Comparative Study of Interactions Between Human Cgas and Inhibitors: Insights from Molecular Dynamics and MM/PBSA Studies

International Journal of Molecular Sciences Article Comparative Study of Interactions between Human cGAS and Inhibitors: Insights from Molecular Dynamics and MM/PBSA Studies Xiaowen Wang 1,2 and Wenjin Li 1,* 1 Institute for Advanced Study, Shenzhen University, Shenzhen 518060, China; [email protected] 2 College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China * Correspondence: [email protected]; Tel.: +86-755-2694-2336 Abstract: Recent studies have identified cyclic GMP-AMP synthase (cGAS) as an important target for treating autoimmune diseases, and several inhibitors of human cGAS (hcGAS) and their structures in complexation with hcGAS have been reported. However, the mechanisms via which these inhibitors interact with hcGAS are not completely understood. Here, we aimed to assess the performance of molecular mechanics/Poisson–Boltzmann solvent-accessible surface area (MM/PBSA) in evaluating the binding affinity of various hcGAS inhibitors and to elucidate their detailed interactions with hcGAS from an energetic viewpoint. Using molecular dynamics (MD) simulation and MM/PBSA approaches, the estimated free energies were in good agreement with the experimental ones, with a Pearson’s correlation coefficient and Spearman’s rank coefficient of 0.67 and 0.46, respectively. In per-residue energy decomposition analysis, four residues, K362, R376, Y436, and K439 in hcGAS were found to contribute significantly to the binding with inhibitors via hydrogen bonding, salt π π···π ···π ···π ···π bridges, and various interactions, such as stacking, cation , hydroxyl , and alkyl interactions. In addition, we discussed other key interactions between specific residues and ligands, Citation: Wang, X.; Li, W. in particular, between H363 and JUJ, F379 and 9BY, and H437 and 8ZM. The sandwiched structures Comparative Study of Interactions of the inhibitor bound to the guanidinium group of R376 and the phenyl ring of Y436 were also between Human cGAS and Inhibitors: consistent with the experimental data. The results indicated that MM/PBSA in combination with Insights from Molecular Dynamics other virtual screening methods, could be a reliable approach to discover new hcGAS inhibitors and and MM/PBSA Studies. Int. J. Mol. thus is valuable for potential treatments of cGAS-dependent inflammatory diseases. Sci. 2021, 22, 1164. https:// doi.org/10.3390/ijms22031164 Keywords: cGAS; inhibitor; MM/PBSA; MD simulation; sandwiched structures Academic Editor: A. Phillip West Received: 22 December 2020 Accepted: 21 January 2021 1. Introduction Published: 25 January 2021 Free DNA in the cytosol is detected by a type of nucleotidyltransferase called cyclic GMP-AMP (cGAMP) synthase (cGAS) [1]. Upon binding to DNA, cGAS is activated and Publisher’s Note: MDPI stays neutral produces cGAMP from GTP and ATP. The second messenger, cGAMP, activates and binds with regard to jurisdictional claims in the stimulator of interferon genes, which subsequently induces the secretion of type I published maps and institutional affil- iations. interferons and triggers the downstream innate immune response [2–5]. While recognition of pathogen DNA is essential for host defense against infections, aberrant activation of cGAS may trigger autoimmune diseases such as systemic lupus erythematosus and Aicardi– Goutières syndrome in the presence of self-DNA [6,7], which may be displaced nuclear or mitochondrial DNA generated as by-products of cellular damage. Hence, cGAS is a Copyright: © 2021 by the authors. vital drug target for treating autoimmune diseases and for preventing autoinflammation in Licensee MDPI, Basel, Switzerland. therapeutic strategies against cancers. This article is an open access article Experimental studies have shown that knockout of the cGAS gene in Trex1−/− mice distributed under the terms and conditions of the Creative Commons dramatically reduces tissue inflammation and hinders autoantibody production [8,9]. Thus, Attribution (CC BY) license (https:// inhibition of cGAS activity is a promising therapeutic strategy for autoimmune diseases. creativecommons.org/licenses/by/ Recently, several inhibitors of human (h) and mouse (m) cGAS have been reported [10–13]. 4.0/). All of them bind to the catalytic center of cGAS and suppress its nucleotidyltransferase Int. J. Mol. Sci. 2021, 22, 1164. https://doi.org/10.3390/ijms22031164 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 1164 2 of 16 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 16 activity. RU.521 is a promising inhibitor (IC50 = 0.11 µM) of mcGAS that can regulate thethroughput levels of screening interferon; study however, showed it binds that th toe hcGAS compounds with lowG108 affinity (containing (IC50 =a 2.94pyrazoleµM) ingroup) spite and of approximately G105 (containing 60% a sequence2-amino pyridine identity ring) between were hcGAS novel andspecific mcGAS inhibitors [14]. Aof high-throughputhcGAS, which targeted screening the study identical showed activation that the loop compounds region G108containing (containing R376 aand pyrazole Y436, group)similar andto the G105 binding (containing pathways a 2-amino of 2,3-cG pyridineAMP [12]. ring) While were G150 novel and specific G108 showed inhibitors high of hcGAS,binding whichaffinities targeted in complexes the identical with hcGAS, activation with loop IC region50 values containing of 10.2 nM R376 and and 27.5 Y436, nM, similarrespectively, to the they binding were pathways both inactivated of 2,3-cGAMP during [12 assessment]. While G150 with and mcGAS G108 showed[12]. Another high bindinghigh-affinity affinities inhibitor, in complexes PF–06928215, with which hcGAS, bind withs to IC the50 hcGASvalues active of 10.2 sites, nM was and shown 27.5 nM, to respectively,have a dissociation they were constant both ( inactivatedKd) of 200 nM during (IC50 = assessment 4.9 µM) using with high-throughput mcGAS [12]. Another screen- high-affinitying assays [11]. inhibitor, Based on PF–06928215, the inactive whichPF–06928215 binds toin thecell-based hcGAS cGAS, active three sites, potent was shown com- topounds, have a18, dissociation S2, and S3 constant (IC50 = 29.88 (Kd) ± of 3.20, 200 nM13.1 (IC± 0.09,50 = 4.9andµ 4.9M) ± using 0.26 µM), high-throughput with highly screeningconsistent assaysbinding [11 modes,]. Based were on theidentified inactive by PF–06928215 Zhao and co-workers in cell-based [13]. cGAS, More threeinformation potent compounds,regarding various 18, S2, hcGAS and S3 (ICinhibito50 = 29.88rs as ±therapeutic3.20, 13.1 ±targets0.09, andof interest 4.9 ± 0.26 areµ presentedM), with highly in Ta- consistentble S1. binding modes, were identified by Zhao and co-workers [13]. More information regardingTo complement various hcGAS and validate inhibitors the as results therapeutic obtained targets using of an interest experimental are presented approach, in Tablecomputational S1. modeling, molecular dynamics (MD) simulations, and molecular mechanics/Poisson–BoltzmannTo complement and solvent-accessible validate the results surface obtained area using (MM/PBSA) an experimental can be approach, used for com-ana- putationallyzing the modeling,real-time dynamic molecular behavior dynamics of (MD)biomolecules simulations, and predicting and molecular the binding mechanics/ free Poisson–Boltzmannenergy. These methods solvent-accessible have been widely surface us areaed for (MM/PBSA) elucidating can the be binding used for mechanism analyzing theand real-time interaction dynamic modes behaviorof protein-inhibitor of biomolecules systems and [15–17]. predicting Recently, the binding virtual free screening energy. Thesewas performed methods for have the been first widelytime, combined used for wi elucidatingth MD and the modeling binding methods, mechanism to identify and in- teractionnew hcGAS-binding modes of protein-inhibitor inhibitors [13]. However, systems complete [15–17]. understanding Recently, virtual regarding screening the wasmo- performedlecular mechanism for the first of hcGAS-inhibitor time, combined withinteractions, MD and especially modeling from methods, the viewpoint to identify of new the hcGAS-bindingdynamics and energy inhibitors of the [13 interactions,]. However, completeis still lacking. understanding regarding the molecular mechanismIn this ofstudy, hcGAS-inhibitor we aimed to interactions, assess the especiallyperformance from of the MM/PBSA viewpoint in of evaluating the dynamics the and energy of the interactions, is still lacking. binding affinity of 10 hcGAS inhibitors (listed in Figure 1 and Table S1). In addition, we In this study, we aimed to assess the performance of MM/PBSA in evaluating the aimed to elucidate the binding modes of hcGAS with these inhibitors in combining MD binding affinity of 10 hcGAS inhibitors (listed in Figure1 and Table S1). In addition, we simulations and per-residue energy decomposition analysis and compare these observa- aimed to elucidate the binding modes of hcGAS with these inhibitors in combining MD sim- tions with those obtained using experimental methods. In combination with existing vir- ulations and per-residue energy decomposition analysis and compare these observations tual screening approaches, our study could provide theoretical guidance in the search for with those obtained using experimental methods. In combination with existing virtual new cGAS inhibitors and thus be helpful in the treatment of autoimmune diseases. screening approaches, our study could provide theoretical guidance in the search for new cGAS inhibitors and thus be helpful in

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