A Binding Site on IL-17A for Inhibitory Macrocycles Revealed by

Feb 6, 2016 - Computational assessment of the IL-17A structure identified two distinct binding pockets, the β-hairpin pocket and the α-helix pocket...
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A Binding Site on IL-17A for Inhibitory Macrocycles Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry Alfonso Espada,*,† Howard Broughton,† Spencer Jones,‡ Michael J. Chalmers,‡ and Jeffrey A. Dodge‡ †

Centro de Investigación Lilly, SA, Avenida de la Industria 30, 28108 Alcobendas, Spain Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, United States

‡

S Supporting Information *

ABSTRACT: Computational assessment of the IL-17A structure identified two distinct binding pockets, the β-hairpin pocket and the α-helix pocket. The β-hairpin pocket was hypothesized to be the site of binding for peptide macrocycles. Support for this hypothesis was obtained using HDX-MS which revealed protection to exchange only within the β-hairpin pocket. This data represents the first direct structural evidence of a small molecule binding site on IL-17A that functions to disrupt the interaction with its receptor.

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INTRODUCTION

IL-17A has been demonstrated to be involved in host defense against both intracellular and extracellular bacterial infections as revealed through IL-17A−/− and IL-17RA−/− mice knockout experiments.17−19 Despite this beneficial capacity, IL-17A is implicated in the pathology of numerous autoimmune diseases including: psoriasis (Ps), rheumatoid arthritis (RA), ankylosing spondylitis (AS), irritable bowel disease (IBD), and multiple sclerosis (MS).1,2,20−23 Indeed, upregulation of IL-17A and IL17A producing cells is observed in patients with rheumatoid arthritis and psoriasis.24 Evidence of a causative relationship between IL-17A and inflammation associated with autoimmunity has been detected in multiple animal models of RA, Ps, MS, and IBD. Clinical evidence for the involvement of IL-17A and IL-17RA in autoimmune diseases has been demonstrated through human studies with anti-IL-17A and anti-IL-17RA monoclonal antibodies. Indeed, antibodies targeting IL-17RA (brodalumab) and IL-17A (ixekizumab, secukinumab) have shown significant efficacy in clinical trials for RA, uvevitis, and Ps.25−27 In addition to biologic agents targeting the IL-17A pathway, researchers have begun to question whether it might be possible to disrupt the IL-17A−IL-17RA protein−protein interaction (PPI) by use of a small molecule inhibitor. In analogy to the antibody approach, this strategy could be accomplished through the targeted development of small

IL-17A is the founding member of a class of pro-inflammatory cytokines involved in host defense and pathogenic autoimmunity.1−3 It is one of six known cytokines in the family (IL17A−F) and exists as a bis-disulfide linked homodimer as well as a heterodimer with IL-17F.4,5 Whereas IL-17A and IL-17F are formed from a range of cell types, they were initially characterized as arising from a subset of CD4+ T helper cells known as Th17 cells.6 Th17 cell differentiation from CD4+ T cells occurs as a result of exposure to a number of cytokines including IL-6, IL-23, IL-1β, and TGF-β.7,8 The homodimers IL-17A and IL-17F and the heterodimer IL-17A−IL-17F induce their biological effect through association with an IL-17RA and IL-17RC heteromultimeric single transmembrane receptor complex that is ubiquitously expressed throughout the body.9−11 Despite signaling through a multimer, the receptor−IL-17A complex is believed to form in a stepwise manner with initial IL-17A−IL-17RA association followed by subsequent recruitment of IL-17RC and an additional copy of IL-17RA.11−14 The association of IL-17A with IL-17RA proceeds with a Kd in the low nanomolar range (1.3 nM), while IL-17F binds IL-17RA with nearly 2 orders of magnitude lower affinity and the IL-17A−IL-17F heterodimer with intermediate affinity.4,5,9 Formation of the cytokine−receptor complex leads to downstream production of transcription factors leading to the upregulation of cytokines, chemokines, and other pro-inflammatory gene products.15,16 © XXXX American Chemical Society

Received: October 30, 2015

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Figure 1. Structures of the macrocyclic peptide inhibitors of IL-17A.

because of the structural features of the IL-17RA receptor protein that are observed to occupy them in the crystal structure of the complex. A more quantitative approach to assessing the various cavities in the protein surface used the SITEMAP34 program to explore the entire surface of IL17-A, identifying and scoring every potential surface cavity. SITEMAP has been shown to be able to identify pockets on a protein surface and in particular to reliably identify pockets which can be occupied by ligands and specifically by drug-like molecules. The two sites mentioned above were selected as being the most druggable according to the SITEMAP scores. The Schrodinger software suite was then used to build docking models for the GLIDE module (see Experimental Section), one centered on the β-hairpin pocket and the other centered on the α-helix pocket in order to evaluate whether the Ensemble compounds would fit well into either pocket. Subjective evaluation of the docked poses and comparison with the published SAR in the Ensemble patent, the magnitude of the docking scores and weak trends relating higher (more negative) docking scores with higher affinity indicated that the β-hairpin pocket was probably the binding site for the Ensemble macrocycle compounds (Figure 2 of the Supporting Information). Although such modeling results cannot be regarded as a definitive method of identifying the binding epitope, they can serve as the basis for a hypothesis that can then be tested experimentally. HDX-MS. To obtain information about the dynamic behavior of IL-17A in solution and to compare this with the crystallographic observations of where this cytokine and its receptor come in contact, HDX-MS experiments were performed. The general workflow for the HDX-MS is depicted in Scheme 1 of the Supporting Information. In short, the proteins were exposed to deuterated buffer for different periods of time (six “on-exchange” time points) to measure the exchange of amides in both the cytokine and the IL-17A−IL17RA complex. A model based on the 4HSA crystal structure of IL-17A−IL-17RA (Figure 3 of the Supporting Information) shows a map of the rate of exchange across IL-17A in the absence of IL-17RA. It is noteworthy that the first 42 amino acids, which comprise residues located in the N-terminus of the cytokine, exchange completely even at the shortest time of 10 s. This rapid deuterium uptake suggests that this part of the protein is disordered and/or dynamic. Two additional regions (residues 84−91 and 130−136) of high exchange rate were also identified.

molecules designed to interact with either IL-17A or IL-17RA, thereby preventing association of the signaling cytokine− receptor complex. A small molecule approach to this problem has the potential to be orally bioavailable and provide increased control over drug exposure, conceivably beneficial, for example, in the event of an opportunistic infection. Recently, Ensemble Therapeutics published a patent application detailing a class of macrocyclic peptide inhibitors of IL-17A (Figure 1).28 Notably, these macrocycles were shown to bind to IL-17A and/or inhibit formation of the IL-17A−IL-17RA complex through an ELISA assay, an HT29-GROα cell based functional assay, a rheumatoid arthritis synovial fibroblast (RASF) assay, and surface plasmon resonance (SPR, Kd < 100 nM) based biophysical binding assessment. Moreover, several of these compounds were reported to have efficacy in several in vivo PD models including a collagen-induced arthritis (CIA) assay and a murine delayed hypersensitivity assay. Whereas the dimeric IL-17A crystal structure has been solved (2.5 Å resolution),29 to date, no small molecule cocrystal structure has been reported in the literature. Our initial attempts to obtain protein cocrystals were unsuccessful, leading us to ask whether it might be possible to determine a plausible binding site and orientation for the Ensemble compounds through a combination of computational methods and hydrogen/deuterium exchange mass spectrometry (HDX-MS) experiments. HDX-MS is a biophysical tool for the characterization of protein dynamics following ligand binding (where the ligand is a small molecule, a peptide, or another protein).30,31 HDX-MS has emerged as a rapid and sensitive approach to interrogate PPIs and transient protein folding states.32,33 Here, we report the use of HDX-MS to characterize interactions of Ensemble macrocycle-peptides with IL-17A. Our results demonstrate that HDX-MS was able to identify the binding epitope for the Ensemble compounds, which was not accessible by conventional structure elucidation techniques. To our knowledge, there is no other structural evidence to indicate the putative site where these compounds bind to IL-17A.

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RESULTS AND DISCUSSION Computational Assessment. We wished to identify the likely binding site for the Ensemble ligands for IL-17A. This protein is a PPI target which upon inspection of the IL17A−IL-17RA complex crystal structure can be seen to possess several surface cavities and grooves and in particular two deep feature-rich pockets that are referred to as the β-hairpin pocket and α-helix pocket (Figure 1 of the Supporting Information) B

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Figure 2. Deuterium uptake plots (complex with IL-17RA ectodomain vs Apo IL17A) of selected representative peptides and their location in the IL-17A structure: 48−57 (interaction of CAT2200 with IL-17A), 58−72 (exterior wall of the β-hairpin site), 90−101 (β-hairpin site), and 104−116 (α-helix site). The crystal structure shows IL-17A (pdb 4HSA) in complex with its receptor IL-17RA.

Figure 3. Deuterium uptake plots of peptides 90−101, 92−99 originating in the β-hairpin pocket and 103−114, 104−116 originating in the α-helix pocket. Colored regions represent the difference in deuterium uptake (complex with Ensemble 159 vs Apo): reduced (called protection) in blue and green and enhanced (called deprotection) in yellow. The crystal structure shows IL-17A (pdb 4HSA).

The results of the differential HDX analysis in the absence and presence of the receptor are shown in Figure 2. Our HDX results, in agreement with X-ray data, indicated that binding of IL-17A to IL-17RA induces large conformational changes in the IL-17A dimer. As a consequence, protection to deuterium exchange was observed across the cytokine with the exception

of two regions the N-terminus (covered by two peptides, 1−25 and 1−27) and the region (72−91) adjacent to the β-hairpin pocket. The N-terminal region peptides in both IL17A alone and in complex with IL-17RA are fully exchanged within 10 s, indicating that this region remains unstructured/dynamic. This is consistent with the crystal structures of both complexed C

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the interaction between the macrocycle and the cytokine. Consistent with our computational assessment of the cytokine, protection to H/D exchange was observed upon binding of Ensemble compounds 159, 182, and 453. A concomitant increase in exchange was observed in the α-helix pocket. Taken together, the data indicate that binding of the peptide macrocycles occurs in the β-hairpin pocket, resulting in significant conformational changes across the cytokine. To our knowledge, these data represents the first direct structural evidence of an inhibitory small molecule binding site that functions to disrupt the interaction between IL-17A and its cognate receptor.

(4HSA) and free (4HR9) IL-17A in which these N-terminal residues are not resolved crystallographically. The region containing residues 72−91 did not exhibit a difference in deuterium uptake, suggesting that the rigidity of this region does not change upon IL-17RA binding. Regions that undergo major conformational changes upon IL-17RA binding were observed in the residues located in an area where the CAT2200 antibody35 has been observed crystallographically to interact with IL-17A (e.g., residues 48−57, Figure 2), as well as with the exterior wall of the β-hairpin pocket (e.g., residues 58− 72, Figure 2) and the β-hairpin and α-helix pockets. Representative HDX uptake plots for peptides covering those protected regions are shown in Figure 2. In short, the data from HDX-MS is consistent with the crystal structure of both the unbound IL-17A dimer and the complex IL-17A−IL-17RA.29 Once the epitope mapping of IL-17RA against IL-17A was completed, the mode of interaction between the Ensemble compounds and the cytokine was studied by HDX-MS. Differential HDX analysis of IL-17A in the presence and absence of the compound Ensemble 15928 revealed that this macrocycle led to both reduced and enhanced deuterium uptake in different parts of the two pockets of IL-17A. As is depicted in Figure 3, peptides 90−101 and 92−99 covering the β-hairpin pocket showed protection from exchange upon binding to Ensemble 159. The major difference in deuterium uptake was detected at the 10, 30, 60 and 300 s time points. These differences almost disappear at the longer two time points (900 and 3600 s). The magnitude and pattern of protection recurs in two other peptides (92−101 and 99−103) covering the structural region of the β-hairpin site. On the other hand, moderate but significant protection was also observed for two peptides, residues 58−71 and 58−72, located in the exterior wall of the β-hairpin pocket (HDX plots not shown). In contrast, peptides 103−114 and 104−116, which comprise residues located in the α-helix pocket region, exhibited a noticeable increase in deuterium uptake. The deuterium uptake increases over time and the major difference was detected at the longest time points, 900 and 3600 s. The observed enhanced deuterium uptake was confirmed by three other peptides (100−116, 102−116, and 102−120) from the structural region of the α-helix pocket. It is worth mentioning that HDX-MS studies with the Ensemble macrocycles 182 and 453 revealed similar exchange effects in the same druggable pockets and therefore suggest the same mode of interaction with IL-17A (data not shown). Thus, the HDX profiles allowed us to characterize the binding of the Ensemble compounds and to support our prediction of the most probable binding site as the β-hairpin pocket. Furthermore, the HDX results revealed how part of the cytokine was deprotected (α-helix region) against deuterium incorporation. This provides a reasonable hypothesis as to why the crystallization of this specific compound in complex with the cytokine has proven so difficult, at least in our hands.

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EXPERIMENTAL SECTION

Proteins and Reagents. Recombinant human IL-17A was purchased from R&D Systems Inc. IL-17RA was made as reported.29 All reagents were purchased from Sigma-Aldrich unless otherwise specified. Computational. Computational studies were carried out using the Schrodinger suite (Schrodinger, Inc., Portland, Oregon, release 2014.4). The IL-17RA molecule was removed from the structure of the IL-17A−IL-17RA complex (4HSA, obtained from the Protein Data Bank) to create a model for IL-17A. The structure was treated according to the standard protocol within the Schrodinger package. The “Protein Prepwizard” tool was applied, with missing side chains being constructed using PRIME, caps being applied to N- and Cterminal groups, and protein protonation state being modeled at pH7.4 using propka, with otherwise default settings; all available steps, including the restrained minimization using OPLS2.1, were taken. All the sites on the surface of the resulting structure of IL-17A were evaluated using the SITEMAP module; SITEMAP scores were used to identify the most “ligandable” and most “druggable” pockets using the criteria described in the software manuals. Analysis of the interactions between the IL-17A and IL-17RA molecules in the 4HSA structure was used to generate constraints for the docking program during GLIDE grid generation for the α-helix and β-hairpin pockets; the relevant segments of the IL-17RA protein (i.e., the β-hairpin and αhelical segments taken from the 4HSA structure) were used to define the regions of interest for these pockets for this step. Conformations of the ligands of interest were generated both on-the-fly during the docking and by using the Monte Carlo multiple minimum/low mode conformational search algorithm available in the MACROMODEL module of the Schrodinger package, using default parameters and the OPLS2.1 force field with the GBSA water solvent model. Docking of the ligands was carried out using GLIDE in SP mode, Poses were tested postdocking and were rejected unless they showed at least one hydrophobic and one hydrogen-bonding interaction that appeared to be important in IL-17A−IL-17RA recognition, as observed in the crystal structure. Docking poses that satisfied several such constraints were preferred over those that satisfied only the minimum requirement. HDX-MS. HDX-MS experiments were performed with an in-house automated system (LEAP Technologies, Carrboro, NC) interfaced with an orbitrap mass spectrometer (Q-Exactive, Thermo Electron, San Jose, CA). Prior to HDX experiments, the digestion and separation conditions of IL-17A were optimized to generate a set of peptides that covers the entire sequence of this cytokine (Figure 3 of the Supporting Information). Samples for HDX were prepared in Hepes buffer (adjusted to pH 7.5) at 10 μM and diluted into D2O buffer of an equivalent composition for 10, 30, 60, 300, 900, and 3600 s. Following this on-exchange, samples were quenched with 3 M urea solution containing 500 mM TCEP (pH 4) and passed across an inhouse generated immobilized pepsin column. The resulting peptides were trapped on a 2 mm × 2 cm C8 trap column and eluted across a 5 cm × 2.1 mm C18 analytical column into the mass spectrometer. The automated system operated under control of custom “Macros” written and executed within CTC Cycle Composer (CTC Analytics). All time

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CONCLUSION IL-17A is an important pro-inflammatory cytokine playing a key role in pathogenic autoimmunity. The recently described macrocyclic peptide inhibitors of IL-17RA signaling have been shown to be active in a number of biophysical and cell based functional assays. To our knowledge, no structural data exists detailing the binding site between this class of macrocycle and the IL-17A cytokine. Therefore, we employed a combination of computational studies and HDX-MS to profile D

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points were acquired in triplicate, and the order of analysis was randomized. HDX MS data were processed with HDX-Workbench.36

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge via the Internet at The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jmedchem.5b01693. Surface representation of the IL-17A homodimer showing the two druggable sites; docking score vs activity in ELISA II assay described in WO2013116682; HDX ribbon representation of the structure of IL-17A; HDX peptide map of IL-17A; general workflow for the HDX-MS experiment (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+34) 916633461. E-mail: [email protected]. Author Contributions

All authors have contributed to the manuscript and given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Kristine Kikly for help in selecting, obtaining, and biochemically characterizing suitable commercial protein reagents, and Marc Rutter and Danalyn Manglicmot for generation of IL-17RA. We also thank Patrick Griffin and Venkat Dharmarajan at Scripps-Florida, for helpful discussions.

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ABBREVIATIONS USED IL17A, interleukin 17A; IL17RA interleukin 17A receptor; CD4, cluster of differentiation 4; Th17, T helper 17 cell; TGFβ, transforming growth factor beta; PPI, protein−protein interaction; HDX-MS, hydrogen−deuterium exchange mass spec-trometry; TCEP, tris(2-carboxyethyl)phosphine; hepes, 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid

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REFERENCES

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