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Highly Selective Inhibition of IMPDH2 Provides the Basis Of 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 2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1706778114 Liao et al.
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