Discovery of a novel small inhibitor RJ19 targeting to human Hsp90

Discovery of a novel small inhibitor RJ19 targeting to human Hsp90 1 equal first-author 2 equal 3 4 5 6 corresp Corresponding author. E-mail address: Jian.li@uky.edu; Corresponding author. E-mail address: Jian.li@uky.edu; Jian.li@uky.edu 4 corresp hejianhua@sinap.ac.cn. hejianhua@sinap.ac.cn. hejianhua@sinap.ac.cn Shaanxi Key Laboratory of Ischemic Cardiovascular Disease; Institute of Basic & Translational Medicine, Xi’an Medical University, Xi’an 710021, China Shaanxi Key Laboratory of Ischemic Cardiovascular Disease; Institute of Basic & Translational Medicine, Xi’an Medical University, Xi’an 710021, China University of Chinese Academy of Sciences, Beijing 100049, China University of Chinese Academy of Sciences, Beijing 100049, China Key Laboratory of Protein Modification and Tumor, Hubei Polytechnic University School of Medicine, Huangshi 435003, China. Key Laboratory of Protein Modification and Tumor, Hubei Polytechnic University School of Medicine, Huangshi 435003, China. Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201204, China Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201204, China Biotech & Biomedicine Science Co. Ltd., Shenyang 110000, China Biotech & Biomedicine Science Co. Ltd., Shenyang 110000, China Department of Molecular & Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY 40508, USA Department of Molecular & Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY 40508, USA Heat shock protein 90 (Hsp90) can promote growth and proliferation of cancer cells by helping in folding, conformational maturation and activation of various client proteins. Therefore, Hsp90 has been paid more attention to as an anti-cancer drug target. Reported Hsp90 inhibitors have several limitations such as poor solubility, limited bioavailability, hepatotoxicity. Here, a novel small inhibitor RJ19has been designed using fragment-based drug discovery and synthesized. Additionally, a crystal structure of Hsp90 N -RJ19was determined by X-ray diffraction (resolution limit, 2.0 Å, PDB code 4L90). The crystal structure of Hsp90 N -RJ19 was analyzed in detail and compared with that of native Hsp90 N , Hsp90 N -ATP and Hsp90 N - GDM, respectively. It was indicated that RJ19 interacted with Hsp90 N at the ATP-binding pocket, which suggests that RJ19 may replace nucleotides to bind with Hsp90 N to result inchaperone function failure of Hsp90. RJ19, therefore, has emerged as a promising anti-cancer lead compound. Rearrangement and displacement of L2 Loop in Hsp90 N -RJ19 plays key role in the function failure, which also makes the pocket wider and longer facilitating structure modification of RJ19 later. The complex crystal structure and interaction between RJ19 and Hsp90 N provide a rational basis for the design and optimization of novel anti-cancer drugs. Heat shock protein 90 Drug target Inhibitor X-ray diffraction Complex crystal structure 1 Introduction: 1 As the most dominant technique, X-ray diffraction (XRD) determined 89.56% structures (116,200) of Protein Data Bank (PDB, total structures 129,745, data on May 2nd, 2017, http://www.Rcsb.org/pdb). Nowadays, XRD is a routine way to determine how a small molecular inhibitor interacts with its target and how to improve it based on ligand-target complex crystal structure, which provides detailed interactions in an active state for drug design and structure modification. As a result, XRD has become a predominant standard technique for drug discovery area [ 1 1 – 3 3 1–3 ]. The heat shock protein 90 (Hsp90, 90 kD) is an α-α or β-β homodimer in the cytosolic, and each monomer consists of three domains. The C-terminal domain plays a key role for dimerization of Hsp90, while the central domain possesses a large hydrophobic surface to bind client proteins and facilitate them folding. The N-terminal domain with highly conserved residues across species contains an extraordinary ATP-binding pocket that is responsible for ATP-binding and hydrolysis, which is indispensable for the chaperone function of Hsp90[ 4 4 – 6 6 4–6 ]. The first crystal structure of human Hsp90 N from Stebbins et al . revealed the α/β sandwich pocket in detail[ 7 7 ]. The pocket is about 15 Å deep, 12 Å in diameter for entrance and 8 Å for baseshapedasa flat-bottomed cone. The base comprises of eight antiparallel β sheets (three central β sheets, S3, S4 and S7), while the wall is consisted of three helices and a loop (H2-H4-H7-L1). From the entrance toward the bottom, the pocket becomes increasingly hydrophobic[ 4 4 – 8 8 4–8 ]. As a chaperone, Hsp90 has dual chaperone functions participating not only in the cellular stress response but also in folding or refolding, conformational maturation and activation of a wide range of client proteins[ 7 7 – 11 11 7–11 ]. More than 300Hsp90-dependent client proteins have been identified including varying eukaryotic protein kinases and nuclear hormone receptors related to multiple signaling pathways[ 12 12 ]. Of these, 48 client proteins are directly related to oncogenesis[ 10 10 ]. Hsp90 is overexpressed to facilitate these client proteins to fold and maintain their activity in malignant cells. Inhibitors of Hsp90 caninterfereinHsp90-mediated chaperone function to make the client proteins being an immature or misfolding state, which will be captured and degraded by proteasomes. With degraded proteins accumulated, multiple signaling pathways can be derailed simultaneously, which can hinder proliferation and progression of cancer[ 9 9 – 12 12 9–12 ]. Hsp90, therefore, has been paid more attention to as an anti-cancer drug target. Hsp90 inhibitors contain C-terminal inhibitors such as novobiocin, and mainly N-terminal inhibitors such as geldanamycin (GDM) and radicicol (RDC)[ 12 12 – 14 14 12–14 ]. As a naturally occurring antitumor antibiotic, anticancer mechanism of GDM was unclear until GDM-Hsp90 N crystal structure was determined [ 7 7 ]. It was suggested that GDM bound in the pocket to hinder ATP binding to Hsp90 N , resulting in Hsp90-mediated folding or refolding, conformational mature or activation of client proteins failed. The growth and progression of cancer, therefore, can be inhibited [ 7 7 , 12 12 – 15 15 12–15 ]. Another naturally occurring antitumor antibiotic radicicol acts in the similar way[ 12 12 – 15 15 12–15 ].Several derivatives of GDM have entered clinical studies, for instance, 17-AAG[ 16 16 ], 17-DMAG[ 17 17 ] and IPI-504[ 18 18 ]. They, nevertheless, have several potential limitations, such as poor solubility, limited bioavailability and hepatotoxicity [ 16 16 – 18 18 16–18 ]. This leads to significant efforts to discover novel inhibitors of Hsp90. It is a major challenge in the drug discovery to find novel compounds as starting points for optimization. Fragment-based Drug Discovery (FBDD) has become a powerful technique to find novel compounds, which was put forward by William P Jencks in 1981 for the first time [ 19 19 ]. It develops based on advantages combining random screening and Structure-based Drug Discovery (SBDD). Active fragments are discovered by random screening of built fragment library. Followed, promising compounds can be obtained by growth, jointing and self-assembly of active fragments. Finally, novel lead compounds will be got by several rounds of crystal structure-directed optimization. Compared with traditional method, FBDD has outstanding merits as follows: (1) To explore more chemical spaces with less fragments; (2) To find promising compounds with higher chances; (3) To find promising compounds with fine affinity and druggability with higher chances; (4) To avoid false positive results [ 19 19 – 21 21 19–21 ]. Here, a novel small inhibitor RJ19 has been designed using FBDD and synthesized. In addition, the complex crystals of RJ19 and Hsp90 N were grown and a high resolution crystal structure was determined by XRD, which was deposited in PDB with accession code 4L90. The crystal structure of Hsp90 N -RJ19 was analyzed in detail and compared with the crystal structure of Hsp90 N , ATP-Hsp90 N , GDM-Hsp90 N , respectively. The complex crystal structure and the interaction between RJ19 and Hsp90 N provide a rational basis for the design and optimization of novel anti-cancer drugs targeting Hsp90 N with superior quality. 2 Experimental Section: 1 2.1 Synthesis of small molecule 2 As show in Fig.1 Fig.1 , Compound 1 reacted with methanol via Oxalyl chloride to afford compound 2, then followed by formylation with α,α-dichloromethyl methyl ether to obtain Compound 3. Construction of compound 5 utilized intramolecular ring-closing from compound 4 that was obtained by reacting with 5-Bromo-2-hydrazinopyridine and compound 3.Compound 5 washydrolysed with Lithium hydroxide inaqueous solutions of methanol to get Compound 6. Compound 6 was condensated with 1- methylpiperazine and deprotected by boron tribromide to obtain the target Compound 8(RJ19). 2.2 Protein purification and crystallization 2 The gene of human Hsp90α N-terminal domain including residues 9-236 was cloned into a pET28a vector, which was transformed into E.coli Rosetta (DE3) pLysSfor over-expression of drug target Hsp90 N (Invitrogen, Carlsbad, America). The engineering bacteria were grown in 1,000 mL Luria-Bertani broth (LB) at 310 K to OD 600 value of 0.6-0.8 and followed induced by 200 μM isopropyl β -D-thiogalacto pyranoside (IPTG) for 3-5 h at 303K. The express bacteria were collected by centrifugation at 10,000 g for 10 min (CF16RX, Hitachi, Japan). The precipitate was re-suspended in buffer A (pH 7.5, 0.1 M Tris-HCl, 0.3 M NaCland 5% glycerol) and followed crushed by Ultra-high Pressure Cell Disrupter (JN-02C, JNBIO, China). For preliminary purification, the product was centrifuged at 30,000 g for 30 minat277 K and the supernatant was loaded onto a 5mL Ni 2+ -nitrilotriacetate column (Ni-NTA, GE Healthcare, America).The target protein was eluted with buffer B (pH 7.5, 0.1 M Tris-HCl, 0.3M NaCl, 0.1M imidazole and 5% glycerol). The primary purified protein was concentrated and the buffer was exchanged to buffer C(pH 7.5, 0.1 M Tris-HCl, 0.15 M NaCl and 5% glycerol) using an 10,000 Mr cut-off centrifugal concentrator (Amicon Ultra-15, Millipore, America). For fine purification, the impurities were removed by a gelfiltration column (Superdex 75 PG, GE Healthcare, America) with buffer C. The purified protein was analyzed by a 15% SDS-PAGE to determine the purity. The fine purified protein was concentrated to a concentration of about 20 mg/mL with the 10 000 Mrcut-off centrifugal concentrator again and its concentration was determined using bicinchoninic acid method (BCA).The fine purified Hsp90 N was flash-frozen with liquid nitrogen and stored at 193 K. For crystallization, the initial concentration of target protein Hsp90 N was approximately20 mg/mL. The small molecular inhibitorRJ19 was added to protein with 5:1 molar ratio and the mixture was incubated for 30 min at 277 K. And then the mixture was centrifuged for 10 min at 3,000 g and the supernate was for crystallization. Co-crystallization was conducted at 277 Kfor 3-7 days using the hanging drop vapor diffusion method in an incubator controlled by a bath circulator (PolyScience 9712, PolyScience, USA).The co-crystals were grown under the condition: pH 6.5, 0.2 M magnesium chloride, 0.1 M sodium cacodylate, 20%-25%(w/v) polyethylene glycol 2000 monomethyl ether (PEG2000 MME)[ 7 7 ]. 2.3 Data collection, structure determination and refinement 2 Co-crystals were mounted with cryo-loop (Hampton research, America) and flash-frozen in liquid nitrogen for XRD data collection. All data sets were collected at 100 K on Macromolecular Crystallography Beamline17U1 (BL17U1) at Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China) using an ADSC Quantum 315r CCD detector [ 22 22 ]. All the data were integrated and merged using the HKL 2000 software package [ 23 23 ]. The structures were determined by molecular replacement with PHENIX software [ 24 24 ], using the structure of Hsp90 N (PDB code 3T0H) as the research model [ 5 5 ]. The program Coot was used to rebuild the initial model [ 25 25 ]. The models were refined to resolution limit 2.0 Å using the PHENIX software. The superimposed contrast was analyzed with PyMOL software package [ 26 26 ].The complete data collection and refinement statistics are shown in Table 1 Table 1 . The coordinates and structure factors have been deposited in PDB (PDB code 4L90). Values in parentheses are for the highest-resolution shell. 1 1 PDB code 1 1 4L90 1 1 Synchrotron 1 1 SSRF 1 1 Beam line 1 1 BL17U1 1 1 Wavelength (Å) 1 1 0.97920 1 1 Space group 1 1 I222 1 1 a , b , c (Å) 1 1 69.88, 88.59, 97.98 1 1 α , β , γ (º) 1 1 90.00,90.00, 90.00 1 1 Resolution (Å) 1 1 2.00(2.03-2.00) 1 1 R -merge (%) 1 1 9.2(63.2) 1 1 Mean I / σ (I) 1 1 25.2/3.1 1 1 Completeness (%) 1 1 99.8(100.0) 1 1 Redundancy 1 1 4.9(4.9) 1 1 Resolution (Å) 1 1 36.54-2.00 1 1 R work / R free (%) 1 1 18.80/21.94 1 1 Atoms 1 1 1751 1 1 Ligand 1 1 RJ19 1 1 Mean temperature factor (Å 2 ) 1 1 27.7 1 1 Bond lengths (Å) 1 1 0.007 1 1 Bond angles (º) 1 1 1.097 3 RESULTS AND DISCUSSION 1 3.1 Purification, crystallization and structure determination of Hsp90 N -RJ19 2 Hsp90 N was purified to apparent homogeneity using metal chelating chromatography, followed by gel filtration chromatography. The single elution volume peak accords with monomeric Hsp90 N with a molecular weight of 25 kDa and showed a good purity which was assessed by SDS-PAGE to be 98% (as shown in Fig. S1). After 3-5 days, complex crystals of Hsp90 N -RJ19 were obtained by the hanging-drop method at 277 K. The average dimension of the crystals was approximately 200 μm×100 μm×50 μm, as shown in Fig. 2 Fig. 2 . The structure of Hsp90 N -RJ19 was determined by molecular replacement using Hsp90 N (PDB code 3T0H) as there search model [ 5 5 ]. The complete data collection and refinement statistics are shown in Table 1 Table 1 . The coordinates have been deposited in PDB (PDB code 4L90). Diffraction data were collected to 2.0Åresolutionand indexed in space groups P 2 1 2 1 2 1 . The unit cell parameters were as follows: a = 69.88Å, b = 88.59 Å, c = 97.98 Å; α = β = γ = 90.00º. The refined model contains residues Val17-Lys224 and no electron density was observed for residues Asp9-Glu16 and Glu225-Glu236 in the structure. It is conceivable that the missing residues at the N- and C-termini of the model could be disordered electron density. 3.2 Analysis on crystal structure and interaction of Hsp90 N -RJ19 2 It was indicated from the complex crystal structure that RJ19bound to the ATP-binding pocket completely and its intact electron density has been captured (as shown in Fig.S2).The N-terminal domain of human Hsp90 contains a highly conserved ATP-binding pocket about 15 Å deep with α/β sandwich structure. Eight uninterrupted antiparallel β-sheets constitute the pocket base. The pocket walls are formed by three α-helices and a loop, H2-H4-H7-L1[ 7 7 ]. The H4-L2-H5-H6 secondary structure elements (residues 99-134) is called ATP lid. The ATP lid has open or close conformation which is responsible for regulating pocket size and accessibility. Only when the pocket is in an open conformation, Hsp90 can bind with ATP and interact with its client proteins to accomplish folding or refolding, conformational maturation and activation of client proteins [ 7 7 – 12 12 7–12 ]. A superimposing correlation was analyzed with PyMOL software between the crystal structure of Hsp90 N -RJ19 and Hsp90 N , ATP-Hsp90 N or GDM-Hsp90 N . It was indicated that the refined crystal structure of Hsp90 N -RJ19 (PDB code 4L90) differed from that of Hsp90 N (PDB code 3T0H), Hsp90 N -ATP (PDB code 3T0Z) or Hsp90 N - GDM (PDB code 1YET) in resolution level, as shown in Fig. 4 Fig. 4 . Their crystal packing patterns are of difference too, Hsp90 N -RJ19 ( P 2 1 2 1 2 1 ), Hsp90 N ( I 222), Hsp90 N -ATP ( P 21) and Hsp90 N - GDM ( P 12 1 1). As can be seen from Fig. 3(a) Fig. 3(a) , there are some subtle differences from residues Ala101 to Phe138 in the crystal structures of Hsp90 N -RJ19 and native Hsp90 N . Fig. 3(c) Fig. 3(c) shows that the complex crystal structure of Hsp90 N -RJ19 differs from Hsp90 N -ATP from residues Asp102 to Phe138. It is different from residues Ile104 to Gly137 in the complex crystal structure of Hsp90 N -RJ19 and Hsp90 N - GDM, as shown in Fig. 3(e) Fig. 3(e) . The superimposition of crystal structure of Hsp90 N -RJ19 with native Hsp90 N ( Fig.3 (b) Fig.3 (b) ) shows that the H4 helix(residues from Thr99 to Leu107) and H5 helix(residues from Thr114 to Ala124) of Hsp90 N undergo a conformational rearrangement after binding RJ19, which impels the L2 loop (residues from Leu107 to Thr114) in Hsp90 N -RJ19rearrangingand displacing to substitute portion of H4 helix as the pocket wall. L2 loop, therefore, acts as a switch to constrain the pocket entrance in diameter from 12 Å to 8 Å. Then, the pocket turns to a close conformation, resulting in disabled ATP-binding and failed chaperone function. The rearrangement and displacement of L2 plays an important role in function failure of Hsp90. The result is in accordance with that of Stebbins’s [ 7 7 ]. The superimposition of Hsp90 N- RJ19 and Hsp90 N -ATP ( Fig.3(d) Fig.3(d) ), Hsp90 N –GDM ( Fig.3(f) Fig.3(f) ) indicates that there are differences in H4 helix, H5 helix or L2 loop, although RJ19, ATP and GDM all bind in the pocket, which imply their binding ways are different. Figure.4 Figure.4 shows the electrostatic potential surface distribution surrounding the active pocket of Hsp90 N ( Fig. 4(a) Fig. 4(a) ), Hsp90 N -ATP( Fig. 4(b) Fig. 4(b) ), Hsp90 N -RJ19( Fig. 4(c) Fig. 4(c) ) and Hsp90 N -GDM ( Fig. 4(d) Fig. 4(d) ).Compared with native Hsp90 N , changes have been taken place in the electrostatic potential surface surrounding the active pocket of Hsp90 N responsible for binding of ATP, RJ19 or GDM. In common with the structure of ATP-Hsp90 N , RJ19or GDM binding site of Hsp90 is located in the ATP-binding pocket. It is interesting that RJ19 binding induces structural rearrangements of L2loop and makes the ATP binding pocket wider and longer, which provides a larger room for structural modification of RJ19 later. Figure 5 Figure 5 shows the crystal structural superimposition of Hsp90 N -RJ19 (PDB code 4L90) with Hsp90 N -GDM (PDB code 1YET), Hsp90 N -17-DMAG (PDB code 1OSF), Hsp90 N -Onalespib (PDB code 2XJX), Hsp90 N -NMS-E973 (PDB code 4BQG) and Hsp90 N -NVP (PDB code 5JZN). It is revealed that the RJ19, GDM, 17-DMAG, Onalespib, NMS-E973 and NVP occupied the same location and there are some tiny differences in the chemical group. It is notable that in the complex of Hsp90 N -GDM, GDM nearly occupies the whole binding pocket and there is no much room for structure modification. It is, therefore, significant changes were rare in solubility, bioavailability and hepatotoxicity in derivatives of GDM. The situation, however, is different for RJ19 and there is enough room for redesigning and modification of RJ19 to obtain derivatives with high efficiency and low toxicity. The pocket is mostly polarat its entrance, so hydrophilic groups, which can interact with polar amino acids, can be added to RJ19 to increase the solubility of small molecule. While it becomes predominantly hydrophobic near the pocket base, derivatives with increased affinity with Hsp90 may be yielded if groups with non-hydrogen atoms are added to RJ19 and hydrogen bond complementarity is increased. The ATP lid consists of the H4-L2-H5-H6 secondary structure elements. It has open or close two different conformations which are responsible for regulating pocket size and accessibility [ 7 7 ]. The pocket in open conformation is about12 Å in diameter near its entrance. When small molecular inhibitor RJ19 binds to the pocket, H4 and H5 helixes undergo helix-to-coil and coil-to-helix transitions, which impels L2 loop rearranging, rolling-over 180 ° and moving toward the pocket to replace portion of H4 helix as one of the pocket wall. The L2 loop, therefore, acts as a switch to constrict the pocket entrance from a width of 12 Å to 8 Å. Then, the pocket changes into closed conformation, which results in disabled ATP-binding and chaperone function failure. The rearrangement and displacement of L2 loop play a key role in chaperone function failure. Various client proteins being immature or misfolding state, therefore, can be captured and degraded by proteasomes. And then, the cancer cells can be inhibited. RJ19, as a result, become a hopeful lead compound. As can be seen from Fig. 6(b) Fig. 6(b) , there are two hydroxyls in the benzene of RJ19. The 4-hydroxyl near the isopropyl forms water-mediated hydrogen bonds with residue Ser52. The 2-hydroxyl forms a direct hydrogen bond with residue Asp93 (distance, 2.4Å) and three water-mediated hydrogen bonds with residue Asp93, Gly97 and Thr184, respectively. The carbonyl of RJ19 forms a direct hydrogen bond with residue Thr184 (distance, 2.8 Å) and three water-mediated hydrogen bonds with residue Asp93, Gly97 and Thr184, respectively. The hydrogen bonds or water-mediated hydrogen bonds play dominant role in keeping fine affinity between RJ19 and Hsp90 N . In addition, extensive surface complementarity between RJ19 and the pocket results in a high density of van der Waals contacts, which also contributes to firmly binding of RJ19 and Hsp90 N . The complex structure of Hsp90 N -RJ19and interaction provide the structural basis to further investigate the activated conformational change of Hsp90 after RJ19 binding, also for redesign and structure modification of RJ19. 4 Conclusion 1 (1) A novel anti-cancer small inhibitor RJ19 has been designed using FBDD and synthesized. (2) Hsp90 N -RJ19crystal structure was determined by XRD (2.0 Å), which was deposited in PDB with accession codes 4L90. (3) RJ19 interacts with Hsp90 N at the ATP-binding pocket, which suggests that RJ19 may replace nucleotides to bind with Hsp90 N to disable chaperone function of Hsp90 to inhibit growth and progression of cancer. RJ19, therefore, can be a potential anti-cancer lead compound. (4) The rearrangement and displacement of L2 loop in Hsp90 N -RJ19 play an important role in function failure of Hsp90, which also makes ATP-binding pocket wider and longer and provides a larger room for redesign and structural modification of RJ19 later. (5) The complex crystal structure and the interaction between RJ19 and Hsp90 N provide a scientific basis for the design and optimization of novel anti-cancer drugs targeting Hsp90 N . References J. Ren , M. Yang , H.C. 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(Science Press), Shanghai Institute of Applied Physics, the Chinese Academy of Sciences, ChineseNuclear Society and Springer Nature Singapore Pte Ltd. 2017 This is a post-peer-review, pre-copyedit version of an article published in Nuclear Science and Techniques. The final authenticated version is available online at: http://dx.doi.org/10.1007/s41365-017-0300-1 http://dx.doi.org/10.1007/s41365-017-0300-1 . 2017 Nuclear Science and Techniques 10 28