International Journal of Molecular Sciences

Article A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Adenylyltransferase HYPE/FICD

1 1, 1, 1 2 Ali Camara , Alyssa George †, Evan Hebner †, Anika Mahmood , Jashun Paluru and Seema Mattoo 1,3,4,* 1 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA; [email protected] (A.C.); [email protected] (A.G.); [email protected] (E.H.); [email protected] (A.M.) 2 William Henry Harrison High School, West Lafayette, IN 47906, USA; [email protected] 3 Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, IN 47907, USA 4 Purdue Institute for Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907, USA * Correspondence: [email protected] These authors contributed equally. †  Received: 27 August 2020; Accepted: 25 September 2020; Published: 27 September 2020 

Abstract: The covalent transfer of the AMP portion of ATP onto a target —termed adenylylation or AMPylation—by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore—Fl-ATP—we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE’s enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.

Keywords: HYPE/FICD; AMPylation/adenylylation; post-translational modification; BiP/GRP78/ HSPA5; α-synuclein; fluorescence polarization; high-throughput screening; drug discovery; assay development

1. Introduction Recent years have witnessed a surge in interest of a once-underappreciated post-translational modification (PTM) termed adenylylation (AMPylation) [1,2]. AMPylation entails the breakdown of an triphosphate (ATP) co-substrate for the covalent transfer of (AMP) to a hydroxyl sidechain-containing residue of a target protein. Among these adenylyltransferases (AMPylases) are the Fic (filamentation induced by cyclic AMP) family of , whose members are present in all domains of life [3]. Fic proteins are characterized by a structurally conserved Fic domain that houses a canonical FIC motif—HXFX(D/E)(G/A)N(G/K)RXXR—where the invariant is required for catalysis [1,2,4]. Early reports on Fic proteins VopS (Vibrio outer

Int. J. Mol. Sci. 2020, 21, 7128; doi:10.3390/ijms21197128 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 21 proteins, whose members are present in all domains of life [3]. Fic proteins are characterized by a structurally conserved Fic domain that houses a canonical FIC motif—HXFX(D/E)(G/A)N(G/K)RXXR—where the invariant histidine is required for catalysis

[1,2,4].Int. J. Mol. Early Sci. 2020 , 21reports, 7128 on Fic proteins VopS (Vibrio outer protein S) and IbpA2 of 20 (immunoglobulin-binding protein A)—from Vibrio parahaemolyticus and Histophilus somni, respectively—revealed that the AMPylation of small Rho promotes bacterial pathogenesis [1,2,5,6].protein Consequently, S) and IbpA previous (immunoglobulin-binding high-throughput screening protein A)—from (HTS) effortsVibrio have parahaemolyticus aimed to identifyand small-moleculeHistophilus somni inhibitors, respectively—revealed of bacterial Fic-mediated that the AMPylation AMPylation of small [7]. Rho GTPases promotes bacterial pathogenesisA single [1Fic,2, 5protein,,6]. Consequently, called HYPE previous (Huntingtin high-throughput yeast-interacting screening partner (HTS) e ffE)/FICD,orts have exists aimed in to humansidentify small-molecule[3,8]. HYPE is inhibitorstopologically of bacterial organized Fic-mediated as an N-terminal AMPylation transmembrane [7]. (TM) domain, followedA single by two Fic tetratricopeptide protein, called HYPE(TPR) domains, (Huntingtin and yeast-interacting a C-terminal Ficpartner domain E) (Figures/FICD, exists1A and in S8A) [9]. [3,8]. HYPE is topologically organized as an N-terminal transmembrane (TM) domain, followed by two tetratricopeptide (TPR) domains, and a C-terminal Fic domain (Figure1A and Figure S8A) [9].

FigureFigure 1. 1. FluorescenceFluorescence polarization polarization (FP) (FP) of Fl-ATP of Fl-ATP monitors monitors HYPE-mediated HYPE-mediated AMPylation. AMPylation. (A) Schematic(A) Schematic depiction depiction of the protein of the domain protein architec domainture of architecture HYPE showing of HYPEthe TM showing(transmembrane), the TM TPR(transmembrane), (tetratricopeptide), TPR and (tetratricopeptide), Fic (filamentation and induced Fic (filamentation by cAMP) domains. induced AMP by cAMP)denotes domains. sites of autoAMPylation.AMP denotes sitesThe glutamate-cont of autoAMPylation.aining inhibitory Theglutamate-containing motif and histidine-containing inhibitory Fic motif motif andare shownhistidine-containing in blue Fic motifand are shownred, inrespectively. blue and red, respectively.(B) Structure (B) Structure of ofFl-ATP Fl-ATP (N6-(6-amino)hexyl-ATP-5(N6-(6-amino)hexyl-ATP-5-carboxyl-fluorescein).-carboxyl-fluorescein). (C()C FP) AMPylationFP AMPylation assay design.assay Whendesign. unattached When unattachedto wild-type to (WT) wild-type HYPE or(WT) compound-inhibited HYPE or compound-inhibited E234G HYPE (purple),E234G HYPE Fl-ATP (purple), undergoes Fl-ATP rapid undergoesrotation to rapid depolarize rotation plane-polarized to depolarize plane-polari light, and azed basal light, FP and signal a basal is generated. FP signal Attachment is generated. of AttachmentFl-AMP via of autoAMPylation Fl-AMP via autoAMPylation (E234G HYPE (E234G or compound-activated HYPE or compound-activated WT HYPE) allows WT lightHYPE) to allows remain lightpolarized to remain for ahigh-FP polarized signal. for (aD )high-FP Proof-of-concept signal. (D of) FPProof-of-concept AMPylation assay of FP comparing AMPylation WT, E234G,assay or E234G/H363A versions of HYPE103-456 (∆102 HYPE) with or without 10X molar excess of substrates comparing WT, E234G, or E234G/H363A versions of HYPE103-456 (Δ102 HYPE) with or without 10X (BiP or α-syn). All samples were normalized to buffer controls. Data are represented as the mean +/ molar excess of substrates (BiP or α-syn). All samples were normalized to buffer controls. Data are− SEM of four replicates. Unpaired t-tests were performed: * = p < 0.05. represented as the mean +/− SEM of four replicates. Unpaired t-tests were performed: * = p < 0.05. The TM domain anchors HYPE to the endoplasmic reticulum (ER) membrane, while the The TM domain anchors HYPE to the endoplasmic reticulum (ER) membrane, while the lumenal-facing TPR and Fic domains are involved in substrate binding and catalysis, respectively [2,9–11]. lumenal-facing TPR and Fic domains are involved in substrate binding and catalysis, respectively Mutation of HYPE’s His363 to Ala (H363A) within its Fic motif renders the inactive [2,5,9]. [2,9–11]. Mutation of HYPE’s His363 to Ala (H363A) within its Fic motif renders the enzyme inactive Additionally, HYPE AMPylation is regulated by an inhibitory , in which a conserved [2,5,9]. Additionally, HYPE AMPylation is regulated by an inhibitory alpha helix, in which a glutamate sterically obstructs catalytically productive ATP binding in the Fic active site [12]. Mutation of conserved glutamate sterically obstructs catalytically productive ATP binding in the Fic active site Glu 234 to Gly (E234G) relieves this intrinsic auto-inhibition, generating a constitutively active AMPylase [12]. HYPE is a regulator of ER stress via reversible AMPylation of the heat shock chaperone BiP (Binding immunoglobulin Protein) [9,13,14]. Interestingly, HYPE catalyzes both the AMPylation and deAMPylation of BiP, in response to as-yet-unknown physiological signals [15]. During homeostasis, HYPE-directed AMPylation locks BiP in an inactive state marked by reduced co-chaperone Int. J. Mol. Sci. 2020, 21, 7128 3 of 20

(J-protein)-assisted ATPase activity and the resultant decrease in chaperoning misfolded proteins [10,14]. Accumulation of misfolded proteins in the ER triggers deAMPylation of BiP by HYPE, with the concomitant induction of the unfolded protein response (UPR) in an attempt to restore homeostasis [15]. Maintenance of ER homeostasis is a critical aspect of cell fate during several diseases, making the AMPylation state of BiP of paramount importance. Indeed, a prominent molecular strategy toward the treatment of hyper-proliferating cancer cells is activation of the UPR, where BiP has been shown to serve as a sentinel for controlling [16]. Likewise, upregulation of the UPR plays a critical role during diabetes and proper folding of insulin [17]. Recent reports also indicate that upregulated BiP can escape the ER to be used as binding and cell entry receptors for SARS-CoV-2 (etiological agent for COVID-19 (corona virus disease 2019)) spike proteins [18,19]. As the cellular stress enacted by cancer triggers the UPR and BiP expression, this underlying disease could synergize with SARS-CoV-2 infection to worsen health outcomes for COVID-19 patients. Whether BiP’s AMPylation status impacts this binding or ER exit awaits further study. Given its role in regulating protein folding and the UPR via BiP, it comes as no surprise that genetically manipulating the AMPylation status of a HYPE ortholog (Fic-1 in C. elegans) leads to enhanced organismal survival under neurodegenerative conditions caused by aggregating proteins such as amyloid-β, α-synuclein (α-syn), or Huntingtin implicated in Alzheimer’s, Parkinson’s, and Huntington’s diseases, respectively [20]. In addition, we have shown that E234G HYPE can directly AMPylate α-syn in vitro and impede disease-linked phenotypes like fibril formation and lipid membrane permeability [21]. A role for another HYPE ortholog (dFic in D. melanogaster) has also been established in blindness [22,23]. The canon of HYPE linkage to disease has been further broadened by omics investigations [24–26]. A study tracking highly differentially expressed transcripts under glucolipotoxicity in primary beta islets cells showed that the HYPE gene, FICD, is both upregulated and methylated [25]. Proteomic research on AMPylated neuronal proteins has also mapped out a role for HYPE in neurogenesis, suggesting that HYPE-promoted AMPylation remodeling is required for proper differentiation of human neurons [26]. Moreover, this same study introduces an ever-expanding portfolio of novel, putative HYPE substrates, the functions of which are being elucidated [26]. Thus, the ability to manipulate HYPE’s enzymatic activity presents an attractive therapeutic target toward the treatment of diseases including cancer, COVID-19, and neurodegeneration. Despite being positioned at a therapeutic nexus of various disease pathways, HYPE has yet to be targeted for high-throughput drug discovery efforts. Previous work using purified HYPE to immunize alpacas resulted in HYPE-specific nanobodies capable of manipulating AMPylation [27]. However, the therapeutic potential of these macromolecules is restricted by their high cost of production, lack of drug-like qualities, and low biological activity [27]. Thus, there remains an imminent need for the discovery of HYPE-targeted drugs. Wild-type (WT) HYPE’s adenylyltransferase activity is intrinsically inhibited but, as mentioned earlier, can be de-repressed via an E234G mutation [2,5,9,12,27]. Exploiting these enzymatically distinct states, we thus set out to discover small-molecule activators and inhibitors of WT and E234G HYPE, respectively. To this end, we developed a dual screen based on the fluorescence polarization (FP) of an ATP analog, N6-(6-amino)hexyl-ATP-5-carboxyl-fluorescein (Fl-ATP) (Figure1B) [ 7,28]. When unattached to WT HYPE or compound-inhibited E234G HYPE, Fl-ATP undergoes rapid rotation to depolarize plane-polarized light, and a basal FP signal is generated. Attachment of Fl-AMP via autoAMPylation (E234G HYPE or compound-activated WT HYPE) allows light to remain polarized for a high-FP signal (Figure1C). We challenged this assay with four diverse chemical libraries (covering nearly 10,000 compounds for each screen) to discover the first small-molecule modulators of HYPE-mediated autoAMPylation. These hits were then assessed for their dose dependencies and validated via orthogonal biochemical counterscreens. Int. J. Mol. Sci. 2020, 21, 7128 4 of 20

2. Results and Discussion

2.1. Assay Design WT HYPE exhibits basal levels of AMPylation in vitro [2,5,9,27], while an E234G mutation results in a robust, constitutively active adenylyltransferase [12]. Therefore, we selected WT HYPE as our negative control for AMPylation and E234G HYPE as our positive control. To overcome the challenge posed by HYPE’s hydrophobic N-terminus in obtaining soluble recombinantly purified full-length protein, we purified two truncated forms of HYPE: (1) ∆45 HYPE, which encodes aa 46–456 of HYPE, thus deleting its TM domain, and has previously been characterized extensively [9]; and (2) ∆102 HYPE, which can be purified to higher yields, and corresponds to what was used previously for solving the crystal structures of HYPE [29,30]. To note, ∆102 HYPE eliminates two of the four identified sites of HYPE autoAMPylation [9,24]. However, this deletion (Figure1A) did not result in a discernable reduction in autoAMPylation signal relative to the longer ∆45 HYPE (Figure S1). Using Fl-ATP as a source, we monitored changes in autoAMPylation between the previously described HYPE constructs. When free in solution, the relatively small Fl-ATP is subject to rapid Brownian motion, enabling it to depolarize plane-polarized light. However, in the event of covalent attachment to a large protein (i.e., AMPylation), Fl-AMP undergoes slow rotation in which the plane-polarized light remains polarized. This latter condition allows for detection and quantification of AMPylation events. Further, the readout is homogenous and amenable to HTS processing using a standard microplate format and automated liquid handling [7,28].

2.2. Assay Development and Optimization We next tested our FP assay’s ability to distinguish between the AMPylation levels of hypo-active WT and hyper-active E234G HYPE. To ensure we were detecting authentic AMPylation and not the stable, noncovalent binding of Fl-ATP to HYPE, we also assessed the FP signal of catalytically inactive E234G/H363A HYPE. Enzymatically dead mutants are known to serve as in vitro substrate traps, a property used in the past for pulling down HYPE’s interacting partner, BiP [9,31]. We reasoned that if the stable binding of Fl-ATP to inactive HYPE occurs at HYPE’s ATP binding pocket within the Fic domain, a false positive signal would be detected. Additionally, we incubated our with known in vitro substrates α-syn and BiP [9,21]. As expected, we saw a significantly higher FP signal from E234G HYPE than WT or E234G/H363A (Figure1D), indicating a lack of adenylyltransferase activity or stable Fl-ATP binding. Of note, a ten times molar excess of substrate failed to significantly improve total AMPylation over auto-AMPylation (Figure1D). This finding is likely due to the higher proximity-dependent availability of auto-AMPylation sites relative to those of substrates. To assess conditions under which optimal AMPylation would occur, we compared two well-established buffers routinely used in AMPylation reactions [9,15,21,30,32]. These included a minimal buffer containing 50 mM HEPES (pH 7.5) and 1 mM MnCl2; and a complete buffer with 50 mM HEPES (pH 7.4), 150 mM KCl, 1 mM CaCl2, 10 mM MgCl2, and 0.1% Triton-X 100. AMPylation signals remained consistently high for E234G HYPE and low for WT HYPE, irrespective of buffer choice (Figure2A). There did appear to be an enhancement in WT HYPE AMPylation under complete buffer conditions, but these levels were still basal relative to E234G. Of the various buffer components, only Mn2+, Ca2+, and Mg2+ are reported to be indispensable for AMPylase activity [9], owing to their ability to coordinate ATP’s α and β phosphates in the Fic catalytic pocket [29,30]. Our previous findings suggested a hierarchy of AMPylation efficiencies among divalent cations, with Mn2+ being the preferred metal, followed by Mg2+ [9]. We therefore conducted a direct comparison between Mn2+ and Mg2+ buffers using our FP assay. This resulted in no significant difference in E234G HYPE AMPylation, while WT HYPE levels remained basal (Figure2B). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 21

2+ 2+ betweenInt. J. Mol. Sci.Mn2020 and, 21, 7128Mg buffers using our FP assay. This resulted in no significant difference5 of in 20 E234G HYPE AMPylation, while WT HYPE levels remained basal (Figure 2B).

Figure 2.2. AMPylationAMPylation buffer buffer assessment. assessment. (A ()A )Comparison Comparison of of Δ∆102102 WT WT and and E234G E234G HYPE autoAMPylation in in complete complete (Com) (Com) or or minimal minimal (Min) (Min) buffer buff (seeer (see Methods). Methods). (B) Comparison (B) Comparison of Δ102 of WT∆102 and WT E234G and E234G HYPE HYPE autoAMPylati autoAMPylationon with withbuffer bu containingffer containing Mn2+ Mn or 2Mg+ or2+. Mg (C)2 +Comparison.(C) Comparison of Δ102 of WT∆102 or WT E234G or E234G HYPE HYPE autoAMPylation autoAMPylation with with or wi orthout without 1% 1% DMSO DMSO in ina minimal a minimal buffer buffer background. background. (D) ComparisonComparison of of∆ 102Δ102 WT WT or E234Gor E234G HYPE HYPE autoAMPylation autoAMPylation with orwith without or without 1% DMSO 1% inDMSO a complete in a completebuffer background. buffer background. All samples All were samples normalized were norm to bualizedffer controls. to buffer Data controls. represented Data asrepresented the mean +/as– theSEM mean of four +/– replicates.SEM of four Unpaired replicates.t-tests Unpaired were performed: t-tests were * performed:= p < 0.05;*** * == p p< <0.05;0.001. *** = p < 0.001.

DMSO (dimethyl(dimethyl sulfoxide) sulfoxide) is theis standardthe standard vehicle vehicle for solvating for solvating the polar the and nonpolarpolar and compounds nonpolar compoundsfound in chemical found libraries. in chemical Despite libraries. widespread Despite usage, widespread DMSO is notusage, without DMSO its drawbacks,is not without and hasits drawbacks,been documented and has to been interfere documented with the to enzymatic interfere activitieswith the enzymatic of multiple activities proteins of [33 multiple]. In anticipation proteins [33].of our In HTSanticipation campaign, of our we addedHTS campaign, DMSO to we minimal added and DMSO complete to minimal buffers and to assesscomplete its impactbuffers onto assessAMPylation. its impact Under on AMPylation. both buffer Under conditions, both thebuffer AMPylation conditions, signal the AMPylation for E234G HYPEsignal for decreased E234G HYPEsignificantly decreased (Figure significantly2C,D), an (Figure observation 2C,D), consistent an observation with previous consistent reports with ofprevious DMSO-mediated reports of DMSO-mediatedenzyme inhibition enzyme [33]. inhibition [33]. To ensureensure we we were were catching catching the reactionthe reaction in its linearin its range, linear we range, performed we performed a time-course a AMPylationtime-course AMPylationexperiment usingexperiment constant using concentrations constant concentr of E234Gations HYPE of E234G and Fl-ATP. HYPE These and Fl-ATP. data suggested These data that suggesteda 10 min timepointthat a 10 wouldmin timepoint give a suitable would signalgive a (Figure suitable3A,B) signal while (Figure allowing 3A,B) su whilefficient allowing time to sufficientprocess successive time to process plates bysuccessive automated plates liquid by handlersautomated during liquid HTS. handlers We next during ran reactions HTS. We at next various ran reactionsconcentrations at various of Fl-ATP concentrations to determine of Fl-ATP the signal to detectiondetermine threshold. the signal This detection step was threshold. especially This critical step wasconsidering especially the critical expansive considering sizes of chemical the expansive libraries, sizes and of the chemical relatively libraries, high cost and of the Fl-ATP relatively (compared high to other reaction components). Further, we wanted to select a sub-K concentration of Fl-ATP to avoid cost of Fl-ATP (compared to other reaction components). Further,m we wanted to select a sub-Km concentrationbiasing our assay of against Fl-ATP compounds to avoid that arebiasing (Fl-)ATP-competitive. our assay against Accordingly, compounds we chose anthat Fl-ATP are (Fl-)ATP-competitive.concentration of 25 nM Accordingly, to conduct the we screening chose an assay Fl-ATP (Figure concentration3C). of 25 nM to conduct the screening assay (Figure 3C).

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Figure 3. Assessment of AMPylation kinetic parameters. (A) Representative fluorescence image of Δ102 E234G HYPE autoAMPylation time-course. (B) Quantification of two independent experiments as in (A). Data represented as the mean +/− SEM. (C) Fluorescence image (top) of Δ102 E234G HYPE autoAMPylation at various Fl-ATP concentrations and corresponding Coomassie image (bottom).

Before challenging compound libraries with our assay, we first needed to evaluate its robustness and scalability. For this, we turned to an assay quality analysis [34], in which 384-well Figure 3. A Figure 3. AssessmentAssessment ofof AMPylationAMPylation kinetickinetic parameters.parameters. (A) RepresentativeRepresentative fluorescencefluorescence imageimage ofof plates∆102 were E234G loaded HYPE with autoAMPylation either WT or time-course.E234G HYPE (B) for Quantification 10 min in minimal of two independent AMPylation experiments buffer. At our Δ102 E234G HYPE autoAMPylation time-course. (B) Quantification of two independent experiments standardas in (Aenzyme). Data represented concentration as the of mean 200 +/nM,SEM. we (Cobserved) Fluorescence excellent image Z’ (top) (i.e., of ∆1 102> Z’ E234G ≥ 0.5) HYPE and S/B as in (A). Data represented as the mean +/−− SEM. (C) Fluorescence image (top) of Δ102 E234G HYPE (signal/background)autoAMPylation at variousvalues: Fl-ATP0.75 concentrationsand 3.87, respectively and corresponding (Figure Coomassie 4A). Doubling image (bottom). the enzyme concentrationautoAMPylation to 400 at nM various resulted Fl-ATP in furtherconcentratio extensionns and ofcorresponding our assay window Coomassie (Figure image 4B). (bottom). BeforeTo further challenging evaluate compound the validity libraries of our with assay, our we assay, reasoned we first that needed if Fl-ATP to evaluate truly mimics its robustness ATP in Before challenging compound libraries with our assay, we first needed to evaluate its andAMPylation scalability. mediated For this, by we E234G-HYPE, turned to an then assay addition quality analysisof unlabeled [34], ATP in which would 384-well collapse plates the E234G were robustness and scalability. For this, we turned to an assay quality analysis [34], in which 384-well loadedHYPE-associated with either FP WT signal or E234G to that HYPE of WT for HYPE. 10 min We in tested minimal this AMPylation assumption bubyff increasinglyer. At our standard titrating plates were loaded with either WT or E234G HYPE for 10 min in minimal AMPylation buffer. At our enzymeunlabeled concentration ATP into our of 200 standard nM, we observedreaction. excellentIndeed, we Z’ (i.e., saw 1 >a Z’concentration-dependent0.5) and S/B (signal/background) inhibitory standard enzyme concentration of 200 nM, we observed excellent≥ Z’ (i.e., 1 > Z’ ≥ 0.5) and S/B values:effect (Figure 0.75 and S2), 3.87, indicating respectively the (Figure ability4 A).of our Doubling assay theto detect enzyme bona concentration fide chemical to 400 modulators nM resulted of (signal/background) values: 0.75 and 3.87, respectively (Figure 4A). Doubling the enzyme inAMPylation. further extension of our assay window (Figure4B). concentration to 400 nM resulted in further extension of our assay window (Figure 4B). To further evaluate the validity of our assay, we reasoned that if Fl-ATP truly mimics ATP in AMPylation mediated by E234G-HYPE, then addition of unlabeled ATP would collapse the E234G HYPE-associated FP signal to that of WT HYPE. We tested this assumption by increasingly titrating unlabeled ATP into our standard reaction. Indeed, we saw a concentration-dependent inhibitory effect (Figure S2), indicating the ability of our assay to detect bona fide chemical modulators of AMPylation.

Figure 4. Assay reproducibility assessment. (A) 200 nM ∆102 WT and E234G HYPE autoAMPylation Figure 4. Assay reproducibility assessment. (A) 200 nM Δ102 WT and E234G HYPE autoAMPylation reactions were measured for FP on separate 384-well microplates. Z’ = 0.749 and S/B = 3.875. Each dot reactions were measured for FP on separate 384-well microplates. Z’ = 0.749 and S/B = 3.875. Each dot represents a single AMPylation reaction in a separate well. Black dash lines represent three standard represents a single AMPylation reaction in a separate well. Black dash lines represent three standard deviations plus or minus the control means. (B) As in (A) but the mean of two independent 400 nM deviations plus or minus the control means. (B) As in (A) but the mean of two independent 400 nM ∆102 HYPE plates. Mean Z’ = 0.884 and mean S/B = 4.598. Δ102 HYPE plates. Mean Z’ = 0.884 and mean S/B = 4.598. To further evaluate the validity of our assay, we reasoned that if Fl-ATP truly mimics ATP in

AMPylationFigure 4. mediatedAssay reproducibility by E234G-HYPE, assessment. then (A addition) 200 nM Δ of102 unlabeled WT and E234G ATP would HYPE autoAMPylation collapse the E234G HYPE-associatedreactions wereFP measured signal tofor that FP on of separate WT HYPE. 384-well We testedmicroplates. this assumption Z’ = 0.749 and by S/B increasingly = 3.875. Each titratingdot unlabeledrepresents ATP a into single our AMPylation standard reaction. reaction in Indeed, a separate we sawwell.a Black concentration-dependent dash lines represent three inhibitory standard e ffect (Figure deviations S2), indicating plus or minus the ability the control of our means. assay to(B) detect As in ( bonaA) but fide the chemical mean of two modulators independent of AMPylation. 400 nM Δ102 HYPE plates. Mean Z’ = 0.884 and mean S/B = 4.598.

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2.3. WT HYPE Activator High-Throughput Screen 2.3. WT HYPE Activator High-Throughput Screen We used our optimized AMPylation assay to screen four diverse chemical libraries—LOPAC1280 1280 (LibraryWe usedof Pharmacologically our optimized AMPylation Active Compounds), assay to screen Spectrum, four diverse MEGx, chemical and NATx—totaling libraries—LOPAC 9680 compounds(Library of (Table Pharmacologically 1). Active Compounds), Spectrum, MEGx, and NATx—totaling 9680 compounds (Table1). Table 1. Compound library information. Table 1. Compound library information. Library Name Company Number of Compounds Chemical Properties LibraryLOPAC Name Sigma-Aldrich Company Number 1280 of Compounds Bioactive, Chemical FDA-approved Properties SpectrumLOPAC Microsource Sigma-Aldrich 2400 1280 Bioactive Bioactive, and natural FDA-approved compounds NATxSpectrum AnalytiCon Microsource Discovery 5000 2400 Synthe Bioactivetics derived and natural from natural compounds products MEGxNATx AnalytiCon AnalytiCon Discove Discoveryry 1000 5000 Synthetics derived Natural from products natural products TotalMEGx AnalytiCon- Discovery 9680 1000 Natural - products Total - 9680 - In the first portion of our dual screen, we challenged these libraries against WT HYPE toward the discoveryIn the first of portionAMPylation of our activators. dual screen, These we screen challengeds yielded these an libraries initial overall against hit WT rate HYPE of 0.44% toward at anthe empirically discovery ofdetermined AMPylation cut-off activators. of 20% activation These screens relative yielded to controls an initial (Figure overall 5A). hit Moreover, rate of 0.44% the screenat an empiricallymaintained determinedsuitable Z’ cut-oand S/Bff of values 20% activation throughout, relative with tominimal controls plate-to-plate (Figure5A). Moreover,variability (Figurethe screen 5B) maintained From these suitableinitial hits, Z’ and we Sthen/B values manu throughout,ally eliminated with any minimal compounds plate-to-plate possessing variability outlier parallel(Figure 5intensities,B) From these to yield initial a final hits, HTS we then hit rate manually of 0.32%. eliminated This measure any compounds was taken to possessing control for outlier false positiveparallel intensities,compounds to capable yield a of final auto-fluorescing HTS hit rate of or 0.32%. quenching This measurefluorescence was emanating taken to control from Fl-ATP. for false Whilepositive our compounds 20% activation capable threshold of auto-fluorescing was empirically or determined quenching fluorescenceto produce a emanatingmanageable from number Fl-ATP. of hits,While it ourdid 20% conform activation to standard threshold assay wasempirically window requirements determined to(i.e., produce greater a manageable than three numberstandard of deviationshits, it did conformabove tothe standard negative assay control window requirementsmean) [34]. (i.e.,As greaterexpected, than threelibraries standard enriched deviations in pharmacologicallyabove the negative active control compounds mean) [34]. (LOPAC As expected, and Sp librariesectrum) enriched displayed in pharmacologicallymore hits relative to active the lesscompounds bioactive (LOPAC natural and products Spectrum) from displayed the MEGx more library hits relative (Table toS1). the Strikingly, less bioactive while natural most products of the synthesizedfrom the MEGx compounds library (Table in NATx S1). Strikingly, failed to whileactiva mostte AMPylation, of the synthesized several compounds hits stemming in NATx from failed the sameto activate source AMPylation, plate were severalclustered hits together. stemming Ind fromeed, the upon same visual source inspection plate were of clustered these NATx together. hit structures,Indeed, upon there visual was inspectiona clear emergence of these of NATx several hit common structures, functional there was groups a clear (Figure emergence 7F), suggestive of several ofcommon a similar functional mechanism groups of action. (Figure 7F), suggestive of a similar mechanism of action.

FigureFigure 5. WTWT HYPEHYPE activator activator high-throughput high-throughput screen. screen. (A) ∆(A102) Δ WT102 HYPE WT HYPE FP autoAMPylation FP autoAMPylation reaction reactionincubated incubated with compounds with compounds from either from LOPAC, either LOPAC, Spectrum, Spectrum, MEGx, orMEGx, NATx or libraries. NATx libraries. All samples All sampleswere normalized were normalized to ∆102 WT to andΔ102 E234G WT HYPEand E234G controls HYPE on the controls same plate on inthe DMSO-containing same plate in DMSO-containingbuffer. Each dot represents buffer. Each a diff erentdot represents compound a incubated different withcompound a single incubated AMPylation with reaction a single in a separate well. Black dashes represent +/ 3 SD from the mean. Red dashes are the 20% hit definition, AMPylation reaction in a separate well. −Black dashes represent +/− 3 SD from the mean. Red dashes arewith the all 20% dots hit above definition, this threshold with all being dots hitabove compounds. this threshold Dots withbeing red hit circlescompounds. show hits Dots selected with red for follow-up, orthogonal validation. (B) Plate-to-plate variability in the positive (E234G HYPE) and circles show hits selected for follow-up, orthogonal validation. (B) Plate-to-plate variability in the negative (WT HYPE) DMSO controls from WT HYPE high-throughput screening (HTS). Left and right positive (E234G HYPE) and negative (WT HYPE) DMSO controls from WT HYPE high-throughput y-axes are for Z’ and S/B values, respectively. Each dot represents the calculated Z’ or S/B values from screening (HTS). Left and right y-axes are for Z’ and S/B values, respectively. Each dot represents the 24 E234G HYPE positive controls and 24 WT HYPE negative controls within a single plate. calculated Z’ or S/B values from 24 E234G HYPE positive controls and 24 WT HYPE negative controls within a single plate.

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2.4.2.4. WTWT HYPEHYPE ActivatorActivator MicroplateMicroplate ValidationValidation WeWe next next validated validated our our cherry-picked cherry-picked activator activator hits hits in anin independentan independent replication replication of the of same the same HTS assay.HTS assay. Many Many of our of previously our previously discovered discovered hits failed hits this failed second-pass this second-pass selection, selection, having an having R2 value an ofR2 0.17value (Figure of 0.176A). (Figure This could6A). This be attributed could be attributed to a relatively to a permissiverelatively permissive 20% hit definition, 20% hit definition, as selection as ofselection a more of stringent a more criterionstringent ofcriterion 50% activation of 50% activation more than more doubled than thedoubled R2 value the (FigureR2 value S3). (Figure Of note, S3). groupingOf note, grouping hits based hits on their based libraries on their of originlibraries revealed of origin a high revealed correlation a high among correlation LOPAC among and Spectrum, LOPAC whileand Spectrum, the NATx while compounds the NATx showed compounds low confirmation showed lo (Figurew confirmation6B–E). This (Figure finding 6B–E). is consistent This finding with is theconsistent notion thatwith bioactives the notion are that likely bioactives to have high,are likely reproducible to have activitieshigh, reproducible when compared activities to novel,when syntheticcompared molecules to novel, [synthetic35]. molecules [35].

FigureFigure 6.6. WTWT HYPEHYPE activatoractivator validation.validation. ((AA)) ComparisonComparison betweenbetween initialinitial HTSHTS compoundcompound activityactivity (x(x-axis)-axis) andand second-passsecond-pass validationvalidation (y(y-axis)-axis) withwith thethe samesame FP FP AMPylation AMPylation assay. assay. AllAll validationvalidation compoundscompounds werewere assayedassayed onon thethe samesame plateplate andand normalizednormalized toto internalinternal positivepositive (E234G(E234G HYPE)HYPE) andand negativenegative (WT(WT HYPE)HYPE) DMSODMSO controls.controls. EachEach dotdot representsrepresents thethe samesame compoundcompound incubatedincubated inin twotwo independentindependent AMPylation AMPylation reactions. reactions. Dots Dots with with red circles red showcircles hits show selected hits forselected follow-up, for orthogonalfollow-up, validation.orthogonal ( Bvalidation.–E) Data reprocessed (B–E) Data from reprocessed (A) to show from compound (A) to show validation compound among validation the various among libraries. the various libraries. Using biological activity, chemical diversity, and commercial availability as selection criteria, we advancedUsing biological six activators activity, (A1 –chemicalA6) for follow-up diversity, validation and commercial (Figures availability5A,6A and7 asA–F, selection Table2 ).criteria, we advanced six activators (A1–A6) for follow-up validation (Figures 5A, 6A, and 7A–F, Table 2). Table 2. WT HYPE activator hit information.

Compound Chemical % Mean MW (Da.) Chemical Name Library % Act. 1 % Act. 2 ID Formula Act.

A1 C31H23Cl7N2O 688 Calmidazolium LOPAC 177 114 146 A2 C18H39NO2 302 DL-erythro-dihydrosphingosine LOPAC 144 77 111 A3 C21H45N3 340 Hexetidine Spectrum 71 50 61 Cetyltrimethylammonium A4 C H BrN 364 Spectrum 47 38 43 19 42 Bromide A5 C32H55BrN4O 592 Thonzonium Bromide Spectrum 45 24 35 A6 C31H39FN4O2 519 NAT14–350426 NATx 129 18 74

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Figure 7. Chemical structures of selected WT HYPE activators. (A) Compound A1.(B) Compound A2. Figure 7. Chemical structures of selected WT HYPE activators. (A) Compound A1. (B) Compound (C) Compound A3.(D) Compound A4.(E) Compound A5.(F) Compound A6. A2. (C) Compound A3. (D) Compound A4. (E) Compound A5. (F) Compound A6. Concentration–response curves were generated for these hits using minimal buffer with or without Table 2. WT HYPE activator hit information. 0.1% Triton X-100 detergent, to control for false positive FP signals arising from nonspecific aggregation. In the absence of detergent, we observed robust dose-dependencies among all activators,% % with% low Compound Chemical MW Chemical Name Library Act. Act. Mean micromolarID EC50Formulavalues (Figure(Da.)8A–E; A6 data not available). With the addition of detergent, however, the putative activators saw a drastic decrease in signal and were only active at1 high2 micromolar Act. concentrationsA1 C (Figure31H23Cl7N S4A–F).2O 688 In fact, A3 reported Calmidazolium no dose-dependency LOPAC at all, even 177 at concentrations 114 146 up A2 C18H39NO2 302 DL-erythro-dihydrosphingosine LOPAC 144 77 111 to 1000 µM (Figure S4C). Given their long alkyl chains (Figure7, compounds A2–A5), these compounds A3 C21H45N3 340 Hexetidine Spectrum 71 50 61 may have been aiding in light polarization via aggregation-assisted trapping of Fl-ATP, rather than Cetyltrimethylammonium A4 C19H42BrN 364 Spectrum 47 38 43 Int.true J. Mol. AMPylation Sci. 2020, 21 [,36 x FOR]. PEER REVIEW Bromide 10 of 21 A5 C32H55BrN4O 592 Thonzonium Bromide Spectrum 45 24 35 A6 C31H39FN4O2 519 NAT14–350426 NATx 129 18 74

Concentration–response curves were generated for these hits using minimal buffer with or without 0.1% Triton X-100 detergent, to control for false positive FP signals arising from nonspecific aggregation. In the absence of detergent, we observed robust dose-dependencies among all activators, with low micromolar EC50 values (Figure 8A–E; A6 data not available). With the addition of detergent, however, the putative activators saw a drastic decrease in signal and were only active at high micromolar concentrations (Figure S4A–F). In fact, A3 reported no dose-dependency at all, even at concentrations up to 1000 μM (Figure S4C). Given their long alkyl chains (Figure 7, compounds A2–A5), these compounds may have been aiding in light polarization via aggregation-assisted trapping of Fl-ATP, rather than true AMPylation [36].

FigureFigure 8. WTWT HYPE HYPE activator activator concentration–response curve. curve. (A (A––EE) )FP FP plots plots from from Δ∆102102 WT WT HYPE HYPE AMPylationAMPylation reactions incubatedincubated withwith 00 toto 200 200µ μMM of of DMSO-dissolved DMSO-dissolved activators. activators. All All data data were were fitted fitted to toEquation Equation (5) (5) (see (see Methods) Methods) to determine to determine EC50 ECvalues.50 values. All reactionsAll reactions were were done indone detergent-less in detergent-less buffer. buffer.

2.5. WT HYPE Activator In-Gel Fluorescence Counterscreen

To further validate these hits, we turned to an orthogonal biochemical assay. In addition to detecting FP, the Fl-ATP fluorophore is amenable to in-gel fluorescence AMPylation assays [7,28]. Unlike the FP-based assay—in which AMPylation is indirectly inferred via reduced rotation of the fluorophore—in-gel assays track the direct modification of proteins separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Using our typical Δ102 construct, we confirmed two of the putative activators (A1 and A4) (Figure 9A). In the presence of detergent, however, A4 was unable to induce AMPylation above the DMSO control (Figure 9B). As seen in the concentration–response curve (Figure S4D), addition of detergent is likely reversing the aggregative effect of A4.

Figure 9. WT HYPE activator validation using in-gel fluorescence. (A) Quantification of Δ102 WT HYPE in-gel fluorescence autoAMPylation reaction in minimal buffer without detergent. (B) As in (A), but with 0.1% Triton X-100. (C) As in (A), but with Δ45 WT HYPE. (D) As in (A), but with Δ45 WT HYPE and 0.1% Triton X-100. Quantified data represented as the mean +/− SEM of three independent experiments. Unpaired t-tests were performed: * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

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Figure 8. WT HYPE activator concentration–response curve. (A–E) FP plots from Δ102 WT HYPE AMPylation reactions incubated with 0 to 200 μM of DMSO-dissolved activators. All data were fitted Int. J.to Mol. Equation Sci. 2020 ,(5)21 ,(see 7128 Methods) to determine EC50 values. All reactions were done in detergent-less10 of 20 buffer.

2.5.2.5. WT WT HYPE HYPE Activator Activator In-Gel In-Gel Fluorescence Fluorescence Counterscreen Counterscreen ToTo further validate these hits, we turned to an orthogonal biochemical as assay.say. In In addition addition to to detectingdetecting FP, FP, the the Fl-ATP Fl-ATP fluorophore fluorophore is is amenable amenable to to in-gel in-gel fluorescence fluorescence AMPylation AMPylation assays assays [7,28]. [7,28]. UnlikeUnlike the the FP-based FP-based assay—in assay—in which which AMPylation AMPylation is indirectly is indirectly inferred inferred via viareduced reduced rotation rotation of the of fluorophore—in-gelthe fluorophore—in-gel assays assays track track the thedirect direct modi modificationfication of ofproteins proteins separated separated by by SDS-PAGE SDS-PAGE (sodium(sodium dodecyl dodecyl sulfate sulfate polyacrylamide polyacrylamide gel gel electrophoresis). electrophoresis). Using Using our ourtypical typical Δ102∆ 102construct, construct, we confirmedwe confirmed two twoof the of theputative putative activators activators (A1 (A1 andand A4A4) (Figure) (Figure 9A).9A). In In the the presence presence of detergent, however,however, A4A4 waswas unable unable to to induce induce AMPylation AMPylation above above the the DMSO DMSO control control (Figure (Figure 99B).B). As seen in the concentration–responseconcentration–response curve curve (Figure (Figure S4D), S4D), addition addition of of detergent detergent is is likely likely reversing reversing the the aggregative aggregative effecteffect of of A4A4..

FigureFigure 9. WTWT HYPE HYPE activator activator validation validation using using in-gel in-gel fluorescence. fluorescence. ( (AA)) Quantification Quantification of of Δ∆102102 WT WT HYPEHYPE in-gel fluorescencefluorescence autoAMPylationautoAMPylation reaction reaction in in minimal minima bul bufferffer without without detergent. detergent. (B) ( AsB) inAs ( Ain), (butA), withbut with 0.1% 0.1% Triton Triton X-100. X-100. (C) As(C) inAs ( Ain), ( butA), but with with∆45 Δ WT45 WT HYPE. HYPE. (D) ( AsD) inAs ( Ain), ( butA), but with with∆45 Δ WT45 HYPE and 0.1% Triton X-100. Quantified data represented as the mean +/ SEM of three independent WT HYPE and 0.1% Triton X-100. Quantified data represented as the− mean +/− SEM of three experiments. Unpaired t-tests were performed: * = p < 0.05; ** = p < 0.01; *** = p < 0.001. independent experiments. Unpaired t-tests were performed: * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Crystallographic analysis of WT and E234G HYPE indicates a compact, primarily alpha-helical structure [29,30]. These studies, however, were performed with ∆102 constructs; no structures of ∆45 HYPE have been published. We therefore sought to confirm the efficacy of our hits with the larger, more physiologically relevant construct. To this end, we repeated the previous in-gel AMPylation assay with ∆45 HYPE in the presence and absence of Triton X-100. Without detergent, A1 was the only active compound (Figure9C); however, the inclusion of detergent led to a significant increase in bioactivity of A1 and A4–A6 (Figure9D). With all of their Hill’s coe fficients being greater than one, the emergence of bioactivity in the larger ∆45 HYPE could result from allosteric cooperativity (Table S2) [37]. Of all the putative WT HYPE activators, A1 (calmidazolium; Figure7) displayed the strongest FP signal and the most consistent in-gel fluorescence AMPylation signal. Strikingly, this very compound was discovered in a previous HTS campaign as an inhibitor of the bacterial Fic adenylyltransferase VopS [7]. Here, calmidazolium had mechanisms of inhibition noncompetitive to ATP and competitive to its substrate Cdc42 [7]. Given the high evolutionary conservation among Fic domain-containing proteins, we hypothesized a broader interaction between calmidazolium and the Fic domain writ large. Accordingly, we challenged another bacterial Fic AMPylase, IbpA-Fic2, with our A1 activator. Like VopS, IbpA-Fic2 preferentially acts on a Q61L mutant of the human GTPase Cdc42 during bacterial pathogenesis [1,2,6]. We thus predicted calmidazolium to outcompete Cdc42 for IbpA-Fic2 and perturb AMPylation. Alternatively, calmidazolium could enhance IbpA-Fic2 AMPylation in a manner Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 21

Crystallographic analysis of WT and E234G HYPE indicates a compact, primarily alpha-helical structure [29,30]. These studies, however, were performed with Δ102 constructs; no structures of Δ45 HYPE have been published. We therefore sought to confirm the efficacy of our hits with the larger, more physiologically relevant construct. To this end, we repeated the previous in-gel AMPylation assay with Δ45 HYPE in the presence and absence of Triton X-100. Without detergent, A1 was the only active compound (Figure 9C); however, the inclusion of detergent led to a significant increase in bioactivity of A1 and A4–A6 (Figure 9D). With all of their Hill’s coefficients being greater than one, the emergence of bioactivity in the larger Δ45 HYPE could result from allosteric cooperativity (Table S2) [37]. Of all the putative WT HYPE activators, A1 (calmidazolium; Figure 7) displayed the strongest FP signal and the most consistent in-gel fluorescence AMPylation signal. Strikingly, this very compound was discovered in a previous HTS campaign as an inhibitor of the bacterial Fic adenylyltransferase VopS [7]. Here, calmidazolium had mechanisms of inhibition noncompetitive to ATP and competitive to its substrate Cdc42 [7]. Given the high evolutionary conservation among Fic domain-containing proteins, we hypothesized a broader interaction between calmidazolium and the Fic domain writ large. Accordingly, we challenged another bacterial Fic AMPylase, IbpA-Fic2, with our A1 activator. Like VopS, IbpA-Fic2 preferentially acts on a Q61L mutant of the human GTPase Int. J. Mol. Sci. 2020, 21, 7128 11 of 20 Cdc42 during bacterial pathogenesis [1,2,6]. We thus predicted calmidazolium to outcompete Cdc42 for IbpA-Fic2 and perturb AMPylation. Alternatively, calmidazolium could enhance IbpA-Fic2 resemblingAMPylation its in interaction a manner with resembling HYPE. However, its intera we detectedction with no manipulationHYPE. However, of AMPylation we detected by any no of themanipulation tested compounds of AMPylation relative by to theany DMSO of the controltested compounds (Figure S5). Therelati activityve to the of calmidazoliumDMSO control (Figure against HYPES5). The and activity VopS of is likelycalmidazolium attributable against to its HYPE vast biological and VopS promiscuity, is likely attributable rather than to its some vast Fic-specific biological mechanismpromiscuity, [38 rather–40]. than some Fic-specific mechanism [38–40].

2.6. E234G HYPE Inhibitor High-Throughput Screen Holding all reaction parameters static from our WT HYPE activator HTS, we screened the same fourfour librarieslibraries (Table1 1)) for for inhibition inhibition of of ∆Δ102102 E234GE234G HYPE. We sawsaw aa highhigh overalloverall inhibitioninhibition raterate ofof 0.98% atat a 50%a inhibition50% inhibition cut-off, evencut-off, after adjustmenteven after for auto-fluorescentadjustment for and auto-fluorescent fluorescent-quenching and moleculesfluorescent-quenching (Figure 10A). molecules We therefore (Figure chose 10A). a more We stringent therefore 50% chose threshold a more tostringent narrow 50% our hitsthreshold down toto anarrow more manageableour hits down level. to a The more greater manageable rate of inhibitorlevel. The hits greater relative rate to of activators inhibitor hits is as relative expected, to givenactivators the easeis as with expected, which enzymaticgiven the activityease with can which be disrupted enzymatic versus activity enhanced can [be41 ].disrupted Consistent versus with theenhanced activator [41]. screen, Consistent the LOPAC, with Spectrum,the activator and sc NATxreen, librariesthe LOPAC, yielded Spectrum, more hits and than NATx the syntheticlibraries compoundsyielded more from hits MEGxthan the (Figure synthetic 10A). compounds To assess compound from MEGx activity (Figure reproducibility, 10A). To assess we compound randomly selectedactivity reproducibility, a plate from the we NATx randomly library. selected This plate a plate showed from strong, the NATx positive library. correlation This plate (Figure showed S6), thusstrong, demonstrating positive correlation the high reproducibility(Figure S6), thus of ourdemonstrating assay. Further, the wehigh report reproducibility suitable Z’ and of our S/B valuesassay. throughoutFurther, we thereport screen suitable (Figure Z’ 10andB). S/B values throughout the screen (Figure 10B).

Figure 10.10.E234G E234G HYPE HYPE inhibitor inhibitor high-throughput high-throughput screen. (Ascreen.) ∆102 E234G(A) HYPEΔ102 FPE234G autoAMPylation HYPE FP reactionautoAMPylation incubated reaction with compoundsincubated with from compou eithernds LOPAC, from Spectrum,either LOPAC, MEGx, Spectrum, or NATx MEGx, libraries. or AllNATx samples libraries. were All normalizedsamples were to normalized∆102 WT andto Δ102 E234G WT HYPEand E234G controls HYPE on controls the same on the plate same in DMSO-containing buffer. Black dashes represent +/ 3 SD from the mean. Red dashes are the plate in DMSO-containing buffer. Black dashes represent− +/− 3 SD from the mean. Red dashes are the 50% hit definition, with all dots above this threshold being hit compounds. Each dot represents a different compound incubated with a single AMPylation reaction in a separate well. Dots with red circles show hits selected for follow-up, orthogonal validation. (B) Plate-to-plate variability shows no difference between the positive (∆102 E234G HYPE) and negative (∆102 WT HYPE) DMSO controls. Left and right y-axes are for Z’ and S/B values, respectively. Each dot represents the calculated Z’ or S/B values from 24 E234G HYPE positive controls and 24 WT HYPE negative controls within a single plate.

2.7. E234G HYPE Inhibitor Microplate Validation Using an independent replication of the same HTS assay, we next validated our E234G HYPE inhibitor hits. These hits saw much greater correlation than the activators—as determined by a higher R2 value—likely due to their more stringent hit definition of 50% inhibition (Figure 11A). These correlations were generally consistent across libraries (Figure 11B–E). Additionally, correlation among LOPAC compounds was enriched relative to other libraries, possibly resulting from their exclusively bioactive identity (Table1). In accordance with the findings of our unlabeled ATP assay (Figure S2), we discovered multiple chemical structures analogous to ATP (data not shown). Indeed, one of our top inhibitors, the ATP analog diadenosine tetraphosphate (Ap4A), was previously identified in a screen targeting the bacterial Fic effector protein VopS [7]. Of note, E234G HYPE-mediated AMPylation of its putative substrate Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 12 of 21

50% hit definition, with all dots above this threshold being hit compounds. Each dot represents a different compound incubated with a single AMPylation reaction in a separate well. Dots with red circles show hits selected for follow-up, orthogonal validation. (B) Plate-to-plate variability shows no difference between the positive (Δ102 E234G HYPE) and negative (Δ102 WT HYPE) DMSO controls. Left and right y-axes are for Z’ and S/B values, respectively. Each dot represents the calculated Z’ or S/B values from 24 E234G HYPE positive controls and 24 WT HYPE negative controls within a single plate.

2.7. E234G HYPE Inhibitor Microplate Validation Using an independent replication of the same HTS assay, we next validated our E234G HYPE inhibitor hits. These hits saw much greater correlation than the activators—as determined by a Int.higher J. Mol. R Sci.2 value—likely2020, 21, 7128 due to their more stringent hit definition of 50% inhibition (Figure12 11A). of 20 These correlations were generally consistent across libraries (Figure 11B–E). Additionally, histonecorrelation H3 was among shown LOPAC to be inhibitedcompounds by Ap4Awas enrichedin vitro [ 7relative], presumably to other via libraries, an ATP-competitive possibly resulting mode offrom action. their exclusively bioactive identity (Table 1).

Figure 11. E234G HYPE inhibitor validation. (A) Comparison between initial HTS compound activity Figure 11. E234G HYPE inhibitor validation. (A) Comparison between initial HTS compound activity (x-axis) and second-pass validation (y-axis) with the same FP AMPylation assay. All validation (x-axis) and second-pass validation (y-axis) with the same FP AMPylation assay. All validation compounds were assayed on the same plate and normalized to internal positive (E234G HYPE) and compounds were assayed on the same plate and normalized to internal positive (E234G HYPE) and negative (WT HYPE) DMSO controls. Each dot represents the same compound incubated in two negative (WT HYPE) DMSO controls. Each dot represents the same compound incubated in two independent AMPylation reactions. Dots with red circles show hits selected for follow-up, orthogonal independent AMPylation reactions. Dots with red circles show hits selected for follow-up, validation. (B–E) Data reprocessed from (A) to show compound validation among the various libraries. orthogonal validation. (B–E) Data reprocessed from (A) to show compound validation among the Employingvarious libraries. similar selection criteria as with the activators, we isolated eight inhibitors (I1–I8) for Int.follow-up J. Mol. Sci. investigation 2020, 21, x FOR PEER (Figures REVIEW 10A, 11A and 12A–H, Table3). 13 of 21 In accordance with the findings of our unlabeled ATP assay (Figure S2), we discovered multiple chemical structures analogous to ATP (data not shown). Indeed, one of our top inhibitors, the ATP analog diadenosine tetraphosphate (Ap4A), was previously identified in a screen targeting the bacterial Fic effector protein VopS [7]. Of note, E234G HYPE-mediated AMPylation of its putative substrate H3 was shown to be inhibited by Ap4A in vitro [7], presumably via an ATP-competitive mode of action. Employing similar selection criteria as with the activators, we isolated eight inhibitors (I1–I8) for follow-up investigation (Figures 10A, 11A, and 12A–H, Table 3).

Figure 12. Chemical structures of selected E234G HYPE inhibitors. (A) Compound I1.(B) Compound Figure 12. Chemical structures of selected E234G HYPE inhibitors. (A) Compound I1. (B) Compound I2.(C) Compound I3.(D) Compound I4.(E) Compound I5.(F) Compound I6.(G) Compound I7.(H) I2. (C) Compound I3. (D) Compound I4. (E) Compound I5. (F) Compound I6. (G) Compound I7. (H) Compound I8. Compound I8.

Table 3. E234G HYPE inhibitor hit information.

Compound Chemical MW % % % Mean Chemical Name Library ID Formula (Da.) Inh. 1 Inh. 2 Inh. I1 C34H24N6Na4O14S4 961 Evan’s Blue Spectrum 109 109 109 Aurintricarboxylic I2 C22H14O9 422 LOPAC 107 106 107 Acid I3 C34H24N6Na4O16S4 993 Chicago Sky Blue Spectrum 106 109 108 I4 C16H14O6 302 Hematoxylin Spectrum 105 30 68 I5 C15H10O8 318 LOPAC 99 86 93 I6 C18H25N3O6 379 NAT2–252122 NATx 106 102 104 I7 C27H36N4O2 449 NAT28–408090 NATx 94 102 98 I8 C24H36N4O2 413 NAT28–405040 NATx 61 61 61

For this, we performed concentration–response experiments in the presence of detergent. Under these conditions, only three of the compounds (I1–I3) displayed dose-dependency (Figure 13A). After fitting the concentration–response data of I1–I3 to the IC50 equation (Equation (6), see Methods), we obtained a low micromolar inhibitory potency for I2, while I1 and I3 were in the mid-micromolar range (Figure 13B–D). To assess whether the reduced bioactivity of I1 and I3 is a function of aggregative forces, we repeated this experiment in the absence of detergent. Unlike in the activator screen—where compound aggregation can sequester Fl-ATP to produce a false positive signal—aggregation of putative inhibitors poses a different threat. Here, aggregative compounds can form unwanted colloidal complexes with the enzyme, thus restricting catalysis [36,42]. Indeed, I1′s IC50 value dropped substantially in detergent-free solution; the IC50 value for I2 remained in the low micromolar range (Figure S7A–C). The activity of I3, however, saw no improvement over its initial mid-micromolar range, indicating an impartial response to detergent.

Int. J. Mol. Sci. 2020, 21, 7128 13 of 20

Table 3. E234G HYPE inhibitor hit information.

Compound Chemical % Mean MW (Da.) Chemical Name Library % Inh. 1 % Inh. 2 ID Formula Inh.

I1 C34H24N6Na4O14S4 961 Evan’s Blue Spectrum 109 109 109 I2 C22H14O9 422 Aurintricarboxylic Acid LOPAC 107 106 107 I3 C34H24N6Na4O16S4 993 Chicago Sky Blue Spectrum 106 109 108 I4 C16H14O6 302 Hematoxylin Spectrum 105 30 68 I5 C15H10O8 318 Myricetin LOPAC 99 86 93 I6 C18H25N3O6 379 NAT2–252122 NATx 106 102 104 I7 C27H36N4O2 449 NAT28–408090 NATx 94 102 98 I8 C24H36N4O2 413 NAT28–405040 NATx 61 61 61

For this, we performed concentration–response experiments in the presence of detergent. Under these conditions, only three of the compounds (I1–I3) displayed dose-dependency (Figure 13A). After fitting the concentration–response data of I1–I3 to the IC50 equation (Equation (6), see Methods), we obtained a low micromolar inhibitory potency for I2, while I1 and I3 were in the mid-micromolar range (Figure 13B–D). To assess whether the reduced bioactivity of I1 and I3 is a function of aggregative forces, we repeated this experiment in the absence of detergent. Unlike in the activator screen—where compound aggregation can sequester Fl-ATP to produce a false positive signal—aggregation of putative inhibitors poses a different threat. Here, aggregative compounds can form unwanted colloidal complexes with the enzyme, thus restricting catalysis [36,42]. Indeed, I10s IC50 value dropped substantially in detergent-free solution; the IC50 value for I2 remained in the low micromolar range (Figure S7A–C). The activity of I3, however, saw no improvement over its initial mid-micromolar range,Int. J. Mol. indicating Sci. 2020, 21 an, x impartialFOR PEER REVIEW response to detergent. 14 of 21

FigureFigure 13.13.E234G E234G HYPE HYPE inhibitor inhibitor concentration–response concentration–response curve. curve. (A) FP(A) plots FP plots from ∆from102 E234GΔ102 E234G HYPE µ B D AMPylationHYPE AMPylation reactions reactions incubated incubated with 0 with to 200 0 toM 200 of μ DMSO-dissolvedM of DMSO-dissolved inhibitors. inhibitors. ( – )(B Data–D) fromData (A) fitted to Equation (6) (see Methods) to determine IC50 values. All reactions were done in buffer from (A) fitted to Equation (6) (see Methods) to determine IC50 values. All reactions were done in containing 0.1% Triton X-100. buffer containing 0.1% Triton X-100. 2.8. E234G HYPE Inhibitor In-Gel Fluorescence Counterscreen 2.8. E234G HYPE Inhibitor In-Gel Fluorescence Counterscreen To confirm their inhibitory potencies via an assay that measures direct protein AMPylation, we subjectedTo confirmI1– I8theirto ourinhibitory in-gel fluorescence potencies via counterscreen. an assay that Further, measures we direct wanted protein to assess AMPylation, the impact we of detergentsubjected onI1– theirI8 to activities.our in-gel Asfluorescence predicted, counterscreen. of our five initially Further, confirmed we wanted inhibitors, to assess only the two impact of them of detergent on their activities. As predicted, of our five initially confirmed inhibitors, only two of them (I2 and I5) survived detergent treatment (Figure 14A,B). This trend was largely reproduced using the Δ45 HYPE construct, with I2 retaining consistently high efficacy (Figure 14C,D). Using molecular docking of I2 to a solved crystal structure of HYPE, we sought to gain insight into its preferred orientation and binding affinity. Indeed, we find that I2 consistently binds to the Fic active site in silico with favorable energetics in the top five searches (Figure S8A–C, Table S4). Our biochemical data were further corroborated by the weak binding affinity and inconsistent docking of our negative control, I8 (Figure S8D, Table S4). Given its reproducible bioactivity across assays, assay conditions, and target variants, I2 (aurintricarboxylic acid) holds promise as both a therapeutic intervention and as a research tool for manipulating AMPylation in vitro. Moreover, aurintricarboxylic acid satisfies Lipinski’s rule of five, indicating its drug-likeness [43]. However, much like our top activator (calmidazolium), aurintricarboxylic acid suffers from broad target promiscuity [44–46]. The potential of aurintricarboxylic acid as a HYPE-specific therapeutic agent, therefore, depends on subsequent hit-to-lead optimization.

Int. J. Mol. Sci. 2020, 21, 7128 14 of 20

(I2 and I5) survived detergent treatment (Figure 14A,B). This trend was largely reproduced using the Int.∆45 J. Mol. HYPE Sci. 2020 construct,, 21, x FOR with PEERI2 REVIEWretaining consistently high efficacy (Figure 14C,D). 15 of 21

Figure 14. E234G HYPE inhibitor validation using in-gel fluorescence. (A) Quantification of ∆102 E234G HYPE in-gel fluorescence autoAMPylation reaction in minimal buffer without detergent. (B) As Figure 14. E234G HYPE inhibitor validation using in-gel fluorescence. (A) Quantification of Δ102 in (A), but with 0.1% Triton X-100. (C) As in (A), but with ∆45 E234G HYPE. (D) As in (A), but with E234G HYPE in-gel fluorescence autoAMPylation reaction in minimal buffer without detergent. (B) ∆45 E234G HYPE and 0.1% Triton X-100. Quantified data represented as the mean +/ SEM of three As in (A), but with 0.1% Triton X-100. (C) As in (A), but with Δ45 E234G HYPE. (D) −As in (A), but independent experiments. Unpaired t-tests were performed: * = p < 0.05; ** = p < 0.01. with Δ45 E234G HYPE and 0.1% Triton X-100. Quantified data represented as the mean +/− SEM of threeUsing independent molecular experiments. docking of UnpairedI2 to a solved t-testscrystal were performed: structure * of= p HYPE, < 0.05; ** we = p sought < 0.01. to gain insight into its preferred orientation and binding affinity. Indeed, we find that I2 consistently binds to the Fic activeUsing siteour intop silico three with inhibitors favorable (I1 energetics–I3), we next in the assessed top five whethe searchesr this (Figure compound-mediated S8A–C, Table S4). disruptionOur biochemical of AMPylation data were could further be corroborated a pan-Fic phen by theomenon. weak bindingFor this, a weffinity turned and inconsistentto our bacterial docking Fic IbpA-Fic-2of our negative and control,its substrateI8 (Figure Q61L S8D, Cdc42. Table Interestingly, S4). Given itsthe reproducible HYPE inhibitors bioactivity failed across to inhibit assays, AMPylationassay conditions, of Q61L and Cdc42 target by variants, IbpA-Fic2I2 (aurintricarboxylic (Figure S9). Given acid) the high holds degree promise of ashomology both a therapeutic between theintervention Fic domains and of as HYPE a research and IbpA-Fic2, tool for manipulating our data suggest AMPylation these inhibitorsin vitro. Moreover, are HYPE-specific aurintricarboxylic and do notacid target satisfies a common Lipinski’s Fic active rule of site. five, indicating its drug-likeness [43]. However, much like our top activator (calmidazolium), aurintricarboxylic acid suffers from broad target promiscuity [44–46]. 3. Materials and Methods The potential of aurintricarboxylic acid as a HYPE-specific therapeutic agent, therefore, depends on subsequent hit-to-lead optimization. 3.1. Protein Expression and Purification Using our top three inhibitors (I1–I3), we next assessed whether this compound-mediated disruptionRecombinant of AMPylation His6-SUMO-tagged could be a pan-Ficproteins phenomenon. were purified For as this, previously we turned described to our bacterial [21]. Specifically,Fic IbpA-Fic-2 HYPE and constructs its substrate (WT, Q61L E234G, Cdc42. and Interestingly,E234G/H363A) the and HYPE WT inhibitorsBiP proteins failed were to cloned inhibit intoAMPylation pSMT3 plasmids of Q61L Cdc42and expressed by IbpA-Fic2 in E. (Figurecoli BL21-DE3-RILP S9). Given the (Stratagene) high degree in of LB homology medium betweencontaining the 50Fic μ domainsg/mL of kanamycin of HYPE and to IbpA-Fic2,an optical density our data of suggest A600 = 0.6. these Protein inhibitors expression are HYPE-specific was induced and with do 0.4 not mMtarget IPTG a common for 12–16 Fic h active at 18 site.°C. Lysis was performed on frozen pellets dissolved in lysis buffer (50 mM Tris, 250 mM NaCl, 5 mM imidazole, 1 mM PMSF, pH 7.5). Lysed cells were centrifuged at 15,000×3. Materials g for and50 min. Methods Supernatants were poured over cobalt resin. Resin was washed with wash buffer (50 mM Tris, 250 mM NaCl, 15 mM imidazole, pH 7.5). Tagged proteins were eluted with 3.1. Protein Expression and Purification elution buffer (50 mM Tris, 250 mM NaCl, 350 mM imidazole, pH 7.5). The His6-SUMO tags were cleavedRecombinant by incubating His6 pr-SUMO-taggedoteins with ULP1 proteins overnight were purified at 4 °C. as The previously protein described mixture [was21]. Specifically,diluted in washHYPE buffer constructs without (WT, imidazole E234G, and re-applied E234G/H363A) into a and cobalt WT column. BiP proteins The flow-through were cloned intocontaining pSMT3 cleavedplasmids protein and expressed was further in E.purified coli BL21-DE3-RILP by size-exclusion (Stratagene) chromatography in LB medium in a buffer containing containing 50 µg 100/mL mMof kanamycin Tris and to100 an mM optical NaCl, density pH of7.5. A 600Fractions= 0.6. Protein containing expression HYPE was were induced verified with for 0.4 purity mM IPTG by SDS-PAGE and pooled together. Protein concentrations were measured spectrophotometrically at A280. Proteins were flash-frozen and stored at −80 °C in storage buffer [50 mM Tris, 300 mM NaCl, 10% (v/v) glycerol, pH 7.5]. His6-tagged WT α-syn was expressed and purified similarly as described above with the following modifications. WT α-syn was cloned into a pT7-7 plasmid and expressed in E. coli

Int. J. Mol. Sci. 2020, 21, 7128 15 of 20

for 12–16 h at 18 ◦C. Lysis was performed on frozen pellets dissolved in lysis buffer (50 mM Tris, 250 mM NaCl, 5 mM imidazole, 1 mM PMSF, pH 7.5). Lysed cells were centrifuged at 15,000 g for × 50 min. Supernatants were poured over cobalt resin. Resin was washed with wash buffer (50 mM Tris, 250 mM NaCl, 15 mM imidazole, pH 7.5). Tagged proteins were eluted with elution buffer (50 mM Tris, 250 mM NaCl, 350 mM imidazole, pH 7.5). The His6-SUMO tags were cleaved by incubating proteins with ULP1 overnight at 4 ◦C. The protein mixture was diluted in wash buffer without imidazole and re-applied into a cobalt column. The flow-through containing cleaved protein was further purified by size-exclusion chromatography in a buffer containing 100 mM Tris and 100 mM NaCl, pH 7.5. Fractions containing HYPE were verified for purity by SDS-PAGE and pooled together. Protein concentrations were measured spectrophotometrically at A . Proteins were flash-frozen and stored at 80 C in 280 − ◦ storage buffer [50 mM Tris, 300 mM NaCl, 10% (v/v) glycerol, pH 7.5]. His6-tagged WT α-syn was expressed and purified similarly as described above with the following modifications. WT α-syn was cloned into a pT7-7 plasmid and expressed in E. coli BL21-DE3-RILP (Stratagene) in LB medium containing 100 µg/mL of ampicillin to an optical density of A600 = 0.6. Protein expression was induced with 0.4 mM IPTG for 3–4 h at 30 ◦C. Lysis was performed on frozen pellets dissolved in lysis buffer. Lysed cells were centrifuged at 15,000 g for 50 min. Supernatants were × poured over cobalt resin. Resin was washed with wash buffer. Tagged proteins were eluted with elution buffer. Eluted protein was further purified by size-exclusion chromatography in a buffer containing 100 mM Tris and 100 mM NaCl, pH 7.5. Fractions containing WT α-syn were verified for purity by SDS-PAGE and pooled together. Protein concentrations were measured spectrophotometrically at A280. Proteins were flash-frozen and stored at 80 C in storage buffer. − ◦ GST-tagged WT IbpA-Fic2 and Q61L Cdc42 were bacterially expressed and purified as previously described [5].

3.2. Assay Development

3.2.1. In-Gel Kinetics For the E234G HYPE time-course experiment, 0.2 µM of ∆102 HYPE enzyme was incubated with 25 nM Fl-ATP in minimal AMPylation buffer (50 mM HEPES, 1 mM MnCl2, pH 7.5) at 37 ◦C in the dark for the specified times. Reactions were stopped with 4X SDS loading dye, boiled for 5 min at 95 ◦C, and run on 12% SDS-PAGE gels. Gels were imaged on a Typhoon 9500 FLA imager (General Electric, Chicago, IL, USA) for fluorescence at 473 nm. Imaged gels were then stained with , destained, and imaged for protein concentration. The Fl-ATP concentration experiment was performed as previously described, but with specified concentrations of Fl-ATP, and at a 10 min timepoint.

3.2.2. Microplate Optimization Buffer comparison experiments were performed by incubating 0.2 µM of ∆102 HYPE enzyme with 25 nM Fl-ATP (final concentrations) in minimal or complete (50 mM HEPES (pH 7.4), 150 mM KCl, 1 mM CaCl2, 10 mM MgCl2, 0.1% Triton-X 100) AMPylation buffer in the dark at room temperature for 10 min in 384-well black-bottom, black-walled microplates (20 µL reaction volume). Where specified, buffers were supplemented with 1% DMSO. Microplates were snap-centrifuged at 1000 g for 10 s. × Microplates were loaded onto a BioTek BioStack NEO2 (Agilent, Winooski, VT, USA) plate-reader and assessed for fluorescence polarization with 485/530 nm filters and a 55/50 gain adjustment. Where specified, enzymes were incubated with a 10 molar excess of substrate. × A Multidrop 384 reagent dispenser (ThermoFisher, Waltham, MA, USA) was used to add 0.2 or 0.4 µM ∆102 HYPE, then 25 nM Fl-ATP (final concentrations) to 384-well black/black microplates (20 µL reaction volume). Both enzyme and nucleotide were dissolved in minimal AMPylation buffer. AMPylation reactions were incubated for 10 min at room temperature in the dark. Microplates were snap-centrifuged at 1000 g for 10 s, then transferred to a BioTek BioStack × Int. J. Mol. Sci. 2020, 21, 7128 16 of 20

NEO2 plate-reader. Reactions were assessed for fluorescence polarization with 485/530 nm filters and a 55/50 gain adjustment. Z’ and S/B (signal/background) values were determined by fitting the data to Equations (1) and (2), respectively,   3 σp + σn Z = 1 (1)

0 − µp µn − µp S/B = (2) µn where µp and µn are the means of the positive (E234G HYPE) and negative (WT HYPE) controls, respectively; and σp and σn are the standard deviations of the positive and negative controls, respectively.

3.3. High-Throughput Screen An Echo Liquid Handler (Labcyte, San Jose, CA, USA) was used to transfer 200 nL of DMSO- dissolved compounds via acoustic-coupled ejection from 10 mM source plates into 384-well black, flat-bottom, black-walled microplates, for a final compound concentration of 10 µM. Compounds were sourced from the following libraries: LOPAC1280 (Sigma Aldrich), The Spectrum Collection (Microsource Discovery Systems), MEGx (Analyticon Discovery), and NATx (Analyticon Discovery). The compounds went into columns 3–22 of each plate, while columns 1, 2, 23, and 24 were reserved for an equivalent volume of 100% DMSO controls. A Multidrop 384 reagent dispenser was used to pipette 0.4 µM of ∆102 WT HYPE (final concentration) dissolved in minimal AMPylation buffer into columns 1–22 of the activator plates, and columns 1 and 2 of the inhibitor plates as positive controls. Then, 0.4 µM of ∆102 E234G HYPE was similarly added to columns 3–24 of the inhibitor plates, and columns 23 and 24 of the inhibitor plates as positive controls. Enzymes were then incubated with either compounds or DMSO for 10 min at room temperature. A Multidrop 384 reagent dispenser was used to pipette 25 nM of Fl-ATP (final concentration) into all wells of each plate, giving a final reaction volume of 20 µL. Plates were then incubated for 10 min at room temperature in the dark, followed by a snap-centrifugation at 1000 g for 10 s. Microplates were loaded × onto a BioTek BioStack stacker and read in succession for fluorescence polarization with 485/530 nm filters and a 55/50 gain adjustment. Fluorescence polarization values were converted to fractional activation (FA) or fractional inhibition (FI) values according to Equations (3) or (4), respectively:

(x µn) FA = − (3) µn   X µp FI = − (4) µp where µp and µn are the means of the positive (E234G HYPE) and negative (WT HYPE) controls, respectively; and x is the measured value of fluorescence polarization. Microplate “cherry-picking” validations were performed as described above.

3.4. Concentration–Response Curves A Multidrop 384 reagent dispenser (ThermoFisher, Waltham, USA) was used to pipette 0.4 µM of ∆102 WT or E234G HYPE (final concentrations) dissolved in minimal AMPylation buffer with 0.1% Triton X-100 (final concentration) into designated microplate wells. In addition, 10 µM of DMSO-dissolved compounds (final concentrations) or an equivalent volume of 100% DMSO was manually pipetted into their specified wells. Enzymes were incubated with compounds or DMSO for 10 min at room temperature. The reagent dispenser was then used to pipette 25 nM of Fl-ATP (final concentration) into each well, followed by a 10 min incubation at room temperature in the dark. Plates were snap-centrifuged at 1000 g for 10 s, then transferred to the BioTek BioStack × Int. J. Mol. Sci. 2020, 21, 7128 17 of 20

NEO2 plate-reader. Reactions were assessed for fluorescence polarization with 485/530 nm filters and a 55/50 gain adjustment. EC50 and IC50 values were determined by fitting polarization values to Equations (5) and (6), respectively,

 n Ymax X [I] Y = + Y (5)  n n min [EC50] + [I]

 n Ymax X [I] Y = (6)  n n [IC50] + [I] where Ymax is the maximum polarization signal; I is the µM concentration of activator or inhibitor; n is the Hill slope; EC50 or IC50 are the inflection point concentrations; and Ymin is the baseline polarization response.

3.5. Fluorescence in-Gel Counterscreen Here, 1 µM of ∆102 or ∆45 WT HYPE (final concentration) were preincubated with 10 µM of DMSO-dissolved compound (final concentration) or an equivalent volume of 100% DMSO at room temperature for 10 min. Reactions were run for 1 h at 37 ◦C in the dark, and began with the addition of 1 µM Fl-ATP (final concentration). Alternatively, 0.4 µM of ∆45 or ∆102 E234G HYPE were preincubated with 10 µM of DMSO-dissolved compound (final concentration) or an equivalent volume of 100% DMSO at room temperature for 10 min. Reactions were run for 1 h at 37 ◦C in the dark, and began with the addition of 25 nM Fl-ATP (final concentration). All reactions were performed in minimal AMPylation buffer (50 mM HEPES, 1 mM MnCl2, pH 7.5) with or without 0.1% Triton X-100 (final concentration) as specified. All reactions were quenched with 4X SDS loading dye and boiled for 5 min at 95 ◦C, and the samples were run on 12% SDS-PAGE gels. Gels were imaged on a Typhoon 9500 FLA imager (General Electric) for fluorescence at 473 nm. Imaged gels were then stained with Coomassie Brilliant Blue, destained, and imaged for protein concentration. All bacterial Fic specificity assays were conducted with 1 µM GST-tagged IbpA-Fic2, 10 µM GST-tagged Q61L Cdc42, and 25 nM Fl-ATP. All other reaction parameters were kept as per HYPE AMPylation assays.

3.6. Molecular Docking Molecular docking to predict protein-compound co-structure and ∆G was done with the SwissDock (http://www.swissdock.ch/) web server. Following program requirements, a protein structure and ZINC (ZINC is not commercial) database accession number were provided [47]. Apo WT HYPE (amino acids 103–445; PDB: 4u04 chain A) was selected due to its free active site and similar overall structure to E234G HYPE. Resultant co-structures were sorted by the top five docking clusters, and the most optimal predicted ∆G values were imaged with the UCSF (University of California San Francisco) Chimera program [48].

4. Conclusions Despite residing at the regulatory intersection of several disease pathways, HYPE has yet to be targeted for an HTS campaign. Here, we present a novel, robust, dual screening assay capable of monitoring changes in HYPE autoAMPylation in vitro. Further, this assay has been optimized for high-throughput scale and demonstrates reproducibility in identifying target hits. In conjunction with our post-HTS counterscreens, we have established a reliable pipeline for hit validation. Indeed, we have discovered at least two promising modulators of HYPE’s adenylyltransferase activity—calmidazolium (A1) and aurintricarboxylic acid (I2). Though their activities against multiple known human targets restrict usage for therapeutic intervention, these compounds nevertheless remain promising candidates Int. J. Mol. Sci. 2020, 21, 7128 18 of 20 for hit-to-lead optimizations. Moreover, the use of these hits as research tools will aid in the mechanistic elucidation of HYPE’s cellular role in the UPR and other disease-associated pathways. Ongoing follow-up studies on remaining HTS hits not initially counterscreened are sure to provide additional activators and inhibitors of HYPE-mediated AMPylation. The high-quality assay described herein is also fully scalable for HTS processing and lends itself to the screening of larger, more diverse chemical libraries with more drug-like potential.

Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/19/7128/s1. Author Contributions: Conceptualization, A.C. and S.M.; methodology, A.C. and S.M.; validation, A.C. and S.M.; formal analysis, A.C. and S.M.; investigation, A.C. and S.M.; resources, S.M.; data curation, A.C., A.G., E.H., A.M., J.P. and S.M.; writing—original draft preparation, A.C., A.G. and E.H.; writing—review and editing, A.C. and S.M.; visualization, A.C. and S.M.; supervision, A.C. and S.M.; project administration, S.M.; funding acquisition, S.M. and A.C. All authors have read and agreed to the published version of the manuscript. Funding: This publication was made possible with partial support from the National Institute of General Medical Sciences of the National Institute of Health (R01GM10092), an Indiana Clinical and Translational Research Grant (CTSI-106564), and a Purdue Institute for Inflammation, Immunology, and Infectious Disease Core Start Grant (PI4D-209263) to S.M.; and partial support from Grant # UL1TR002529 (A. Shekhar, PI), 5/18/2018–4/30/2023, and Grant # TL1TR002531 (T. Hurley, PI), 5/18/2018–4/30/2023, from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award to A.C. Acknowledgments: The authors thank Lan Chen and Li Wu (Purdue Institute for Drug Discovery) for their technical advice regarding fluorescence polarization assays. We also thank Andrew Mesecar (Purdue Biochemistry) and Daniel Flaherty (Purdue Medicinal Chemistry and Molecular Pharmacology) for their insightful analysis of chemical structures. Finally, we are grateful to members of the Mattoo laboratory for their helpful discussions. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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