Acs Jcim 0c00171

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Pharmaceutical Modeling
In Silico Screening-based Discovery of Novel Inhibitors
of Human Cyclic GMP-AMP Synthase: A Cross-validation
Study of Molecular Docking and Experimental Testing
Wenfeng Zhao, Muya Xiong, Xiaojing Yuan, Minjun Li, Hongbin Sun, and Yechun Xu
J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.0c00171 • Publication Date (Web): 27 May 2020
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Page 1 of 60 Journal of Chemical Information and Modeling
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In Silico Screening-based Discovery of Novel
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Inhibitors of Human Cyclic GMP-AMP
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13 Synthase: A Cross-validation Study of Molecular
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16 Docking and Experimental Testing
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Wenfeng Zhao†,‡,§,#, Muya Xiong‡,§,#, Xiaojing Yuan‡,§, Minjun Li⊥, Hongbing Sun*,†,
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22 Yechun Xu*,‡,§
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26 †Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease and State Key
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29 Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing
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210009, P.R. China
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35 ‡CAS Key Laboratory of Receptor Research, Drug Discovery and Design Center,
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38 Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
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41 201203, China
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44 §University of Chinese Academy of Sciences, Beijing 100049, China
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48 ⊥Shanghai Synchrotron Radiation Facility, Shanghai Advanced Research
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51 Institute, Chinese Academy of Sciences, Shanghai 201210, China
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60 ACS Paragon Plus Environment
Journal of Chemical Information and Modeling Page 2 of 60
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4 KEYWORDS: cGAS, crystal structures of complexes, inhibitors, molecular docking,
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7 virtual screening, MD simulations.
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11 ABSTRACT: Cyclic GMP-AMP synthase (cGAS) is recently uncovered to be a
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14 promising therapeutic target for immune-associated diseases. Up to now, only a
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17 few inhibitors were identified through high-throughput screening campaigns. Here,
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19 we reported the discovery of novel inhibitors for the catalytic domain of human
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22 cGAS (h-cGASCD) by virtual screening for the first time. To generate a reliable
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25 docking mode, we first obtained a high-resolution crystal structure of h-cGASCD in
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complex with PF-06928215, a known inhibitor of h-cGAS, followed by MD
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30 simulations on this complex structure. Four fragment hits were identified by the
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33 virtual screening together with a thermal shift assay. Crystal structures of these four
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36 compounds in complex with h-cGASCD were subsequently determined and the
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38 binding modes of the compounds were similar to those predicted by molecular
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41 docking, supporting the reliability of the docking model. In addition, an enzyme
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44 activity assay identified compound 18 (IC50 = 29.88 ± 3.20 μM) from the compounds
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predicted by the virtual screening. A similarity search of compound 18 followed by
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49 a second virtual screening led to the discovery of compounds S2 (IC50 =13.1 ± 0.09
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52 μM) and S3 (IC50 =4.9 ± 0.26 μM) as h-cGAS inhibitors with improved potency.
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55 Therefore, the present study not only provides the validated hit compounds for
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4 further development of h-cGAS inhibitors but also demonstrates a cross-validation
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7 study of virtual screening, in vitro experimental assays and crystal structures
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10 determination.
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14 1. INTRODUCTION
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17 The innate immunity senses pathogen-related molecular patterns (PAMPs) and
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19 damage-associated molecular patterns (DAMPs) through different pattern
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22 recognition receptors (PRRs), and initiates innate immune response.1 Cyclic GMP-
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25 AMP synthase (cGAS) has emerged as the dominant sensor of cytosolic double-
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stranded DNAs (dsDNAs) in a sequence-independent manner,2–5 which are
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30 canonical DAMPs derived from a variety of danger signals and tissue damage, such
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33 as virus invasion, chromosome damage, mitochondrial damage, aging or other
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36 abnormal conditions.6,7 Upon binding with cytoplasmic dsDNAs, cGAS promotes
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38 cyclization of adenosine 5´-triphosphate (ATP) with guanosine 5´-triphosphate
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41 (GTP) into cyclic [G(2´,5´)pA(3´,5´)p] (cGAMP), which acts as a second messenger
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44 to activate an endoplasmic reticulum membrane-anchored adaptor protein, named
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stimulator of interferon genes (STING).2, 8–11 Activated STING initiates a signaling
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49 cascade by recruiting and activating the TANK-binding kinase 1 (TBK1) to
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52 phosphorylate the transcription factor family of interferon regulatory factors 3
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4 (IRF3), resulting in gene expression of type-I interferons (IFN-I) to trigger further
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7 immune cascade reactions.8, 12–14
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10 Owing to the high immunogenicity of cytoplasmic dsDNAs, it has been reported
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12 that abnormal activation of the cGAS pathway is associated with various
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15 autoimmune disorders. Functional loss of three-prime repair exonuclease 1
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18 (TREX1) is a potential pathogenic factor for Aicardi–Goutières syndrome and
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systemic lupus erythematosus (SLE), as TREX1 is the primary cellular exonuclease
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23 in cytoplasms to prevent cells from excessive existence of dsDNAs and cGAS
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26 activation.15–19 Polyarthritis/fetal and neonatal anemia were also identified as a type
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29 I interferonopathy due to loss-of-function mutations in DNase II, another enzyme
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31 digesting cytoplasmic DNA.20 Activation of cGAS by cytoplasmic escaping
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34 mitochondrial DNAs (mtDNAs) was reported as an essential etiology of age-related
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37 macular degeneration.21 Apart from these autoinflammatory and autoimmune
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diseases, acute pancreatitis,22 myocardial infarction23 and other inflammatory
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42 diseases were also associated with dysfunction of cGAS-STING signaling
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45 pathways.24, 25 Notably, the deletion of cGAS in some autoimmune disease models
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48 showed remissions of symptoms. Together, inhibition of cGAS has been regarded
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50 as a promising therapeutic strategy for inflammatory and autoimmune diseases.24
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4 The promising therapeutic benefits of cGAS inhibition prompt a considerable
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7 amount of interest in the design of cGAS inhibitors (Figure 1). Through a virtual
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10 screening targeting the site of mouse cGAS (m-cGAS) interacting with DNA (PDB:
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12 4LEZ), a series of antimalarial drugs (AMDs) were found to disturb the interactions
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15 between cGAS and DNA, reduce the production of cGAMP and inhibit the dsDNA
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18 sensing in THP-1 cells.26, 27 However, these AMDs have been proven to bind to
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DNA rather than cGAS. Suramin, an essential drug for the treatment of river
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23 blindness and African sleeping sickness, was disclosed as a cGAS inhibitor which
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26 blocked the binding between cGAS and DNA.28 RU.521 and G150 were lately
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29 developed on the basis of the hits resulted from high throughput screenings, and
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31 both compounds occupied the cGAS active site.29,30 By far, RU.521 and G150 are
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34 the most potent inhibitors of m-cGAS and human cGAS (h-cGAS), and their cellular
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37 efficacies have been demonstrated in mouse RAW264.7 cells and human THP-1
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cells, respectively. In addition, Hall et al. at Pfizer reported the first h-cGAS selective
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42 inhibitor (PF-06928215) which possessed a high affinity with the catalytic site and
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45 was identified by a fragment screening via NMR followed by a fragment evolution.
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48 Nevertheless, PF-06928215 displayed no activity in cell-based cGAS assays and
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50 the underlying mechanism remains unclear.31
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4 Virtual screening has proven great advantage in identifying new chemical hits,32
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7 in particular for the case of cGAS to which only a few inhibitors are available. We
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10 thereby utilized the virtual screening campaigns together with in vitro assays and
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12 crystal structure determination to discover novel inhibitors of h-cGAS. First, we
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15 determined a high-resolution (1.8 Å) crystal structure of the catalytic domain of h-
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18 cGAS (h-cGASCD) in complex with PF-06928215, which was the only validated
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inhibitor of h-cGAS when we started this project. Then, a virtual screening campaign
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23 was subsequently carried out based on this complex structure and several novel-
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26 scaffold hits as h-cGAS inhibitors were successfully identified. Moreover, the crystal
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29 structures of h-cGASCD in complex with these newly discovered hits were
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31 determined, providing structural basis for further development of novel inhibitors.
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34 As an example, a similarity search upon one of these hits (compound 18) resulted
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37 in the discovery of new inhibitors with improved potency.
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25 Figure 1. Chemical structures of reported cGAS inhibitors.
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28 2. METHODS
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31 2.1 h-cGAS Expression and Purification. A gene encoding full-length human
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34 cGAS (Fl-h-cGAS) (amino acids 1-522 of human MB21D1 (Uniprot ID: Q8N884))
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36 was synthesized at synbio-tech and cloned into pET-15b for expression of Fl-h-
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39 cGAS with an N-terminal 6×His-SUMO2 fusion protein, as described by Zhou, W.
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42 et al. previously.33 Based on this plasmid, a construct for expression of the catalytic
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domain of h-cGAS (amino acids: 157-522, h-cGASCD) was further generated by
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47 molecular cloning. The sequences of two plasmids were confirmed by sequencing.
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50 Rosetta 2 (DE3) (Sigma-Aldrich) was transformed with the above plasmids and
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53 cultured in LB media at 37 °C to reach an OD600 of 0.8. Then the culture temperature
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57 7
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4 was reduced to 16 °C and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added
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7 with a final concentration of 0.5 mM for induction. After 8-10 h, bacteria were
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10 harvested by centrifugation at 4 °C, washed with PBS, flash-frozen in liquid
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12 nitrogen, and stored at -80 ℃ until lysis for purification.
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15 The protein purification protocol followed the reported procedure33 with
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18 modifications. Cell pellets resulted from 2 L LB culture were lysed in a lysis buffer
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(20 mM HEPES, pH 7.5, 400 mM NaCl, 1 mM TCEP) with DNase I using a high-
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23 pressure homogenizer. The solution was centrifuged at 12,000 g for 30 min, and
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26 the supernatant was slowly passed through a manually packed nickel-bead column
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29 (GE) which was pre-equilibrated with the lysis buffer. The column was washed with
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31 a wash buffer (20 mM HEPES, pH 7.5, 400 mM NaCl, 50 mM imidazole, 1 mM
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34 TCEP), and then eluted with an elution buffer (20 mM HEPES, pH 7.5, 400 mM
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37 NaCl, 500 mM imidazole, 1 mM TCEP). The eluted fractions were pooled, diluted
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with an equal volume buffer without salt (20 mM HEPES, pH 7.5, 1 mM TCEP),
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42 supplemented with ~250 μg of human SENP2 protease (residues 364–589 with an
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45 M497A mutation), and dialyzed overnight at 4 ℃. Untagged cGAS was further
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48 purified by binding to two 5-mL Heparin HP ion-exchange columns connected in
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50 tandem (GE Healthcare) and eluted with the buffer (20 mM HEPES, pH 7.5, 1 mM
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53 TCEP) containing a gradient of 300-1000 mM NaCl. The eluted protein was
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4 concentrated and injected into a size-exclusion chromatography column (16/600
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7 Superdex S75, GE Healthcare) equilibrated with the gel filtration buffer (20 mM
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10 HEPES, pH 7.5, 250 mM KCl, 1 mM TCEP, 10% glycerine). The S75 pools from
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12 each peak were separately collected, concentrated to 8-9 mg/ml, aliquoted and
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15 flash frozen in liquid nitrogen. Fl-h-cGAS was used for the enzyme activity assay,
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18 and h-cGASCD was used for crystallization as well as the thermal shift assay.
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2.2 Thermal Shift Assay. Thermal shift assays were performed on a 7500 Fast
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23 Real-Time PCR System (Thermo Fisher Scientific) with 96-well white plates
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26 (Thermo Fisher Scientific). Each well contained 20 μL 5 μM h-cGASCD and 5×
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29 Protein Thermal Shift dye (Thermo Fisher Scientific) in a buffer (20 mM HEPES, pH
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31 7.5, 250 mM KCl, 1 mM TCEP, 10% glycerine). Compounds were tested at a final
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34 concentration of 200 μM or 400 μM in 5% (v/v) DMSO. Each plate was sealed with
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37 an optically clear foil and centrifuged for 1 min at 1000 rpm. Then the plates were
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heated from 25-95 °C at approximately 1 °C min−1. The fluorescence intensity was
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42 measured with λex = 480 nm and λem = 580 nm. The melting temperature (Tm) of the
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45 protein with or without the compound was obtained by determining the minimum of
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48 the first derivative curve of the melt curve. The thermal shift (ΔTm) was determined
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50 by calculating the difference between the Tm of the protein in the presence of and
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53 in the absence of the tested compound.
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4 2.3 Pyrophosphatase-coupled cGAS Activity Assay. The enzymatic activity of
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7 cGAS was measured using a pyrophosphatase-coupled assay developed by
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10 Richard and Jungsan34 with some modifications. Briefly, a final concentration of 200
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12 nM Fl-h-cGAS, 50 nM E.coli pyrophosphatase (Sigma-Aldrich I5907), 1 mM ATP,
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15 0.3 mM GTP, 5 μg/ml HT-DNA (Sigma-Aldrich) in 40 μL reaction buffer (10 mM
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18 HEPES, 140 mM NaCl, 0.01% Tween-20, 5 mM MgCl2, pH 7.5) in the presence or
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absence of tested compounds were incubated for 90 min at room temperature.
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23 Then, 40 μL quenched solutions (50 mM EDTA) were mixed with 20 μL malachite
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26 green solution and incubated for 45 min at room temperature. The absorbance of
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29 each well at ~620 nm was measured and compared to the negative control (without
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31 HT-DNA) and the positive control (DMSO instead of the compound).
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34 The amount of phosphoric acid represents the amount of pyrophosphoric acid
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37 generated by the reaction, allowing for the determination of the equivalent amount
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of cGAMP generated in the reaction. The inhibition ratio of a compound was
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42 normalized against the positive control and the negative control as follows: inhibition
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45 % = 1- (Abssample−Absaverage negative control)/(Absaverage positive control − Absaverage negative
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48 control).
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50 2.4 Crystallization and Structure Determination. Crystals of h-cGASCD were
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53 obtained by a hanging-drop vapor diffusion method using 1.5 μL protein (8-9 mg/ml)
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4 plus 1.5 μL reservoir containing PEG 3350 (17.5-19% v/v) and 0.2 M ammonium
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7 citrate (pH 7-7.5) at 4 °C. Crystals of complexes were obtained via soaking apo
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10 crystals of h-cGASCD with compounds at a concentration of 10 mM in the mother
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12 liquor overnight. Crystals were flash frozen in liquid nitrogen in the presence of the
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15 mother liquor supplemented with 20% glycerol.
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18 The diffraction data of crystals were collected at 100 K on the beamline BL17U1
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or BL19U1 at the Shanghai Synchrotron Radiation Facility (SSRF) and were
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23 processed with the HKL3000 software packages.35 The structures were solved by
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26 molecular replacement, using the program CCP436 with a search model of PDB
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29 code 4LEV.5 The structures were refined using PHENIX.37 With using the program
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31 Coot,38 compounds and water molecules were fitted into the initial Fo-Fc map. Data
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34 collection and refinement statistics of six determined structures are shown in Table
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37 S1.
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2.6 Molecular Dynamics (MD) Simulations. MD simulations were performed to
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42 observe the key residues interacting with PF-06928215 by using AMBER 14
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45 package,39 which provides a hint for picking compounds after the virtual screening.
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48 Our crystal structure of h-cGASCD in complex with PF-06928215 and the protein
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50 structure solely (PDB code: 6LRC) were used for setting up the model of MD
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53 simulations. The parameters of PF-06928215 were calculated using the B3LYP
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4 method at the 6-31G* level with Gaussian 09.40 Its restricted electrostatic potential
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7 (RESP) was generated by Antechamber41 and assigned the general amber force
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10 field.42 For the protein, we fixed the missing side chains and loops, deleted Zn2+
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12 ions, and the ff14SB force field was used.43 To mimic the stable interactions with
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15 the Zn2+ ion, four residues (H390, C396, C397, and C404) interacting with Zn2+ were
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18 restricted in all the stages of MD simulations. Next, the protein or the complex were
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solvated with an 8-Å TIP3P water box. The whole system was neutralized by adding
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23 counterions (Cl- or Na+). Two successive energy minimizations were carried out
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26 using the steepest descent and the conjugate gradient algorithm in the Sander
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29 module. First, hydrogen atoms and water molecules were minimized in 2500 steps
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31 using the steepest descent algorithm combined with the conjugate gradient one.
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34 Second, another 5000 steps of the steepest-descent minimization followed by 5000
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37 cycles of the conjugated gradient one were performed to minimize all atoms of the
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systems except a restriction on four residues that coordinate with the Zn2+ ion.
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42 Then, the system was gradually heated from 0 to 100 K and 100 to 300 K separately
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45 in the NVT ensemble over 5 ps and 100 ps, respectively, with a smaller restriction
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48 on protein and ligand. Then, the system was equilibrated at 300 K and 1 atm without
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50 any restriction except for the four residues mentioned above. The periodic boundary
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53 conditions were utilized. The SHAKE algorithm44 was applied to constrain the
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4 hydrogen atoms. The long-range electrostatic interactions were calculated by the
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7 particle mesh Ewald (PME) method.45 Finally, a 100-ns productive MD trajectory
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10 was generated for each model at a constant temperature and pressure using the
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12 PMEMD module46 implemented in the AMBER 14. The coordinates of the protein
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15 or the protein-ligand complex in the MD trajectory were saved every 10 ps.
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18 2.7 Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA)
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Calculations. The energy decomposition was calculated based on the 100-ns MD
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23 trajectories in order to assess the contribution of each residue to the total binding
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26 free energy of PF-06928215 with h-cGASCD. The binding free energy and energy
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29 decomposition calculations were implemented using the MM/PBSA method47 in
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31 AMBER 14. The binding free energy calculations were performed on 20 snapshots
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34 extracted from the 100 ns trajectories. In MM-PBSA, the binding free energies
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37 (ΔGbind) are calculated by the following equations:
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ΔGbind = Gcomplex – (Greceptor + Gligand) (1)
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42 ΔGbind = ΔEMM + ΔGsolvation – TΔS (2)
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45 where Gcomplex, Greceptor and Gligand are the representations of the free energies of
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48 complex, receptor and ligand, respectively; ΔEMM is the gas phase molecular
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50 mechanical energy; ΔGsolvation is the desolvation free energy; -TΔS represents the
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53 conformational entropy upon binding of ligand at a temperature of T. The binding
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4 free energies (ΔGbind) are calculated using equation 1 and each free energy is
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7 calculated as the sum of the MM energy EMM, the solvation free energy Gsolvation and
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10 the entropy contribution S, respectively (equation 2). The MM energy is
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12 decomposed into EMM, EMM, and EMM,
complex, rec lig. The solvation free energy
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15 represents the sum of the electrostatic solvation free energy and nonpolar solvation
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18 free energy. The entropy contribution was neglected due to the cost of the
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computational process is huge for large protein-ligand systems.
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23 2.8 Virtual Screening. Our crystal structure of h-cGASCD in complex with PF-
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26 06928215 (PDB code: 6LRC) was used as the receptor structure. Three water
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29 molecules forming more than 3 hydrogen bonds (H-bonds) to ligand or/and residues
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31 were reserved. The docking grid was centered on the centroid of PF-06928215.
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34 The receptor was then prepared using the Protein Preparation Wizard and Grid
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37 Preparation tools. The OPLS3 force field was used for minimization and grid
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generation.48
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42 We used tools implemented in the Schrödinger suite to screen the ChemDiv
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45 database. We first prepared the 3D structures of ~1,500,000 compounds from the
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48 database with LigPrep.49 The output structures were then supplied for the virtual
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50 screening workflow. The overall workflow includes filtering the compounds with
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53 Propfilter on QikProp properties and docking them to the receptor with Glide.50 In
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4 the first step, we filtered out compounds that are not compatible with lead-like rules
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7 (250<MW≤350, LogP≤3.5) or Lipinski’s Rule of 5. In the docking step, we carried
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10 out Standard Precision (SP) calculations with default settings. The top 10% of the
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12 best compounds were reserved for the post-processing analysis. The OPLS3 force
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15 field was used for docking. The survivors were then imported into Canvas for
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18 clustering analysis using the Leader−Follower method.51 Ultimately, the top-ranking
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compounds were visually analyzed and 59 compounds with diverse structures were
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23 purchased for biological testing.
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26 2.9 Similarity Searching. In the similarity searching of compound 18, we used
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29 tools implemented in the Schrödinger suite to screen the ChemDiv database. We
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31 first prepared the 3D structures of ~1,500,000 compounds from the database with
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34 LigPrep. The workflow of ligand-based virtual screening includes filtering the
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37 compounds with the Shape Screening and docking them to the receptor with
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Glide.52 First, the compounds were filtered out with a similarity score below 0.6
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42 using the structure of compound 18 as a shape query. Second, we carried out
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45 Standard Precision (SP) calculations with default settings to dock the remaining
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48 compounds to the receptor. The top 10% of the best compounds were reserved for
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50 the post-processing analysis. The OPLS3 force field was used for docking. The
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53 survivors were then imported into Canvas for clustering analysis using the
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57 15
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4 Leader−Follower method. Ultimately, 7 compounds with different structures were
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7 purchased for biological testing.
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12 3. RESULTS AND DISCUSSION
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15 3.1 A High Resolution Crystal Structure of h-cGASCD in Complex with PF-
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18 06928215. In order to set up the structural biological platform for drug design
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targeting cGAS, we first tried to optimize the crystallization conditions of apo h-
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23 cGASCD to obtain the crystals for inhibitors soaking. And we determined the crystal
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26 structure of h-cGASCD bound with PF-06928215 at a resolution of 1.8 Å (PDB code:
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29 6LRC), which is the highest resolution one among the reported crystal structures of
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31 h-cGASCD (Figure 2 and Table S1). Similar to the crystal structure of h-cGAS-PF-
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34 06928215 (PDB code: 6NAO) determined previously at a resolution of 3.2 Å,31 the
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37 pyrazolopyrimidine of PF-06928215 is sandwiched between the guanidinium group
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of R376 and the aromatic ring of Y436, mimicking the nucleobase. The benzene is
41
42 anchored in a hydrophobic sub-pocket lined by the side chains of F488 and L490.
43
44
45 Nevertheless, we observe that the position of PF-06928215 in our structure is
46
47
48 slightly shifted compared to the one in 6NAO (RMSD =0.73 Å) so that the
49
50 carboxylate headpiece not only interacts with R376 and K36231 but also makes
51
52
53 contact with R302 through two water molecules (Figure 2c). The carbonyl of the
54
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56
57 16
58
59
1
2
3
4 amide forms an H-bond with the hydroxyl of S434 (Figure 2b), which is not found in
5
6
7 the previous structure owing to a larger distance of the acceptor to the donor.
8
9
10 Additionally, owing to the high resolution, more water molecules have been seen in
11
12 our structure and they mediate multiple interactions between PF-06928215 and
13
14
15 residues such as R302, L377, S378, F379, S380, E383, S435, Y436, and N482
16
17
18 (Figure 2c). Together, the crystal structure of h-cGASCD/PF-06928215 determined
19
20
21
at 1.8 Å reveals more distinct protein-ligand interactions and provides a good
22
23 starting point for the design of new inhibitors.
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38 Figure 2. Crystal structures of h-cGASCD in complex with PF-06928215. (a) The
39
40
41 2Fo–Fc electron density map of the bound PF-06928215 contoured at 1.6 σ level.
42
43
44 (b) The structure of PF-06928215 in the high-resolution crystal structure (PDB code:
45
46 6LRC, yellow) superimposed to that in 6NAO (green). (c) Intermolecular contacts
47
48
49 among PF-06928215, water molecules and amino acids lining the binding pocket
50
51
52 of h-cGASCD.
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57 18
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59
1
2
3
4 3.2 MD Simulations of h-cGASCD in Complex with PF-06928215. In order to
5
6
7 better understand the binding mode of PF-06928215 with h-cGAS, the high-
8
9
10 resolution crystal structure (PDB code: 6LRC) mentioned above was subjected to
11
12 MD simulations. The C atom RMSD and RMSF of each residues along the 100-
13
14
15 ns MD trajectories show a relative stable structure of h-cGASCD during MD
16
17
18 simulations (Figure S1). The 100-ns MD trajectories were then used to calculate
19
20
21
the binding free energy and energy decomposition utilizing the MM/PBSA method.
22
23 While the entropic term is ignored because of its huge cost of computation, the
24
25
26 calculated binding energy indicates that the binding enthalpy can be used as an
27
28
29 good indicator of the ligand-receptor binding and hit selection.53 The results from
30
31 the MM/PBSA calculations show that the contribution of electrostatic interactions
32
33
34 (EELE= -11.16 kcal/mol) is much smaller compared to the van der Waals interactions
35
36
37 (EVDM= -41.43 kcal/mol), a major contribution to the binding of PF-06928215 with h-
38
39
40
cGAS (Table S2). In accordance with this, the nonpolar interactions (EVDM +
41
42 GNPOLAR) are more significant compared with the polar interaction (EELE + GPB),
43
44
45 indicating that compounds forming more nonpolar interactions with h-cGAS in the
46
47
48 following virtual screening could be selected for biological evaluation.
49
50 To find out which residues count a great deal in the receptor-ligand binding,
51
52
53 energy decomposition calculations were performed. As presented in Figure 3, PF-
54
55
56
57 19
58
59
1
2
3
4 06928215 forms the most robust Van der Waal interactions with Y436 as well as
5
6
7 R376 and great electrostatic interactions with R376 in the active site. It also shows
8
9
10 that E383 establishes distinct electrostatic interactions with PF-06928215 but with
11
12 a high deviation. However, in the crystal structure, it makes contact with PF-
13
14
15 06928215 through two water molecules (Figure 2c), although the representative
16
17
18 snapshots extracted from the MD trajectory display that the pyrazolopyrimidine
19
20
21
interacts directly with E383 without the aid of water molecules (Figure S2).
22
23 Considering the inconsistency between the crystal structure and the calculation
24
25
26 together with the high deviation, the electrostatic interactions of compounds with
27
28
29 E383 are not taken as a key factor for hit selection. In addition, most of the residues
30
31 in the active-site pocket tend to form nonpolar interactions with the ligand, such as
32
33
34 K362, L377, S378, F379, S434, H437, N482, F488, L490, and L495. Accordingly,
35
36
37 on the basis of energy decomposition calculations on the h-cGASCD-PF-06928215
38
39
40
complex, the compounds establishing more nonpolar or polar interactions with
41
42 residues indicated in Figure 3b would be favored in hit selection.
43
44
45
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48
49
50
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57 20
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7
8
9
10
11
12
13
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15
16
17
18 Figure 3. Energy decomposition calculations on the h-cGASCD-PF-06928215
19
20
21
complex. (a) The contributions of van der Waals and electrostatic interactions of
22
23 residues (171-510) to the binding of PF-06928215 with h-cGASCD. (b) Contributions
24
25
26 of these residues positioned less than 5 Å of PF-06928215.
27
28
29
30
3.3 Virtual Screening. The ChemDiv database containing ~1,500,000 compounds
31
32 was applied in our virtual screening with an aim of identifying novel scaffolds of
33
34
35 cGAS inhibitors. As mentioned above, the crystal structure of h-cGASCD in complex
36
37
38 with PF-06928215 (PDB codes: 6LRC and 6NAO) determined at 1.8 and 3.2 Å,
39
40 respectively, were tested for molecular docking. The RMSD of docked conformation
41
42
43 of PF-06928215 compared to it in 6LRC and 6NAO is 1.53 and 1.71 Å (Figure S3),
44
45
46 respectively, suggesting no significant difference was seen between the docking of
47
48
49
two crystal structures to regenerate the binding pose of the ligand. Only our crystal
50
51 structure (PDB code: 6LRC) was thus used for the following virtual screening. In
52
53
54 addition, in the docking model, three water molecules were kept as they established
55
56
57 21
58
59
1
2
3
4 more than 3 H-bonds to ligand or/and residues (Figure S3a). The RMSD of PF-06928215
5
6
7 between the docked conformation and its crystal structure without water molecules
8
9
10 is 5.16 Å, much larger than the one (1.53 Å) with three water molecules (Figure
11
12 S3a, b). The workflow of the virtual screening and the protocol of molecular docking
13
14
15 are described in Material and Methods. Compared with the docking scores of
16
17
18 ligands from the crystal structures, the compounds with docking scores greater than
19
20
21
-6.4 were retained, resulting in 3000 compounds. To ensure the structural diversity
22
23 of compounds, a clustering analysis based on the Leader−Follower method was
24
25
26 applied to these 3000 compounds so as to decrease the number of compounds with
27
28
29 similar scaffolds. According to the binding free energy and energy decomposition,
30
31 the compounds forming the Van der Waals interactions with Y436 and R376, and
32
33
34 the electrostatic interactions with R376 were retained. In addition, those featuring
35
36
37 more nonpolar interaction with more negative-energy residues listed in Figure 3b
38
39
40
were also selected. In the end, 59 compounds were purchased for biological testing.
41
42 3.4 Evaluation of Virtual Screening Hits via the Thermal Shift Assay (TSA). When
43
44
45 gradually heated, the protein unfolds and its hydrophobic core is exposed, providing
46
47
48 more hydrophobic regions for fluorescence probes binding and resulting in a rise of
49
50 fluorescence intensity. The binding of a ligand to the protein will increase or reduce
51
52
53 the melting temperature (Tm) by stabilizing or destabilizing the protein, respectively.
54
55
56
57 22
58
59
1
2
3
4 The difference between the Tm of the protein binding with and without the ligand
5
6
7 indicates the thermal shift (ΔTm).54 The value of ΔTm indicates the binding affinity of
8
9
10 compounds with a targeting protein and such an assay is widely used to screening
11
12 compounds or verify the binding of compounds with a protein.55
13
14
15 We first explored if the TSA was feasible for evaluation of the binding of
16
17
18 compounds with h-cGASCD as such an assay had not been reported for cGAS
19
20
21
inhibitors screening or binding validation. During the process of protein purification
22
23 in our study as well as others, two peaks of h-cGASCD were eluted from the size-
24
25
26 exclusion chromatography, suggesting that h-cGASCD can form dimer without
27
28
29 dsDNA.34 Therefore, we measured the melting temperatures of the purified
30
31 monomer as well as dimer, and the Tm of the dimer is nearly 3 ℃ higher than that
32
33
34 of the monomer (Figure 4a, b). In addition, the high-resolution crystal structure of
35
36
37 the h-cGASCD-PF-06928215 complex was solved by soaking the inhibitor to the
38
39
40
crystals of apo h-cGASCD dimer. Accordingly, the dimeric h-cGASCD was used for
41
42 the following TSA.
43
44
45 Adding of 50 μM PF-06928215 to the dimeric h-cGASCD protein solution results
46
47
48 in 7℃ change in transition temperature (∆Tm) (Figure 4c, d), which means the
49
50 binding of ligands such as PF-06928215 to the active site of h-cGASCD affects the
51
52
53 thermal stability of h-cGASCD. Therefore, we applied the TSA to test the binding of
54
55
56
57 23
58
59
1
2
3
4 59 compounds resulted from the virtual screening with h-cGASCD. As a result, 4
5
6
7 compounds (3, 17, 23, and 40) at a concentration of 400 μM shifted the melting
8
9
10 temperature of h-cGASCD (ΔTm > 1 °C, Table 1, Figure S4). Interestingly, these four
11
12 compounds all belong to N-tricyclic heterocyclic carboxylic acids and two carboxyl
13
14
15 groups present in compounds 3 and 17 with high ΔTm values (2.50 °C and 2.90 °C
16
17
18 for 3 and 17, respectively).
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34 Figure 4. The melting temperature of h-cGASCD with or without compounds
35
36
37 measured by the thermal shift assay. (a, b) The melting curve of monomer (gray)
38
39
40
and dimer (blue) of h-cGASCD. (c, d) The melting curve of the dimeric h-cGASCD in
41
42 the absence (blue) and presence of PF-06928215(orange).
43
44
45
46 Table 1. The melting temperature of h-cGASCD shifted upon the binding of
47
48
49
compounds
50
51
52 Compound Structure MW. ∆Tm (℃) Inhibition radio
53
54
55
56
57 24
58
59
1
2
3
4 s （100 μM）
5
6 H
7 O N O
8
3 285 2.50 39.9 %
9
10 HO OO OH
11 O
12 OH
13 N
17 316 2.90 29.9 %

N O
14 N
N
15 HO
16 O
17
18 N
HO
23 314 1.77 21.8 %

H
19 N N N
H
N O
O
20 N
21
22
N
23 O
40 H
295 1.14 16.8 %
24 N N OH
25
26
27 3.5 Crystal Structures of h-cGASCD in Complex with Four Hits. To reveal the
28
29
30 binding features of the four positive hits from the TSA, crystal structures of h-
31
32
33 cGASCD in complex with these compounds were determined (Figures 5, S5 and S6).
34
35 Remarkably, similar to the docking predicted conformations, the heterocyclics of
36
37
38 the four hit compounds are sandwiched between R376 and Y436 (Figure 5), and it
39
40
41 is consistent with the previous finding that this site could accommodate a variety of
42
43
44
2- or 3-ring aromatic systems.56 The isoquinoline-1, 3(2H)-dione head of compound
45
46 3 (PDB code: 6LRE) interacts with residues E383 and S378 through water
47
48
49 molecules, which resembles the interactions of the pyrazolopyrimidine or the
50
51
52 hydroxyl group of PF-06928215 with surrounding residues. While no π-π interaction
53
54 is formed between 3 and F488, the binding of 17, 23 and 40 (PDB code: 6LRI, 6LRJ
55
56
57 25
58
59
1
2
3
4 and 6LRK, respectively) all result in a side-chain flip of F488 and the π-π
5
6
7 interactions of these compounds with F448 are thus established. In addition, the
8
9
10 carboxyl groups in four compounds form H-bond (s) or/and salt bridge with
11
12 surrounding residues, although their location and orientation are different. For
13
14
15 example, both carboxyl groups of compound 3 form an H-bond to S434, while the
16
17
18 carboxyl group of 17 either contacts with S378 through a direct H-bond or with H437
19
20
21
via a water molecule. A salt bridge is established between the carboxyl group of 23
22
23 and R376, while the carboxyl group of 40 sitting on the chair-like configuration of
24
25
26 the piperidine constructs both the salt bridge and water-mediated H-bonds to two
27
28
29 positive-charged residues R376 and K362. For compounds 17 and 23, their
30
31 carboxyl groups are linked to the N-tricyclic heterocyclic ring through a relatively
32
33
34 long and flexible chain, leading to the partial missing of electron density for the chain
35
36
37 (Figure S5 and S6). Overall, the sandwich interactions of the N-tricyclic heterocyclic
38
39
40
ring with R376 and Y436 act as an anchor for the binding of the four compounds,
41
42 while the accommodation of the diverse location or orientation of these carboxyl
43
44
45 groups by the ligand binding pocket of h-cGAS reveals that this pocket possesses
46
47
48 a relatively open and polar character. Moreover, the docked conformation of the
49
50 ligand is well superimposed to it in the crystal structure for all four cases, supporting
51
52
53 that our docking model as well as the workflow of the virtual screening is reliable to
54
55
56
57 26
58
59
1
2
3
4 discover novel h-cGAS inhibitors. These crystal structures also suggest that the
5
6
7 TSA is a reliable assay for the screening of h-cGAS inhibitors, although the
8
9
10 determined ∆Tm values are relatively low. Moreover, we also find the water molecule
11
12 next to R376 can be seen in three crystal structures of complexes except that of
13
14
15 compound 17, although three water molecules including this one were conserved
16
17
18 in the docking model (Figure S7). It is thus suggested that the water molecule next
19
20
21
to R376 may play a pivotal role in h-cGAS-inhibitor binding, providing a better model
22
23 for future virtual screening.
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57 27
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38 Figure 5. Superposition of the docked conformation (gray) and bound conformation
39
40
41 of compounds 3 (a), 17 (b), 23 (c), and 40 (d) (PDB codes: 6LRE, 6LRI, 6LRJ, and
42
43
44 6LRK, respectively) in their crystal structures with h-cGASCD. The RMSD of the
45
46 ligand between the docked conformation and the crystal structure is 1.50 Å, 1.26 Å,
47
48
49 0.48 Å, and 1.30 Å, for 3, 17, 23, and 40, respectively.
50
51
52
53
54
55
56
57 28
58
59
1
2
3
4 3.6 Evaluation of Virtual Screening Hits via the PPiase-coupled Assay. To
5
6
7 measure inhibitory activities of the 59 compounds against h-cGAS, we applied a
8
9
10 pyrophosphatase (PPiase)-coupled assay34 with some modifications. Briefly, cGAS
11
12 links ATP and GTP to generate cGAMP with two inorganic pyrophosphates (PPi)
13
14
15 which are digested by a pyrophosphatase to generate phosphoric acid. The activity
16
17
18 of cGAS is reflected by the amount of phosphoric acid in the reaction system. With
19
20
21
this assay, we first tested the activity of the reported inhibitors PF-06928215 and
22
23 RU.521 against the full-length human cGAS (FL-h-cGAS). PF-06928215 inhibited
24
25
26 the catalytic activity of FL-h-cGAS with an IC50 of 2.08 ± 0.32 μM, which is close to
27
28
29 the previously reported value (2.0 μM) using a mass spectrometry assay (Figure
30
31 S8a).31 RU.521 inhibited the activity of the enzyme with an IC50 of 5.72 ± 0.32 μM
32
33
34 (Figure S8b), which is also consistent with the data reported lately.30 Subsequently,
35
36
37 the inhibitory activities of the 59 compounds obtained from the virtual screening
38
39
40
were tested at a concentration of 50 μM (poor solubility) or 100 μM, and the results
41
42 are shown in Figure 6a and Table S3. Unexpectedly, although compounds 3, 17,
43
44
45 23, and 40 show a positive result in the TSA and their crystal structures with h-
46
47
48 cGASCD were determined, they do not show an apparent inhibition on FL-h-cGAS
49
50 in this enzymatic activity assay. Instead, compound 18 (Figure 6b), with an IC50 of
51
52
53 29.88 ± 3.20 μM, was identified as the best inhibitor among the 59 compounds. We
54
55
56
57 29
58
59
1
2
3
4 tried to solve the crystal structure of h-cGASCD in complex with 18 but failed, mainly
5
6
7 due to the low solubility of the compound.
8
9
10 The inconsistent results from two assays may be ascribed to two factors. First,
11
12 different protein samples of h-cGAS were used in two assays, since h-cGASCD and
13
14
15 FL-h-cGAS were used for the TSA and the PPiase-coupled assay, respectively.
16
17
18 Second, the use of HT-DNA in the PPiase-coupled assay might contribute to this
19
20
21
inconsistency. We tried to apply the FL-h-cGAS for the TSA but resulted in an
22
23 uncharacteristic melting curve of the protein, while the curve of h-cGASCD shown in
24
25
26 Figure 4 suggests that h-cGASCD is more suitable than FL-h-cGAS to detect the
27
28
29 binding of compounds with h-cGAS. However, it has been reported that because of
30
31 the cGAS liquid phase separation the catalytic activity of h-cGASCD is weaker than
32
33
34 that of FL-h-cGAS. To be closer to the natural conditions, FL-h-cGAS is more
35
36
37 suitable for the h-cGAS activity assay.57 Nevertheless, the binding of four positive
38
39
40
hits (compounds 3, 17, 23, and 40) with h-cGASCD has been verified by the crystal
41
42 structure determination. Probably, the binding affinity of these four compounds with
43
44
45 h-cGAS is too weak to exert a significant inhibition on the enzyme’s activity. In
46
47
48 addition, because of the low solubility of compound 18, the highest concentration
49
50 used in the TSA is 200 μM and the resulting ΔTm is 0.9 °C.
51
52
53
54
55
56
57 30
58
59
1
2
3
4
5
6
7
8
9
10
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15
16
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18
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26
27
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29
30
31
32
33 Figure 6. Inhibition of the FL-h-cGAS by 59 compounds measured by the PPiase-
34
35
coupled assay. (a) Inhibition of the FL-h-cGAS by the 59 compounds at a
36
37
38 concentration of 100 μM or 50 μM (for compounds with a low solubility). (b)
39
40
41 Chemical structure and dose-dependent inhibition of compound 18 against the FL-
42
43
44 h-cGAS.
45
46
47
48
49 3.7 Similarity Search of Compound 18. A similarity searching of compound 18
50
51
52 was carried out in order to improve the solubility as well as the potency of the
53
54
55 inhibitor. To this end, we first used the structure of 18 as a shape query to search
56
57 31
58
59
1
2
3
4 new compounds with a similarity score above 0.6, and 1400 compounds from the
5
6
7 ChemDiv database were retained. Given the reliability of our virtual screening
8
9
10 described above, the molecular docking was then applied as the second filtration.
11
12 Afterward, 607 compounds with a docking score above -5.0 were kept while the
13
14
15 docking score of compound 18 was -6.95. These survivors were then imported into
16
17
18 Canvas for clustering analysis using the Leader−Follower method. Finally, 24
19
20
21
compounds passed through a variety of filtering and 21 commercially available ones
22
23 were purchased and tested (Table S4).
24
25
26 We applied the PPiase-coupled assay on these 21 compounds at a concentration
27
28
29 of 62.5 μM (Figure 7a). Two compounds were identified to be more potent than
30
31 compound 18. Compound S2, with a carboxyl group attached to benzene in 18
32
33
34 changed from the ortho to meta position, has IC50 of 13.1 ± 0.09 μM (Figure 7b). In
35
36
37 addition, the IC50 value of compound S3 is 4.9 ± 0.26 μM (Figure 7b), showing a 6-
38
39
40
fold increase in potency compared to compound 18 and a potency similar to that of
41
42 PF-06928215 and RU.521. The similarity scores (Table S5) based on Modified
43
44
45 Tanimoto Distance measure between three novel compounds (compound S2, S3
46
47
48 and 18) identified and two existing ones (PF-06928215 and RU.521) showed that
49
50 there are several varied structural features. These relatively rigid compounds can
51
52
53 be divided into three parts: the aromatic, amide and ring region. In the first part, 2H-
54
55
56
57 32
58
59
1
2
3
4 3,4'-bi(1,2,4-triazole) of three compounds replaced 7-hydroxy-5-
5
6
7 phenylpyrazolo[1,5-a]pyrimidine and 4,5-dichloro-1H-benzo[d]imidazole in PF-
8
9
10 06928215 and RU.521, respectively. Regarding RU.521, the difference is slightly
11
12 larger as its 3-methyl-1H-pyrazol-5-ol group plays the same role as the amide does
13
14
15 in other compounds. Especially for the third part, the aromatic benzoic acid in
16
17
18 compound 18 and S2 is distinguished from the aliphatic carboxylic acid in PF-
19
20
21
06928215. Moreover, 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile of
22
23 compound S3 shows the largest variety in comparison to RU.521 and PF-06928215.
24
25
26 Together, these results demonstrate the high efficiency of the similarity searching
27
28
29 for the discovery of new and more potent inhibitors of h-cGAS.
30
31
32
33
34
35
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37
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40
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57 33
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33
34 Figure 7. Inhibition of the FL-h-cGAS by 21 compounds measured by the PPiase-
35
36
37 coupled assay. (a) Inhibition of the FL-h-cGAS by 21 compounds (at 100 μM)
38
39
40
resulted from the similarity searching of compound 18. (b) Chemical structure and
41
42 dose-dependent inhibition of compounds S2 and S3 against the FL-h-cGAS.
43
44
45
46
47
48 3.8 Binding Modes of Compounds 18, S2 and S3 with h-cGASCD Characterized
49
50
51
by Crystal Structure together with Molecular Docking. After the discovery of
52
53 compounds S2 and S3, we tried again to determine the crystal structures of h-
54
55
56
57 34
58
59
1
2
3
4 cGASCD in complex with these two compounds. We failed with compound S3 but
5
6
7 solved the S2 bound structure of h-cGASCD (PDB code: 6LRL). Figure 8a shows the
8
9
10 electron density of the bi(1,2,4-triazole) region of S2 is visible, while the meta-
11
12 benzoic acid moiety is blurred. On the basis of this crystal structure, molecular
13
14
15 docking of compounds 18, S2 and S3 to h-cGAS were performed again to reveal
16
17
18 the binding mode of these compounds (Figure 8b-e). It should be noted that the
19
20
21
electron density of compound S2 in the crystal structure especially the carboxyl is
22
23 not clear, leading the uncertainty in placement of compound S2 in the crystal
24
25
26 structure. Thus, the RMSD of S2 between the crystal structure and the predicted
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29 docking conformation is relatively large (3.02 Å), but it could be 1.57 Å if the
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31 carboxyl is omitted. The results indicated that three inhibitors with a similar scaffold
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34 formed H-bonds with R376 and N482, a water-bridge H-bond with S380, π-π
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37 interactions with Y436, and cation-π interactions with R376, providing the structural
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basis underlying their potency to h-cGAS (Figure 8). Among these three inhibitors,
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42 compound 18 is less potent than S2 and S3, mainly due to the high possibility of
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45 forming an intra-molecular H-bond between two adjacent groups of the benzene,
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48 the carboxyl and amide groups. Such an intra-molecular H-bond may also account
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50 for the low solubility of 18. As a comparison, compound S2 with a very similar
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53 structure to 18 possesses a better solubility and potency because such an intra-
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57 35
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1
2
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4 molecular H-bond can not be established in S2. Nevertheless, the binding of the
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7 most potent S3 slightly moves towards the interior of the binding pocket. The other
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10 change is the hydrophobic interactions formed between the 4,5,6,7-
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12 tetrahydrobenzo[b]thiophene of S3 and the hydrophobic sub-pocket consisting of
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15 H437, F488, L490, and L495 (Figure 8c). These findings also verify the key
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18 contribution of the nonpolar interactions to the cGAS-inhibitor binding predicted by
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MD simulations.
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48 Figure 8. The binding modes of three inhibitors (compounds 18, S2 and S3) with h-
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50 cGAS revealed by a crystal structure together with molecular docking. (a) The 2Fo–
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53 Fc electron density map of the bound S2 contoured at 1.6 σ level (PDB code: 6LRL).
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57 36
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4 (b-d) Superimposition of docked conformations of three inhibitors, compounds 18
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7 (c), S2 (d) and S3 (e).
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9
10
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13 4. CONCLUSION
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15 Based on the high-resolution crystal structure (1.8 Å, PDB code: 6LRC), the
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18 binding model and the key residues of h-cGAS interacting with PF-06928215 were
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21 studied using MD simulations. On this basis, virtual screening was carried out to
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23 discover novel scaffolds of h-cGAS inhibitors, and 59 compounds were purchased.
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26 Then we performed the TSA on these compounds and found 4 hits that were further
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29 evaluated via the crystal structure determination. The bound poses of four hits in
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32
the crystal structures are highly consistent with the docking poses. Subsequently,
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34 a PPiase-coupled assay was used to evaluate the inhibitory activities of the 59
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37 compounds, and compound 18 was found to have a good inhibitory activity. After
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40 that, the similarity search on compound 18 brought compounds S2 and S3 with
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42 improved inhibitory activities. Compound S3 shows the highest potency among the
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45 compounds resulted from the present study (IC50: 4.9 ± 0.26 μM). Finally, we
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48 determined the crystal structure of compound S2 bound to h-cGASCD and explored
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the binding model of this series of compounds by molecular docking.
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57 37
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4 To sum up, although the open and polar property of the catalytic pocket of h-
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7 cGAS poses a great challenge for virtual screening, several novel-scaffold hits of
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10 h-cGAS inhibitors were successfully obtained through virtual screening. Meanwhile,
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12 the crystal structures of h-cGASCD bound with these ligands indicate various binding
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15 models of inhibitors with cGAS, providing important hints for further development of
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18 cGAS inhibitors. In addition, our molecular docking and experimental data support
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each other, presenting a good example to the discovery of novel ligands of a target
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23 by such a cross-validation approach.
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29
30
31 ASSOCIATED CONTENT
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33
Supporting Information
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35
36 The Supporting Information is available free of charge on the ACS Publications
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39 website at DOI:
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42
Structures of the compounds, experimental assays, molecular docking, and
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44 MD simulations results.
45
46
47 AUTHOR INFORMATION
48
49
50 Corresponding Authors
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52
*E-mail: ycxu@simm.ac.cn (Y. X).
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57 38
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4 *E-mail: hongbinsun@cpu.edu.cn (H. S).
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6
7
8 ORCID
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10
Yechun Xu: 0000-0002-1581-6155
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12
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14 Hongbin Sun: 0000-0002-3452-7674
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16
17
18 Author Contributions
19
20 #These authors contributed equally to this work.
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22
23 Notes
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25
26 The authors declare no competing financial interest.
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29
ACKNOWLEDGMENTS
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31 We acknowledge the financial support by the National Key R&D Program of China
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34 (No. 2017YFB0202604) and the National Natural Science Foundation of China
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37 (21877122).
38
39 ABBREVIATIONS
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41
42 h-cGAS, human Cyclic GMP-AMP synthase; h-cGASCD, the catalytic domain of
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44
45 human cGAS; HEPES, 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid;
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47
MD, molecular dynamics; MM/PBSA, Molecular Mechanics Poisson-Boltzmann
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50 Surface Area.
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52
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18 (56) Hall, J.; Ralph, E. C.; Shanker, S.; Wang, H.; Byrnes, L. J.; Horst, R.; Wong, J.;
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Brault, A.; Dumlao, D.; Smith, J. F.; Dakin, L. A.; Schmitt, D. C.; Trujillo, J.; Vincent, F.;
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23 Griffor, M.; Aulabaugh, A. E. The Catalytic Mechanism of Cyclic GMP-AMP Synthase
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26 (CGAS) and Implications for Innate Immunity and Inhibition: Catalytic Mechanism of
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29 CGAS. Protein Sci. 2017, 26 (12), 2367–2380.
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31 (57) Du, M.; Chen, Z. J. DNA-Induced Liquid Phase Condensation of CGAS Activates
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6 For Table of Contents Use Only
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Inhibitors of Human Cyclic GMP-AMP
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Synthase: A Cross-validation Study of Molecular
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17 Docking and Experimental Testing
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20 Wenfeng Zhao†,‡,§,#, Muya Xiong‡,§,#, Xiaojing Yuan‡,§, Minjun Li⊥, Hongbing Sun*,†,
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Yechun Xu*,‡,§
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24 †Jiangsu
25 Key Laboratory of Drug Discovery for Metabolic Disease and State
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27 Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing
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30 210009, P.R. China
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‡CAS Key Laboratory of Receptor Research, Drug Discovery and Design
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Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
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39 Shanghai 201203, China
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§University of Chinese Academy of Sciences, Beijing 100049, China
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46 ⊥Shanghai
47 Synchrotron Radiation Facility, Shanghai Advanced Research
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49 Institute, Chinese Academy of Sciences, Shanghai 201210, China
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3 *Corresponding Author.
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24 Figure 1. Chemical structures of reported cGAS inhibitors.
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36 Figure 2. Crystal structures of h-cGASCD in complex with PF-06928215. (a) The 2Fo–Fc electron density
37 map of the bound PF-06928215 contoured at 1.6 σ level. (b) The structure of PF-06928215 in the high-
38 resolution crystal structure (PDB code: 6LRC, yellow) superimposed to that in 6NAO (green). (c)
39 Intermolecular contacts among PF-06928215, water molecules and amino acids lining the binding pocket of
h-cGASCD.
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19 Figure 3. Energy decomposition calculations on the h-cGASCD-PF-06928215 complex. (a) The contributions
20 of van der Waals and electrostatic interactions of residues (171-510) to the binding of PF-06928215 with h-
21 cGASCD. (b) Contributions of these residues positioned less than 5 Å of PF-06928215.
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18 Figure 4. The melting temperature of h-cGASCD with or without compounds measured by the thermal shift
19 assay. (a and b) The melting curve of monomer (gray) and dimer (blue) of h-cGASCD. (c and d) The melting
20 curve of the dimeric h-cGASCD in the absence (blue) and presence of PF-06928215(orange).
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38 Figure 5. Superposition of the docking conformation (gray) and bound conformation of (a) compounds 3, (b)
39 17, (c) 23 and, (d) 40 (PDB code: 6LRE, 6LRI, 6LRJ, and 6LRK, respectively) in their crystal structures with
40 h-cGASCD.
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31 Figure 6. Inhibition of the FL-h-cGAS by 59 compounds measured by the PPiase-coupled assay. (a)
32 Inhibition of the FL-h-cGAS by the 59 compounds at a concentration of 100 μM or 50 μM (for compounds
with a low solubility). (b) Chemical structure and dose-dependent inhibition of compound 18 against the FL-
33 h-cGAS.
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35 Figure 7. Inhibition of the FL-h-cGAS by 21 compounds measured by the PPiase-coupled assay. (a)
36 Inhibition of the FL-h-cGAS by 21 compounds (at 100 μM) resulted from the similarity searching of
37 compound 18. (b) Chemical structure and dose-dependent inhibition of compounds S2 and S3 against the
38 FL-h-cGAS.
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1045x927mm (96 x 96 DPI)
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28 Figure 8. The binding modes of three inhibitors (compounds 18, S2 and S3) with h-cGAS revealed by a
29 crystal structure together with molecular docking. (a) The 2Fo–Fc electron density map of the bound S2
30 contoured at 1.6 σ level (PDB code: 6LRL). (b-d) Superimposition of docked conformations of three
31 inhibitors, compounds 18 (c), S2 (d) and S3 (e).
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