Accessing anti-HIV activity through the attenuation of USP18 activity: novel insights from molecular dynamic simulations, free-energy profiling, and multi-cellular inhibition assays

Accessing anti-HIV activity through the attenuation of USP18 activity: novel insights from molecular dynamic simulations, free-energy profiling, and multi-cellular inhibition assays | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Accessing anti-HIV activity through the attenuation of USP18 activity: novel insights from molecular dynamic simulations, free-energy profiling, and multi-cellular inhibition assays View ORCID Profile Lester T Sigauke , Jonathan Bvunzawabaya , Gabrielle Le Bury , Godwin A. Dziwornu , Saikat Boliar , David W Gludish , David G Russell , View ORCID Profile Krishna Govender , Grace Mugumbate , Nyaradzo Chigorimbo-Murefu doi: https://doi.org/10.1101/2024.06.23.600290 Lester T Sigauke 1 Division of Medical Virology, Department of Pathology, University of Cape Town , Rondebosch, South Africa Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lester T Sigauke Jonathan Bvunzawabaya 2 Department of Chemical Sciences, Faculty of Science and Technology, Midlands State University , Gweru, Zimbabwe Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gabrielle Le Bury 3 Microbiology and Immunology, College of Veterinary Medicine, Cornell University , Ithaca, New York, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site Godwin A. Dziwornu 4 Department of Chemistry, University of Cape Town , Rondebosch, South Africa Find this author on Google Scholar Find this author on PubMed Search for this author on this site Saikat Boliar 3 Microbiology and Immunology, College of Veterinary Medicine, Cornell University , Ithaca, New York, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site David W Gludish 3 Microbiology and Immunology, College of Veterinary Medicine, Cornell University , Ithaca, New York, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site David G Russell 3 Microbiology and Immunology, College of Veterinary Medicine, Cornell University , Ithaca, New York, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site Krishna Govender 5 Department of Chemical Sciences, University of Johannesburg, Doornfontein Campus , Johannesburg, South Africa Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Krishna Govender Grace Mugumbate 2 Department of Chemical Sciences, Faculty of Science and Technology, Midlands State University , Gweru, Zimbabwe Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nyaradzo Chigorimbo-Murefu 1 Division of Medical Virology, Department of Pathology, University of Cape Town , Rondebosch, South Africa 6 International Centre for Genetic Engineering and Biotechnology , Cape Town, South Africa Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: nyaradzo.chigorimbotsikiwa{at}icgeb.org Abstract Full Text Info/History Metrics Preview PDF Abstract The feasibility of achieving anti-HIV activity from the attenuation of USP18 activity was explored for the first time. A cheminformatic survey demonstrated that the current known USP18 isopeptidase inhibitors are derivatives of a bis-aryl pyranone scaffold that possesses undesirable toxicity profiles. Molecular modelling approaches applied to these active bis-aryl pyranones isolated the likely mechanism that perturbs the isopeptidase activity of USP18. Molecular dynamic simulations and free-energy profiling showed that induced-fit effects on the catalytic triad and the IBB-1 domain residues of USP18 drive a reversible non-competitive isopeptidase inhibition mechanism. Proof-of-concept multi-cellular HIV inhibition assays demonstrate the utility of achieving anti-HIV-1 activity from attenuating the activity of USP18 using small molecules. This study motivates for the pursuit of scaffolds that target the allosteric site of USP18, fine-tuning the IFN response as a strategy to enhance the natural control mechanisms that lead to an antiviral state potentially curing viral infection. Introduction Although certain interferon stimulated genes (ISG) exist for the express purpose of dampening inducible interferon (IFN) responses as a natural control mechanism to prevent systemic inflammation, HIV-1 also attenuates this IFN signal cascade rendering it insufficient to resolve infection ( 1 – 4 ). The role of IFN signaling in HIV infection is thus complex and interventions that target the IFN response regulating ISGs remain underexplored. It has been speculated that fine-tuning of the IFN induced restriction factors that counter viral replication could potentially provide a pathway to the long-term control of HIV if their upregulation could be achieved in a limited focused way such as is demonstrated by IFNα2 ( 1 ). Achieving this effect through sustained expression of IFN however has thus far not yielded positive results in chronic HIV patients despite the lack of detrimental effects associated with the risk of increased systemic immune ( 5 , 6 ). As interferon therapy studies in chronic Hepatitis B virus have demonstrated distinct antiviral activities from different IFNα subtypes it is thought that perturbing the IFN pathway in HIV patients could lead to a similar antiviral state without the detrimental effects of sustained IFN expression ( 7 ). Computer-aided drug discovery approaches such as molecular dynamics have contributed to the rapid search of small molecule candidates as well as the isolation of likely mechanisms that inhibitors use to perturb known targets ( 8 – 10 ). We aim to perturb the activity of Ubiquitin-specific peptidase 18 (USP18) because it has been identified as a dominant ISG that regulates the IFN response ( 11 ). USP18 is a 43 kDa cysteine isopeptidase that serves a complicated role of reversing the ISGylation of viral proteins as well as a non-catalytic function that regulates the inflammatory response ( 12 – 14 ). The non-catalytic function is due to USP18 interactions with interferon α/β receptor 2 (IFNAR2) which leads to the attenuation of IFN signaling preventing the detrimental impacts that arise from elevated and sustained inflammation ( 15 – 17 ). The catalytic USP18 role is related to a reversal of the post-translational modification of proteins by ISG15 (ISGylation). ISGylation regulates the activity of target proteins as part of the type I IFN mediated response that initiates the ubiquitin-proteasome system to trigger the anti-viral response ( 12 , 18 , 19 ). The catalytic role of USP18 reverses this ISGylation and affects the IFN response by dampening the anti-viral response ( 11 ). The inter-play of the ubiquitin-proteasome system is observed in SARS-CoV, where viral proteins mimic and suppress ubiquitin while other viral proteases have been shown to reverse the ISGylation of viral proteins reducing viral clearance ( 13 , 19 ). Inhibiting the expression of USP18 has been shown to impact the survival of HIV-1 infected CD4 T cells, while an anti-viral IFN effect in liver cells infected with Hepatitis B and C resulted in viral clearance ( 20 – 22 ). This multifaceted function of USP18 as a deISGylating enzyme and a modulator of the IFN response has led us to hypothesize that approaches that selectively target either the catalytic isopeptidase or the non-catalytic activity of USP18 will lead to therapeutic strategies that can be applied in various human diseases such as HIV. Bis-aryl pyranone scaffolds have been shown to have nonselective and unclassified USP18 inhibitory potential in antineoplastic applications ( 23 ). Cersosimo et al (2015) synthesized two series of a previously identified non-selective isopeptidase inhibitor (N-SII) G5 . The first series of the 12 compounds are based on the bis(arylidene)tetrahydrothiapyran-4-one 1,1-dioxide scaffold of G5 with variations in the substituents on the aromatic rings. The second series are eight nitrobenzylidene pyranones acquired from a substitution of the G5 sulfone group by different groups leaving the 4-nitro substituents on the aromatic rings fixed. From these synthesized G5 analogues, only 2c and the G5 were tested in their cell based USP18 assays against the isopeptidase activity. The absence of the accumulation of free cleaved GFP led them to suggest that the observed USP18 inhibition was due to USP18’s reduced isopeptidase activity on the GFP tagged ISG15 chimera substrate. Both 2c and G5 were shown to inhibit the USP18 deISGylation of ISG15 tagged substrates with IC 50 of 21.20 and 33.23 μM, respectively. We report the first attempt at acquiring anti-HIV activity from agonists tuned to dampen the IFN response through the perturbation of specific ISG factors such as USP18. Anti-HIV therapeutics that target ISGs such as USP18 have the potential to serve as a natural control mechanism for inflammation and cell activation by enhancing end stage IFN signalling. To date this is the first study that has attempted to pursue this approach to anti-HIV activity. Materials and methods Synthesis of compounds Compounds were synthesized as previously described and the chemical reagents were procured from Combi-Blocks Inc ( 23 ). All solvents were anhydrous or of analytical grade. Cheminformatics of the pyranone series Absorption, distribution, metabolism, and excretion (ADME) properties of 13 pyranones were profiled using SwissADME server ( 24 , 25 ). The evaluation included parameters such as physicochemical properties, lipophilicity, water-solubility, pharmacokinetics, drug-likeness, and medicinal chemistry properties. The Brain Or IntestinaL EstimateD permeation method (BOILED-Egg) from SwissADME was implemented to graphically show the passive gastrointestinal absorption (GI) and blood-brain barrier penetrant (BBB) properties of the compounds respectively ( 25 ). ADMETlab 2.0 was used to compute toxicity properties such as hERG blockage, Human hepatotoxicity (H-HT), drug induced liver injury (DILI), Ames toxicity, rat oral acute toxicity (ROA) and carcinogenicity of the pyranones ( 26 ). Metabolic transformation products predictions were done using BioTransformer 3.0 ( 27 ). Metalated USP18 model building In the absence of a crystal structure of the human USP18 protein, a human model of the USP18 protein was obtained from the SWISS model database ( 24 ). Model Q9UMW8 which had a 74.4 % sequence similarity to the mouse 5CHV crystal structure model was downloaded for this study. This USP18 homology model structure was submitted to the Metal-Ion Binding Site server to estimate the binding site region for the Zn 2+ ions and the pose of the metal ion to acquire the metalated USP18 ( 28 , 29 ). new Molecular docking The surface of the USP18 model was treated with the SiteMap tool excluding shallow solvent exposed pockets from its search to identify pockets that can be perturbed by small molecules ( 30 ). The SiteMap algorithm ranked the identified sites according to a SiteScore scoring function. The degree of discrimination of each site was determined by setting up grids for virtual screening of all the ligands at the centre of each of the sites. The 13 pyranone small molecule derivatives were constructed using the Marvin Sketch web tool and the ligands were prepared by the LigPrep workflow using the following parameters (hydrogens added and pH of 7.0 and a maximum of 32 stereoisomers for each ligand) ( 31 , 32 ). The docking methodology relied on the use of XP Glide docking followed by a Quantum Polarized ligand docking (QPLD) workflow ( 33 – 35 ). The QPLD docking workflow made use of the Jaguar Accurate method for the calculation of the ligand charges of each pose during the redocking stage. The QPLD output was limited to a maximum of 5 poses for each ligand from a generous RMSD threshold of 2Å to separate the different conformers. The total number of poses for each docked ligand using this threshold to evaluate the number of modes indicated the degree of discrimination of each site for each ligand. The 5.2 million ZINC15 compounds that satisfied the following criteria: MW greater than 325 g/mol and less than 425 g/mol, possessing 3D representations obtained at pH 7.4, anodyne reactivity to eliminate PAINS assay patterns, and purchasable compounds that are charged between +2 and -2. These ligands/compounds were downloaded in tranches from the online server using download scripts ( 36 , 37 ). These ligands were prepared using LigPrep before Glide XP docking with a grid box centered on the favored allosteric pocket of site 2. The ligands were ranked according to their Glide score and the top 200 candidates were selected for filtering based on the ZINC15 vendor databases to identify purchasable compounds. An in-house scoring metric ranked the purchasable compounds based on 29 features acquired from the SwissADME portal describing their solubility, pharmacokinetics, drug-likeness, and medicinal chemistry properties to prioritize compounds for purchasing. Single-trajectory molecular dynamics simulations Prior to all molecular dynamics simulations Induced-Fit Docking (IFD) of the ligands in the metalated receptor with grid boxes centred on the allosteric pocket of site 2 was used to prepare the starting conformations for the systems ( 38 , 39 ). The following steps were carried out during the IFD workflow: Protein Preparation (RMSD 0.18), Glide Docking (SP, 20 poses per ligand, OPLS2005), Determine Residues for Refinement (Distance cut-off of 5.0 Å), PRIME Active Site Optimization (OPLS2005), Scoring and Filtering (filter using Prime Energy, energy window of 30 kcal/mol, keep maximum of 20 poses per ligand), Glide Docking2 (XP, 1 pose per ligand, OPLS2005), and Scoring (Prime Energy). Complexes with the most optimal IFDScore were extracted and used as the starting configuration for the single trajectory simulations for each system. Desmond single trajectory molecular dynamics simulations under constant pressure were performed to interrogate the protein-ligand interactions of the systems in solution ( 40 ). The systems were prepared in an orthorhombic box of buffer size 10 Å, solvated by TIP4P waters, and neutralized by adding sodium and chloride ions when necessary. The OPLS2005 forcefield was used for system preparation and production ( 41 ). Prior to the start of the 100 ns of NPT unrestrained production simulations, 160 ps of a standard NPT relaxation protocol was used to get each system to a temperature of 300 K and an isotropic pressure of 1.013 bar. Multi-trajectory molecular dynamics simulations A multi-trajectory approach to interrogating the free-energy profile of the complexes made use of biased pulling simulations that were sent through to umbrella sampling of coordinates that trace the pulling reaction coordinate ( 42 ). These simulations were performed in GROMACS (version 2018.8) and made use of a CHARMM36 (version July 2021) forcefield for the atom parameters ( 43 , 44 ). Molecular topologies were acquired from single trajectory protein-ligand systems. The ligand topologies were acquired from the automated CHARMM General Force Field program (version 1.0.0) using valence-based bond orders ( 45 – 47 ). Stream files of the CHARMM36 protein topologies and the CGenFF ligand topologies were converted to GROMACS format and merged manually before the system was solvated using TIP4P waters, and neutralized using chloride ions ( 46 ). The systems were minimized and equilibrated before steered/biased NVT production simulations were propagated. The steered pulling simulations were performed on a box that was extended along the z axis, allowing the ligand to be pulled away from the receptor at a rate of 0.01 nm ps -1 with a force of 1000 kJ mol -1 nm -2 until greater than 5 nm separation was achieved. Windows separated by center of mass (COM) distances between the ligand and protein of either 0.2 nm or 0.12 nm defined the configurations that sample the reaction coordinate from the biased unbinding trajectories. For each system about 20 configuration files were selected from these windows as the steps along the bind reaction path. These files/steps were the starting points for the multi-trajectory simulations with equilibration being performed before NVT production simulations of 1 ns at each step. For each system these simulations gave rise to an ensemble of overlapping configurations that traced the reaction path. A WHAM analysis applied over the entire ensemble of configurations for each of the systems gave the potential of mean force (PMF) that models the unbinding path ( 48 ). For each ensemble, these PMF curves gave access to the free-energy of bind (ΔG) of the binding/unbinding path from the extremes of the reaction path traced by the trajectory. The slope of this PMF curve and the COM windows gave the friction plots and compared the dissociation time in an analogous manner to the residence time. Multi-cellular HIV inhibition assays Virus production An HIV-1 infectious molecular clone, pWT/BaL (HXB3/BaL, Catalog 11414), was obtained from an NIH AIDS Reagent Program. The plasmid encoding HIV-1 molecular clone NL43-IRES-mCherry with R5-tropic env (BaL) was generated by replacing the EGFP sequence with that of Gaussia luciferase (gLuc) in the original plasmid pBR43IeG-nef+ R5env (a kind gift from Thorsten R. Mempel, Massachusetts General Hospital, Boston). Replication-competent VSV-G pseudotyped HIV-1 virus was prepared by co-transfecting 293FT cells with the respective HIV-1 molecular clone and VSV-G expression plasmid using Lipofectamine 3000 reagent (Life Technologies). Transfection media was replaced with fresh antibiotic free DMEM media 8 h post-transfection. After 72 h, cell culture supernatant containing HIV-1 virus was harvested, centrifuged to remove cell debris, passed through 0.45-µm filter, and stored at −80 °C in aliquots. Multiplicity of infection 0.05 of virus was used to infect human MDMs. Human monocyte derived macrophage differentiation Human monocytes were obtained from peripheral blood mononuclear cells of healthy individuals by counter current centrifugal elutriation with an average purity of >97% (Elutriation Core Facility, University of Nebraska Medical Center). Monocytes were maintained in DMEM media supplemented with 10% human serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen). Cells were cultured at 37 °C with 6% CO2 for 7 – 8 days to fully differentiate into macrophages (MDMs). Results and discussion The goal of this study was to demonstrate whether the anti-USP18 inhibition strategy could fine-tune the IFN response by enhancing natural control mechanisms that lead to viral clearance. Illuminating the structural dynamics of USP18 inhibition by the known actives will assist in the development of new anti-HIV therapeutics with greater potency. Presenting the USP18 active bis-aryl pyranones 2c and G5 Although the activity of compounds 2c and G5 are known the mechanisms by which they perturb USP18 have not been determined. In the absence of a crystal structure of the human USP18 protein in complex with an active USP18 inhibitor it was pertinent to first predict the likely binding site of the active anti-USP18 pyranones G5 and 2c . This would enable elucidation of the mechanism employed by these antagonists to inhibit USP18. Molecular modelling approaches coupled with an interrogation of the free-energy profile of binding at this site were used to guide the selection of this site and propose a mechanism based on the known activities of these pyranones. Isolating the binding site of the USP18 active bis-aryl pyranones using docking The SiteMap workflow identified and ranked according to their SiteScore 5 pockets on USP18 that are accessible to small molecule inhibitors ( Fig 1 ). Download figure Open in new tab Fig 1: Docking on USP18. A. Ranked docking sites on USP18 identified by SiteMap. B. Ribbon structure of USP18 with map of small molecule docking sites (pseudo atoms are blue spheres). Small molecules shown are docked in site 2. C. Binding modes from poses of QPLD docking that satisfy the RMSD threshold for the 13 pyranone ligands on each of the 5 USP18 sites. Of the 5 sites identified by SiteMap, site 2 (1.029) was the pocket most optimal for small molecule access ( Fig 1 A ). Grids for QPLD of the 13 proposed pyranone ligands were placed at the centre of these 5 sites identified by SiteMap. The centre of site 2 overlapped with the centroid of residues Ala299 , Asp124 , Gln122 and Tyr348 of the human USP18 sequence from the Q9UMW8 homology model. The binding modes from QPLD of the 13 pyranone ligands showed that site 2 (QPLD 2) was the most discriminatory site. This site had discriminatory potential expressed as achieving less than 5 poses, the maximum allowed number of poses, for 9 ligands while site 1 discriminated for 6 of the 13 ligands ( Fig 1 C ). For the known USP18 actives site 2 biased 2c to 1 binding mode and 3 modes for G5. Site 1 however had 5 or more modes for ligand 2c and G5 . The SiteMap rank and its ability to restrict the binding modes affirms the suspicion that site 2 is a binding pocket able to bias active ligands into active modes and is thus the binding pocket of these 2 active inhibitors. Structural studies such as saturation transfer difference NMR (STD-NMR) of the ligands in solution with USP18 would confirm these observations in the absence of solved crystal structures of the protein-ligand complexes. Using the free-energy profiles of the USP18 active bis-aryl pyranones to guide site selection To understand how binding at site 2 impacts the functioning of USP18, complexes of USP18 docked with the two active pyranone ligands at this site were studied using molecular dynamics simulations. Based on the known activity of 2c on USP18 compared to the activity of G5 , there was a need to find a plausible explanation for the slightly improved activity of 2c over G5 . IFD of these 2 ligands at site 2 gave access to starting structures for Desmond simulations and a stabilized conformer from these simulations were used for multi-trajectory umbrella simulations using GROMACS to evaluate the free-energy profiles of binding/unbinding. The ligand interaction networks from the Desmond simulations showed that throughout the 100 ns of production simulation G5 directly made use of 7 intermediary water molecules in its binding at site 2 whereas ligand 2c recruited only a single water molecule in its interaction ( Fig 2 A, B ). The reliance on fewer water molecules for the 2c interaction reflected on its stable ligand conformation maintained during the simulation compared to that of G5 ( Fig 2 C, D ). This observation is consistent with the QPLD binding mode observation that showed that 2c bound at site 2 had only one binding mode whereas G5 had 3. Download figure Open in new tab Fig 2: Ligand interaction network of ligand 2c (A) and ligand G5 (B) on site 2 during 100 ns of NPT Desmond simulations. RMSD plots of the ligand and the protein backbone and protein side chains of the USP18 complexes with ligand 2c (C) and ligand G5 (D) bound to site 2 during the 100ns of NPT Desmond simulations. Interrogations of the free-energy profiles of the ligand complexes highlight the differences in the stabilities of the interactions of the 2 known USP18 actives at site 2 ( Fig 4 ). Ligand 2c (red line) dissociates from the active site later than ligand G5 (blue) line ( Fig 3 A, B ). This difference in the dissociation is reflected by the PMF curves which estimated the free-energy of the binding reaction. Ligand 2c (-10.55 kcal mol -1 ) has a slightly larger free-energy of binding than ligand G5 (-10.08 kcal mol -1 ) ( Fig 3 C, D ). When taken together, the recruitment of intermediary waters from the ligand interaction network, the stability of the binding conformation from the RMSD, the dissociation time and the free-energy of binding, confirm that site 2 binding of 2c and G5 is responsible for the anti-USP18 activity observed by Cersosimo et al., 2015. Download figure Open in new tab Fig 3: Free-energy profile plots. A., B. COM positions of trajectories from steered dynamics simulations of the ligand pulled away from USP18. Location of the asymmetric coordinate files that trace the ligand pulling trajectory for umbrella sampling of the dissociation path. Blue dash line shows the point of dissociation for ligand G5, Red shows the point of dissociation for ligand 2c. Arrows show the shift from the competing ligand. C., D. Potential of mean force (PMF) plots obtained from the WHAM analysis across the ensemble of configurations that trace the pulling trajectory of the dissociation path for ligand 2c and G5. ΔG d 2c = -2.50 + (-7.55) = -10.55 kcal mol -1 ; ΔG d G5 = -3.38 + (- 6.70) = -10.08 kcal mol -1 ; ΔG d 2c >= ΔG d G5 . Download figure Open in new tab Download figure Open in new tab Download figure Open in new tab Fig 4: Cheminformatic analysis of pyranone derivatives. A. G5 analogues proposed by Cersosimo et al., 2015 used in this study. Series 1 analogues are based on variations in the substituents on the positions of the nitro group (red) of G5. Series 2 are based on the substitution of the sulfone group (blue) of G5. B. BOILED-egg model of the bis-aryl pyranones from the SwissADME server. The white region highlights compounds with high probability of GI absorption and the yellow highlights those with a high probability of BBB penetration. PGP-compounds are non substrates for P-gp. Compounds outside the grey region are not likely to be absorbed by GI and are not BBB permeant. C. PAINS and Brenk substructural alerts on 2c and G5. The ene_one_ene_A group (orange) is the only PAINS alert while the Brenk alerts present are the Michael acceptor 1(red), the oxygen_nitrogen single bond (green) and the nitro_group (blue). Presentation of the bis-aryl pyranone scaffold and its potential for pharmaceutical applications It was pertinent to determine whether the bis-aryl pyranone scaffold was ideal for pharmaceutical applications. QSAR and machine learning derived models were used to predict the pharmacokinetic and toxicity profiles of the 12 Cersosimo derivatives of bis(arylidene)tetrahydrothiapyran-4-one 1,1-dioxide (bis-aryl pyranone), G5 . All the bis-aryl pyranones that we examined possess two substituted aromatic benzene rings that are connected to a substituted cyclohexanone by double bond linkers. This connectivity is planar and reduces the fraction of sp 3 -hybridized carbons and the torsions within the small molecules reducing their flexibility ( Fig 5 A ). The 13 compounds have physicochemical properties that adhere to Lipinski and Ghose drug-like rules (Supplementary Table 1) ( 49 , 50 ). Although 1i , 1d and G5 have TPSA values that disobey Veber, Egan and Muegge properties the rest of the pyranones have ideal bioavailability scores which would translate to good oral bioavailability ( 51 – 53 ). All our compounds have low and acceptable LogP values ( LogP o/w < 5) with some being classified as soluble or moderately soluble (Supplementary Table 2). Download figure Open in new tab Download figure Open in new tab Download figure Open in new tab Fig 5: Perturbed USP18 domains. A, B Human USP18 surface. Green: Zinc finger domain, Red: IBB-1 domain, Blue: Catalytic triad domain, Yellow: location of Site2 pocket with surface around pseudo ligands from SiteMap. C. Overlap of IBB-1 domain residues in ligand bound with 2c conformation (Red) and ligand unbound conformation (cpk). D. Overlap of catalytic triad residues in ligand bound with 2c conformation (Blue) and ligand unbound conformation (cpk). Purple lines trace some of the bond lengths monitored from trajectories of the NPT simulations. Apart from 1i , 2d and G5 , the ADME profiles of these compounds predict high gastro-intestinal (GI) absorption ( Fig 5 B ). With exceptions to 1a , 1b , 1f and 1g , they have low polar surface areas (TPSA <90) and are not likely to penetrate through the blood brain barrier (BBB) ( 54 ) BBB permeant compounds such as 1a , 1b , 1f , and 1g , should be avoided as they carry the possibility of undesired pharmacological events associated with potential neurotoxicity ( 54 ) None of the pyranones are P-glycoprotein (P-gp) substrates, therefore their bioavailability cannot be affected by P-gp flux transporters ( 56 ). Unfortunately, all 13 compounds inhibit at least one of the Cytochrome P450 (CYP450) human isoforms CYP1A2, CYP2C19, CYP2C9, and CYP3A4 with none inhibiting the CYP2D6 isoform. Of this set 1c , 1i , 2b , 2e and G5 have the best metabolic profiles as they inhibit only one CYP450 isoform. As CYP450 enzymes are responsible for metabolizing 80 % of drugs, therefore any compound that inhibits P450s is likely to be hepatotoxic ( 57 ). All 13 pyranones possess the ene_one _ene _A PAIN alert. Compounds 1a, 1b 1c, 1f and 1g contain the michael_acceptor_1 as a Brenk alert while the nitro groups on 1i, G5 and 2a-2f are flagged as additional Brenk alerts ( Fig 5 C). These features are used to predict compounds that show false positive activity, metabolically instability, putative toxicity, chemically reactivity and poor pharmacokinetic properties ( 58 , 59 ). Although the G5 analogs had low cardiotoxicity, they had a high probability of triggering drug-induced liver injury, while the nitro containing derivatives were flagged for mutagenicity and hepatotoxicity from the ADMETlab toxicity predictions (Supplementary Table 3)( 60 , 61 ). Although several approved drugs in clinical use contain the nitro group this group is avoided due to its likelihood of triggering toxicity considerations ( 62 ). Overall, despite these compounds being druglike and orally bioavailable the pyranone scaffold makes them not ideal for medicinal and pharmaceutical applications. The metabolic and toxicity profiles of these compounds show that they possess CYP450 inhibition, carcinogenicity, and hepatotoxicity. Compound G5 and 2c possess significant structural alerts that would need removal if these scaffolds were to be pursued for further development. Undeterred by this negative outlook, this class of compounds however enables us to probe the role that antagonism of USP18 plays in the development of novel anti-HIV therapies due to the documented isopeptidase activity of G5 and 2c . Description of anti-USP18 activity which leads to anti-HIV-1 activity Based on the observed anti-HIV activity ( Fig 9 ) 8 compound systems were selected to guide the development of a hypothesis that explains the role that USP18 activity could have on anti-HIV activity. The ligand systems selected were ligand 1a , 2a , 2c , 2d , G5 , Chem46 (acquired from ChemDiv), Mol33 and Mol71 (acquired from Molport). Allosteric inhibition The USP18 active ligands ( 2c and G5 ) are hypothesized to have an allosteric perturbation of USP18 owing to the proximity of their preferred binding pocket, site 2, to the residues that affect USP18 activity ( Fig 6 ). It is possible that the allosteric impact could arise from changes within the IBB-1 domain or changes within the catalytic triad region brought on by binding of these active small molecules at the proposed allosteric site 2. If the activity is due to impacts on the IBB-1 domain, these changes will need to be significant enough to successfully impede the binding of USP18 to the ISGylated target substrate prior to proteolysis. Where the activity is due to changes to the triad residues, such perturbations would need to stall or reduce the proteolysis reaction that cleaves the substrate after the ISGylated target binds successfully to the inhibitor bound USP18. An additional allosteric non-enzymatic impact could result in limiting the upstream binding of USP18 to INFAR2 and STAT2 that serves to dampen the interferon cascade ( Fig 1 ). It is, however, not immediately apparent how limiting this interaction would result in the observed viral clearance. Current understanding, instead, is that binding of USP18 to IFNAR2 reduces IFN signaling and dampens expression of antiviral ISGs such as USP18 and ISG15 resulting in a decrease in the rate of ISGylation and thus viral clearance. Isolating the likely mechanism employed by these ligands will assist in the development of inhibitors that enhance the perturbations that are leading to viral clearance. Download figure Open in new tab Download figure Open in new tab Download figure Open in new tab Fig 6: Statistical analysis of Cα residue fluctuations of residues in the USP18 domains during 100ns of simulation. A. Heatmap plot of RMSF of the IBB-1 residues. B. Heatmap plot of RMSF of the triad residues. C. Correlative PCA analysis of IBB-1 residues. D. Clustering PCA analysis of IBB-1 residues. E. Correlative PCA analysis of triad residues. F. Clustering PC analysis of triad residues. By extracting residue fluctuations and measuring concerted changes in atom distances between residues in the domains during the NPT molecular dynamic simulations the induced-fit effect on critical residues of either the IBB-1 domain or the catalytic triad was monitored ( Figs 6 and 7 ). The trajectories from the NPT simulations of the 8 ligand systems were compared to those of the unbound apo protein. PCA plots show that complexes with 2c and Mol33 cluster their IBB-1 domain residue fluctuations in the same quadrant ( Fig 6 A, C and D). The plots of the triad domain show an anti-correlative clustering of 2c with Mol33 or the apo systems in contradiction to that observed with the IBB-1 domains ( Fig 6 B, E and F). Interestingly ligand 2c is having an impact on residues in both the IBB-1 domain and the catalytic triad, whereas G5 IBB-1 fluctuations overlap the apo protein. This observation strengthens the case that the source of USP18 activity of 2c and G5 is likely due to impacts on the triad residues. It is likely that the position in the active pocket that 2c occupies relative to Mol33 and G5 as well as the type of interactions that 2c initiates in its binding lead to the observations of the differences in its impacts on the 2 domains we interrogated. Download figure Open in new tab Download figure Open in new tab Fig 7: Total changes of atom distances within domain. A. Concerted IBB-1 domain atom distance changes. B. Concerted catalytic triad domain atom distance changes. The distance changes between specific atoms of domain residues were collected as a single observable, total perimeter, allowing us to monitor the concerted distance change of the conformations at each time step. This analysis is analogous to measuring the changes in the gaps between the top and bottom bellows of an accordion over time. This observable allows us to deconvolute the relationships between the systems obtained from the multivariate PCAs. From the concerted total perimeter measurements, the domain residues of the systems show a correlation with ligand binding at the allosteric site ( Fig 7 ). The IBB-1 residues show that conformations in ligand bound systems decrease the size of the total perimeter of this domain when compared to the unbound apo system ( Fig 7 A). Ligand 2c and Mol33 that cluster in the same quadrant from their PCA ( Fig 6 C and D), have the largest decrease in the total perimeter between these residues with the mean of Mol33 (24.81 nm) lower than that of 2c (26.25 nm) ( Fig 7 A). The USP18 active ligand 2c demonstrates a greater decrease in the mean of the total perimeter than G5 (27.40 nm) consistent with the reported superior isopeptidase activity. These simulations put forward the role of allosteric inhibition in manipulating the IBB-1 domain from the induce-fit effect of ligand binding as the source of the isopeptidase activity. Mol33 , which has a noticeable impact on the IBB-1 domain does appear to influence viral turnover to the same degree as 2c from the anti-HIV-1 assays. The binding of all the small molecules has an impact on this IBB-1 domain and as such it is plausible that the anti-USP18 isopeptidase activity of 2c and G5 occurs due to impaired interactions with the ISGylated substrate. Binding affinity surface plasmon resonance studies, beyond the scope of this study, conducted in the presence of inhibitor could further elucidate whether the mechanism of impaired USP18 binding to ISGylated substrate is the source of the observed isopeptidase activity of 2c and G5 . Close inspection of the triad residues suggests that the differences in the fluctuations of the 2c system ( Fig 6 B, E and F) occur due to the expansion of the catalytic triad ( Fig 7 B). This opening results in a greater separation of the residues in the triad reflected in an increase in the RMSFs for the 2c system not seen in the other ligand systems. Based on the total perimeter ( Fig 7 B) Mol33 , shared similarities with 2d and G5 (b), whereas when the magnitudes of the residue fluctuations were interrogated independent of direction and orientation through PCA, it overlapped with 1a ( Fig 6 E, F). Therefore, the magnitude of the triad contractions of the systems gives more resolution on the nature of the perturbation, than is shown by the PCA clustering. Analysis of the simulations show that the induced-fit effect on the IBB-1 domain residues due to allosteric binding of all the ligands has a greater potential to explain the USP18 inhibition. Although the anti-HIV-1 assays did not equivocally confirm our hypothesis, it is possible that the low anti-HIV-1 activity we are observing is due to low anti-USP18 activity as is seen in 2c and G5 . We propose that in instances where USP18 deISGylation of ISGylated substrate viral proteins is inhibited successfully by small molecules, a decrease in the viability of critical viral proteins would be expected to result in the amplified detection of anti-HIV-1 activity from the assays. While the focus has been on the impact of the induced-fit effect on USP18’s isopeptidase activity, it is also possible that the pronounced induced-fit effect observed at 2c at the allosteric site contributes to interferon signaling by reducing the IFNAR2 receptor binding. This would have the impact of influencing the USP18 function in an isopeptidase independent function. This interaction cannot be ruled out as residues on USP18 responsible for binding to STAT2 overlap with the residues of the catalytic triad domain that regulate isopeptidase activity (63). In this scenario however, if inhibitors such as 2c cause a reduction in the binding of USP18 to IFNAR2 they would serve to reduce the non-enzymatic IFN dampening function of USP18. This reduction would allow the IFN cascade to proceed, as the phosphorylation of STAT1 and STAT2 would be prolonged resulting in the expression of antiviral chemokines and genes such as ISG15 as well as pro-viral USP18 (64). However, mutation studies using selective inhibition of USP18 isopeptidase activity have repeatedly led to enhanced viral resistance in mice due to increased ISGylation with minimal risk of severe side effects (65). Whether the isopeptidase inhibition is due to the allosteric induced-fit effect on the triad residues, the IBB-1 domain or is due to the reduction in IFN dampening is further complicated by the observation that it could be a combination of all three or two phenomenon. Elucidating the precise source of the elimination of viral proteins from these assays is beyond the scope of this study owing to the low activity of the USP18 inhibitors we have access to currently. It is possible that microarray studies of these phenomenon could confirm our suspicions and shed light on the mechanism of viral control due to allosteric binding (66). Free-energy profiling Multi-trajectory simulations allowed free-energy profiling of the binding of the ligands at the allosteric site 2 of USP18. These profiles would assist in confirming whether patterns that explain the increased USP18 activity of 2c over G5 could be extrapolated to the other candidates based on their relationship with G5 ( Fig 9 ). It is also possible that the proposed mechanism of allosteric binding and the free-energy profiles of the small molecules could account for differences in the domain fluctuations ( Fig 6 ) and the anti-HIV-1 activity observed ( Fig 9 E). Although 1a (10.80 kcal mol -1 ) has a comparable PMF to that of 2c (10.40 kcal mol -1 ) ( Fig 8 D), the friction plot shows that 2c dissociates from the protein later than 1a ( Fig 8 C). This later dissociation accounts for the increased induced-fit effects on the IBB-1 domain and the triad residues by 2c ( Fig 7 ). Although the PMF of Mol33 (8.20 kcal mol -1 ) is less than 2c , it has a comparable dissociation time. These multi-trajectory free-energy profiles show that 2c has a comparable profile to that of 1a , 2d and Mol33 . The superior isopeptidase activity of 2c over G5 can be attributed to combination of its superior binding affinity and the induced-fit effect of 2c on the IBB-1 domain residues of USP18 which would be expected to translate into an improved anti-HIV-1 activity over G5 . A combination of these traits is reflected across the set of compounds that we have evaluated in this study. Download figure Open in new tab Download figure Open in new tab Fig 8: Free-energy profiling of ligand systems. A. Ligand G5 dissociation path with protein conformation at initial. B. Ligand G5 dissociation path with protein conformation after a few steps. C. PMF changes with COM distance. Solid lines are for systems obtained from 0.12 nm separation. D. Maximum PMF measured during dissociation for the 8 ligand systems. Boxes with borders show the systems obtained from 0.12 nm separation. Proof of concept for anti-viral activity with HIV-infected primary macrophages The 5.2 million ZINC15 compounds that satisfied the criteria of MW between 325 and 425 g/mol, 3D representations obtained at pH 7.4, anodyne reactivity patterns, purchasable, and charged between +2 and -2 were screened against USP18 affinity using Glide XP with a grid box centered on site 2. The top 200 ligands were prioritized by ranking the compounds according to their Glide scores. For this study, 18 of the top 200 compounds that were immediately available for purchase from vendors at a satisfactory quality and located within ideal physicochemical space were purchased as the active ZINC15 set of compounds. Anti-HIV assays were performed on the 31 compounds (13 pyranone derivatives and the 18 USP18 active ZINC15 compounds) in human monocyte derived macrophages (hMDMs). From the anti-HIV-1 assays, the bis-aryl pyranone compounds G5 , 2c and 2d recorded activity of up to 60 % after 3 days post infection (dpi) and 70 % after 6 dpi for 2d ( Fig 9 B). This activity demonstrated the first proof of concept observation of the hypothesis, that anti-USP18 activity can lead to anti-HIV-1 activity. When the activity in the Alamar Blue assay was interrogated the 2d activity coincided with a drop in cell viability whereas the activity of 2c and G5 was attributed to viral clearance ( Fig 9 D). Inhibition of viral activity of 40 % by G5 and 2c was more significant earlier in the infection cycle (3 dpi) and dropped to 20 % later in the infection cycle (6 dpi). Dose dependent plots after 3 dpi highlight how the cytotoxic effect of the pyranone derivatives (blue line) correlates with the anti-HIV activity (red line) observed ( Fig 9 E). The ZINC filtered compounds on the other hand had modest inhibition activities of up to 20 % but eliminated the cytotoxicity of the pyranone derived compounds. The HIV screening assay demonstrates the utility of resolving genuine, non-cytotoxic, HIV inhibition. The promising activities serve as a proof of concept for the pursuit of anti-HIV small molecules targeted towards anti-USP18 activity. The impact of binding Mol33 to the allosteric USP18 site on the fluctuations of key residues, and the concerted perturbations and expansions of functional domains, coupled with its binding free-energy profile, and non-cytotoxic anti-HIV inhibition underscore the need to explore the potential of accessing anti-HIV activity through attenuation of USP18 activity. Download figure Open in new tab Fig 9: Compound screening for anti-HIV-1 activity in human monocyte-derived macrophages (hMDM). A. Timeline for compound screening B. Inhibition of HIV-1 viral production of 12 compounds D1-D12 measured by the inhibition of Gaussia release 3- or 6-days post infection (dpi). C-D. Inhibition of HIV-1 viral production measured by the inhibition of Gaussia release (C), and the cell viability compared to DMSO measured by the Alamar Blue Assay (D) of 4 compounds at 3 or 6 days post-infection (dpi) with 1 donor and 3 technical replicates. E. Dose dependent curves showing the inhibition of Gaussia release (in red) and Alamar Blue (in blue) for uninfected (circles) and HIV-infected macrophages (dots) recorded after 3 dpi and 2 days of bisaryl pyranones compounds G5, 2c, 2d, 1a and 2a; and best ZINC filtered compounds Chem46, Mol33 and Mol71. One to 2 donors are represented. Although we have modest anti-HIV-1 activities, trends from molecular docking, free-energy profiles, and the influence of the induced-fit effect on the IBB-1 domain and the catalytic triad residues show that binding of small molecules at the allosteric site of USP18 impact its function and have the potential to direct the anti-HIV-1 activity. We postulate that the most dominant influence of anti-HIV-1 activity is the induced-fit perturbations on the USP18 IBB-1 domain residues arising from ligand binding at the allosteric site. We are not able to eliminate the contribution that large perturbations on the triad group due to expansion of this triad as this is beyond the scope of this study. The combined influence of these perturbations affects the activity of USP18 justifying the reported isopeptidase inhibition. We anticipate that inhibition in the early stages of infection enhance viral clearance by triggering ubiquitin-proteosome degradation of ISGylated critical viral proteins. Conclusions We report the first attempt at acquiring anti-HIV activity from small molecules that target the IFN response by the perturbation of USP18, a key ISG factor in the interferon pathway. Bis-aryl pyranones with low micromolar USP18 activity were repurposed for anti-HIV activity. These antagonists were used to motivate for the hypothesis that fine-tuning the IFN response enhances natural control mechanisms which can lead to viral clearance. The pyranone derivatives had unfavourable metabolic and toxicity profiles as their scaffold possesses numerous structural alerts that render them unsuitable for medicinal development. Despite these drawbacks this series of compounds gives valuable insights into the role of USP18 antagonism as a promising strategy for novel anti-HIV therapies. A human USP18 model was acquired and metalated with Zn 2+ . This model was mapped for potential small molecule perturbation sites and an allosteric site that had ideal characteristics for small molecule binding was identified. The centre of this site overlapped with the centroid of residues Ala299 , Asp124 , Gln122 and Tyr348 on the human USP18 sequence from the Q9UMW8 homology model. Molecular dynamics studies highlighted the impact of ligand binding to this site. The analysis of reported active USP18 agonists that had anti-HIV-1 activity suggested that these inhibitors possess a reversible non-competitive inhibitory effect due to an induced-fit effect on domain residues that regulate its activity. Although the binding of active inhibitors at the allosteric site results in the expansion of the catalytic triad it is thought that the main driver for the isopeptidase inhibition is likely perturbation of the IBB-1 domain. The adoption of this conformation would reduce the turnover of cleaved ISG15 products within the cytosol resulting in an increase in ISG15 tagged proteins which was observed by Cersosimo (2015). It is our opinion that these ISG15-tagged proteins trigger the ubiquitin code resulting in the rapid degradation of the targeted viral proteins. Multi-trajectory simulations that allow the computation of the relative binding free-energy showed that the anti-HIV-1 activity of these anti-USP18 inhibitors was not attributable to a distinctively superior affinity to the USP18 allosteric pocket. The resulting anti-HIV-1 activity of these USP18 antagonists is thus due to a combination of induced-fit effects on the catalytic triad domain and the IBB-1 domain due to binding of small molecules at the allosteric site. Novel anti-HIV scaffolds that possess potent anti-USP18 activity would need to be developed as the pyranone scaffolds possess undesirable toxicity profiles (Supplementary Information). This study suggests that a compound screening approach that considers the induce-fit effect is critical. This is a key determinant on anti-HIV-1 activity due to the impact of the catalytic functions of USP18 on the interferon signal cascade. Although inhibitors that mediate the type I IFN anti-viral response have been pursued in the past with limited success, this is the first study that has elucidated the specific role that USP18 inhibition and the ubiquitin-proteasome could play in this application. The allosteric inhibition of USP18 gives rise to an induced-fit effect with potential to influence both the enzymatic and non-enzymatic activities of USP18. This study shows that targeting the catalytic USP18 activity is a viable strategy to fine-tune the IFN response to enhance natural control mechanisms that lead to clearance of viral infection and a potential cure if targeted to reservoir cells. Acknowledgements We acknowledge the Centre for High Performance Computing (CHPC), South Africa, for providing computational resources to this research project. References 1. ↵ Netanya Utay AS , Douek DC . Interferons and HIV Infection: The Good, the Bad, and the Ugly . Pathogens and Immunity . 2016 ; 1 ( 1 ). 2. 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