(RANKL) - Receptor Activator of Nuclear

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Development of Small Molecules Targeting Receptor Activator of Nuclear Factor-#B Ligand (RANKL) - Receptor Activator of Nuclear Factor-#B (RANK) Protein-Protein Interaction by Structure-Based Virtual Screening and Hit Optimization Min Jiang, lei peng, Kai Yang, tianqi wang, Xueming Yan, Tao Jiang, Jianrong Xu, jin qi, hanbing zhou, niandong qian, qi zhou, bo chen, xing xu, Lianfu Deng, and Chunhao Yang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b02027 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 13, 2019

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Journal of Medicinal Chemistry

Development of Small Molecules Targeting Receptor Activator of Nuclear Factor-κB Ligand (RANKL) - Receptor Activator of Nuclear Factor-κB (RANK) Protein-Protein Interaction by Structure-Based Virtual Screening and Hit Optimization Min Jiang a‡, Lei Peng b, c‡, Kai Yang a, Tianqi Wang a, Xueming Yan a, Tao Jiang a, Jianrong Xu d, Jin Qi a, Hanbing Zhou a, Niandong Qian a, Qi Zhou a, Bo Chen a, Xing Xu a*, Lianfu Deng a*, Chunhao Yang b, c*

a Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Shanghai Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 2nd Ruijin Road, Shanghai 200025, China

b State Key Laboratory of Drug Research, Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu

Chong Zhi Road, Shanghai 201203, China

c School of Pharmacy, University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

d Department of Pharmacology and Chemical Biology, Institute of Medical Sciences, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China. ‡ These authors contributed equally to this work.

Abstract: Targeting RANKL/ RANK offers the possibility of developing novel therapeutic approaches to treat bone metabolic diseases. Multiple efforts have been made to inhibit RANKL. For example, marketed monoclonal antibody drug Denosumab could inhibit the maturation of osteoclasts by binding to RANKL. This study is an original approach aimed at discovering small-molecule inhibitors impeding RANKL/RANK protein interaction. We identified compound 34 as a potent and selective RANKL/RANK inhibitor by performing structure-based virtual screening and hit optimization. The disruption of the RANKL/RANK interaction by 34 effectively inhibits RANKL-induced osteoclastogenesis and bone resorption. The expressions of osteoclast marker genes were also suppressed by treatment of 34. Furthermore, 34 markedly blocked the NFATc1/ c-fos pathway. Thus our current work demonstrates that the chemical tractability of the difficult PPI (RANKL/RANK) target by a small molecule compound 34 offers a potential lead compound to facilitate the development of new medications for bone-related diseases.

Key words: Osteoclasts, RANKL, RANK, Therapeutics, Osteoclastogenesis


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INTRODUCTION Bone remodeling is crucial for maintaining the normal skeletal structure1. To maintain bone homeostasis, bone metabolism consists of a life-long remodeling process which involves a cross-talk between osteoclasts and osteoblasts2-5. Osteoblasts are mononucleated bone-forming cells derived from mesenchymal stem cells (MSCs). Osteoclasts are cells derived from bone marrow-derived macrophages cells (BMMs) and play an important role in bone resorption. Enhanced osteoclastogenesis is a common pathological feature in many age-associated bone diseases, such as osteoporosis, rheumatoid joint destruction, cancer induced bone metastasis and Paget's disease6-9. Receptor activator of NF-kB ligand (RANKL) which belongs to the tumor necrosis factor (TNF) receptor-ligand family is expressed by osteoblasts, stromal cells and osteocytes10. RANKL plays multiple functions in immune system11, hormone-derived breast cancer development12 and especially in bone remodeling. RANKL plays an essential role in osteoclast differentiation, acting through its binding to receptor activator of NF-kB (RANK) and then associating with TNF receptor associated family members to trigger downstream signaling events such as NF-κB pathway and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1)/c-fos pathway13-15. Osteoprotegerin (OPG) functions as a decoy receptor for RANKL and inhibits osteoclastogenesis by preventing RANKL/RANK interaction16, 17.



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Since RANKL/RANK/OPG are recognized critical proteins for the bone metabolism, targeting this system offers the possibility of developing novel therapeutic approaches to treating bone metabolic diseases18-20. Denosumab, a human monoclonal antibody against RANKL, is developed and indicated for treating postmenopausal osteoporosis and bone metastasis from solid tumors21-24. It is the first RANKL inhibitor approved by FDA and its total sales is more than 3 billion dollars in 2016. However, there are inadequate responses to denosumab in the treatment of osteoporosis and bone-metastatic cancers25. In addition, its application is limited partially for their high price. Besides, recombinant proteins such as Fc-OPG and RANK-Fc 27.

have

also

been

developed

as

therapeutics

for

osteoporosis26,

Alternatively, OPG (OP3-4 peptide) was developed based on the structure of the

loop in the third cysteine-rich domain of OPG (OP3 site) to mimic the soluble osteoprotegerin (OPG) and showed inhibitory activity against the RANKL-induced osteoclastogenesis and bone resorption28, 29. One of eRANK (ectodomain of mouse RANK) mutants, the cyclic peptide L3-3 was proved as a potent RANKL inhibitor which blocked RANKL-induced osteoclast differentiation more efficiently than OP3-425. ITC (isothermal titration calorimetry) analysis of L3-3 and OP3-4 showed that the binding affinity (KD) with RANKL were 17.3 μM and 284.4 μM respectively25. Although multiple efforts have been made to inhibit RANKL, the therapeutic applications of biologics in the clinic were hindered for some of drawbacks, including


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high cost, unfavorite intravenous administration etc. An alternative approach to inhibit RANKL is to utilize nonpeptidic small organic molecules, which, in general, have better pharmacokinetic properties. To date, there are just a handful of nonpeptidic small molecular inhibitors targeting RANKL and RANK protein interaction. SPD304, T8, T23 were proven to directly bind to RANKL with Kd values of 13.8 μM, 6.3 μM, 7.3 μM respectively. Despite their promising effectiveness, the agents are not specific in inhibiting RANKL-RANK interaction. It has been demonstrated that SPD304, T8, T23 have poor selectivity on TNF and RANKL. Among them, SPD304 and T23 displayed high toxicity30, 31. Development of small molecular inhibitors of RANKL with low toxicity, high potency and selectivity remains inadequate. The binding modes between RANKL/RANK and RANKL/OPG were well-characterized, providing molecular foundations for the development of small molecules targeting RANKL-RANK interface25,

32, 33.

Here, based on virtual screening, we identified a

new scaffold compound 1 as a candidate small molecule that may target RANKL and RANK

protein

interaction.

The

binding

to

RANKL/RANK

and

the

anti-osteoclastogenesis activity of 1 were confirmed in surface plasmon resonance (SPR) analysis and RANKL-induced osteoclastogenesis assay respectively. Based on these promising results, we considered 1 as a hit compound and further chemical optimization resulted in four novel compounds (2, 7, 29, and 34) with good activities against osteoclastogenesis. 2, 7, 29, and 34 were also determined by SPR binding assays and displayed high affinity for RANKL with KD values of 2.7, 3.1, 3.5 μM



and 614 nM respectively. Moreover, 34 was further validated to inhibit RANKL-induced bone resorption by downregulating the c-fos/NFATc1 signaling pathway and osteoclastogenesis marker genes. Thus our current work offers an attractive starting point for facilitating the PPI (protein/protein interaction, RANKL/RANK) target strategy by small molecular compounds and helps further development of new medications for osteoporosis and bone-related diseases discovery progress. RESULTS and DISCUSSION Discovery of a Lead compound, through Virtual Screening Structure based virtual screening is an effective method in hit compound identification and has been widely used in the process of lead compound discovery. In this study, we used a molecular docking method with Schrodinger (Schrödinger, LLC.) standard precision mode to screen SPECS database (http://www.specs.net), which contains more than 200,000 compounds, to identify the RANKL inhibitors. Based on the analysis of the interaction surface of published RANKL crystal structures, the crystal structure with concave pocket (Protein Data Bank PDB ID 3URF) mostly suitable molecular docking was chosen as receptor structure for virtual screening. 3URF includes two parts, RANKL and its decoy receptor OPG. Three amino acids, Glu93, Ile92 and Glu95 from OPG probe deeply into the concave surface of RANKL that defines the main binding pocket for small molecular inhibitors targeting RANKL/RANK interaction. The top 109 compounds were


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overlaid with complex structure for visual inspection based on the three criteria: (1) Compounds must not have high strained motifs. (2) Compounds must occupy the deep pocket surrounding residues Glu93, Ile94 and Glu95 of OPG. (3) Compounds must have a certain ratio of hydrophobic fragments participating in key interactions. Finally, 70 compounds were selected and purchased from SPECS (Fig. 1). These virtual screening hits were also filtered for Pan Assay Interference Compounds (PAINS)34. The ranking of prioritized 109 compounds is given in Supporting Information. Inhibition

of

RANKL-induced

Osteoclastogenesis

and

RANKL/RANK

interaction by Compound 1 Since tartrate-resistant acid phosphatase (TRAP) is an established marker for osteoclast differentiation and bone resorption35, the 70 candidate molecules selected by virtual screening were tested for TRAP activity assay on RANKL-induced osteoclastic activation and differentiation from BMMs to quickly determine their osteoclastogenesis inhibitory activity. According to TRAP activity assay, we chose compound 1 as a lead compound since its inhibition on TRAP activity was >50% (54.9 %) at the concentration of 5 μM (Fig. 2A). After RANKL binding to its receptor RANK on osteoclast precursors, various downstream signalings are triggered to stimulate osteoclast differentiation and activity. Then the inhibitory effect of 1 was evaluated against RANKL-induced osteoclast differentiation on BMMs. BMMs were induced to become mature osteoclasts by M-CSF and RANKL, and



compound 1 with different indicated concentrations were incubated with BMMs for 5 days. As shown in Fig. 2B-C, treatment of 1 significantly reduced osteoclast formation in a dose-dependent manner especially at the concentrations of 5 μM (50.0 %) and 10 μM (93.3 %). Compound 1 inhibited RANKL-induced osteoclast differentiation with IC50 = 6.6 μM (Figure S1, Supporting Information). In response to RANKL-RANK interaction, up-regulation of specific genes has been observed36. Real time PCR was used to assess whether 1 can regulate RANKL-induced gene expression levels during osteoclastogenesis. The results were consistent with TRAP activity and osteoclast formation assays. The master regulators of osteoclast NFATc1, as well as bone resorption-related genes TRAP and Cathepsin K were significantly and dose-dependently inhibited by 1 at day 3 of culture (Fig. 2D-F). In addition, MTT assay against BMMs

showed the anti-osteoclastogenic effects of 1 are not attributable to cellular toxicity (Fig. 2G). To further validate the inhibitory effect on osteoclastogenesis of 1 was donated by targeting RANKL and RANK protein interaction, SPR analysis was conducted for characterizing the binding properties of 1 for RANKL. According to SPR analysis, 1 has higher binding affinity for RANKL (Kd = 9.8 μM) than OP3-4 and L3-3 (Kd = 284.4 μM and 17.3 μM respectively). The binding affinity of RANKL for RANK was reported as 0.11 nM37. We retested this value (0.1 nM, Figure S2, Supporting Information) and compound 1 inhibited RANKL binding to its receptor RANK in a dose dependent manner with IC50 = 26.2 μM. The equilibrium binding


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curve fits are shown in Fig. 2H-I. This result indicated the direct binding of 1 for RANKL and 1 has the property of impeding RANKL/RANK protein interaction. Analysis of the Binding Mode of Compound 1 The proposed binding mode of 1 was presented in Fig. 3. The PDB ID of crystal structure used for molecular modeling is 3URF in Protein Data Bank. 1 binds partly in the pocket of RANKL occupied by three residues from OPG (93-95) with a phenyl group. The phenyl group is surrounded by hydrophobic side chains of His180, Lys181, Tyr241 and Glu237. The indole and dimethylphenyl group bind in clefts extending from the RANKL-OPG interaction site. The two bridging amides bind with Leu236, Gln237 and Asn295 through strong hydrogen bonds. The indole NH interacts with Asn295 amide through hydrogen bond too (Fig. 3). Chemical Modification of Compound 1 and SAR Exploration To explore the SAR and improve the potency of compound 1, 15 compounds (compounds 2-16) were firstly synthesized to investigate the structure−activity relationship (SAR) for the carbamate part of compound 1 (Scheme 1). (Scheme 1) Then we evaluated their osteoclastogenesis inhibitory activity by a tartrate-resistant acid phosphatase (TRAP) activity assay. Testing compounds at 5 μM were added to BMMs in the presence of RANKL (50 ng/mL) and M-CSF (20 ng/mL) and incubated for 3 days. The activity results were summarized in Table 1. When the atom O in CBZ group of compound 1 was replaced by methylene group (X position, compound 2),



the inhibitory activity was remarkably increased. Then, para- or meta-substituents like ﬂuorine atom or methoxyl group on the benzene ring (R1 position) led to the reduced inhibitory of TRAP activity (compounds 3-6, Table 1), while para-substituted chlorine atom on the benzene ring in the R1 position (compound 7, Table 1) showed more potent inhibitory activity than compound 1, and meta-substituted chlorine atom displayed the same potency as compound 1 (compound 8, Table 1). The introduction of heteroaromatic ring, such as pyridyl and thienyl, in the R1 position showed the same potency or a little decrease (compounds 9-12, Table 1), while replacement by 2-thiazolyl in the R1 position (compound 13, Table 1) could increase the inhibitory activity. As for cycloalkanes, the introduction of cyclohexyl, cyclopentyl, or cyclopropyl in the R1 position decreased the inhibitory activity (compounds 14-16, Table 1). Thus compound 2 was the most potent for the first-round optimization. (Table 1) Next, modification was focused on 2,6-dimethyl aniline while 3-phenyl propanoic acid amide was kept in the structure. Different substituents were introduced to the benzene ring of aniline structure, like methyl, fluoride, methoxyl, dimethylamino, morpholinyl, methylsulfonyl, trifluoromethyl (compounds 17-26, Table 2), however, the inhibitory activities of these compounds generally decreased than that of compound 2. (Table 2)


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Afterwards, the substitution of the indole ring at the R5 and R6 position were investigated (compounds 27-29, Table 3). Fluorine atom and methoxyl showed comparable potency to compound 2. The results of compounds 30-32 showed that increase or decrease of the chain length was unfavorable to the inhibitory potency (compounds 30-32, Table 3). While replacement of carbonyl group with methylene group in the Y position could increase the inhibitory activity of TRAP slightly (33 vs 30, 34 vs 2, 35 vs 31, 36 vs 32, Table 3). Compound 34 was slightly superior to compound 2 against TRAP. Furthermore, replacement of carbonyl group with methylene group in the Z position turned out that the inhibitory activity against TRAP decreased dramatically (37 vs 2). (Table 3) Inhibition of RANKL-induced Osteoclastogenesis by Compounds 2, 7, 29 and 34 Among all of the synthesized compounds, 2, 7, 29, and 34 performed better than others as illustrated by more than 90% or close to 90% inhibition at 5 μM. Cell survival test based on MTT assay suggested that antiosteoclastogenic effects of 2, 7, 29, and 34 are not attributable to cellular toxicity on BMMs (Table 4). The enantiomers of compound 2 showed a similar inhibitory effect (Figure S3, Supporting Information). In view of the relatively high inhibitory activity on TRAP and low cytotoxicity,

we

further

evaluated

their

effects

on

RANKL-induced

osteoclastogenesis. M-CSF and RANKL induced the formation of TRAP-positive multinucleated cells, while treatment of compounds 2, 7, 29, and 34 (5.0 μM) even completely inhibited osteoclast formation. In addition, BMMs challenged with or



without compounds were double stained with rhodamine phalloidin and DAPI (4',6-diamidino-2-phenylindole). The results showed that M-CSF and RANKL induced the formation of typical osteoclasts morphology such as the formation of F-actin ring and numerous nuclei. In contrast, treatment with compounds had smaller size and fewer nuclei numbers of F-actin ring formation. The results above indicated that compounds strongly inhibited osteoclast differentiation in a dose dependent manner especially at the concentrations of 2.5 μM and 5.0 μM based on TRAP staining and F-actin staining (Fig. 4A). Representative pictures of 34 were shown in Fig. 4B-C. The binding activity to RANKL of compounds 2, 7, 29, and 34 were also determined by SPR binding assays and they displayed much higher affinity for RANKL with KD values of 2.7, 3.1, 3.5 μM and 614 nM respectively (Table 5). Interestingly, 34 displayed weak affinity for TNF (93.6 μM, Figure S4, Supporting Information). As to its highest affinity to RANKL, 34 was chosen as a prototype for further examination in our subsequent studies. (Table 4) (Table 5) Inhibition of RANKL-induced Bone resorption by Compound 34 It is well known that bone resorption is a characteristic feature of osteoclasts during bone remodeling. RANKL/RANK interaction could induce the bone resorption activity of mature osteoclasts, thus we evaluated the effect of 34 on RANKL-induced bone resorption subsequently. As shown in Fig. 5A, almost no resorption was detected in bone slices treated with 5 μM 34 while obvious resorbing pits were


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formed in the RANKL-treated group as visualized by toluidine blue staining. 34 (1, 2.5 μM) also significantly decreased the percentage of resorbed area. Besides, the calculated bone resorption shown in Fig. 5B is consistent with Fig. 5A. Inhibition of osteoclastogenesis-related gene expression and c-Fos/NFATc1 signaling pathway by Compound 34 We used RT-PCR to examine the effects of 34 on RANKL-induced osteoclast-specific gene expressions during osteoclastogenesis. BMMs were treated with different concentrations of 34 for 3 days. TRAP, cathepsin K and NFATc1 were increased in response to RANKL stimulation, while 34 strongly decreased the expressions of these genes even at 2.5 μM (Fig. 6A-C). c-fos/NFATc1 signaling pathway serves as a major regulatory axis downstream of RANKL. They trigger RANKL-induced osteoclast formation through regulating the expressions of related genes such as TRAP and cathepsin K. We thus conducted WB to verify the effects of 34 on the protein expressions of c-fos and NFATc1. As shown in Fig. 6D, 34 could effectively downregulate the c-fos/NFATc1 signaling in BMMs in a dose-dependent manner. CONCLUSION The development of potent RANKL inhibitors is currently a hot topic of bone metabolic diseases drug discovery. Promising results were obtained such as recombinant proteins (RANK-Fc, Fc-OPG, and anti-RANKL antibodies) and small peptides (OP3-4 and L3-3). However, the application of large macromolecules is



limited partially by high cost and difficulties in administration. There are also some drawbacks of small peptides such as poor tissue penetration, oral bioavailability. Currently there is only a handful of nonpeptidic small molecular inhibitors targeting RANKL and RANK protein interaction. Among them, most of compounds displayed high toxicity, poor potency and selectivity. It thus seems urgent to explore small molecular inhibitors with high potency and selectivity preventing RANKL/RANK interaction. Here, based on structure-based virtual screening and hit optimization, we reported 2-amino-3-(1H-indol-3-yl)-N-phenylpropanamides as a novel scaffold possessing high binding affinity to RANKL targeting RANKL-RANK protein-protein interaction. Four compounds exhibited high binding affinity to the RANKL (KD for RANKL

(RANKL) - Receptor Activator of Nuclear

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