Highly selective inhibition of IMPDH2 provides the basis of antineuroinflammation therapy

Abstract

Inosine monophosphate dehydrogenase (IMPDH) of human is an attractive target for immunosuppressive agents. Currently, smallmolecule inhibitors do not show good selectivity for different IMPDH isoforms (IMPDH1 and IMPDH2), resulting in some adverse effects, which limit their use. Herein, we used a small-molecule probe specifically targeting IMPDH2 and identified Cysteine residue 140 (Cys140) as a selective druggable site. On covalently binding to Cys140, the probe exerts an allosteric regulation to block the catalytic pocket of IMPDH2 and further induces IMPDH2 inactivation, leading to an effective suppression of neuroinflammatory responses. However, the probe does not covalently bind to IMPDH1. Taken together, our study shows Cys140 as a druggable site for selectively inhibiting IMPDH2, which provides great potential for development of therapy agents for autoimmune and neuroinflammatory diseases with less unfavorable tolerability profile.nosine monophosphate dehydrogenase (IMPDH) is a major rate-limiting enzyme involved in guanosine and deoxyguanosine biosynthesis and widely expressed in immunocytes (1). There exist two IMPDH isoforms (IMPDH1 and IMPDH2), which are encoded by distinct genes (2, 3). Many inflammation-relevant diseases have been specially characterized by the high expression of isoform II of IMPDH (IMPDH2) in rapidly proliferating immunocytes, rather than the "housekeeping" type I isoform (IMPDH1) in normal human leukocytes and lymphocytes (4, 5). Therefore, selective targeting of IMPDH2 with small molecules is an attractive topic for development of antiinflammation agents with low side effects.Both IMPDH isoforms contain two major domains: the catalytic domain for substrate interaction and the Bateman domain, which is not required for catalytic activity but exerts an important allosteric regulation effect on IMPDH activity by communicating with the catalytic domain (6, 7). By influencing catalytic domain activity, the Bateman domain can regulate IMPDH function and further blocks the downstream-of-inflammation signaling pathways (8, 9). Currently, IMPDH inhibitors are divided into two major categories. One kind of inhibitor, including 6-chloropurine riboside ribavirin and mizoribine, targets the binding pocket of the natural substrate, inosine monophosphate (IMP). Another kind of inhibitor (e.g., mycophenolic acid and thiazole-4-carboxamide adenine dinucleotide) targets the site of the cofactor, NAD + / NADH, which usually leads to low selectivity or even side effects in clinical trials, such as diarrhea and leukopenia (10, 11). Moreover, a third ligand has been speculated to bind to a possible site far from the IMP and NAD + pockets as an allosteric inhibitor. However, an allosteric site for designing selective IMPDH2 inhibitors has been largely unexplored.Natural small molecules remain promising drug sources (12, 13). In the present study, we report that a natural small-molecule probe, sappanone A (SA, Fig.1A), demonstrated significant inhibitory effects on neuroinflammation by directly targetin...

Highly Selective Activation of Heat Shock Protein 70 by Allosteric Regulation Provides an Insight into Efficient Neuroinflammation Inhibition

“…The vehicle and handelin-coupled beads were subsequently mixed with cell lysates and then incubated overnight at 4 °C. The bead-captured proteins were obtained by incubating the mixture and separated to 10% SDS-PAGE, visualized by silver staining and then identified by LC-MS/MS using a nano-HPLC-tandem LTQ-Orbitrap Velos pro mass spectrometer (Thermo Fisher Scientific) as our previous study reported ( Liao et al, 2017 ).…”

“…Then, the GSH-handelin complex was analyzed by liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometer (LC-Q-trap-MS) method. The protocol was according to previous report ( Liao et al, 2017 ) excepting that the mobile phase consisting of water (0.1% formic acid, A) and acetonitrile (B), was delivered at a constant flow rate of 0.4 mL/min with a gradient elution of 5–20% B at 0–1 min, 20–100% B at 1–3 min.…”

“…Recombinant Hsp70 protein was incubated with handelin for overnight at 4 °C, and the mixture was purified using an ultrafiltration tube. For the identification of handelin-binding site on Hsp70, the nano-LC-MS/MS experiments were performed using an EASY-nLC II system and a LTQ-Orbitrap velos pro mass spectrometer according to our previous study ( Liao et al, 2017 ). Mass spectrometric data were analyzed with Proteome Discoverer (1.4) software with the SEQUEST search engine (Thermo Fisher Scientific) using the following criteria: taxonomy, human; enzyme, trypsin; missed cleavage sites, 2; variable modifications, methionine oxidation (+ 15.995 Da), cysteine carbamidomethylation (+ 57.021 Da), cysteine binding with Hsp70 (+ 552.272 Da); precursor mass tolerance as 10 ppm, fragment mass tolerance as 0.6 Da; and the false discovery rate (FDR) at 0.01.…”

Elucidating Direct Kinase Targets of Compound Danshen Dropping Pills Employing Archived Data and Prediction Models

“…Using this strategy, a series of targets for active components of TCM have been successfully identified. For example, Tu research group constructed probe for sumitone [47] and chrysanthema lactone [48], successively identified the target of sumitone a as inosine monophosphate dehydrogenase 2 (IMPDH2), and the target of chrysanthema lactone with anti-inflammatory effect was heat shock protein 70 (HSP70). All of them revealed the mechanism of action for active components in TCM from the origin.…”

Ensemble cryo-EM structures demonstrate human IMPDH2 filament assembly tunes allosteric regulation

“…IMPDH plays a particularly important role in the immune response, where T-cell activation is dependant on increased production of purine nucleotides, and is associated with IMPDH filament assembly (Gu et al 2000; Zimmermann et al 1998; Duong-Ly et al 2018; Calise et al 2018). As a result, IMPDH is the target of several drugs used in immunosuppressive treatment of both autoimmune disease and organ transplant rejection, and is considered a promising target for antineoplastic agents (Shu & Nair 2008; Liao et al 2017; Bergan et al 2016).…”