– Phe59. RMSF graph is shown in Figure 2b. MD Simulation of livin was performed for 25 ns in the presence of zinc. The BIR domain of livin spans residues 90–155. The RMSD initially increased to 0.6 nm and then stabilized (Figure 1c). It became stable around 15 ns and remained so till the end (25 ns) of the simulation. The amino acids interacting with SMAC and SMAC-based peptides are shown in Table 1. Similar to XIAP-BIR3 and survivin, livin was also highly mobile. We observed that most of the mobile regions are in the dimeric interface and few residues are involved in SMAC interaction. Asp120 is involved in both SMAC and caspase interaction. Except Cys124 and Cys127 other two zinc co-ordinated residues are in the mobile region. Stable region includes Leu108, Phe113, Val122–Cys124 and Cys127. These regions correspond to residues involved in SMAC mimetic interactions and zinc coordination. RMSF graph is shown in Figure 2c.
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Figure 1. RMSD of three IAPs (a) RMSD of XIAP-BIR3 (BIR3 domain encompasses 265–330 shown in graph as 13–78) (b) RMSD of survivin (BIR domain encompasses 18–88 shown in graph as 14–84) (c) RMSD of livin (BIR domain encompasses 90–155 shown in graph as 20–85). Simulations performed in the presence of zinc are shown in green and those in the absence of zinc are shown in red.
Figure 2. RMSF of three IAPs (a) RMSF of XIAP-BIR3 (BIR3 domain encompasses 265–330 shown in graph as 13–78) (b) RMSF of survivin (BIR domain encompasses 18–88 shown in graph as 14–84) (c) RMSF of Livin (BIR domain encompasses 90–155 shown in graph as 20–85). Simulations performed in the presence of zinc are shown in green and those in the absence of zinc are shown in red.
Table 1 Functional sites of interest of the three IAPs Protein name
SMAC binding region
Caspase binding region / Mutational studies
Other protein interactions
Zinc binding residues
IBM binding groove
XIAPBIR3
296, 297, 299, 306–308, 310, 314, 315, 318, 319, 323–326, 3439 54, 64, 65, 67, 76, 8029
275, 277, 279, 308, 310, 311, 314, 319, 323, 325, 326, 343, 34410,31–33
ARTs (272–277)33
300, 303, 320, 327
314, 319, 3232
Kinases Cdk1, aurora kinase B and plk1 phosphorylate Thr34, 38; Thr117 and Ser 20 respectively of the survivin29
57, 60, 77, 84
76, 71, 802
116, 120–124, 130–134, 138, 143–144, 147, 14835,36
87, 88, 12034
121, 124, 143, 147
138, 143, 1472
Survivin
Livin
A common theme in all the simulations was, SMAC binding residues, which overlap with caspase binding residues/IBM interacting groove, showed higher mobility. Exclusively SMAC binding
residues are relatively stable in all the simulations. In all three IAPs, throughout the course of simulations, we found a pattern of residues mapping to strand-1, -2,turn-2 and part of helix-3 being
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moderately stable (i.e.) with low fluctuations. Strand-2 plays a major role in SMAC and zinc ion binding. Turn-2 does not have any direct importance in SMAC or caspase binding, but comprises of a conserved ‘AGF’ pattern hosting a hydrophobic environment (Figure 3). The functional importance of helix-3 is not yet reported. However, based on its strong hydrogen bond interaction with turn-4 of all three IAPs in SMAC-bound state (PDB structures), we propose that it may be important for the function or structural stability of these IAPs. MD Simulation for XIAP-BIR3 was performed for 25 ns in the absence of zinc. It became stable around 15 ns and remained stable thereafter. The RMSD initially increased to 0.6 nm and then stabilized (Figure 1a). In the absence of zinc, XIAP-BIR3 was less fluctuating than in its presence (as deciphered through RMSF) (Figure 2a). Secondary structure was not retained after 1 ns. We were able to observe the low fluctuations of strand-1, -2 based on RMSF throughout the course of simulation. MD Simulation for survivin was performed for 10 ns in the absence of zinc. It became stable around 5 ns and remained stable thereafter. The RMSD initially increased to 1.1 nm and then stabilized (Figure 1b). In the absence of zinc, similar kinds of fluctuations were observed as seen with zinc (as evidenced through RMSF) (Figure 2b) and there was a partial reformation of secondary structure after 4.5 ns. In this simulation also we observed the stability of strand-1 and -2 throughout the course of simulation both structurally and also based on RMSF. MD Simulation for livin was performed for 25 ns in the absence of zinc. It became stable around 2 ns and was stable till the end of
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the simulation. The RMSD initially increased to 0.62 nm and then stabilized (Figure 1c). Fluctuations in some regions were similar to that seen with zinc (Figure 2c). Similar to XIAP-BIR3, even in the absence of zinc also we were able to observe the stability of strand-1,-2 throughout the course of simulation. Stability of zinc coordinating residue in strand-2 of survivin and livin even in the absence of zinc indicates that, coordination by zinc is perhaps not the only factor responsible for the stability. Multiple Sequence alignment of XIAP, survivin and livin was performed to generate a conserved motif of functional significance (Figure 3). The motif ‘[QGP]DX[VTA]XCF[FH]CXXXLXXW’ was specific to XIAP-BIR3, survivin-BIR and livin-BIR. It had a precision of 95.2% and recall of 76.9%. The motif spans residues 295–310 in the case of XIAP-BIR3, residues 52–67 in the case of survivin and residues 119–134 in the case of livin. This includes turn-3, strand-2,-3 and turn-4 (which connects the strand-2 and -3) of BIR domain of all three IAPs. On correlating the motif with MD simulations, we find that in each of the three proteins, zinc-coordinating residue in strand-2 is stable in the simulations performed in the presence and absence of zinc. Additionally strand-2, which overlaps with the motifs, was also structurally stable in both sets of simulations of survivin and stable in the presence of zinc in the case of livin. SMAC is a common binding partner of all three IAPs. Hence, compounds similar to available lead SMAC non-peptido-mimetics were screened from Chembank database. We found five compounds similar to 40d (one of the lead SMAC-non-peptidomimetics) satisfying the criterion of tanimoto coefficient > 0.8 (details shown in
Figure 3. Multiple sequence alignment of XIAP-BIR3, survivin-BIR and livin-BIR. (1) A conserved ‘AGF’ hydrophobic pattern, (2) sequence based novel motif.
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Supplementary Table S1). Interestingly N,N-dimethyl-D-valyl-D-valyl-N-methyl-L-valyl-D-prolyl-N-benzyl-D-prolinamide (NCIMech_ 000654), a compound related to cemadotin was retrieved as a compound similar to 40d. Cemadotin (N,N-dimethyl-L-valyl-Lvalyl-N-methyl-L-valyl-L-prolyl-N-benzyl-L-prolinamide) is a synthetic analogue of dolastatin-15 (tubulin inhibitor) that has
anti-proliferative and antitumor activity.25 Four compounds, dolastatin-15, cemadotin, NCIMech_000654 and compound 40d26 were docked against all three IAPs. We find that interactions of all these compounds with the BIR domain of the three IAPs were in the novel motif region described in this study. Schematic representations of these four compounds are shown in Figure 4. The docked
Figure 4. Schematic representation of four compounds: 40d, NCIMech_000654, dolastatin-15 and cemadotin.
Figure 5. Representation of docked complexes of the three IAPs with NCIMech_000654. (a) and (b) shows hydrogen bond interactions of XIAP and survivin, (c) shows pi interactions of livin with NCIMech_000654. (d–f) are 2D plots of interactions between NCIMech_000654 and XIAP, survivin and livin, respectively.
J. Jayakumar, S. Anishetty / Bioorg. Med. Chem. Lett. 24 (2014) 2098–2104 Table 2 Interacting amino acid residues and binding energies of the docked complexes of IAPs with various ligands Docked complex
Amino acids having hydrogen bond interaction with the ligand
XIAP-BIR3–NCIMech_000654 XIAP-BIR3–Dolastatin-15 XIAP-BIR3-40d XIAP-BIR3–Cemadotin Survivin–NCIMech_000654 Survivin–Dolastatin-15 Survivin-40d Survivin–Cemadotin Livin–NCIMech_000654 Livin–Dolastatin-15
Lys 297, Gly 306 Lys 297 Lys 299 Lys 299, Gly 306 Gln 56 Gln 56 Glu 63 Glu 63, Lys 62 Arg 123 (Pi interaction) Arg 123 (Pi interaction), Tyr 128 (Pi interaction) Gly 130 Arg 123 (Pi interaction), Gly 130
Livin-40d Livin–Cemadotin
Binding energy (kcal/mol) 4.31 5.38 2.86 4.23 4.64 0.04 6.34 5.49 5.83 1.72 4.86 4.86
complexes of the three IAPs with NCIMech_000654 are shown in Figure 5 and details of interactions with 40d, cemadotin, NCIMech_000654 and dolastatin-15 are provided in Table 2. XIAP, survivin and livin are considered to be potential therapeutic targets due their exclusive over-expression in many types of cancer. XIAP-BIR3 and livin have similar mode of binding with SMAC/SMAC mimetics. Experimental studies have shown that survivin binds to SMAC through its strand-3.29 Among the three IAPs, XIAP-BIR3 has the highest binding affinity towards SMAC. Livin inhibits caspase-9 less potently than XIAP-BIR3,30 but there is no evidence for interaction of survivin with caspase-9. SMAC, caspase and IBM (IAP binding motif) interacting groove residues of the three IAPs were compared with each other through MD simulations to gain insights into their behaviour. We observed that there was an overlap in SMAC and caspase/ IBM-interacting groove residues in the three IAPs. These interacting residues were mobile in XIAP-BIR3, survivin and livin. Exclusively SMAC binding residues were less mobile and relatively
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stable in all three IAPs. SMAC binding lysine residues were moderately stable (Lys299 of XIAP-BIR3, Lys62 of survivin and Lys121 of livin) in all three IAPs. Helix-5 of XIAP-BIR3 is necessary for caspase-9 interaction and it had least similarity at a sequence level with the corresponding helix of livin.30 Helix-5 of XIAP-BIR3 was relatively more stable compared to helix-5 of livin. There was a higher mobility of helix-5 of livin probably because of more hydrophilic residues in this region. All three IAPs behave in different ways, yet they have a similar pattern of maintaining the stability of part of alpha-helix-3, strand2 and turn-2 till the end of the simulation. A novel sequence based motif specific to all three IAPs was designed. This motif does not overlap with the known IBM interacting groove (Figure 6). From our simulations we propose that the motif region provides a relatively stable scaffold of possible functional significance. The novel motif has two zinc binding residues, along with a few caspase and SMAC binding residues. BIR domains coordinate with zinc ion which may be important for structural or functional stability. Since caspases use cysteine and histidine in their catalytic mechanism, they can be inhibited by metals like zinc which is a potent inhibitor of caspases.37,38 It is possible that metal binding by BIR domain may have a role in caspase inhibition mechanism of IAPs.39 Design of a small molecule or a mimetic which can target the motif region of IAPs can help caspase enter the apoptotic pathway. We chose NCIMech_000654 from our hits, since it is related to cemadotin which is a compound with known antitumor activity. We sought to find if NCIMech_000654 and related compounds can bind with XIAP, survivin and livin. Our docking studies with cemadotin, NCIMech_000654, dolastatin-15 and compound 40d show that all these in fact bind to all three IAPs and these interactions are within the novel motif region designed in this study. Among these, NCIMech_000654, compound 40d and cemadotin were found to exhibit a higher binding affinity with all three IAPs while dolastatin-15 had good affinity for XIAP-BIR3. We hypothesize that NCIMech_000654/dolastatin-15/cemadotin derivatives may help in the inhibition of these three IAPs.
Figure 6. Representation of the novel motif and IBM groove. The novel motif is shown in white with the ends marked as p and q.
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In summary, IAPs are now being increasingly recognized as therapeutic targets in cancer. Several groups are exploring small molecules for inhibition of these IAPs. In the current study, we have gained insights into the dynamics of XIAP-BIR3, survivin and livin and identified regions of potential importance which can be utilized for rational drug design. Using molecular docking, we have shown that NCIMech_000654, natural peptide dolastatin-15 and its derivative cemadotin interact with the motif region. Based on this analysis, we propose that NCIMech_000654/cemadotin/dolastatin derivatives may have a potential in inhibiting XIAPBIR3, survivin and livin. Acknowledgments The authors thank DBT, Government of India for support (BT/ 01/COE/07/01). The authors also thank BTIS, Department of Biotechnology for computational facilities. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03. 046. References and notes 1. Lamkanfi, M.; Festjens, N.; Declercq, W.; Vanden Berghe, T.; Vandenabeele, P. Cell Death Differ. 2004, 14, 44. 2. Eckelman, B. P.; Salvesen, G. S.; Scott, F. L. EMBO Rep. 2006, 7, 988. 3. Schimmer, A. D. Cancer Res. 2004, 64, 7183. 4. Obiol-Pardo, C.; Granadino-Roldán, J. M.; Rubio-Martinez, J. J. Mol. Recognit. 2008, 21, 190. 5. Chiou, S. K.; Jones, M. K.; Tarnawski, A. S. Med. Sci. Monit. 2003, 9, 125. 6. Pennati, M.; Folini, M.; Zaffaroni, N. Carcinogenesis 2007, 28, 1133. 7. Kasof, G. M.; Gomes, B. C. J. Biol. Chem. 2001, 276, 3238. 8. Du, C.; Fang, M.; Li, Y.; Li, L.; Wang, X. Cell 2000, 102, 33. 9. Liu, Z.; Sun, C.; Olejniczak, E. T.; Meadows, R. P.; Betz, S. F.; Oost, T.; Herrmann, J.; Wu, J. C.; Fesik, S. W. Nature 2000, 408, 1004. 10. Srinivasula, S. M.; Hegde, R.; Saleh, A.; Datta, P.; Shiozaki, E.; Chai, J.; Lee, R. A.; Robbins, P. D.; Fernandes-Alnemri, T.; Shi, Y.; Alnemri, E. S. Nature 2001, 410, 112. 11. Ma, L.; Huang, Y.; Song, Z.; Feng, S.; Tian, X.; Du, W.; Qiu, X.; Heese, K.; Wu, M. Cell Death Differ. 2006, 13, 2079. 12. Ling, B.; Dong, L.; Zhang, R.; Wang, Z.; Liu, Y.; Liu, C. J. Mol. Graph. Model. 2010, 29, 354.
13. Park, I. H.; Li, C. J. Phys. Chem. B 2010, 114, 5144. 14. Seiler, K. P.; George, G. A.; Happ, M. P.; Bodycombe, N. E.; Carrinski, H. A.; Norton, S.; Brudz, S.; Sullivan, J. P.; Muhlich, J.; Serrano, M.; Ferraiolo, P.; Tolliday, N. J.; Schreiber, S. L.; Clemons, P. A. Nucleic Acids Res. 2008, 36, D351. 15. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235. 16. Lindahl, E.; Hess, B.; van-der-Spoel, D. J. Mol. Model. 2001, 7, 306. 17. Miyamoto, S.; Kollman, P. A. J. Comput. Chem. 1992, 3, 952. 18. Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. J. Comput. Chem. 1997, 16, 273. 19. Essman, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. J. Chem. Phys. 1995, 103, 8577. 20. Vriend, G. J. Mol. Graph. 1990, 8, 52. 21. The Universal Protein Resource (UniProt) consortium Nucleic Acids Res. 2008, 36, D190. 22. Thompson, J. D.; Gibson, T. J.; Plewniak, F.; Jeanmougin, F.; Higgins, D. G. Nucleic Acids Res. 1997, 25, 4876. 23. De-Castro, E.; Sigrist, C. J.; Gattiker, A.; Bulliard, V.; Langendijk-Genevaux, P. S.; Gasteiger, E.; Bairoch, A.; Hulo, N. Nucleic Acids Res. 2006, 1, W362. 24. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785. 25. Supko, J. G.; Lynch, T. J.; Clark, J. W.; Fram, R.; Allen, L. F.; Velagapudi, R.; Kufe, D. W.; Eder, J. P. J. R. Cancer Chemother. Pharmacol. 2000, 46, 319. 26. Seneci, P.; Bianchi, A.; Battaglia, C.; Belvisi, L.; Bolognesi, M.; Caprini, A.; Cossu, F.; Franco, E. d.; Matteo, M. d.; Delia, D.; Drago, C.; Khaled, A.; Lecis, D.; Manzoni, L.; Marizzoni, M. Bioorg. Med. Chem. 2009, 17, 5834. 27. Oost, T. K.; Sun, C.; Armstrong, R. C.; Al-Assaad, A. S.; Betz, S. F.; Deckwerth, T. L.; Ding, H.; Elmore, S. W.; Meadows, R. P.; Olejniczak, E. T.; Oleksijew, A.; Oltersdorf, T.; Rosenberg, S. H.; Shoemaker, A. R.; Tomaselli, K. J.; Zou, H.; Fesik, S. W. J. Med. Chem. 2004, 47, 4417. 28. Altieri, D. C. Nat. Rev. Cancer 2008, 8, 61. 29. Sun, C.; Nettesheim, D.; Liu, Z.; Olejniczak, E. T. Biochemistry 2005, 44, 11. 30. Vucic, D.; Franklin, M. C.; Wallweber, H. J.; Das, K.; Eckelman, B. P.; Shin, H.; Elliott, L. O.; Kadkhodayan, S.; Deshayes, K.; Salvesen, G. S.; Fairbrother, W. J. Biochem. J. 2005, 385, 11. 31. Sun, C.; Cai, M.; Meadows, R. P.; Xu, N.; Gunasekera, A. H.; Herrmann, J.; Wu, J. C.; Fesik, S. W. J. Biol. Chem. 2001, 275, 33777. 32. Shiozaki, E. N.; Chai, J.; Rigotti, D. J.; Riedl, S. J.; Li, P.; Srinivasula, S. M.; Alnemri, E. S.; Fairman, R.; Shi, Y. Mol. Cell 2003, 11, 519. 33. Bornstein, B.; Gottfried, Y.; Edison, N.; Shekhtman, A.; Lev, T.; Glaser, F.; Larisch, S. Apoptosis 2011, 16, 869. 34. Vucic, D.; Stennicke, H. R.; Pisabarro, M. T.; Salvesen, G. S.; Dixit, V. M. Curr. Biol. 2000, 21, 1359. 35. Vucic, D.; Deshayes, K.; Ackerly, H.; Pisabarro, M. T.; Kadkhodayan, S.; Fairbrother, W. J. J. Biol. Chem. 2002, 277, 12275. 36. Franklin, M. C.; Kadkhodayan, S.; Ackerly, H.; Alexandru, D.; Distefano, M. D.; Elliott, L. O.; Flygare, J. A.; Mausisa, G.; Okawa, D. C.; Ong, D.; Vucic, D.; Deshayes, K.; Fairbrother, W. J. Biochemistry 2003, 27, 8223. 37. Perry, D.; Smyth, M.; Stennicke, H.; Salvesen, G.; Duriez, P.; Poirier, G.; Hannun, Y. J. Biol. Chem. 1997, 272, 18530. 38. Stennicke, H. R.; Salvesen, G. S. J. Biol. Chem. 1997, 272, 25719. 39. Deveraux, Q. L.; Reed, J. C. Genes Dev. 1999, 13, 239.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Molecular Dynamics simulations of Inhibitor of Apoptosis Proteins and identification of potential small molecule inhibitors Jayanthi Jayakumar, Sharmila Anishetty ⇑ Centre for Biotechnology, Anna University, Chennai 600 025, India
a r t i c l e
i n f o
Article history: Received 20 December 2013 Revised 13 March 2014 Accepted 17 March 2014 Available online 26 March 2014 Keywords: Inhibitor of Apoptosis Proteins (IAPs) Cancer SMAC MD simulations Small molecule inhibitors
a b s t r a c t Chemotherapeutic resistance due to over expression of Inhibitor of Apoptosis Proteins (IAPs) XIAP, survivin and livin has been observed in various cancers. In the current study, Molecular Dynamics (MD) simulations were carried out for all three IAPs and a common ligand binding scaffold was identified. Further, a novel sequence based motif specific to these IAPs was designed. SMAC is an endogenous inhibitor of IAPs. Screening of ChemBank for compounds similar to lead SMAC-non-peptidomimetics yielded a cemadotin related compound NCIMech_000654. Cemadotin is a derivative of natural anti-tumor peptide dolastatin-15; hence these compounds were docked against all three IAPs. Based on our analysis, we propose that NCIMech_000654/dolastatin-15/cemadotin derivatives may be investigated for their potential in inhibiting XIAP, survivin and livin. Ó 2014 Elsevier Ltd. All rights reserved.
One of the main players in apoptosis is a family of cysteine dependent aspartate specific proteases called caspases.1 Caspase activity can be abolished either by its inhibition or through ubiquitinilation and subsequent degradation. Studies indicate that Inhibitor of Apoptosis Proteins (IAPs) are capable of fulfilling both these functions.2 IAPs are anti apoptotic and are endogenous inhibitors of caspases. Under cancerous conditions, cells escape apoptosis and one of the crucial factors contributing to this is a higher level of expression of IAPs. Currently, there are eight known human IAPs: X-linked inhibitor of apoptosis (XIAP), cIAP1, cIAP2, NAIP (NLR family, apoptosis inhibitory protein), livin, ILP2 (IAP-like protein 2), BRUCE and survivin.3 They are characterized by the presence of 1 to 3 copies of a zinc binding Baculoviral IAP Repeat (BIR) domain at their N terminus, each of which is approximately 70 amino acids in length. Some IAPs have a RING finger domain at the C terminus.3 XIAP, survivin and livin are highly expressed in several types of cancer. Of these, XIAP is the most well characterized IAP. It inhibits caspases-3, -7 and -9, and is the only IAP capable of blocking caspase activation both at the initiation and the execution phases.2 Survivin (BIRC5) is the smallest known IAP.4 It is said to inhibit caspases-3 and -95 and is associated with higher tumor grade, advanced stage of cancers and chemo-resistance. It is expressed only in cancer cells or during embryonic development, but not in ⇑ Corresponding author. Tel.: +91 44 22350772; fax: +91 44 22350299. E-mail addresses: (S. Anishetty).
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http://dx.doi.org/10.1016/j.bmcl.2014.03.046 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
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normal cells.5,6 Livin also called as KIAP or ML-IAP (Melanoma IAP) is not detected in most adult tissues but is highly expressed in melanoma cancer cells.7 It has a single BIR domain (through which it interacts with caspases-3, -7 and -9) and a carboxy terminal RING finger domain. SMAC/DIABLO (Second mitochondrial derived activator of caspase, direct inhibitor of apoptosis binding protein with low pI) is a mitochondrial protein which is released into the cytosol along with cytochrome c when the cell undergoes apoptosis. SMAC is an endogenous IAP inhibitor.8 Structural studies indicate that SMAC binds to BIR2 and BIR3 domains of XIAP and inhibits its activity.9 It competes with caspase-9 in binding to BIR3 domain of XIAP.10 SMAC binds to survivin and indirectly inhibits apoptosis.5 Livin can also bind to SMAC.11 XIAP, survivin and livin are amongst the therapeutic targets for cancer. Any molecular level insights into these proteins will be valuable for development of drugs against them. MD simulation studies of XIAP and survivin in complex with SMAC and various inhibitors have provided insights into protein interaction and drug binding modes.4,12,13 In this study, we have performed two sets of Molecular Dynamics (MD) simulations (in the presence and absence of zinc ions) of the three IAPs: XIAP-BIR3, survivin and livin, in order to gain insights into the dynamics of BIR domains and identify a scaffold for rational drug design. We chose BIR3 domain of XIAP since it has been shown to be the minimal region of XIAP that can inhibit caspase-9. To our knowledge this is the first report on MD simulations of livin. Additionally, a comparative MD study of IAPs in the presence and absence of zinc has not been
J. Jayakumar, S. Anishetty / Bioorg. Med. Chem. Lett. 24 (2014) 2098–2104
reported in literature. A sequence based novel motif, specific to XIAP-BIR3, survivin, livin has been designed. Further, compounds similar to lead SMAC non peptidomimetics were screened from ChemBank database14 and their affinity towards the three IAPs was assessed using molecular docking tools. The crystal structure of XIAP-BIR3 (PDB ID|A:3HL5), survivin (PDB ID|A:2RAW) and livin (PDB ID|A:1OXN) were retrieved from PDB database.15 Since IAPs are zinc metallo-proteins, two sets of simulations were performed for each of these proteins: one in the presence and the other in the absence of zinc ion. The function genrestr from GROMACS version 4 was used to impose distance restraints on the zinc ion such that the protein remains zinc bound. In the other set of simulations, zinc ion was removed before subjecting the proteins to MD simulation. Apart from these two steps all other parameters and setup was similar for both sets of simulations. All atom MD simulations were performed using GROMACS.16 Proteins were solvated with explicit solvent SPC water in a cubic box, which left 0.3 nm space around the solute for XIAP-BIR3, survivin and livin for simulations performed with and without zinc. To neutralize the simulation system, counter ions in the form of Na+ and Cl ions were added as required. Energy minimization was performed using steepest descent method. The system was weakly coupled to an external bath using Berendson’s method with the reference temperature fixed at 300 K. All bonds were constrained with LINCS.17,18 Energy calculations were performed using G43a1 force field in GROMACS. During the simulation, grid type neighbor searching was done and long-range electrostatics was handled using PME.19 Position restrained MD simulation was performed for 50 ps for all the energy minimized structures for each of the six simulations. This was followed by full scale all atom MD simulation. A time step of 2 fs was used. Coordinates were written every 0.5 ps to the trajectory. Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) were computed using built in tools found in GROMACS. Xmgrace was used to plot the graphs [http://plasma-gate.weizmann.ac.il/Grace/]. Residues with RMSF of 0.2 nm and above were considered mobile. Inter-atomic contacts of PDB complexes of XIAP-BIR3 and caspase-9 (PDB ID: 1NW9), XIAP-BIR3 and SMAC (PDB ID: 1G3F), livin-SMAC mimetic (PDB ID:1OXN) were computed using WHAT IF server.20 Representative sequences of XIAP, survivin and livin with UniProt IDs P98170, Q60989, Q9R0I6, Q8JGN5, Q4R1J6, Q50L39, Q804H7,Q28ER3, Q6J1J1, Q8I009, Q6I6F4, O15392, O70201, Q9GLN5, Q5RAH9, Q9JHY7, Q96CA5 and A2AWP0 were retrieved from UniProtKB.21 Multiple sequence alignment was performed using ClustalX22 and a conserved motif was designed. This was scanned against the UniProtKB/SwissProt using ScanProsite tool.23 The precision and recall of the motif was computed taking into consideration the reviewed sequences from UniprotKB. Lead SMAC-non-peptidomimetics were retrieved from literature and compounds similar to these were obtained by searching ChemBank database based on tanimoto coefficient > 0.8 (Supplementary Table S1). Docking of small molecules with the three IAPs was performed using AutoDock-4.24 The standard docking protocol was used for rigid protein and flexible ligand where default grid spacing was 0.375 Angstroms. The IAP family proteins XIAP, survivin and livin have conserved BIR domains. They are zinc-binding proteins with three conserved cysteines and one histidine coordinated to a zinc ion. IAPs contain IAP binding motif (IBM) surface groove that can bind to IBM containing proteins. The IBM is located at the N-terminal region of caspases and pro-apoptotic IAP-antagonists like SMAC. We performed two sets of MD simulations for these three proteins (one in the presence and the other in the absence of zinc ions). Simulation of XIAP-BIR3 was performed for 25 ns in the presence of zinc. The RMSD initially increased to 0.4 nm and remained
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the same till 10 ns. After 10 ns it gradually increased to 0.82 nm and then became stabile around 20 ns and maintained its stability till the end of the simulation (Figure 1a). The protein on the whole was mobile. Residue wise fluctuations are shown in Figure 2a. XIAP binds to caspase-9 and inhibits its activity, while SMAC can bind to XIAP and relieve this inhibition. The BIR3 domain utilizes a two site binding mechanism for caspase binding, one of which is an exosite, the IBM interacting groove. Additionally, a helix distal to the BIR3 domain which packs into the dimer interface of caspase-9, prevents it from going into an active dimeric conformation.2 Caspase-9 and SMAC bind to XIAP-BIR3 in a mutually exclusive way.10 The residues important for interaction with caspase-9, SMAC and IBM groove are tabulated in Table 1. We find that some of the residues (308, 310, 314, and 323) that bind to caspase-9 and SMAC overlap and these were mobile in the simulation. Stable region includes Asn280–Glu282, Phe289–Tyr290, Val298–Phe301, Glu318–His320 and Leu330–Leu331. This includes part of ARTs (antagonist of XIAP) binding interface (Phe272– Leu292) and the zinc coordinated residues His320 and Cys300 of sheet-2. Lys299 of hydrophobic pocket (Val297–Lys299) with a high affinity for SMAC/SMAC mimetics binding27 was also in the stable region. This region binds exclusively to SMAC. Survivin is an important drug target in cancer as it is a nodal protein orchestrating the integration of several pathways related to tumor cell viability and maintenance. It has a single BIR domain, spanning residues 18–88 and a long carboxy terminal alpha helix. It is associated with regulators of cytokinesis, Aurora B kinase, INCENP and Borealin.28 All functional sites of interest are tabulated in Table 1. SMAC inhibits survivin and this results in an indirect method of caspase inhibition wherein binding of SMAC to other IAPs is prevented.5 Leu54, Leu64, Glu65, Trp67, Glu76, and His80 of survivin are said to be important for binding to SMAC.29 MD simulation of survivin was performed for 10 ns in the presence of zinc. The RMSD initially increased to 1.4 nm and then stabilized. The protein became stable around 3.5 ns and remained so till the end of the simulation (Figure 1b). Most part of the protein showed high mobility. The high deviation in RMSD is due to the mobility of its C-terminal helix. This region encompassing residues 97–141 is involved in dimerization and interaction with borealin, a key regulator of chromosome segregation and cytokinesis.28 The other flexible regions include Thr5–Cys33, Pro47–Asp53 and Cys59–Leu96. We observed a similar pattern of certain exclusively SMAC interacting residues (Leu54, Lys62) showing low mobility, compared to residues common to IBM interaction groove and SMAC interaction. Except Cys57 of strand-2 other zinc co-ordinated residues are in the mobile region. Stable region includes Thr34–Cys46 and Leu54– Phe59. RMSF graph is shown in Figure 2b. MD Simulation of livin was performed for 25 ns in the presence of zinc. The BIR domain of livin spans residues 90–155. The RMSD initially increased to 0.6 nm and then stabilized (Figure 1c). It became stable around 15 ns and remained so till the end (25 ns) of the simulation. The amino acids interacting with SMAC and SMAC-based peptides are shown in Table 1. Similar to XIAP-BIR3 and survivin, livin was also highly mobile. We observed that most of the mobile regions are in the dimeric interface and few residues are involved in SMAC interaction. Asp120 is involved in both SMAC and caspase interaction. Except Cys124 and Cys127 other two zinc co-ordinated residues are in the mobile region. Stable region includes Leu108, Phe113, Val122–Cys124 and Cys127. These regions correspond to residues involved in SMAC mimetic interactions and zinc coordination. RMSF graph is shown in Figure 2c.
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Figure 1. RMSD of three IAPs (a) RMSD of XIAP-BIR3 (BIR3 domain encompasses 265–330 shown in graph as 13–78) (b) RMSD of survivin (BIR domain encompasses 18–88 shown in graph as 14–84) (c) RMSD of livin (BIR domain encompasses 90–155 shown in graph as 20–85). Simulations performed in the presence of zinc are shown in green and those in the absence of zinc are shown in red.
Figure 2. RMSF of three IAPs (a) RMSF of XIAP-BIR3 (BIR3 domain encompasses 265–330 shown in graph as 13–78) (b) RMSF of survivin (BIR domain encompasses 18–88 shown in graph as 14–84) (c) RMSF of Livin (BIR domain encompasses 90–155 shown in graph as 20–85). Simulations performed in the presence of zinc are shown in green and those in the absence of zinc are shown in red.
Table 1 Functional sites of interest of the three IAPs Protein name
SMAC binding region
Caspase binding region / Mutational studies
Other protein interactions
Zinc binding residues
IBM binding groove
XIAPBIR3
296, 297, 299, 306–308, 310, 314, 315, 318, 319, 323–326, 3439 54, 64, 65, 67, 76, 8029
275, 277, 279, 308, 310, 311, 314, 319, 323, 325, 326, 343, 34410,31–33
ARTs (272–277)33
300, 303, 320, 327
314, 319, 3232
Kinases Cdk1, aurora kinase B and plk1 phosphorylate Thr34, 38; Thr117 and Ser 20 respectively of the survivin29
57, 60, 77, 84
76, 71, 802
116, 120–124, 130–134, 138, 143–144, 147, 14835,36
87, 88, 12034
121, 124, 143, 147
138, 143, 1472
Survivin
Livin
A common theme in all the simulations was, SMAC binding residues, which overlap with caspase binding residues/IBM interacting groove, showed higher mobility. Exclusively SMAC binding
residues are relatively stable in all the simulations. In all three IAPs, throughout the course of simulations, we found a pattern of residues mapping to strand-1, -2,turn-2 and part of helix-3 being
J. Jayakumar, S. Anishetty / Bioorg. Med. Chem. Lett. 24 (2014) 2098–2104
moderately stable (i.e.) with low fluctuations. Strand-2 plays a major role in SMAC and zinc ion binding. Turn-2 does not have any direct importance in SMAC or caspase binding, but comprises of a conserved ‘AGF’ pattern hosting a hydrophobic environment (Figure 3). The functional importance of helix-3 is not yet reported. However, based on its strong hydrogen bond interaction with turn-4 of all three IAPs in SMAC-bound state (PDB structures), we propose that it may be important for the function or structural stability of these IAPs. MD Simulation for XIAP-BIR3 was performed for 25 ns in the absence of zinc. It became stable around 15 ns and remained stable thereafter. The RMSD initially increased to 0.6 nm and then stabilized (Figure 1a). In the absence of zinc, XIAP-BIR3 was less fluctuating than in its presence (as deciphered through RMSF) (Figure 2a). Secondary structure was not retained after 1 ns. We were able to observe the low fluctuations of strand-1, -2 based on RMSF throughout the course of simulation. MD Simulation for survivin was performed for 10 ns in the absence of zinc. It became stable around 5 ns and remained stable thereafter. The RMSD initially increased to 1.1 nm and then stabilized (Figure 1b). In the absence of zinc, similar kinds of fluctuations were observed as seen with zinc (as evidenced through RMSF) (Figure 2b) and there was a partial reformation of secondary structure after 4.5 ns. In this simulation also we observed the stability of strand-1 and -2 throughout the course of simulation both structurally and also based on RMSF. MD Simulation for livin was performed for 25 ns in the absence of zinc. It became stable around 2 ns and was stable till the end of
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the simulation. The RMSD initially increased to 0.62 nm and then stabilized (Figure 1c). Fluctuations in some regions were similar to that seen with zinc (Figure 2c). Similar to XIAP-BIR3, even in the absence of zinc also we were able to observe the stability of strand-1,-2 throughout the course of simulation. Stability of zinc coordinating residue in strand-2 of survivin and livin even in the absence of zinc indicates that, coordination by zinc is perhaps not the only factor responsible for the stability. Multiple Sequence alignment of XIAP, survivin and livin was performed to generate a conserved motif of functional significance (Figure 3). The motif ‘[QGP]DX[VTA]XCF[FH]CXXXLXXW’ was specific to XIAP-BIR3, survivin-BIR and livin-BIR. It had a precision of 95.2% and recall of 76.9%. The motif spans residues 295–310 in the case of XIAP-BIR3, residues 52–67 in the case of survivin and residues 119–134 in the case of livin. This includes turn-3, strand-2,-3 and turn-4 (which connects the strand-2 and -3) of BIR domain of all three IAPs. On correlating the motif with MD simulations, we find that in each of the three proteins, zinc-coordinating residue in strand-2 is stable in the simulations performed in the presence and absence of zinc. Additionally strand-2, which overlaps with the motifs, was also structurally stable in both sets of simulations of survivin and stable in the presence of zinc in the case of livin. SMAC is a common binding partner of all three IAPs. Hence, compounds similar to available lead SMAC non-peptido-mimetics were screened from Chembank database. We found five compounds similar to 40d (one of the lead SMAC-non-peptidomimetics) satisfying the criterion of tanimoto coefficient > 0.8 (details shown in
Figure 3. Multiple sequence alignment of XIAP-BIR3, survivin-BIR and livin-BIR. (1) A conserved ‘AGF’ hydrophobic pattern, (2) sequence based novel motif.
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Supplementary Table S1). Interestingly N,N-dimethyl-D-valyl-D-valyl-N-methyl-L-valyl-D-prolyl-N-benzyl-D-prolinamide (NCIMech_ 000654), a compound related to cemadotin was retrieved as a compound similar to 40d. Cemadotin (N,N-dimethyl-L-valyl-Lvalyl-N-methyl-L-valyl-L-prolyl-N-benzyl-L-prolinamide) is a synthetic analogue of dolastatin-15 (tubulin inhibitor) that has
anti-proliferative and antitumor activity.25 Four compounds, dolastatin-15, cemadotin, NCIMech_000654 and compound 40d26 were docked against all three IAPs. We find that interactions of all these compounds with the BIR domain of the three IAPs were in the novel motif region described in this study. Schematic representations of these four compounds are shown in Figure 4. The docked
Figure 4. Schematic representation of four compounds: 40d, NCIMech_000654, dolastatin-15 and cemadotin.
Figure 5. Representation of docked complexes of the three IAPs with NCIMech_000654. (a) and (b) shows hydrogen bond interactions of XIAP and survivin, (c) shows pi interactions of livin with NCIMech_000654. (d–f) are 2D plots of interactions between NCIMech_000654 and XIAP, survivin and livin, respectively.
J. Jayakumar, S. Anishetty / Bioorg. Med. Chem. Lett. 24 (2014) 2098–2104 Table 2 Interacting amino acid residues and binding energies of the docked complexes of IAPs with various ligands Docked complex
Amino acids having hydrogen bond interaction with the ligand
XIAP-BIR3–NCIMech_000654 XIAP-BIR3–Dolastatin-15 XIAP-BIR3-40d XIAP-BIR3–Cemadotin Survivin–NCIMech_000654 Survivin–Dolastatin-15 Survivin-40d Survivin–Cemadotin Livin–NCIMech_000654 Livin–Dolastatin-15
Lys 297, Gly 306 Lys 297 Lys 299 Lys 299, Gly 306 Gln 56 Gln 56 Glu 63 Glu 63, Lys 62 Arg 123 (Pi interaction) Arg 123 (Pi interaction), Tyr 128 (Pi interaction) Gly 130 Arg 123 (Pi interaction), Gly 130
Livin-40d Livin–Cemadotin
Binding energy (kcal/mol) 4.31 5.38 2.86 4.23 4.64 0.04 6.34 5.49 5.83 1.72 4.86 4.86
complexes of the three IAPs with NCIMech_000654 are shown in Figure 5 and details of interactions with 40d, cemadotin, NCIMech_000654 and dolastatin-15 are provided in Table 2. XIAP, survivin and livin are considered to be potential therapeutic targets due their exclusive over-expression in many types of cancer. XIAP-BIR3 and livin have similar mode of binding with SMAC/SMAC mimetics. Experimental studies have shown that survivin binds to SMAC through its strand-3.29 Among the three IAPs, XIAP-BIR3 has the highest binding affinity towards SMAC. Livin inhibits caspase-9 less potently than XIAP-BIR3,30 but there is no evidence for interaction of survivin with caspase-9. SMAC, caspase and IBM (IAP binding motif) interacting groove residues of the three IAPs were compared with each other through MD simulations to gain insights into their behaviour. We observed that there was an overlap in SMAC and caspase/ IBM-interacting groove residues in the three IAPs. These interacting residues were mobile in XIAP-BIR3, survivin and livin. Exclusively SMAC binding residues were less mobile and relatively
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stable in all three IAPs. SMAC binding lysine residues were moderately stable (Lys299 of XIAP-BIR3, Lys62 of survivin and Lys121 of livin) in all three IAPs. Helix-5 of XIAP-BIR3 is necessary for caspase-9 interaction and it had least similarity at a sequence level with the corresponding helix of livin.30 Helix-5 of XIAP-BIR3 was relatively more stable compared to helix-5 of livin. There was a higher mobility of helix-5 of livin probably because of more hydrophilic residues in this region. All three IAPs behave in different ways, yet they have a similar pattern of maintaining the stability of part of alpha-helix-3, strand2 and turn-2 till the end of the simulation. A novel sequence based motif specific to all three IAPs was designed. This motif does not overlap with the known IBM interacting groove (Figure 6). From our simulations we propose that the motif region provides a relatively stable scaffold of possible functional significance. The novel motif has two zinc binding residues, along with a few caspase and SMAC binding residues. BIR domains coordinate with zinc ion which may be important for structural or functional stability. Since caspases use cysteine and histidine in their catalytic mechanism, they can be inhibited by metals like zinc which is a potent inhibitor of caspases.37,38 It is possible that metal binding by BIR domain may have a role in caspase inhibition mechanism of IAPs.39 Design of a small molecule or a mimetic which can target the motif region of IAPs can help caspase enter the apoptotic pathway. We chose NCIMech_000654 from our hits, since it is related to cemadotin which is a compound with known antitumor activity. We sought to find if NCIMech_000654 and related compounds can bind with XIAP, survivin and livin. Our docking studies with cemadotin, NCIMech_000654, dolastatin-15 and compound 40d show that all these in fact bind to all three IAPs and these interactions are within the novel motif region designed in this study. Among these, NCIMech_000654, compound 40d and cemadotin were found to exhibit a higher binding affinity with all three IAPs while dolastatin-15 had good affinity for XIAP-BIR3. We hypothesize that NCIMech_000654/dolastatin-15/cemadotin derivatives may help in the inhibition of these three IAPs.
Figure 6. Representation of the novel motif and IBM groove. The novel motif is shown in white with the ends marked as p and q.
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In summary, IAPs are now being increasingly recognized as therapeutic targets in cancer. Several groups are exploring small molecules for inhibition of these IAPs. In the current study, we have gained insights into the dynamics of XIAP-BIR3, survivin and livin and identified regions of potential importance which can be utilized for rational drug design. Using molecular docking, we have shown that NCIMech_000654, natural peptide dolastatin-15 and its derivative cemadotin interact with the motif region. Based on this analysis, we propose that NCIMech_000654/cemadotin/dolastatin derivatives may have a potential in inhibiting XIAPBIR3, survivin and livin. Acknowledgments The authors thank DBT, Government of India for support (BT/ 01/COE/07/01). The authors also thank BTIS, Department of Biotechnology for computational facilities. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03. 046. References and notes 1. Lamkanfi, M.; Festjens, N.; Declercq, W.; Vanden Berghe, T.; Vandenabeele, P. Cell Death Differ. 2004, 14, 44. 2. Eckelman, B. P.; Salvesen, G. S.; Scott, F. L. EMBO Rep. 2006, 7, 988. 3. Schimmer, A. D. Cancer Res. 2004, 64, 7183. 4. Obiol-Pardo, C.; Granadino-Roldán, J. M.; Rubio-Martinez, J. J. Mol. Recognit. 2008, 21, 190. 5. Chiou, S. K.; Jones, M. K.; Tarnawski, A. S. Med. Sci. Monit. 2003, 9, 125. 6. Pennati, M.; Folini, M.; Zaffaroni, N. Carcinogenesis 2007, 28, 1133. 7. Kasof, G. M.; Gomes, B. C. J. Biol. Chem. 2001, 276, 3238. 8. Du, C.; Fang, M.; Li, Y.; Li, L.; Wang, X. Cell 2000, 102, 33. 9. Liu, Z.; Sun, C.; Olejniczak, E. T.; Meadows, R. P.; Betz, S. F.; Oost, T.; Herrmann, J.; Wu, J. C.; Fesik, S. W. Nature 2000, 408, 1004. 10. Srinivasula, S. M.; Hegde, R.; Saleh, A.; Datta, P.; Shiozaki, E.; Chai, J.; Lee, R. A.; Robbins, P. D.; Fernandes-Alnemri, T.; Shi, Y.; Alnemri, E. S. Nature 2001, 410, 112. 11. Ma, L.; Huang, Y.; Song, Z.; Feng, S.; Tian, X.; Du, W.; Qiu, X.; Heese, K.; Wu, M. Cell Death Differ. 2006, 13, 2079. 12. Ling, B.; Dong, L.; Zhang, R.; Wang, Z.; Liu, Y.; Liu, C. J. Mol. Graph. Model. 2010, 29, 354.
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