Highly Selective Inhibition of IMPDH2 Provides the Basis Of

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Highly selective inhibition of IMPDH2 provides the PNAS PLUS basis of antineuroinflammation therapy

Li-Xi Liaoa,1, Xiao-Min Songa,1, Li-Chao Wanga,b,1, Hai-Ning Lva, Jin-Feng Chena, Dan Liuc,GeFua, Ming-Bo Zhaoa, Yong Jianga, Ke-Wu Zenga,2, and Peng-Fei Tua,2

aState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China; bState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; and cProteomics Laboratory, Medical and Healthy Analytical Center, Peking University Health Science Center, Beijing 100191, China

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved June 7, 2017 (received for review April 28, 2017) Inosine monophosphate dehydrogenase (IMPDH) of human is an of IMPDH2. Interestingly, SA selectively targets and inactivates attractive target for immunosuppressive agents. Currently, small- IMPDH2 but not IMPDH1. The selectivity is explained by differ- molecule inhibitors do not show good selectivity for different IMPDH ential substituent groups of amino acids in two IMPDH isoforms. isoforms (IMPDH1 and IMPDH2), resulting in some adverse effects, The thiol in cysteine 140 of IMPDH2 can lead to irreversible co- which limit their use. Herein, we used a small-molecule probe valent binding via the Michael addition to the α,β-unsaturated car- specifically targeting IMPDH2 and identified Cysteine residue 140 bonyl in SA. However, the corresponding amino acid in IMPDH1 is (Cys140) as a selective druggable site. On covalently binding to serine with a weaker nucleophilic group hydroxyl, resulting in a Cys140, the probe exerts an allosteric regulation to block the catalytic weakened covalent binding effect of SA. The selective modification pocket of IMPDH2 and further induces IMPDH2 inactivation, leading of SA on IMPDH2 caused an allosteric effect on its catalytic domain to an effective suppression of neuroinflammatory responses. How- to narrow the substrate combination space in the catalytic pocket, ever, the probe does not covalently bind to IMPDH1. Taken together, which led to a suppression of IMPDH2 activity and IMPDH2- our study shows Cys140 as a druggable site for selectively inhibiting dependent neuroinflammatory response without obvious hemato- IMPDH2, which provides great potential for development of therapy logical side effects. agents for autoimmune and neuroinflammatory diseases with less These findings indicate that cysteine 140 is a druggable binding

unfavorable tolerability profile. site for selectively targeting IMPDH2. Small molecules binding PHARMACOLOGY to cysteine 140 of IMPDH2 can exert an effective antineuroin- IMP dehydrogenase-2 | druggable site | covalent binding | allosteric flammation therapy in clinical trials with fewer side effects. regulation | immunosuppression Results nosine monophosphate dehydrogenase (IMPDH) is a major IMPDH2 Is Selectively Targeted by SA. First, we found that sap- Irate-limiting enzyme involved in guanosine and deoxyguanosine panone A (SA) was a potent inhibitor of microglial activation. biosynthesis and widely expressed in immunocytes (1). There exist As shown in SI Appendix,Fig.S1A and B,SAsignificantly α two IMPDH isoforms (IMPDH1 and IMPDH2), which are encoded suppressed the releases of NO, TNF- ,IL-6,andPGE2 and by distinct genes (2, 3). Many inflammation-relevant diseases decreased the gene expressions of TNF-α,IL-6,IL-1β, MCP-1, have been specially characterized by the high expression of isoform iNOS, and COX-2 in BV-2 cells. Similarly, SA significantly II of IMPDH (IMPDH2) in rapidly proliferating immunocytes, suppressed the production of NO, TNF-α, and IL-6 in primary rather than the “housekeeping” type I isoform (IMPDH1) in nor- microglia (SI Appendix,Fig.S2). mal human leukocytes and lymphocytes (4, 5). Therefore, selective targeting of IMPDH2 with small molecules is an attractive topic for Significance development of antiinflammation agents with low side effects. Both IMPDH isoforms contain two major domains: the cata- Inosine monophosphate dehydrogenase (IMPDH) is an attractive lytic domain for substrate interaction and the Bateman domain, target for immunosuppressive agents. Currently, small-molecule which is not required for catalytic activity but exerts an important inhibitors do not show good selectivity for different IMPDH iso- allosteric regulation effect on IMPDH activity by communicating forms (IMPDH1 and IMPDH2), resulting in some adverse effects, with the catalytic domain (6, 7). By influencing catalytic domain which limit their use. Here, we identified Cys140 as an isoform- activity, the Bateman domain can regulate IMPDH function and selective druggable binding site for IMPDH2 inhibition but not for further blocks the downstream-of-inflammation signaling path- IMPDH1. We found small-molecule sappanone A directly co- ways (8, 9). Currently, IMPDH inhibitors are divided into two valently targets Cys140 in IMPDH2 to block its activity, resulting in major categories. One kind of inhibitor, including 6-chloropurine neuroinflammatory inhibition with less side effects than pan- riboside ribavirin and mizoribine, targets the binding pocket of IMPDH inhibitor. In summary, our findings reveal Cys140 is a the natural substrate, inosine monophosphate (IMP). Another kind druggable binding site for selectively inhibiting IMPDH2 for neu- of inhibitor (e.g., mycophenolic acid and thiazole-4-carboxamide roinflammatory diseases with less unfavorable tolerability profile. + adenine dinucleotide) targets the site of the cofactor, NAD / NADH, which usually leads to low selectivity or even side effects in Author contributions: K.-W.Z. and P.-F.T. designed research; L.-X.L., X.-M.S., L.-C.W., H.-N.L., J.-F.C., and K.-W.Z. performed research; H.-N.L., J.-F.C., D.L., G.F., M.-B.Z., and clinical trials, such as diarrhea and leukopenia (10, 11). More- Y.J. contributed new reagents/analytic tools; L.-X.L., X.-M.S., L.-C.W., J.-F.C., D.L., G.F., over, a third ligand has been speculated to bind to a possible site and K.-W.Z. analyzed data; and X.-M.S., L.-C.W., K.-W.Z.., and P.-F.T. wrote the paper. + far from the IMP and NAD pockets as an allosteric inhibitor. The authors declare no conflict of interest. However, an allosteric site for designing selective IMPDH2 inhibitors This article is a PNAS Direct Submission. has been largely unexplored. Freely available online through the PNAS open access option. Natural small molecules remain promising drug sources (12, 13). 1L.-X.L., X.-M.S., and L.-C.W. contributed equally to this work. In the present study, we report that a natural small-molecule probe, 2To whom correspondence may be addressed. Email: [email protected] or pengfeitu@ sappanone A (SA, Fig.1A), demonstrated significant inhibitory bjmu.edu.cn. effects on neuroinflammation by directly targeting the conserved This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cysteine residue 140 (Cys140) in the noncatalytic Bateman domain 1073/pnas.1706778114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1706778114 PNAS Early Edition | 1of9 Downloaded by guest on September 27, 2021 Fig. 1. IMPDH2 is selectively targeted by SA. (A) The chemical structures of SA, Cy3-SA, and biotin-SA analogs. (B) Identification of SA target proteins using pull-down technology coupled with stable isotope labeling with amino acids in cell culture (SILAC). (C) Identification of SA target proteins using pull-down technology coupled with shotgun proteomics. The BV-2 lysates were incubated with SA beads or control beads, and then the proteins bound to the beads were resolved by SDS/PAGE, followed by silver staining. (D) SA selectively binds to IMPDH2. SA beads were incubated with BV-2 lysate or the recombinant IMPDH2 (or IMPDH1) protein in the absence or presence of SA for the competitive binding, and then the proteins bound to the beads were detected by Western blot and silver staining. (E) SPR analysis of SA binding to IMPDH2 or IMPDH1. (F) Fluorescence labeling assay of IMPDH2. Recombinant IMPDH2 protein was incubated with Cy3-SA in the absence or presence of SA for competitive binding, and then the products were resolved by SDS/PAGE for detecting Cy3-SA–labeled IMPDH2 protein. (G) Cellular thermal shift assay (CETSA) using BV-2 lysate or intact cells, which were exposed to SA (20 μM). (H)SA promotes target protein IMPDH2 resistant to proteases (DARTS). The values represent the means ± SD of triplicates in an independent experiment.

To explore the pharmacological target of SA, we prepared that IMPDH2 might be as a key target protein (Fig. 1B, Top Right). chemical probes for affinity purification and fluorescent labeling. Next, we performed pull-down assay coupled with shotgun pro- As shown in Fig. 1A, a biotin-tagged SA probe (Biotin-SA) was teomics to further confirm our findings. As presented in Fig. 1C, used to pull down the cellular target of SA, and a Cy3-tagged SA one obvious protein band could be observed between 50 and probe (Cy3-SA) was used for fluorescent labeling of the target 60 kDa in the pull-down group with SA beads; however, a much protein. Both probes retained the ability to inhibit NO release, weaker protein band was found in the group with an excess amount suggesting that the chemical modification did not influence the of SA for competition. LC-MS/MS analysis showed the protein was biological activity of SA (SI Appendix,Fig.S3). Pull-down assay IMPDH2 (56 kDa, SI Appendix,Fig.S4). Western blot also verified coupled with stable isotope labeling with amino acids in cell cul- this protein with a specific anti-IMPDH2 antibody (Fig. 1C). Other ture (SILAC), which is a global protein assay, revealed that the protein bands were identified as heat shock protein 90 and β-actin, SILAC ratio (heavy/light) of IMP dehydrogenase-2 (IMPDH2) which were excluded from the alternative targets of SA (SI Ap- was much higher than other proteins. Therefore, we speculated pendix,Fig.S4). We also confirmed IMPDH2 as the target of SA

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Fig. 2. Cysteine 140 is a covalent binding site of IMPDH2. (A) Irreversible binding of SA to IMPDH2. In the cotreatment group, the recombinant IMPDH2 protein was incubated with SA beads in the absence or presence of SA for competitive binding for 12 h. In the posttreatment group, the recombinant IMPDH2 protein was preincubated with SA beads for 12 h and then further incubated with or without SA for 12 h for competitive binding. (B) Covalent binding of SA to IMPDH2. The recombinant IMPDH2 protein was preincubated with DTT (1 mM) and β-mercaptoethanol (BME, 1 mM) for 2 h (Δ, thermal denaturation) and then further incubated with SA beads at 4 °C for 12 h. (C) LC-MS/MS analysis of the recombinant IMPDH2 protein incubated with (Bottom)orwithout(Top)SAfor6h.C* represents the Cys bound by SA. (D) SA interacts with Cys140 of IMPDH2. Recombinant WT IMPDH2 protein and its mutants were incubated with SA beads at 4 °C for 12 h, and the proteins bound to SA beads were detected by Western blot. (E) Fluorescence labeling Cys140 of IMPDH2 by SA. Recombinant WT IMPDH2 protein and its mutants were incubated with Cy3-SA (20 μM) at 4 °C for 12 h, and the products were resolved by SDS/PAGE for detecting Cy3-SA–labeled IMPDH2 proteins. (F) Cys140 is responsible for SA-mediated IMPDH2 inhibition. Recombinant IMPDH2 proteins (WT or C140A mutant) and IMPDH1 protein (WT) were used as kinases, and IMP was used as the substrate. The mixtures were incubated with different concentrations of SA for 3 h at 37 °C for IMPDH activity assay. (G) A sequence comparison analysis of IMPDH2 from different species and different isoforms was performed.

in primary cultured microglial lysates and the tissue lysate of LPS- Small-molecule inhibitors can perturb protein function and in- injected mouse brains (SI Appendix, Figs. S5 and S6). crease the protein stability via forming a ligand–protein complex To verify the interaction of SA with IMPDH2, we incubated (14). Thus, we attempted to investigate whether SA could bind to BV-2 cell lysates or recombinant IMPDH2 protein with SA IMPDH2 protein and increase its stability in intact cells or lysate beads in the absence or the presence of an excess amount of SA using two target engagement assays (15, 16). From cellular thermal for competitive binding. As shown in Fig. 1D, IMPDH2 was shift assay (CETSA), we found that SA treatment efficiently pro- obviously pulled down by SA beads, which were detected by tected IMPDH2 protein from temperature-dependent degradation Western blot and silver staining. Moreover, an excess amount of (Fig. 1G). Second, DARTS assay was used to monitor target en- SA effectively blocked the binding of IMPDH2 to SA beads. gagement based on SA-induced stabilization of IMPDH2 protein. Meanwhile, we did not detect obvious binding of SA with the Our data also demonstrated a concentration-dependent reduced type I isoform (IMPDH1) (Fig. 1D). SPR analysis revealed that proteolysis with the incubation of SA (Fig. 1H). the target affinity [KD (equilibrium dissociation constant) value] of SA binding to IMPDH2 was 3.944 nM, almost 10 times lower Cysteine 140 Is a Covalent Binding Site of IMPDH2. To investigate than the KD of SA binding to IMPDH1 (29.44 nM, Fig. 1E). the nature of SA interaction with IMPDH2, we first tested Fluorescent labeling assay also showed a specific fluorescent whether SA could covalently bind to IMPDH2. Western blot band around 56 kDa for Cy3-SA–labeled IMPDH2 protein revealed that IMPDH2 was pulled down by SA beads, which (Fig. 1F). were reversed by adding an excess amount of SA. However,

Liao et al. PNAS Early Edition | 3of9 Downloaded by guest on September 27, 2021 when IMPDH2 was preincubated with SA beads, posttreatment S9). Then, we mutated each cysteine residue of IMPDH2 into of an excess amount of SA could not prevent IMPDH2 binding alanine. Pull-down assay with recombinant cysteine-mutated to SA beads (Fig. 2A), indicating a covalent bond formation IMPDH2 proteins further supported that SA covalently mod- between SA and IMPDH2 protein. Because SA contains an ifies Cys140 but not the other cysteines of IMPDH2 (Fig. 2D). α,β-unsaturated carbonyl group, which has a potential to react Additionally, these observations were supported by fluorescent covalently with the thiols of cysteine on IMPDH2 (17), IMPDH2 labeling experiments, which showed that only Cys140-mutated was incubated with SA beads in the absence or the presence of IMPDH2 (C140A) could not be labeled by Cy3-SA (Fig. 2E). β-mercaptoethanol (BME)/DTT for competitive binding to IMPDH2 We further investigated whether Cys140 mutation could impact via thiols (18). As shown in Fig. 2B, BME or DTT completely the inhibitory effect of SA on IMPDH2. As shown in Fig. 2F, abolished IMPDH2 binding to SA beads, suggesting that SA SA significantly inhibited WT IMPDH2 activity, which was might covalently bind to the thiols of cysteine. Additionally, markedly suppressed in Cys140-mutated IMPDH2. Meanwhile, the SA-glutathione (GSH, a thiol donor) complex formed via SA did not show obvious inhibitory effect in WT IMPDH1 protein Michael addition was also accurately confirmed using LC-pMRM activity. A sequence comparison in Fig. 2G suggests that the analysis (SI Appendix,Fig.S7). Cys140 residue in IMPDH2 is conserved among various species. Next, we used BLAST analysis with full-length IMPDH2 protein Interestingly, IMPDH1 does not possess a cysteine (Cys140) at the sequence and found eight conserved cysteine residues (SI Appen- corresponding site, but a serine instead (Fig. 2G). Because hy- dix,Fig.S8). To determine which cysteine residue was attacked by droxyl in serine is a weaker nucleophilic group than the thiol in SA, we incubated IMPDH2 protein with or without SA, followed cysteine, this could explain why SA tends to preferably bind to by LC-MS/MS analysis. Tryptic peptides containing cysteine were IMPDH2 over IMPDH1. evaluated, and Fig. 2C presents a peptide with a calculated mass of 1884.86 Da, which is 284.07 Da larger than the Cysteine140 Cysteine 140 Is Targeted via Michael Reaction. To further explore (Cys140)-containing peptide ARHGFCGIPITDTGR, which has a the structure–activity relationship of SA, we synthesized several calculated mass of 1600.79 Da. The mass difference of 284.07 Da SA derivatives (represented by boldface numerals) including: 1 exactly matches the molecular weight of an SA molecule. MS/MS (esterification of phenolic hydroxyl), 2 (partial destruction of the spectrometry of this peptide revealed that a 284.07 Da mass shift α,β-unsaturated carbonyl), and 3 (complete destruction of the occurred starting from the b5 to the b6 fragment ions, in- α,β-unsaturated carbonyl). NO assay demonstrated that SA and 1 dicating that the Cys140 residue was covalently modified by exhibited similar inhibitory effects on NO release; however, 2 SA. This finding was also confirmed by synthetic peptides de- showed a weak inhibitory effect on NO production, and 3 almost lost rived from human IMPDH2 containing Cys140 (PeptideC140, its ability to suppress NO (Fig. 3A). Next, we investigated the binding ARHGFCGIPIT-His) incubated with SA (SI Appendix,Fig. capacities of SA derivatives to IMPDH2. As shown in Fig. 3B,SA

Fig. 3. Cysteine 140 is targeted via Michael reaction. (A) α,β-unsaturated carbonyl is the major pharmacophore of SA for its inhibitory effect on NO production. BV-2 cells were treated with LPS in the absence or presence of different concentrations of SA and its derivatives (1, 2, and 3) for 24 h. (B)The α,β-unsaturated carbonyl is a major site for IMPDH2 binding with SA (pull-down assay). SA beads were incubated with recombinant IMPDH2 proteins in the presence of SA and its derivatives (1, 2, and 3) at 4 °C for 12 h. (C) α,β-unsaturated carbonyl is a major site for IMPDH2 binding to SA (Cy3-SA labeling assay). Recombinant IMPDH2 proteins were incubated with Cy3-SA (20 μM) in the absence or presence of SA and its derivatives (1, 2, and 3) at 4 °C for 12 h, and then the products were resolved by SDS/PAGE for detecting Cy3-SA–labeled IMPDH2 protein (red). (D) α,β-unsaturated carbonyl contributes to the inhibitory effect of SA on IMPDH2 activity. Recombinant IMPDH2 protein was used as kinase, and IMP was used as the substrate. The mixtures were incubated with SA and its derivatives (1, 2, and 3) for 3 h at 37 °C for the IMPDH2 activity assay. (E) The proposed covalent binding mode of SA to IMPDH2.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1706778114 Liao et al. Downloaded by guest on September 27, 2021 beads effectively pulled down IMPDH2 protein, which was markedly the loop region interacts with the IMP-binding pocket via hydrogen PNAS PLUS reversed by an excess amount of SA and 1, but not 2 or 3.Addi- bonds and enhances the conformational stabilization (Fig. 4D). The tionally, a fluorescent labeling experiment showed that Cy3-SA– variations of hydrogen bond length in simulated movement locus labeled IMPDH2 protein bands were significantly decreased by were shown in SI Appendix,Fig.S10. Additionally, the interaction adding an excess amount of SA or 1, but not 2 or 3 (Fig. 3C). To surface of the loop region contains several hydrophobic amino acids, verify the functional significance of the α,β-unsaturated carbonyl tending to interact with the hydrophobic surface of the IMP catalytic in SA, we performed in vitro kinase assay (19, 20). As shown in pocket (SI Appendix,Fig.S11). These observations were also con- Fig. 3D, both SA and 1 markedly inhibited IMPDH2 activity; firmed by pull-down assay using IMP-coupled beads. We found however, 2 and 3 did not demonstrate any inhibitory effects on IMPDH2 protein could bind to IMP-coupled beads and was inhibited IMPDH2 activity. Overall, we demonstrated that SA directly targets by adding an excess amount of SA (Fig. 4E). and inactivates IMPDH2 protein via the Michael addition of thiol in IMPDH2 protein functions as a tetramer by clustering four cysteine to the α,β-unsaturated carbonyl (Fig. 3E). monomers (7, 21). We tried to evaluate the effect of SA on IMPDH2 clustering by observing the colocalization of GFP-tagged Cysteine 140 Is an Allosteric Regulatory Site of IMPDH2. IMPDH2 protein IMPDH2 (green) and mCherry-tagged IMPDH2 (red). As shown in has a two-domain structure: (i) a catalytic domain (amino acid Fig. 4F, the overlap of green and red fluorescence (yellow) was residues 2–92 and 224–492) forming the core of the active obvious in control cells; however, SA treatment markedly suppressed enzyme; and (ii) a regulatory Bateman domain (amino acid the overlap of green and red fluorescence (Fig. 4F). Moreover, residues 93–223) (Fig. 4A) (7, 9). Molecular dynamics simulation nondenaturing gel electrophoresis and cross-linked whole-cell ex- analysis reveals that SA is deeply embedded in the cleft of the tracts also showed that IMPDH2 tetramers were decreased by SA IMPDH2 Bateman domain and further promotes its bending to treatment (Fig. 4G). catalytic domain (“head-lowering” conformation). Additionally, as showninFig.4B, a strong hydrogen-bonding interaction exists be- NF-κB and p38 MAPK Pathways Contribute to IMPDH2-Dependent tween SA and the residues of IMPDH2, including Thr225, Arg224, Neuroinflammation. GTP is a key cellular metabolite of IMPDH2 and Arg226. Such noncovalent interactions can serve as an initial site- (22). Fig. 5A shows that SA markedly reduced GTP level in recognitionstepwhenSAbindstoIMPDH2andhenceraisesthe BV-2 cells by about 50%. We next sought to elucidate whether the probability of the covalent reaction. Upon SA binding to Cys140, a function of IMPDH2 is required for SA to inhibit microglial protein loop region containing 20 amino acids (amino acid residues activation.AsshowninFig.5B, blockage of IMPDH2 gene

322–342) in catalytic domain moves into the substrate IMP-binding expression using a specific IMPDH2 siRNA significantly reversed PHARMACOLOGY pocket (Fig. 4C), leading to the inactivation of IMPDH2. Moreover, SA-mediated inhibition of NO production. Moreover, we found

Fig. 4. Cysteine 140 is an allosteric regulatory site of IMPDH2. (A) A representative global view of the SA-mediated allosteric effect of IMPDH2 protein. IMPDH2 protein alone (head-raising) was shown by the yellow ribbon model, SA-IMPDH2 complex (head-lowering) was shown by the green ribbon model. (B) Hydrogen bonds between SA and the binding pocket in the Bateman domain were identified and indicated by red lines. (C) A representative view of the conformation change of the catalytic pocket in IMPDH2 protein. The catalytic pocket is shown by gray (IMPDH2 alone) or green color (SA-IMPDH2 complex). IMP is indicated by red color. (D) The hydrogen bonds forming between loop region and neighboring amino acid residues in catalytic pocket. (E) SA inhibits IMPDH2 binding to IMP. Recombinant IMPDH2 protein was incubated with IMP-bound beads (IMP beads) in the absence or presence of SA for competitive binding at 4 °C for 12 h. (F) The effect of SA on IMPDH2 clustering was investigated by dual-label fluorescence analysis (bar = 50 μm). The colocalization ratio of IMPDH2 clusters was calculated. (G) The clusters of IMPDH2 tetramer were detected by nondenaturing gel electrophoresis and cross-linked whole-cell extracts. The values represent the means ± SD of triplicates in an independent experiment. **P < 0.01, compared with the control group.

Liao et al. PNAS Early Edition | 5of9 Downloaded by guest on September 27, 2021 Fig. 5. NF-κB and p38 MAPK pathways contribute to IMPDH2-dependent neuroinflammation. (A) Intracellular GTP concentration was detected by HPLC analysis. (B) IMPDH2 is necessary for SA-mediated antiinflammatory activity. IMPDH2 siRNA-transfected BV-2 cells were treated with LPS in the absence or presence of SA for antiinflammation assay. (C) IMPDH2 is involved in SA-mediated NF-κB pathway suppression. BV-2 cells were treated with LPS in the absence or presence of SA for 30 min. Additionally, BV-2 cells transfected with IMPDH2 siRNA were treated with LPS in the absence or presence of SA for 30 min. (D) IMPDH2 is necessary for SA- dependent NF-κB inactivation. BV-2 cells transfected with IMPDH2 siRNA were further transfected with NF-κB and Renilla reporter plasmids for 24 h, and then the cells were treated with LPS in the absence or presence of SA for 12 h and subjected to luciferase assay. (E) IMPDH2 is involved in SA-mediated p38 pathway suppression. BV-2 cells were treated with LPS in the absence or presence of SA for 30 min. Additionally, BV-2 cells transfected with IMPDH2 siRNA were treated with LPSinthe absence or presence of SA for 30 min. (F) SA derivatives show a stronger inhibitory effect on NO production. (G) SA derivatives show a stronger inhibitory effect on IMPDH2 activity. The values represent the means ± SD of triplicates in an independent experiment. *P < 0.05, **P < 0.01, compared with the control group.

similar inhibitory effects of SA on TNF-α and IL-6 release, which JNK or ERK. Moreover, a specific IMPDH2 inhibition via siRNA was abolished in IMPDH2 siRNA-transfected BV-2 cells. Although substantially reversed the SA-dependent down-regulation of the detailed molecular pathway remains unknown, our present ob- p38 phosphorylation (Fig. 5E), demonstrating that p38 MAPK servation implies that NF-κB inflammation pathway activation is inflammatory signal might function as an essential component preferentially susceptible to suppression by SA (SI Appendix,Fig. downstream of IMPDH2. S12 A and B). We found that SA-dependent down-regulation of the Based on rational drug structure optimization, we next synthe- phosphorylation of IKKβ,IκBα,andNF-κB, as well as NF-κB– sized several SA derivatives (4, 5,and6) by inducing an electron- responsive luciferase activity, were markedly reversed when withdrawing group such as -F, -Br, and -NO2 to elevate the activity IMPDH2 expression was knocked down (Fig. 5 C and D). of α,β-unsaturated carbonyl. NO assay demonstrated that 4, 5,and6 Moreover, mitogen-activated protein kinases (MAPKs) also exhibited stronger inhibitory effects on NO release than SA. No- regulate microglial activation (23). As shown in Fig. 5E,SA tably, the IC50 of 5 is around 620 nM (Fig. 5F). Additionally, we significantly inhibited p38 MAPK phosphorylation, but not on detected the effects of different SA derivatives on IMPDH2 activity.

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Fig. 6. SA shows inhibitory effects on neuroinflammation with low side effects in vivo. (A) Schedule of animal treatments. BALB/c mice were orally treated with 0.5% sodium carboxyl methyl cellulose (vehicle), SA (50 or 100 mg per kg body weight), and MPA (50 or 100 mg per kg body weight). One hour later, the inflammatory responses were activated by one i.p. injection of LPS (2 mg/kg). (B) Differential inflammatory protein expressions in the brain were measured 24 h after LPS challenge by Western blot. (C) Representative sections (cortex and CA1 region) prepared from brain tissues were stained with the specific microglia marker Iba-1, CD11b, and CD68. Arrows indicate the activated microglia. (D) Representative sections (cortex and CA1 region) prepared from the brain tissues were stained with Nissl solutions. (E) Analysis of hematological side effects. BALB/c mice were orally treated with SA or MPA (100 mg per kg body weight) for continuous 10 d (n = 10). **P < 0.01, compared with the control group.

As expected, 4, 5,and6 significantly suppressed IMPDH2 activity, found that oral administration of SA effectively inhibited the in- and the IC50 of 5 is around 750 nM (Fig. 5G). flammatory macrophage infiltration in the brains of mice that received LPS (SI Appendix,Fig.S13). Moreover, as shown in Fig. SA Shows Inhibitory Effects on Neuroinflammation with Low Side Effects 6B, several inflammatory-related proteins in the brain tissue, in- in Vivo. We next assessed the in vivo antineuroinflammatory effect cluding iNOS, COX-2, TNF-α,IL-β, and IL-6, were significantly of SA in LPS-stimulated BALB/c mouse model (Fig. 6A). We decreased following the administration of SA or mycophenolic

Liao et al. PNAS Early Edition | 7of9 Downloaded by guest on September 27, 2021 acid (MPA), which is a non-isoform-selective pan-IMPDH in- in the Bateman domain and further contribute to conformational hibitor. Furthermore, immunohistochemical staining showed changes of the catalytic domain. Collectively, these observations that activated microglia (indicated by specific Iba-1, CD11b, suggest a physiologically important role in the regulatory region and CD68 antibodies) in the cortex and hippocampal CA1 re- outside of the catalytic site of IMPDH2. To our knowledge, SA gions were effectively inhibited by SA or MPA (Fig. 6C). represents the first small-molecule allosteric inhibitor that blocks Meanwhile, SA or MPA effectively protected neurons against IMPDH2 by directly targeting Cys140 residue in the regulatory microglia-mediated neuroinflammatory injuries by Nissl’sstaining Bateman domain. assay (Fig. 6D). It is noteworthy that SA did not show signifi- Notably, S. Lee et al. have reported that SA could inhibit in- cant hematological side effects; however, the same dose of MPA flammation response on murine periphery macrophages via caused significant decrease in several hemogram indexes, includ- Nrf2 and NF-κB pathways (31); however, the direct target of SA is ing whole-blood-cell counts, lymphocyte counts, and monocyte largely unexplored. Here, we showed SA directly targeted Cys140 in counts, suggesting that specific IMPDH2 inhibitor SA can inhibit IMPDH2 to block IKKβ kinase activity, leading to an effective neuroinflammatory responses with high drug safety and less side suppression of the NF-κB pathway. Moreover, we revealed that SA effects (Fig. 6E). showed an inhibitory effect on IMPDH2-mediated guanine nucleotide biosynthesis, which was important for DNA or RNA synthesis. Discussion This could cause the blockage of various inflammation-associated Over the past decade, IMPDH has been viewed as an attractive gene expressions. Thus, we speculate that SA might also inhibit the drug target for the chemotherapy for autoimmune disorders, and expression of heme oxygenase (HO)-1, which was revealed in S. Lee IMPDH inhibitors appeared to act as effective immunosup- et al.’s work (31) by direct inhibition of IMPDH2. Interestingly, SA pressive agents in clinical trials (24). Thus, there has been a was also found to inhibit cellular tyrosinase activity via repressing concerted effort to identify small-molecule inhibitors of IMPDH tyrosinase gene expression in mouse B16 melanoma cells (28). This for inflammation-related diseases (5). However, current small- could be explained by SA-mediated IMPDH2 inhibition and re- molecule inhibitors do not show good selectivity for different sultant guanine nucleotide biosynthesis stagnation, which is impor- IMPDH isoforms (IMPDH1, IMPDH2), resulting in some ad- tant for the tyrosinase genetic transcription process. verse effects, which limit their clinical use. In summary, we discovered Cys140 as a covalent allosteric reg- The druggable target identification is extremely important for ulatory site for selective IMPDH2 inhibition. The small molecules seeking therapy drugs (25–27). To explore the direct cell target of targeting Cys140, such as SA, can induce an allosteric effect on SA (28), we designed a small-molecular probe based on SA struc- catalytic pocket and suppress IMPDH2 activity, leading to antiin- ture and found it selectively targets IMPDH2, but not IMPDH1. flammation and immunosuppressive action. Therefore, Cys140 may Notably, IMPDH2 contains a critical cysteine residue (Cys331) in its represent a promising drug-binding site of IMPDH2 inhibitors to catalytic domain that was targeted by several current inhibitors (29, accelerate clinical drug development for neuroinflammation with 30). Interestingly, SA is selective toward Cys140 in regulatory low side effects. Bateman domain but not Cys331 in catalytic domain. Hitherto, Cys140 has not been reported as a druggable site for IMPDH2 Methods inhibition. Notably, SA showed selectivity to IMPDH2 rather than Cell Survival Assay. The cell survival assay was performed using the MTT IMPDH1. We assume that the molecular geometry of SA and the method. The detailed protocol is found in SI Appendix, SI Methods. chemical environment surrounding the binding pocket might be the key factors. The covalent binding site of SA in IMPDH2 is the thiol Identification of SA Target Proteins. Identification of SA target proteins was of Cys140, which supplies a structural specificity for the recognition based on pull-down technology coupled with SILAC and shotgun protemics analysis. The methods are found in SI Appendix, SI Methods. and binding of SA. However, the corresponding site in IMPDH1 is serine, which contains a hydroxyl group and is reactionless to SA. Determination of the SA-Binding Site on IMPDH2. The SA-binding site on Molecular dynamics (MD) stimulation analysis indicated that the IMPDH2 was detected using LC-MS/MS analysis on LTQ-Orbitrap. The detailed covalent binding of SA to Cys140 induced an allosteric effect on protocol is found in SI Appendix, SI Methods. IMPDH2 by promoting the Bateman domain to bend to the catalytic domain. To our surprise, we found that the IMP-binding site in Molecular Dynamics Simulation. The force field parameters for inhibitor co- the catalytic domain was also affected by the SA-induced allosteric valently bonded to Cys140 residue of IMPDH2 protein were generated by the effect. In the SA-IMPDH2 complex, the IMP-binding site was oc- General AMBER Force Field (GAFF) and Restrained Electrostatic Potential (RESP). cupied by the neighboring loop region and caused a dysfunction of The detailed methodologies for MD are provided in SI Appendix, SI Methods. substrate processing as well as IMPDH2 inactivation. We found All other methods, including cell culture, chemical synthesis, target identi- that SA-induced serpentine flow mainly passes from the Bateman fication, enzyme activity, gene or protein expression, animal experiments, data collection, and so forth, are described in detail in SI Appendix, SI Methods. domain to the IMPDH domain and arrives in the catalytic domain. Notably, Ile461, Leu235, Ser237, and Ala236 play important roles in ACKNOWLEDGMENTS. This work was supported by National Key Technology serpentine flow passing (SI Appendix,Fig.S14). Thus, we speculated R & D Program “New Drug Innovation” of China Grant 2012ZX09301002-002- that IMPDH2 activity might be subject to distinct regulation by SA 002 and Natural Science Foundation of China Grants 81303253 and 30873072.

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