PMC 20201220 pmc.key 4869123 CC BY no 0 0 10.1038/srep26071 srep26071 4869123 27184415 26071 This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ surname:Liu;given-names:Shenping surname:Desharnais;given-names:Joel surname:Feng;given-names:Xidong surname:Griffor;given-names:Matt surname:Jimenez;given-names:Judith surname:Chen;given-names:Gang surname:Tumelty;given-names:David surname:Bhat;given-names:Abhijit surname:Bradshaw;given-names:Curt W. surname:Woodnutt;given-names:Gary surname:Lappe;given-names:Rodney W. surname:Thorarensen;given-names:Atli surname:Sahasrabudhe;given-names:Parag V. surname:Qiu;given-names:Xiayang surname:Withka;given-names:Jane M. surname:Wood;given-names:Lauren D. surname:Jin;given-names:Ping surname:Li;given-names:Wei surname:Oates;given-names:Bryan D. surname:Shanker;given-names:Suman surname:Banker;given-names:Mary Ellen surname:Chrunyk;given-names:Boris A. surname:Song;given-names:Xi TITLE front 6 2016 0 Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide 0.95569223 protein cleaner0 2023-07-20T08:16:49Z PR: IL-17A 0.9845403 protein cleaner0 2023-07-20T08:16:59Z PR: IL-17RA 0.99911207 chemical cleaner0 2023-07-20T09:57:34Z CHEBI: peptide ABSTRACT abstract 73 IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target. 0.9583933 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A protein_type MESH: cleaner0 2023-07-20T08:50:51Z cytokine 0.8298379 protein_type cleaner0 2023-07-20T09:25:45Z MESH: antibodies protein PR: cleaner0 2023-07-20T08:16:50Z IL-17A chemical CHEBI: cleaner0 2023-07-20T08:18:25Z high affinity IL-17A peptide antagonist 0.4951907 chemical cleaner0 2023-07-20T08:18:35Z CHEBI: HAP 0.9906646 residue_range cleaner0 2023-07-20T09:26:24Z DUMMY: 15 residues 0.99955106 experimental_method cleaner0 2023-07-20T09:18:38Z MESH: phage-display screening 0.99955803 experimental_method cleaner0 2023-07-20T09:29:41Z MESH: saturation mutagenesis optimization 0.9994239 experimental_method cleaner0 2023-07-20T09:29:45Z MESH: amino acid substitutions 0.98556024 chemical cleaner0 2023-07-20T08:18:35Z CHEBI: HAP 0.99852103 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A protein_type MESH: cleaner0 2023-07-20T08:50:51Z cytokine protein_type MESH: cleaner0 2023-07-20T09:25:36Z receptor 0.996836 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.9989361 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.97730803 chemical cleaner0 2023-07-20T08:18:35Z CHEBI: HAP protein_type MESH: cleaner0 2023-07-20T08:45:34Z cytokines experimental_method MESH: cleaner0 2023-07-20T09:29:32Z Crystal structure studies 0.9854585 chemical cleaner0 2023-07-20T08:18:35Z CHEBI: HAP 0.9989205 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A 0.99925786 oligomeric_state cleaner0 2023-07-20T09:18:26Z DUMMY: dimer 0.97047263 chemical cleaner0 2023-07-20T08:18:35Z CHEBI: HAP 0.99970937 structure_element cleaner0 2023-07-20T09:45:39Z SO: β-strand 0.99877185 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A 0.99928147 oligomeric_state cleaner0 2023-07-20T08:23:13Z DUMMY: monomers 0.99972767 structure_element cleaner0 2023-07-20T09:45:43Z SO: α helix 0.9988974 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.9989691 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A INTRO paragraph 1118 The family of IL-17 cytokines and receptors consists of six polypeptides, IL-17A-F, and five receptors, IL-17RA-E. IL-17A is secreted from activated Th17 cells, and several innate immune T cell types including macrophages, neutrophils, natural killer cells, and dendritic cells. IL-17A signals through a specific cell surface receptor complex which consists of IL-17RA and IL-17RC. IL-17A’s downstream signaling leads to increased production of inflammatory cytokines such as IL-6, IL-8, CCL-20 and CXCL1 by various mechanisms including stimulation of transcription and stabilization of mRNA. Although various cell types have been reported to express IL-17RA, the highest responses to IL-17A come from epithelial cells, endothelial cells, keratinocytes and fibroblasts. 0.9996173 protein_type cleaner0 2023-07-20T08:20:45Z MESH: IL-17 cytokines protein PR: cleaner0 2023-07-20T08:19:52Z IL-17A-F 0.98434764 protein cleaner0 2023-07-20T09:24:28Z PR: IL-17RA-E protein PR: cleaner0 2023-07-20T08:19:34Z IL-17A 0.9625089 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A protein_type MESH: cleaner0 2023-07-20T09:25:36Z receptor 0.99856997 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99870443 protein cleaner0 2023-07-20T08:20:06Z PR: IL-17RC 0.9706386 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A protein_type MESH: cleaner0 2023-07-20T08:45:34Z cytokines 0.99858046 protein_type cleaner0 2023-07-20T08:20:58Z MESH: IL-6 0.99673206 protein_type cleaner0 2023-07-20T08:21:00Z MESH: IL-8 0.9613981 protein_type cleaner0 2023-07-20T08:21:02Z MESH: CCL-20 0.64120615 protein_type cleaner0 2023-07-20T08:21:05Z MESH: CXCL1 0.98661 chemical cleaner0 2023-07-20T08:21:09Z CHEBI: mRNA 0.9991738 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99757344 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A INTRO paragraph 1890 IL-17A and its signaling is important in host defense against certain fungal and bacterial infections as demonstrated by patients with autoantibodies against IL-17A and IL-17F, or with inborn errors of IL-17 immunity. In addition to its physiological role, IL-17A is a key pathogenic factor in inflammatory and autoimmune diseases. In phase II and III clinical trials, neutralizing monoclonal antibodies against IL-17A (secukinumab and ixekizumab) or its receptor IL-17RA (brodalumab) are highly efficacious in treating moderate to severe plaque psoriasis and psoriatic arthritis. Secukinumab has been approved recently as a new psoriasis drug by the US Food and Drug Administration (Cosentyx™). In addition to psoriasis and psoriatic arthritis, IL-17A blockade has also shown preclinical and clinical efficacies in ankylosing spondylitis and rheumatoid arthritis. protein PR: cleaner0 2023-07-20T08:16:50Z IL-17A 0.94309646 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A 0.9504142 protein cleaner0 2023-07-20T09:24:32Z PR: IL-17F 0.9994386 protein_type cleaner0 2023-07-20T08:20:35Z MESH: IL-17 protein PR: cleaner0 2023-07-20T08:16:50Z IL-17A protein_type MESH: cleaner0 2023-07-20T09:26:14Z antibodies 0.86277634 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A 0.99755496 chemical cleaner0 2023-07-20T08:21:18Z CHEBI: secukinumab 0.99573684 chemical cleaner0 2023-07-20T08:21:23Z CHEBI: ixekizumab 0.95846945 protein_type cleaner0 2023-07-20T09:25:29Z MESH: receptor 0.92973715 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99208236 chemical cleaner0 2023-07-20T08:21:27Z CHEBI: brodalumab 0.9984517 chemical cleaner0 2023-07-20T08:21:19Z CHEBI: Secukinumab 0.9591042 chemical cleaner0 2023-07-20T08:21:33Z CHEBI: Cosentyx™ 0.99504143 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A INTRO paragraph 2757 Among IL-17 cytokines, IL-17A and IL-17F share the highest homology. These polypeptides form covalent homodimers, and IL-17A and IL-17F also form an IL-17A/IL-17F hetereodimer. Structures are known for apo IL-17F and its complex with IL-17RA, for apo IL-17A, its complex with an antibody Fab, and its complex with IL-17RA. In these structures, both IL-17A and IL-17F adopt a cysteine-knot fold with two intramolecular disulfides and two interchain disulfide bonds that covalently link two monomers. 0.99963903 protein_type cleaner0 2023-07-20T08:20:46Z MESH: IL-17 cytokines 0.9972264 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A 0.997863 protein cleaner0 2023-07-20T09:24:38Z PR: IL-17F 0.57897425 protein_state cleaner0 2023-07-20T08:22:03Z DUMMY: covalent 0.99931896 oligomeric_state cleaner0 2023-07-20T08:23:07Z DUMMY: homodimers 0.99898124 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A 0.9990733 protein cleaner0 2023-07-20T09:24:38Z PR: IL-17F complex_assembly GO: cleaner0 2023-07-20T08:23:37Z IL-17A/IL-17F 0.99701786 oligomeric_state cleaner0 2023-07-20T08:23:02Z DUMMY: hetereodimer 0.9988896 evidence cleaner0 2023-07-20T09:40:29Z DUMMY: Structures 0.9996731 protein_state cleaner0 2023-07-20T08:21:58Z DUMMY: apo 0.9991986 protein cleaner0 2023-07-20T09:24:38Z PR: IL-17F 0.99873376 protein_state cleaner0 2023-07-20T08:21:53Z DUMMY: complex with 0.998748 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.9996706 protein_state cleaner0 2023-07-20T08:21:59Z DUMMY: apo 0.99925786 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A 0.99880755 protein_state cleaner0 2023-07-20T08:21:53Z DUMMY: complex with 0.9991448 protein_type cleaner0 2023-07-20T08:47:54Z MESH: antibody 0.99974674 structure_element cleaner0 2023-07-20T08:47:40Z SO: Fab 0.9989835 protein_state cleaner0 2023-07-20T08:21:52Z DUMMY: complex with 0.9982937 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99912375 evidence cleaner0 2023-07-20T09:40:33Z DUMMY: structures 0.99910766 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A 0.99922085 protein cleaner0 2023-07-20T09:24:38Z PR: IL-17F structure_element SO: cleaner0 2023-07-20T08:22:32Z cysteine-knot 0.99184275 ptm cleaner0 2023-07-20T08:22:49Z MESH: disulfides 0.87225294 ptm cleaner0 2023-07-20T08:22:56Z MESH: disulfide bonds 0.99904376 oligomeric_state cleaner0 2023-07-20T08:23:12Z DUMMY: monomers INTRO paragraph 3256 There has been active research in identifying orally available chemical entities that would functionally antagonize IL-17A-mediated signaling. Developing small molecules targeting protein-protein interactions is difficult with particular challenges associated with the large, shallow IL-17A/IL-17RA interfaces. Since IL-17RA is a shared receptor for at least IL-17A, IL-17F, IL-17A/IL-17F and IL-17E, we chose to seek IL-17A-specific inhibitors that may have more defined pharmacological responses than IL-17RA inhibitors. Our initial approach was to identify peptide inhibitors which could serve as leads for the development of anti-inflammatory therapeutics that could be used alone or in combination with other agents. Our efforts resulted in discovery of a high affinity IL-17A peptide antagonist (HAP), which we attempted to increase the functional production and pharmacokinetics after fusing HAP to antibodies for evaluation as a bispecific therapeutic in animal studies. Unfortunately, this past work revealed stability issues of the uncapped HAP in cell culture Here, we provide the details of the discovery and optimization that led to HAP and report the complex structure of IL-17A with HAP, which provides structure based rationalization of peptide optimization and structure activity relationship (SAR). 0.9965642 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A 0.99520123 site cleaner0 2023-07-20T08:39:44Z SO: IL-17A/IL-17RA interfaces protein PR: cleaner0 2023-07-20T08:17:00Z IL-17RA protein_type MESH: cleaner0 2023-07-20T09:25:36Z receptor 0.994631 protein cleaner0 2023-07-20T08:16:50Z PR: IL-17A 0.9799486 protein cleaner0 2023-07-20T09:24:38Z PR: IL-17F complex_assembly GO: cleaner0 2023-07-20T08:23:38Z IL-17A/IL-17F 0.9883621 protein cleaner0 2023-07-20T08:24:20Z PR: IL-17E protein PR: cleaner0 2023-07-20T08:16:51Z IL-17A protein PR: cleaner0 2023-07-20T08:17:00Z IL-17RA chemical CHEBI: cleaner0 2023-07-20T08:24:35Z high affinity IL-17A peptide antagonist 0.9493229 chemical cleaner0 2023-07-20T08:18:35Z CHEBI: HAP 0.87955695 experimental_method cleaner0 2023-07-20T09:29:52Z MESH: fusing 0.8208474 chemical cleaner0 2023-07-20T08:18:35Z CHEBI: HAP 0.54800403 protein_type cleaner0 2023-07-20T09:25:44Z MESH: antibodies protein_state DUMMY: cleaner0 2023-07-20T08:41:30Z uncapped 0.84635955 chemical cleaner0 2023-07-20T08:18:35Z CHEBI: HAP 0.70992947 chemical cleaner0 2023-07-20T08:18:35Z CHEBI: HAP evidence DUMMY: cleaner0 2023-07-20T09:42:22Z complex structure 0.9984722 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9962735 chemical cleaner0 2023-07-20T08:18:35Z CHEBI: HAP 0.8284904 experimental_method cleaner0 2023-07-20T09:29:55Z MESH: peptide optimization 0.76640385 experimental_method cleaner0 2023-07-20T09:30:00Z MESH: structure activity relationship experimental_method MESH: cleaner0 2023-07-20T09:04:27Z SAR RESULTS title_1 4573 Results RESULTS title_2 4581 Identification of IL-17A peptide inhibitors 0.97984284 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A RESULTS paragraph 4625 Peptides specifically binding to human IL-17A were identified from phage panning using cyclic and linear peptide libraries (Supplementary Figure S1). Positive phage pools were then sub-cloned into a maltose-binding protein (MBP) fusion system. Single clones were isolated and sub-cultured in growth medium, and culture supernatants were used in an enzyme-linked immunosorbent assay (ELISA) to identify specific IL-17A-binding clones. The positive binding supernatants were tested for the ability to block biotinylated IL-17A signaling through IL-17RA in an IL-17A/IL-17RA competition ELISA assay where unlabeled IL-17A was used as positive control to inhibit biotinylated IL-17A binding. Approximately 10% of the clones that specifically bound to IL-17A also prevented the cytokine from binding to IL-17RA. Sequences identified from phage clones were chemically synthesized (Supplementary Table 1) and tested for inhibition of IL-17A binding to IL-17RA (Table 1). A 15-mer linear peptide 1 was shown to block IL-17A/IL-17RA binding with an IC50 of 80 nM in the competition ELISA assay (Table 1). This peptide was then tested in a cell-based functional assay wherein production of GRO-α in BJ human fibroblast cells was measured as a function of IL-17A stimulation using 1 ng/ml IL-17A. Peptide 1 was found to be active in this functional assay with an IC50 of 370 nM. 0.9993475 species cleaner0 2023-07-20T08:25:03Z MESH: human 0.9993885 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.999529 experimental_method cleaner0 2023-07-20T09:19:10Z MESH: phage panning experimental_method MESH: cleaner0 2023-07-20T08:26:37Z cyclic and linear peptide libraries experimental_method MESH: cleaner0 2023-07-20T08:26:18Z Positive phage pools 0.9991283 experimental_method cleaner0 2023-07-20T09:30:04Z MESH: sub-cloned experimental_method MESH: cleaner0 2023-07-20T08:26:02Z maltose-binding protein (MBP) fusion system 0.9995176 experimental_method cleaner0 2023-07-20T08:26:46Z MESH: enzyme-linked immunosorbent assay 0.9980411 experimental_method cleaner0 2023-07-20T08:26:51Z MESH: ELISA 0.98475736 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99689853 protein_state cleaner0 2023-07-20T09:15:20Z DUMMY: biotinylated 0.99892646 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99871516 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA complex_assembly GO: cleaner0 2023-07-20T08:28:04Z IL-17A/IL-17RA 0.9978862 experimental_method cleaner0 2023-07-20T08:27:13Z MESH: competition ELISA assay 0.9931461 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99169886 protein_state cleaner0 2023-07-20T09:15:20Z DUMMY: biotinylated 0.99915606 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99945474 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A protein_type MESH: cleaner0 2023-07-20T08:50:51Z cytokine 0.9991376 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.9994713 experimental_method cleaner0 2023-07-20T09:19:24Z MESH: phage clones 0.99924475 experimental_method cleaner0 2023-07-20T09:19:26Z MESH: chemically synthesized 0.9984878 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.999304 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA chemical CHEBI: cleaner0 2023-07-20T08:38:10Z peptide 1 complex_assembly GO: cleaner0 2023-07-20T08:28:05Z IL-17A/IL-17RA 0.9994924 evidence cleaner0 2023-07-20T08:32:48Z DUMMY: IC50 0.99933887 experimental_method cleaner0 2023-07-20T08:27:13Z MESH: competition ELISA assay 0.9960262 experimental_method cleaner0 2023-07-20T08:39:25Z MESH: cell-based functional assay 0.99944496 protein cleaner0 2023-07-20T09:24:45Z PR: GRO-α 0.99842834 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.99569863 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99933106 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A chemical CHEBI: cleaner0 2023-07-20T08:38:10Z Peptide 1 experimental_method MESH: cleaner0 2023-07-20T09:30:35Z functional assay 0.9993832 evidence cleaner0 2023-07-20T08:32:48Z DUMMY: IC50 RESULTS title_2 6000 Optimization of IL-17A peptide inhibitors 0.9963889 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A RESULTS paragraph 6042 A SAR campaign was undertaken to improve the potency of peptide 1. An alanine scan of peptide 2, an analogue of 1 with a lysine to arginine substitution at position 14, was initiated. When alanine was already present (positions 7 and 15), substitution was made with lysine (Table 1, peptides 3–17). Positions 1, 2, 4, 5, 7, 14 and 15 were shown to be amenable to substitution without significant loss (less than 3-fold) of binding affinity as measured by the IL-17A/IL-17RA competition ELISA. In particular, at position 5 (13), substitution of methionine with alanine resulted in a seven fold improvement in potency (80 nM versus 11 nM respectively). In order to rapidly evaluate the effects of substitution of natural amino acids at tolerant positions identified by the alanine scan, the lead sequence was subjected to site-specific saturation mutagenesis using MBP. Each of the seven positions identified by the alanine scan was individually modified while keeping the rest of the sequence constant. Modifications at positions 2 and 14 were shown to display improvement in binding affinity (data not shown). experimental_method MESH: cleaner0 2023-07-20T09:04:27Z SAR chemical CHEBI: cleaner0 2023-07-20T08:38:09Z peptide 1 0.99952424 experimental_method cleaner0 2023-07-20T09:31:46Z MESH: alanine scan chemical CHEBI: cleaner0 2023-07-20T08:38:27Z peptide 2 0.60970217 chemical cleaner0 2023-07-20T08:38:30Z CHEBI: 1 0.99725467 residue_name cleaner0 2023-07-20T09:21:46Z SO: lysine 0.9975005 residue_name cleaner0 2023-07-20T09:21:48Z SO: arginine 0.99908924 experimental_method cleaner0 2023-07-20T09:31:49Z MESH: substitution 0.99520063 residue_number cleaner0 2023-07-20T09:27:33Z DUMMY: 14 0.9989141 residue_name cleaner0 2023-07-20T09:21:52Z SO: alanine 0.9927239 residue_number cleaner0 2023-07-20T09:27:36Z DUMMY: 7 0.99312204 residue_number cleaner0 2023-07-20T09:27:39Z DUMMY: 15 0.9978138 experimental_method cleaner0 2023-07-20T09:31:52Z MESH: substitution 0.99909055 residue_name cleaner0 2023-07-20T09:22:33Z SO: lysine chemical CHEBI: cleaner0 2023-07-20T08:37:43Z peptides 3–17 0.9782874 residue_number cleaner0 2023-07-20T09:27:42Z DUMMY: 1 0.94353265 residue_number cleaner0 2023-07-20T09:27:45Z DUMMY: 2 0.9745618 residue_number cleaner0 2023-07-20T09:27:48Z DUMMY: 4 0.9761025 residue_number cleaner0 2023-07-20T09:27:51Z DUMMY: 5 0.98406637 residue_number cleaner0 2023-07-20T09:27:54Z DUMMY: 7 0.98588353 residue_number cleaner0 2023-07-20T09:27:56Z DUMMY: 14 0.989995 residue_number cleaner0 2023-07-20T09:27:59Z DUMMY: 15 0.9992334 evidence cleaner0 2023-07-20T09:40:48Z DUMMY: binding affinity complex_assembly GO: cleaner0 2023-07-20T08:28:05Z IL-17A/IL-17RA 0.9994817 experimental_method cleaner0 2023-07-20T08:28:33Z MESH: competition ELISA 0.9915571 residue_number cleaner0 2023-07-20T09:28:02Z DUMMY: 5 0.99155676 chemical cleaner0 2023-07-20T08:38:44Z CHEBI: 13 0.99961156 experimental_method cleaner0 2023-07-20T09:32:01Z MESH: substitution 0.99922335 residue_name cleaner0 2023-07-20T09:21:56Z SO: methionine 0.9991792 residue_name cleaner0 2023-07-20T09:21:59Z SO: alanine experimental_method MESH: cleaner0 2023-07-20T09:32:25Z substitution 0.9994993 experimental_method cleaner0 2023-07-20T09:32:30Z MESH: alanine scan 0.99943316 experimental_method cleaner0 2023-07-20T09:32:37Z MESH: site-specific saturation mutagenesis 0.5736163 experimental_method cleaner0 2023-07-20T08:38:58Z MESH: MBP 0.99947476 experimental_method cleaner0 2023-07-20T09:32:42Z MESH: alanine scan 0.9931477 residue_number cleaner0 2023-07-20T09:28:04Z DUMMY: 2 0.992432 residue_number cleaner0 2023-07-20T09:28:07Z DUMMY: 14 0.9993174 evidence cleaner0 2023-07-20T09:40:52Z DUMMY: binding affinity RESULTS paragraph 7158 Peptides with beneficial point mutations at positions 2, 5, and 14 were synthesized and evaluated in the competition ELISA (Table 1). Two of the changes, V2H (18) or V2T (21) displayed improved binding in the competition ELISA. Since the replacement of methionine at position 5 with alanine was beneficial, the additional hydrophobic amino acids isoleucine (24), leucine (25) and valine (26) were evaluated and an additional two-three fold improvement in binding was observed for the valine and isoleucine replacements in comparison with alanine. Introduction of a methionine (27) or a carboxamide (28 and 29) at position 14 was shown to improve the binding affinity of the lead peptide. In general, there was good agreement between the respective binding affinities of the synthesized peptides and their MBP fusion counterparts, except for substitution of valine at position 2 to a tryptophan (22), which resulted in a fivefold loss of affinity, for the free peptide when compared with the MBP fusion. experimental_method MESH: cleaner0 2023-07-20T09:33:09Z point mutations 0.9938885 residue_number cleaner0 2023-07-20T09:28:10Z DUMMY: 2 0.99447745 residue_number cleaner0 2023-07-20T09:28:13Z DUMMY: 5 0.9924971 residue_number cleaner0 2023-07-20T09:28:16Z DUMMY: 14 0.99920183 experimental_method cleaner0 2023-07-20T09:33:13Z MESH: synthesized 0.9996035 experimental_method cleaner0 2023-07-20T08:28:52Z MESH: competition ELISA mutant MESH: cleaner0 2023-07-20T08:32:11Z V2H chemical CHEBI: cleaner0 2023-07-20T08:36:11Z 18 mutant MESH: cleaner0 2023-07-20T08:32:27Z V2T chemical CHEBI: cleaner0 2023-07-20T08:36:20Z 21 0.9995967 experimental_method cleaner0 2023-07-20T08:29:00Z MESH: competition ELISA 0.9990429 experimental_method cleaner0 2023-07-20T09:33:18Z MESH: replacement 0.9991487 residue_name cleaner0 2023-07-20T09:22:03Z SO: methionine 0.9952172 residue_number cleaner0 2023-07-20T09:28:18Z DUMMY: 5 0.998944 residue_name cleaner0 2023-07-20T09:22:05Z SO: alanine 0.9989441 residue_name cleaner0 2023-07-20T09:22:08Z SO: isoleucine chemical CHEBI: cleaner0 2023-07-20T08:36:29Z 24 0.99902976 residue_name cleaner0 2023-07-20T09:22:10Z SO: leucine chemical CHEBI: cleaner0 2023-07-20T08:36:39Z 25 0.99911135 residue_name cleaner0 2023-07-20T09:22:12Z SO: valine chemical CHEBI: cleaner0 2023-07-20T08:36:47Z 26 0.99879634 residue_name cleaner0 2023-07-20T09:22:16Z SO: valine 0.99913895 residue_name cleaner0 2023-07-20T09:22:14Z SO: isoleucine 0.5152386 experimental_method cleaner0 2023-07-20T09:33:22Z MESH: replacements 0.99869436 residue_name cleaner0 2023-07-20T09:22:18Z SO: alanine 0.9977946 experimental_method cleaner0 2023-07-20T09:33:25Z MESH: Introduction 0.9992287 residue_name cleaner0 2023-07-20T09:22:20Z SO: methionine chemical CHEBI: cleaner0 2023-07-20T08:36:59Z 27 0.9798034 chemical cleaner0 2023-07-20T08:41:41Z CHEBI: carboxamide chemical CHEBI: cleaner0 2023-07-20T08:37:09Z 28 chemical CHEBI: cleaner0 2023-07-20T08:37:17Z 29 0.9943374 residue_number cleaner0 2023-07-20T09:28:21Z DUMMY: 14 0.9994836 evidence cleaner0 2023-07-20T09:40:55Z DUMMY: binding affinity 0.99957854 evidence cleaner0 2023-07-20T09:40:59Z DUMMY: binding affinities 0.88407314 experimental_method cleaner0 2023-07-20T08:29:16Z MESH: MBP fusion 0.99947506 experimental_method cleaner0 2023-07-20T09:33:47Z MESH: substitution 0.9991304 residue_name cleaner0 2023-07-20T09:22:23Z SO: valine 0.9938672 residue_number cleaner0 2023-07-20T09:28:24Z DUMMY: 2 0.9991211 residue_name cleaner0 2023-07-20T09:22:27Z SO: tryptophan chemical CHEBI: cleaner0 2023-07-20T08:37:27Z 22 0.86052316 evidence cleaner0 2023-07-20T09:41:03Z DUMMY: affinity 0.97281766 experimental_method cleaner0 2023-07-20T08:29:17Z MESH: MBP fusion RESULTS paragraph 8161 Combining the key amino-acid residues identified by SAR into a single peptide sequence resulted in peptide 30, named high affinity peptide (HAP), that was found to inhibit IL-17A signaling in a BJ human fibroblast cell assay with an IC50 of 17 nM, a more than 20-fold improvement over the phage peptide 1 (Table 2 and Supplementary Figure S2). We also examined the effect of removing the acetyl group at the N-terminus of HAP (which is present in all the peptides made, see Supplementary Material). The un-capped peptide (31) had an IC50 of 420 nM in the cell-based assay. The loss of cellular activity of 31 was most likely due to the degradation of the N-terminus of 31, since peptide 31 was shown to be able to bind to IL-17A with similar affinity as HAP itself. Furthermore, our previous work had reported that in antibody fusions the uncapped peptide was degraded under cell assay conditions with removal of the first 1-3 residues to inactive products with the same N-terminal sequences as peptides 32–34. In this work, 32–34 are capped by protective acetyl group and reflect the same inactivity as reported. C-terminal truncations showed a more gradual reduction in activity (35–37; Table 2). After deletion of three amino acids from the C-terminal end (37), the peptide is no longer active. 0.99909127 experimental_method cleaner0 2023-07-20T09:04:27Z MESH: SAR chemical CHEBI: cleaner0 2023-07-20T08:35:01Z peptide 30 0.94325596 chemical cleaner0 2023-07-20T09:57:42Z CHEBI: high affinity peptide 0.98649454 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP protein PR: cleaner0 2023-07-20T08:16:51Z IL-17A species MESH: cleaner0 2023-07-20T08:25:04Z human 0.995522 evidence cleaner0 2023-07-20T08:32:48Z DUMMY: IC50 experimental_method MESH: cleaner0 2023-07-20T09:01:15Z phage chemical CHEBI: cleaner0 2023-07-20T08:35:59Z peptide 1 0.99545926 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9994185 protein_state cleaner0 2023-07-20T08:41:22Z DUMMY: un-capped chemical CHEBI: cleaner0 2023-07-20T08:35:37Z peptide (31) 0.9992348 evidence cleaner0 2023-07-20T08:32:48Z DUMMY: IC50 0.97057736 experimental_method cleaner0 2023-07-20T09:34:06Z MESH: cell-based assay chemical CHEBI: cleaner0 2023-07-20T08:35:16Z 31 chemical CHEBI: cleaner0 2023-07-20T08:35:17Z 31 chemical CHEBI: cleaner0 2023-07-20T08:35:17Z 31 protein PR: cleaner0 2023-07-20T08:16:51Z IL-17A 0.9978257 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99392676 experimental_method cleaner0 2023-07-20T09:19:48Z MESH: antibody fusions 0.999673 protein_state cleaner0 2023-07-20T08:41:29Z DUMMY: uncapped 0.6190488 chemical cleaner0 2023-07-20T09:57:58Z CHEBI: peptide 0.9911684 experimental_method cleaner0 2023-07-20T09:34:11Z MESH: removal of 0.90224326 residue_range cleaner0 2023-07-20T09:26:32Z DUMMY: first 1-3 residues chemical CHEBI: cleaner0 2023-07-20T08:40:30Z peptides 32–34 0.9917441 chemical cleaner0 2023-07-20T08:40:45Z CHEBI: 32–34 0.95190495 protein_state cleaner0 2023-07-20T08:41:07Z DUMMY: capped 0.883352 experimental_method cleaner0 2023-07-20T09:34:19Z MESH: truncations 0.9986224 chemical cleaner0 2023-07-20T08:40:53Z CHEBI: 35–37 0.9165045 experimental_method cleaner0 2023-07-20T09:34:22Z MESH: deletion of 0.991111 residue_range cleaner0 2023-07-20T09:26:39Z DUMMY: three amino acids 0.94737333 chemical cleaner0 2023-07-20T08:40:08Z CHEBI: 37 RESULTS title_2 9469 Dimerization of HAP can further increase its potency 0.9830784 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP RESULTS paragraph 9522 We reasoned that since the IL-17A protein is almost exclusively present in a dimeric form, dimerizing the IL-17A binding peptides could result in an improvement in binding affinity and inhibitory activity. Homodimers of HAP were made through attachment of polyethylene glycol (PEG) spacers of different lengths at amino acids 4, 7 and 14, as these positions were identified in the alanine scan analysis as not contributing significantly to the activity, and at each N-terminus (Supplementary Table S2). Due to the high reactivity of the pentafluoroester (PFP) group used as the activating group in the PEG, the histidine at position 2 and the lysine at position 15 were replaced with threonine and dimethyllysine respectively to prevent formation of side products, which resulted in peptide 38 that was comparable in activity with HAP. This exercise revealed that several dimeric peptides with the longer PEG21 spacer were significantly more potent than the monomer peptide in the cell-based assay (Supplementary Table S2). Peptide 45, dimerized via attachment of a PEG21 spacer at position 14 (Supplementary Scheme S1 and Figure S3), was the most potent with cellular IC50 of 0.1 nM. This significant improvement in antagonism was not seen in the peptide monomer functionalized with a PEG21 group at position 14 as peptide 48 had an IC50 of 21 nM (Supplementary Scheme S2). 0.9925688 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9993999 oligomeric_state cleaner0 2023-07-20T08:33:39Z DUMMY: dimeric 0.99754333 oligomeric_state cleaner0 2023-07-20T08:33:46Z DUMMY: dimerizing 0.97856927 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.85114086 evidence cleaner0 2023-07-20T09:41:09Z DUMMY: binding affinity 0.99938464 oligomeric_state cleaner0 2023-07-20T08:23:08Z DUMMY: Homodimers 0.995388 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9979036 chemical cleaner0 2023-07-20T08:33:14Z CHEBI: polyethylene glycol 0.99892706 chemical cleaner0 2023-07-20T08:33:18Z CHEBI: PEG 0.99253124 residue_number cleaner0 2023-07-20T09:28:30Z DUMMY: 4 0.99313086 residue_number cleaner0 2023-07-20T09:28:33Z DUMMY: 7 0.992668 residue_number cleaner0 2023-07-20T09:28:39Z DUMMY: 14 0.9994256 experimental_method cleaner0 2023-07-20T09:34:44Z MESH: alanine scan analysis 0.99972945 chemical cleaner0 2023-07-20T08:33:24Z CHEBI: pentafluoroester 0.99973756 chemical cleaner0 2023-07-20T08:33:28Z CHEBI: PFP 0.99917716 chemical cleaner0 2023-07-20T08:33:19Z CHEBI: PEG 0.99935335 residue_name cleaner0 2023-07-20T09:22:40Z SO: histidine 0.99485767 residue_number cleaner0 2023-07-20T09:28:42Z DUMMY: 2 0.9993436 residue_name cleaner0 2023-07-20T09:22:42Z SO: lysine 0.995314 residue_number cleaner0 2023-07-20T09:28:45Z DUMMY: 15 0.99937785 residue_name cleaner0 2023-07-20T09:22:48Z SO: threonine 0.99938715 residue_name cleaner0 2023-07-20T09:22:56Z SO: dimethyllysine chemical CHEBI: cleaner0 2023-07-20T09:23:12Z peptide 38 0.9922083 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9993723 oligomeric_state cleaner0 2023-07-20T08:33:38Z DUMMY: dimeric 0.9179296 chemical cleaner0 2023-07-20T09:58:17Z CHEBI: peptides 0.9063272 chemical cleaner0 2023-07-20T09:58:21Z CHEBI: PEG21 0.9993399 oligomeric_state cleaner0 2023-07-20T09:23:52Z DUMMY: monomer experimental_method MESH: cleaner0 2023-07-20T09:35:09Z cell-based assay chemical CHEBI: cleaner0 2023-07-20T08:34:30Z Peptide 45 0.99911827 oligomeric_state cleaner0 2023-07-20T08:33:53Z DUMMY: dimerized 0.9993223 chemical cleaner0 2023-07-20T09:58:25Z CHEBI: PEG21 0.99526054 residue_number cleaner0 2023-07-20T09:28:48Z DUMMY: 14 0.99950886 evidence cleaner0 2023-07-20T08:32:48Z DUMMY: IC50 0.999368 oligomeric_state cleaner0 2023-07-20T09:23:53Z DUMMY: monomer 0.9993444 chemical cleaner0 2023-07-20T09:58:29Z CHEBI: PEG21 0.99590635 residue_number cleaner0 2023-07-20T09:28:51Z DUMMY: 14 chemical CHEBI: cleaner0 2023-07-20T09:23:29Z peptide 48 0.9994103 evidence cleaner0 2023-07-20T08:32:48Z DUMMY: IC50 RESULTS paragraph 10901 The species cross-reactivity of the dimeric peptide 45 and HAP were assessed in a murine functional cell assay using 15 ng/ml murine IL-17A. Peptide 45 blocked the receptor binding of murine IL-17A although with potency two orders of magnitude weaker than that observed against human IL-17A (IC50 = 41 nM vs IC50 = 0.1 nM, respectively). The monomer HAP was much weaker (IC50 >1 μM) in inhibiting murine IL-17A signaling (Supplementary Figure S4). 0.99936944 oligomeric_state cleaner0 2023-07-20T08:33:39Z DUMMY: dimeric chemical CHEBI: cleaner0 2023-07-20T08:34:30Z peptide 45 0.9996276 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9974878 experimental_method cleaner0 2023-07-20T08:42:45Z MESH: murine functional cell assay 0.9954389 taxonomy_domain cleaner0 2023-07-20T08:42:12Z DUMMY: murine 0.99672765 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A chemical CHEBI: cleaner0 2023-07-20T08:34:30Z Peptide 45 protein_type MESH: cleaner0 2023-07-20T09:25:36Z receptor 0.995782 taxonomy_domain cleaner0 2023-07-20T08:42:12Z DUMMY: murine 0.9957366 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9992908 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.9978351 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.93804055 evidence cleaner0 2023-07-20T08:32:48Z DUMMY: IC50 0.8690941 evidence cleaner0 2023-07-20T08:32:48Z DUMMY: IC50 0.9993467 oligomeric_state cleaner0 2023-07-20T09:23:53Z DUMMY: monomer 0.99963975 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP evidence DUMMY: cleaner0 2023-07-20T08:32:48Z IC50 0.9985342 taxonomy_domain cleaner0 2023-07-20T08:42:11Z DUMMY: murine 0.9880951 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A RESULTS paragraph 11366 Although the dimeric peptide 45 is much more potent than HAP in the cell-based assay, in subsequent studies we decided to focus our efforts solely on characterizations of the monomeric peptide HAP in hopes to identify smaller peptide inhibitors containing the best minimal functional group. 0.9993104 oligomeric_state cleaner0 2023-07-20T08:33:39Z DUMMY: dimeric chemical CHEBI: cleaner0 2023-07-20T08:34:31Z peptide 45 0.9993166 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP experimental_method MESH: cleaner0 2023-07-20T08:42:36Z cell-based assay 0.9989555 oligomeric_state cleaner0 2023-07-20T09:24:03Z DUMMY: monomeric 0.99938715 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP RESULTS title_2 11657 Orthogonal assays to confirm HAP antagonism 0.9997316 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP RESULTS paragraph 11701 To further characterize the interaction of HAP with IL-17A, we set out to determine its in vitro binding affinity, specificity and kinetic profile using Surface Plasmon Resonance (SPR) methods (Fig. 1A). HAP binds to immobilized human IL-17A homodimer tightly (Table 3). It has slightly weaker affinity for human IL-17A/F heterodimer and >10 fold weaker affinity for mouse IL-17A (Table 3). HAP does not show significant binding to immobilized human IL-17F homodimer or IL-17RA at concentrations up to 100 nM. 0.99962664 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9991302 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9748456 evidence cleaner0 2023-07-20T09:41:17Z DUMMY: binding affinity 0.9721006 evidence cleaner0 2023-07-20T09:41:22Z DUMMY: kinetic profile 0.9995649 experimental_method cleaner0 2023-07-20T08:43:31Z MESH: Surface Plasmon Resonance 0.9996598 experimental_method cleaner0 2023-07-20T08:43:35Z MESH: SPR 0.9996233 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99923086 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.99936104 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99927896 oligomeric_state cleaner0 2023-07-20T09:24:10Z DUMMY: homodimer 0.907098 evidence cleaner0 2023-07-20T09:41:26Z DUMMY: affinity 0.999366 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.9975115 complex_assembly cleaner0 2023-07-20T08:43:17Z GO: IL-17A/F 0.9991142 oligomeric_state cleaner0 2023-07-20T09:24:17Z DUMMY: heterodimer 0.99288344 evidence cleaner0 2023-07-20T09:41:30Z DUMMY: affinity 0.9979754 taxonomy_domain cleaner0 2023-07-20T08:42:57Z DUMMY: mouse 0.99932736 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9996487 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9993154 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.99919397 protein cleaner0 2023-07-20T09:24:38Z PR: IL-17F 0.9992888 oligomeric_state cleaner0 2023-07-20T09:24:11Z DUMMY: homodimer 0.9994171 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA RESULTS paragraph 12213 Additionally, we investigated the antagonism of the human IL-17A/IL-17RA interaction by HAP using orthogonal methods including SPR and Förster resonance energy transfer (FRET) competition assays (Fig. 1B,C). In both assays, incubation of IL-17A with HAP effectively blocks the binding of IL-17A to immobilized IL-17RA with similar sub-nM IC50 (Table 3). 0.99922895 species cleaner0 2023-07-20T08:25:04Z MESH: human complex_assembly GO: cleaner0 2023-07-20T08:28:05Z IL-17A/IL-17RA 0.99973136 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9996264 experimental_method cleaner0 2023-07-20T08:43:35Z MESH: SPR experimental_method MESH: cleaner0 2023-07-20T08:44:07Z Förster resonance energy transfer (FRET) competition assays 0.9995489 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9997228 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99951553 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9434454 protein_state cleaner0 2023-07-20T09:55:35Z DUMMY: immobilized 0.9993718 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.9993969 evidence cleaner0 2023-07-20T08:32:49Z DUMMY: IC50 RESULTS title_2 12568 HAP blocks IL-17A signaling in a human primary cell assay 0.9997081 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.93761903 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99930716 species cleaner0 2023-07-20T08:25:04Z MESH: human RESULTS paragraph 12626 While either IL-17A or TNF-α alone can stimulate the release of multiple inflammatory cytokines, when acting together they can synergistically enhance each other’s effects (Supplementary Figure S5). These integrative responses to IL-17A and TNF-α in human keratinocytes have been reported to account for key inflammatory pathogenic circuits in psoriasis. Thus, we chose to study HAP’s efficacy in blocking the production of IL-8, IL-6 and CCL-20 by primary human keratinocytes stimulated by IL-17A in the presence of TNF-α, an assay which may be more disease-relevant. HAP inhibits the production of all three cytokines in a dose-dependent fashion (Fig. 1D). Significantly, the baseline levels of IL-8, IL-6 and CCL-20 stimulated by TNF-α alone are not inhibited by HAP, further indicating the selectivity of HAP (Fig. 1D). Such pharmacological selectivity may be important to suppress inflammatory pathogenic circuits in psoriasis, while sparing the anti-infectious immune responses produced by TNF-α. The relatively high IC50 values in this assay (Table 3) are probably due to the high IL-17A concentration (100 ng/ml) needed for detection of IL-6. As a reference, a commercial anti-IL-17A antibody (R&D Systems) inhibits the production of IL-8 with an IC50 of 13(±6) nM (N = 3). Indeed, the IC50 was 14(±9) nM (N = 12) for HAP inhibition of IL-8 production when only 5 ng/ml IL-17A was used in this assay. In patients, the concentration of IL-17A in psoriatic lesions is reported to be 0.01 ng/ml, well below the EC50 (5–10ng/ml) of IL-17A induced IL-8 production in vitro. 0.9989521 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9993132 protein cleaner0 2023-07-20T08:44:22Z PR: TNF-α protein_type MESH: cleaner0 2023-07-20T08:45:33Z cytokines 0.99909925 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99937916 protein cleaner0 2023-07-20T08:44:23Z PR: TNF-α 0.9993874 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.99958223 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.999372 protein_type cleaner0 2023-07-20T08:44:31Z MESH: IL-8 0.9994655 protein_type cleaner0 2023-07-20T08:44:36Z MESH: IL-6 protein_type MESH: cleaner0 2023-07-20T08:44:54Z CCL-20 0.99926895 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.9993224 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9896974 protein cleaner0 2023-07-20T08:44:23Z PR: TNF-α 0.99960405 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP protein_type MESH: cleaner0 2023-07-20T08:45:34Z cytokines 0.9993946 protein_type cleaner0 2023-07-20T08:44:32Z MESH: IL-8 0.9994555 protein_type cleaner0 2023-07-20T08:44:36Z MESH: IL-6 protein_type MESH: cleaner0 2023-07-20T08:44:55Z CCL-20 0.9222906 protein cleaner0 2023-07-20T08:44:23Z PR: TNF-α 0.99961793 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9995413 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99793345 protein cleaner0 2023-07-20T08:44:23Z PR: TNF-α 0.99960846 evidence cleaner0 2023-07-20T08:32:49Z DUMMY: IC50 0.99871665 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99944514 protein_type cleaner0 2023-07-20T08:44:36Z MESH: IL-6 protein PR: cleaner0 2023-07-20T08:16:51Z IL-17A protein_type MESH: cleaner0 2023-07-20T08:47:54Z antibody 0.99719936 protein_type cleaner0 2023-07-20T08:44:32Z MESH: IL-8 0.999605 evidence cleaner0 2023-07-20T08:32:49Z DUMMY: IC50 0.99960977 evidence cleaner0 2023-07-20T08:32:49Z DUMMY: IC50 0.9996481 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9992235 protein_type cleaner0 2023-07-20T08:44:32Z MESH: IL-8 0.9984741 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9984421 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9857058 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9992984 protein_type cleaner0 2023-07-20T08:44:32Z MESH: IL-8 RESULTS paragraph 14230 Similar to keratinocytes assay results, while HAP inhibits IL-17A stimulated IL-6 production by BJ human fibroblast potently (IC50 of 17 nM), it does not inhibit TNF-α stimulated IL-6 production at concentrations up to 10 μM (Supplementary Figure S2). 0.9993535 experimental_method cleaner0 2023-07-20T08:45:53Z MESH: keratinocytes assay 0.99973947 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP protein PR: cleaner0 2023-07-20T08:16:51Z IL-17A 0.99953794 protein_type cleaner0 2023-07-20T08:44:37Z MESH: IL-6 0.99904794 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.9992768 evidence cleaner0 2023-07-20T08:32:49Z DUMMY: IC50 0.9035897 protein cleaner0 2023-07-20T08:44:23Z PR: TNF-α 0.99948907 protein_type cleaner0 2023-07-20T08:44:37Z MESH: IL-6 RESULTS title_2 14488 Crystallization and structure determination 0.9995088 experimental_method cleaner0 2023-07-20T08:48:17Z MESH: Crystallization and structure determination RESULTS paragraph 14532 Extensive crystallization trials, either by co-crystallization or by soaking HAP into preformed apo IL-17A crystals, failed to lead to an IL-17A/HAP complex crystals. We theorized that HAP binding induced large conformational changes in IL-17A that led to the difficulty of getting an IL-17A/HAP binary complex crystal. It is known that an antibody antigen-binding fragment (Fab) can be used as crystallization chaperones in crystallizing difficult targets. We hypothesized that HAP may target the N-terminal of IL-17A which is known to be more flexible than its C-terminal and conformational changes needed for HAP binding may be more likely there. We designed an antibody Fab known to target the C-terminal half of IL-17A based on a published IL-17A/Fab complex crystal structure, and produced it in HEK293 cells. In an SPR assay HAP and this Fab were able to co-bind IL-17A without large changes in their binding affinities and kinetics, confirming our hypothesis (Supplementary Figure S6). Furthermore, since it binds to an area far away from that of HAP (see below), this Fab should have minimum effects on HAP binding conformation. Crystals of Fab/IL-17A/HAP ternary complex were obtained readily in crystallization screens. 0.99862516 experimental_method cleaner0 2023-07-20T08:48:14Z MESH: crystallization trials 0.9995484 experimental_method cleaner0 2023-07-20T08:48:27Z MESH: co-crystallization 0.99957675 experimental_method cleaner0 2023-07-20T08:48:30Z MESH: soaking 0.9997243 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9996561 protein_state cleaner0 2023-07-20T08:21:59Z DUMMY: apo 0.9994383 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99938405 evidence cleaner0 2023-07-20T08:48:04Z DUMMY: crystals 0.99961644 complex_assembly cleaner0 2023-07-20T08:46:25Z GO: IL-17A/HAP 0.9993255 evidence cleaner0 2023-07-20T08:48:06Z DUMMY: crystals 0.999076 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.999286 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99953806 complex_assembly cleaner0 2023-07-20T08:46:26Z GO: IL-17A/HAP 0.9993813 evidence cleaner0 2023-07-20T08:48:09Z DUMMY: crystal 0.99914825 protein_type cleaner0 2023-07-20T08:47:53Z MESH: antibody 0.99950033 structure_element cleaner0 2023-07-20T08:47:33Z SO: antigen-binding fragment 0.9997451 structure_element cleaner0 2023-07-20T08:47:39Z SO: Fab 0.99963105 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9990602 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99944824 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9992513 protein_type cleaner0 2023-07-20T08:47:54Z MESH: antibody 0.9997279 structure_element cleaner0 2023-07-20T08:47:40Z SO: Fab 0.9996767 structure_element cleaner0 2023-07-20T09:45:49Z SO: C-terminal half 0.9992643 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99960023 complex_assembly cleaner0 2023-07-20T08:46:48Z GO: IL-17A/Fab 0.99963623 evidence cleaner0 2023-07-20T08:48:11Z DUMMY: crystal structure 0.9992395 experimental_method cleaner0 2023-07-20T08:46:37Z MESH: SPR assay 0.9995968 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9997446 structure_element cleaner0 2023-07-20T08:47:40Z SO: Fab 0.9992985 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9995063 evidence cleaner0 2023-07-20T08:48:37Z DUMMY: binding affinities evidence DUMMY: cleaner0 2023-07-20T08:47:19Z kinetics 0.9996674 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9997683 structure_element cleaner0 2023-07-20T08:47:40Z SO: Fab 0.9984326 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99895763 evidence cleaner0 2023-07-20T08:48:34Z DUMMY: Crystals 0.99862987 complex_assembly cleaner0 2023-07-20T08:47:06Z GO: Fab/IL-17A/HAP 0.9885818 experimental_method cleaner0 2023-07-20T09:35:48Z MESH: crystallization screens RESULTS paragraph 15763 Crystallization of IL-17A and its binding partners was accomplished using two forms of IL-17A. These were, respectively, a presumably more homogeneous form of IL-17A that lacked the disordered N-terminal peptide and a full-length form of the cytokine with a full complement of disulfide bonds. (see Method). Crystals of the Fab/truncated IL-17A/HAP complex diffracted to 2.2 Å, and the Fab/full length IL-17A/HAP complex diffracted to 3.0 Å (Supplementary Table S3). Both structures were solved by molecular replacement. Both complexes crystallized in the space group of P321, with half the complex (1 Fab/1 IL-17A monomer/1 HAP) in the asymmetric unit. The intact complex can be generated by applying crystallographic 2-fold symmetry. Electron densities for HAP residues Ile1-Asn14 were readily interpretable with the exception of Lys15, which is disordered. When considering the protein, the complex structure containing the full length IL-17A is identical to that of the truncated IL-17A, with the exception of Cys106 (Ser106 in the truncated IL-17A), which is disordered. Cys106 is covalently linked to Cys10 that resides in the disordered N-terminal peptide in the full length IL-17A. 0.9996735 experimental_method cleaner0 2023-07-20T09:35:54Z MESH: Crystallization 0.99947786 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9995449 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9994836 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99967206 protein_state cleaner0 2023-07-20T09:55:41Z DUMMY: lacked 0.99911493 protein_state cleaner0 2023-07-20T08:50:12Z DUMMY: disordered 0.9991087 structure_element cleaner0 2023-07-20T09:45:53Z SO: N-terminal peptide 0.99956125 protein_state cleaner0 2023-07-20T09:55:47Z DUMMY: full-length protein_type MESH: cleaner0 2023-07-20T08:50:50Z cytokine 0.96609986 ptm cleaner0 2023-07-20T08:22:57Z MESH: disulfide bonds 0.99891126 evidence cleaner0 2023-07-20T09:41:38Z DUMMY: Crystals complex_assembly GO: cleaner0 2023-07-20T08:49:08Z Fab/truncated IL-17A/HAP complex_assembly GO: cleaner0 2023-07-20T08:49:32Z Fab/full length IL-17A/HAP 0.9788112 evidence cleaner0 2023-07-20T09:41:47Z DUMMY: structures 0.99959016 experimental_method cleaner0 2023-07-20T09:38:09Z MESH: molecular replacement 0.8246216 experimental_method cleaner0 2023-07-20T09:38:28Z MESH: crystallized 0.9996136 structure_element cleaner0 2023-07-20T08:47:40Z SO: Fab 0.99951524 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99906343 oligomeric_state cleaner0 2023-07-20T09:23:53Z DUMMY: monomer 0.8256031 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9996556 protein_state cleaner0 2023-07-20T09:56:06Z DUMMY: intact 0.99957204 evidence cleaner0 2023-07-20T09:41:51Z DUMMY: Electron densities 0.85543257 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9728047 residue_range cleaner0 2023-07-20T09:20:04Z DUMMY: Ile1-Asn14 0.99989474 residue_name_number cleaner0 2023-07-20T08:52:46Z DUMMY: Lys15 0.9995851 protein_state cleaner0 2023-07-20T08:50:12Z DUMMY: disordered evidence DUMMY: cleaner0 2023-07-20T09:42:18Z complex structure 0.9996083 protein_state cleaner0 2023-07-20T08:49:57Z DUMMY: full length 0.99952394 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9996094 protein_state cleaner0 2023-07-20T08:50:17Z DUMMY: truncated 0.9994805 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9998896 residue_name_number cleaner0 2023-07-20T08:52:50Z DUMMY: Cys106 0.9998876 residue_name_number cleaner0 2023-07-20T08:52:55Z DUMMY: Ser106 0.9996117 protein_state cleaner0 2023-07-20T08:50:16Z DUMMY: truncated 0.9994609 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.999613 protein_state cleaner0 2023-07-20T08:50:12Z DUMMY: disordered 0.9998901 residue_name_number cleaner0 2023-07-20T08:52:51Z DUMMY: Cys106 0.9998889 residue_name_number cleaner0 2023-07-20T08:53:02Z DUMMY: Cys10 0.9996817 protein_state cleaner0 2023-07-20T08:50:12Z DUMMY: disordered 0.9992804 structure_element cleaner0 2023-07-20T09:45:56Z SO: N-terminal peptide 0.9996056 protein_state cleaner0 2023-07-20T08:49:57Z DUMMY: full length 0.9995246 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A RESULTS title_2 16959 Overall structure of Fab/IL-17A/HAP complex 0.9995376 evidence cleaner0 2023-07-20T09:42:36Z DUMMY: structure complex_assembly GO: cleaner0 2023-07-20T08:47:06Z Fab/IL-17A/HAP RESULTS paragraph 17003 In a similar manner to the published structure of Fab/IL-17A complex, two Fab molecules bind symmetrically to the C-terminal of the cytokine dimer, interacting with epitopes from both monomers (Fig. 2A). Two copies of HAP bind to the N-terminal of the cytokine dimer, also symmetrically, and each HAP molecule also interacts with both IL-17A monomers (Fig. 2). Based on disclosed epitopes of Secukinumab and Ixekizumab, HAP binds to IL-17A at an area that is also different from those of those two antibodies. The N-terminal 5 residues of HAP, 1IHVTI, form an amphipathic β-strand that inserts between β-strand 4 of one IL-17A monomer and β-strand 0 (the first ordered peptide of IL-17A) of the second monomer. This β-strand is parallel to both strands 0 and 4 (Fig. 3B). Strands 0 of two IL-17A monomer are antiparallel, as appeared in other IL-17A structures. The C-terminal 8 residues of the HAP that are ordered in the structure, 7ADLWDWIN, form an amphipathic α-helix interacting with the second IL-17A monomer. Pro6 of HAP makes a transition between the N-terminal β-strand and the C-terminal α-helix of HAP. As a comparison, an IL-17A/IL-17RA complex structure (PDB code 4HSA) is also shown with IL-17A in the same orientation (Fig. 2C). 0.99806315 evidence cleaner0 2023-07-20T09:42:39Z DUMMY: structure 0.9996273 complex_assembly cleaner0 2023-07-20T08:51:15Z GO: Fab/IL-17A 0.9994886 structure_element cleaner0 2023-07-20T08:47:40Z SO: Fab protein_type MESH: cleaner0 2023-07-20T08:50:51Z cytokine 0.9993357 oligomeric_state cleaner0 2023-07-20T09:18:27Z DUMMY: dimer 0.9993024 oligomeric_state cleaner0 2023-07-20T08:23:13Z DUMMY: monomers 0.61343205 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP protein_type MESH: cleaner0 2023-07-20T08:50:51Z cytokine 0.99925727 oligomeric_state cleaner0 2023-07-20T09:18:27Z DUMMY: dimer 0.5626085 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99929076 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9993086 oligomeric_state cleaner0 2023-07-20T08:23:13Z DUMMY: monomers 0.99596775 chemical cleaner0 2023-07-20T08:21:19Z CHEBI: Secukinumab 0.9903983 chemical cleaner0 2023-07-20T08:21:23Z CHEBI: Ixekizumab 0.88919455 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99936366 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9986572 protein_type cleaner0 2023-07-20T09:25:45Z MESH: antibodies 0.99149716 residue_range cleaner0 2023-07-20T09:26:46Z DUMMY: 5 residues 0.55224633 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP chemical CHEBI: cleaner0 2023-07-20T08:52:18Z 1IHVTI 0.7852521 protein_state cleaner0 2023-07-20T08:52:30Z DUMMY: amphipathic 0.99962467 structure_element cleaner0 2023-07-20T09:46:01Z SO: β-strand 0.99964887 structure_element cleaner0 2023-07-20T09:46:05Z SO: β-strand 4 0.9992595 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9992736 oligomeric_state cleaner0 2023-07-20T09:23:53Z DUMMY: monomer 0.99960977 structure_element cleaner0 2023-07-20T08:55:11Z SO: β-strand 0 0.9992409 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99922407 oligomeric_state cleaner0 2023-07-20T09:23:53Z DUMMY: monomer 0.999587 structure_element cleaner0 2023-07-20T09:46:09Z SO: β-strand 0.99940616 structure_element cleaner0 2023-07-20T09:46:12Z SO: strands 0 and 4 0.99930954 structure_element cleaner0 2023-07-20T09:46:16Z SO: Strands 0 0.99928087 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9992731 oligomeric_state cleaner0 2023-07-20T09:23:53Z DUMMY: monomer 0.9980819 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99938834 evidence cleaner0 2023-07-20T09:42:42Z DUMMY: structures 0.98815715 residue_range cleaner0 2023-07-20T09:26:50Z DUMMY: 8 residues 0.7171196 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9983701 evidence cleaner0 2023-07-20T09:42:46Z DUMMY: structure chemical CHEBI: cleaner0 2023-07-20T08:52:00Z 7ADLWDWIN 0.91809016 protein_state cleaner0 2023-07-20T08:52:31Z DUMMY: amphipathic 0.9996877 structure_element cleaner0 2023-07-20T09:46:19Z SO: α-helix 0.9993891 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99920493 oligomeric_state cleaner0 2023-07-20T09:23:53Z DUMMY: monomer 0.9998714 residue_name_number cleaner0 2023-07-20T08:53:09Z DUMMY: Pro6 0.58119506 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9996755 structure_element cleaner0 2023-07-20T09:46:22Z SO: β-strand 0.9996958 structure_element cleaner0 2023-07-20T09:46:25Z SO: α-helix 0.54212296 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99954754 complex_assembly cleaner0 2023-07-20T08:28:05Z GO: IL-17A/IL-17RA evidence DUMMY: cleaner0 2023-07-20T09:42:22Z complex structure 0.99945027 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A RESULTS title_2 18271 Inhibition mechanism of IL-17A signaling by HAP 0.97919995 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9996896 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP RESULTS paragraph 18319 IL-17RA binds IL-17A at three regions on the IL-17A homodimer. HAP binds IL-17A at region I. Region I is formed by residues at the ends of β strands 0 and 4, and from loops 1–2 and 3–4 of IL-17A (Fig. 2). Conformational changes in region I induced by HAP binding alone may allosterically affect IL-17RA binding, but more importantly, the α-helix of HAP directly competes with IL-17RA for binding to IL-17A (Fig. 3). The most significant interactions between the α helix of HAP and IL-17A involve Trp12 of HAP, which binds in a hydrophobic pocket in IL-17A formed by the side chains of Phe110, Tyr62, Pro59 and the hydrophobic portion of the Arg101 side chain (Fig. 3A). The Trp12 side chain of HAP donates a hydrogen bond to the main chain oxygen of Pro69 of IL-17A. The positively charged Arg101 side chain of the IL-17A engages in a charge-helix dipole interaction with the main chain oxygen of Trp12. Additionally, Leu9 and Ile13 of the HAP have hydrophobic interactions with IL-17A, and the Asp8 side chain has hydrogen bond and ion pair interactions with Tyr62 and Lys114 of IL-17A, respectively. 0.99949485 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99935526 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99940854 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99936646 oligomeric_state cleaner0 2023-07-20T09:24:11Z DUMMY: homodimer 0.99929655 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9995146 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9996282 structure_element cleaner0 2023-07-20T08:54:15Z SO: region I 0.99964935 structure_element cleaner0 2023-07-20T08:54:15Z SO: Region I 0.999601 structure_element cleaner0 2023-07-20T09:46:28Z SO: β strands 0 and 4 0.99961 structure_element cleaner0 2023-07-20T09:46:33Z SO: loops 1–2 0.999551 structure_element cleaner0 2023-07-20T09:46:35Z SO: 3–4 0.99930185 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9996196 structure_element cleaner0 2023-07-20T08:54:15Z SO: region I 0.99903524 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99943143 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99970704 structure_element cleaner0 2023-07-20T09:46:39Z SO: α-helix 0.9990969 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9995093 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99942046 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9997335 structure_element cleaner0 2023-07-20T09:46:42Z SO: α helix 0.9985568 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9992655 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9999037 residue_name_number cleaner0 2023-07-20T08:53:16Z DUMMY: Trp12 0.9994375 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99956673 site cleaner0 2023-07-20T09:39:43Z SO: hydrophobic pocket 0.9994507 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99990094 residue_name_number cleaner0 2023-07-20T08:53:21Z DUMMY: Phe110 0.9999037 residue_name_number cleaner0 2023-07-20T08:53:25Z DUMMY: Tyr62 0.9999033 residue_name_number cleaner0 2023-07-20T08:53:30Z DUMMY: Pro59 0.99990165 residue_name_number cleaner0 2023-07-20T08:53:35Z DUMMY: Arg101 0.9998971 residue_name_number cleaner0 2023-07-20T08:53:17Z DUMMY: Trp12 0.9993279 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:18:31Z hydrogen bond 0.9999037 residue_name_number cleaner0 2023-07-20T08:53:42Z DUMMY: Pro69 0.99942374 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99989986 residue_name_number cleaner0 2023-07-20T08:53:36Z DUMMY: Arg101 0.9994616 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:18:31Z charge-helix dipole interaction 0.99990106 residue_name_number cleaner0 2023-07-20T08:53:17Z DUMMY: Trp12 0.99990165 residue_name_number cleaner0 2023-07-20T08:53:50Z DUMMY: Leu9 0.9999014 residue_name_number cleaner0 2023-07-20T08:53:55Z DUMMY: Ile13 0.9982498 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:18:31Z hydrophobic interactions 0.99939984 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9999045 residue_name_number cleaner0 2023-07-20T08:54:00Z DUMMY: Asp8 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:18:31Z hydrogen bond bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:18:31Z ion pair interactions 0.99990094 residue_name_number cleaner0 2023-07-20T08:53:26Z DUMMY: Tyr62 0.99989843 residue_name_number cleaner0 2023-07-20T08:54:06Z DUMMY: Lys114 0.99948734 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A RESULTS paragraph 19432 In region I, an IL-17RA peptide interacts with IL-17A in a very similar fashion to the α-helix of HAP. The IL-17RA peptide has sequences of 27LDDSWI, and part of the peptide is also α-helical (Fig. 3B). Leu7, Trp31 and Ile32 of IL-17RA interact very similarly with the same residues of IL-17A as Leu9, Trp12 and Ile13 of HAP (Fig. 3B). In this sense, the α-helix of HAP with a sequence of 9LWDWI is a good mimetic of the 27LDDSWI peptide of IL-17RA. 0.9996127 structure_element cleaner0 2023-07-20T08:54:15Z SO: region I 0.99875784 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.9995494 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99970263 structure_element cleaner0 2023-07-20T09:46:49Z SO: α-helix 0.9991284 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9990926 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.7019358 chemical cleaner0 2023-07-20T08:54:33Z CHEBI: 27LDDSWI 0.95826167 structure_element cleaner0 2023-07-20T09:46:54Z SO: α-helical 0.99990034 residue_name_number cleaner0 2023-07-20T08:55:19Z DUMMY: Leu7 0.9999012 residue_name_number cleaner0 2023-07-20T08:55:24Z DUMMY: Trp31 0.9999027 residue_name_number cleaner0 2023-07-20T08:55:28Z DUMMY: Ile32 0.99933296 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.999542 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.99989986 residue_name_number cleaner0 2023-07-20T08:53:51Z DUMMY: Leu9 0.9999027 residue_name_number cleaner0 2023-07-20T08:53:17Z DUMMY: Trp12 0.9999013 residue_name_number cleaner0 2023-07-20T08:53:56Z DUMMY: Ile13 0.99930537 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9996958 structure_element cleaner0 2023-07-20T09:46:58Z SO: α-helix 0.99933165 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9877137 chemical cleaner0 2023-07-20T08:54:46Z CHEBI: 9LWDWI 0.28522155 chemical cleaner0 2023-07-20T08:54:33Z CHEBI: 27LDDSWI 0.9992437 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA RESULTS paragraph 19894 The β-strand of HAP has no equivalent in IL-17RA. However, it mimics the β-strand 0 of IL-17A. The amphipathic β-strand of HAP orients the hydrophilic side chains of His2 and Thr4 outwards, and the hydrophobic side chains of Ile1, Val3 and Ile5 inward (Fig. 3A). β-strand 0 in IL-17A is also amphipathic with the sequence of 21TVMVNLNI. In all IL-17A structures obtained to date, β-strand 0 orients the hydrophilic side chains of Thr21, Asn25 and Asn27 outward, and the hydrophobic side chains of Val22, Val24, Leu26 and Ile28 inward. 0.999598 structure_element cleaner0 2023-07-20T09:47:04Z SO: β-strand 0.99895287 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99928015 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99962753 structure_element cleaner0 2023-07-20T08:55:10Z SO: β-strand 0 0.99929506 protein cleaner0 2023-07-20T08:16:51Z PR: IL-17A 0.9992919 protein_state cleaner0 2023-07-20T08:52:31Z DUMMY: amphipathic 0.99960834 structure_element cleaner0 2023-07-20T09:47:02Z SO: β-strand 0.9991375 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9998934 residue_name_number cleaner0 2023-07-20T08:55:43Z DUMMY: His2 0.999887 residue_name_number cleaner0 2023-07-20T08:55:47Z DUMMY: Thr4 0.99989235 residue_name_number cleaner0 2023-07-20T08:55:52Z DUMMY: Ile1 0.999892 residue_name_number cleaner0 2023-07-20T08:55:56Z DUMMY: Val3 0.9998919 residue_name_number cleaner0 2023-07-20T08:56:01Z DUMMY: Ile5 0.9996271 structure_element cleaner0 2023-07-20T08:55:11Z SO: β-strand 0 0.9993189 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.99940896 protein_state cleaner0 2023-07-20T08:52:31Z DUMMY: amphipathic 0.9022796 chemical cleaner0 2023-07-20T08:54:59Z CHEBI: 21TVMVNLNI 0.99885064 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9995154 evidence cleaner0 2023-07-20T09:42:53Z DUMMY: structures 0.9996283 structure_element cleaner0 2023-07-20T08:55:11Z SO: β-strand 0 0.9998964 residue_name_number cleaner0 2023-07-20T08:56:08Z DUMMY: Thr21 0.99989736 residue_name_number cleaner0 2023-07-20T08:56:12Z DUMMY: Asn25 0.99989593 residue_name_number cleaner0 2023-07-20T08:56:16Z DUMMY: Asn27 0.99989736 residue_name_number cleaner0 2023-07-20T08:56:21Z DUMMY: Val22 0.99989736 residue_name_number cleaner0 2023-07-20T08:56:25Z DUMMY: Val24 0.99989676 residue_name_number cleaner0 2023-07-20T08:56:29Z DUMMY: Leu26 0.9998976 residue_name_number cleaner0 2023-07-20T08:56:34Z DUMMY: Ile28 RESULTS paragraph 20444 The binding pocket occupied by either Trp12 of HAP or Trp31 of IL-17RA is not formed in the apo IL-17A structure (Fig. 3C). Conformational changes of IL-17A are needed for both HAP and IL-17RA to bind to that region. Particularly for HAP, β-strands 0 have to shift out of the hydrophobic cleft formed by the main body of the IL-17A by as much as 10 Å between Cα atoms (Fig. 3C). Disruptions of the apo IL-17A structure by HAP binding are apparently compensated for by formation of the new interactions that involve almost the entire HAP molecule (Fig. 3B). 0.99961424 site cleaner0 2023-07-20T09:39:47Z SO: binding pocket 0.99989605 residue_name_number cleaner0 2023-07-20T08:53:17Z DUMMY: Trp12 0.9997621 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99989736 residue_name_number cleaner0 2023-07-20T08:55:25Z DUMMY: Trp31 0.99956274 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99963915 protein_state cleaner0 2023-07-20T08:21:59Z DUMMY: apo 0.99955493 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9995809 evidence cleaner0 2023-07-20T09:42:58Z DUMMY: structure 0.9994552 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9997068 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99957436 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99975723 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.9995387 structure_element cleaner0 2023-07-20T09:47:12Z SO: β-strands 0 0.99959135 site cleaner0 2023-07-20T09:39:50Z SO: hydrophobic cleft 0.99966615 structure_element cleaner0 2023-07-20T09:47:17Z SO: main body 0.99951905 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9996426 protein_state cleaner0 2023-07-20T08:21:59Z DUMMY: apo 0.99947864 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.99891293 evidence cleaner0 2023-07-20T09:43:00Z DUMMY: structure 0.99960774 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99975985 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP RESULTS title_2 21008 Structure basis for the observed SAR of peptides experimental_method MESH: cleaner0 2023-07-20T09:04:27Z SAR RESULTS paragraph 21057 The IL-17A/HAP complex structure obtained is very consistent with the observed SAR of our identified peptide inhibitors, explaining well how the evolution of the initial phage peptide 1 to HAP and 45 improved its potency (Supplementary Figure S7). The important interactions involving Trp12 of HAP explain the >90 times drop in potency of the W12A variant (6 vs 1, Table 1). The amphipathic nature of the HAP β-strand explains the preference of the hydrophilic residues at the 2 and 4 positions of peptides (14, 18, 19, 21 and 23 vs 1 and 22, Table 1). All N-terminal residues of HAP are part of the β-sheet with β-stands 0 and 4 of IL-17A, which explains why removal of the first 1–3 residues completely abolishes the ability of HAP to block IL-17A cell signaling (31,32 and 33, Table 2). The C-terminal Asn14 and Lys15 of HAP are not directly involved in interactions with IL-17A, and this is reflected in the gradual reduction in activity caused by C-terminal truncations (35 and 36, Table 2). Each peptide monomer in 45 may not necessarily be more potent than HAP, but two monomer peptides within the same molecule that can simultaneously bind to IL-17A can greatly improve its potency due to avidity effects. 0.9996457 complex_assembly cleaner0 2023-07-20T08:46:26Z GO: IL-17A/HAP evidence DUMMY: cleaner0 2023-07-20T09:42:22Z complex structure experimental_method MESH: cleaner0 2023-07-20T09:04:27Z SAR experimental_method MESH: cleaner0 2023-07-20T09:01:14Z phage chemical CHEBI: cleaner0 2023-07-20T08:38:10Z peptide 1 0.98668355 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.98723143 chemical cleaner0 2023-07-20T09:58:35Z CHEBI: 45 0.99989724 residue_name_number cleaner0 2023-07-20T08:53:17Z DUMMY: Trp12 0.67716074 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99962044 mutant cleaner0 2023-07-20T09:02:54Z MESH: W12A 0.9936667 protein_state cleaner0 2023-07-20T08:52:31Z DUMMY: amphipathic 0.68529487 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99964666 structure_element cleaner0 2023-07-20T09:47:22Z SO: β-strand 0.9873102 residue_number cleaner0 2023-07-20T09:28:56Z DUMMY: 2 0.9828635 residue_number cleaner0 2023-07-20T09:28:59Z DUMMY: 4 0.41763604 chemical cleaner0 2023-07-20T09:58:41Z CHEBI: 14 0.40545538 chemical cleaner0 2023-07-20T09:58:44Z CHEBI: 18 0.3360236 chemical cleaner0 2023-07-20T09:58:47Z CHEBI: 19 0.3754059 chemical cleaner0 2023-07-20T09:58:49Z CHEBI: 21 0.3401372 chemical cleaner0 2023-07-20T09:58:52Z CHEBI: 23 chemical CHEBI: cleaner0 2023-07-20T09:01:41Z 1 chemical CHEBI: cleaner0 2023-07-20T09:01:51Z 22 0.47717986 chemical cleaner0 2023-07-20T08:18:36Z CHEBI: HAP 0.99965686 structure_element cleaner0 2023-07-20T09:47:25Z SO: β-sheet 0.99937516 structure_element cleaner0 2023-07-20T09:47:28Z SO: β-stands 0 and 4 0.99845576 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.95655215 experimental_method cleaner0 2023-07-20T09:38:42Z MESH: removal of residue_range DUMMY: cleaner0 2023-07-20T09:27:17Z first 1–3 residues 0.5255684 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP protein PR: cleaner0 2023-07-20T08:16:52Z IL-17A chemical CHEBI: cleaner0 2023-07-20T08:35:17Z 31 chemical CHEBI: cleaner0 2023-07-20T09:02:03Z 32 chemical CHEBI: cleaner0 2023-07-20T09:02:12Z 33 0.99989414 residue_name_number cleaner0 2023-07-20T09:21:02Z DUMMY: Asn14 0.9998921 residue_name_number cleaner0 2023-07-20T08:52:46Z DUMMY: Lys15 0.7684114 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.99879616 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.5781626 experimental_method cleaner0 2023-07-20T09:38:48Z MESH: truncations chemical CHEBI: cleaner0 2023-07-20T09:02:24Z 35 chemical CHEBI: cleaner0 2023-07-20T09:02:35Z 36 0.9993181 oligomeric_state cleaner0 2023-07-20T09:23:53Z DUMMY: monomer 0.96388507 chemical cleaner0 2023-07-20T09:58:56Z CHEBI: 45 0.5796342 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.99935955 oligomeric_state cleaner0 2023-07-20T09:23:53Z DUMMY: monomer 0.9987038 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A RESULTS paragraph 22280 HAP targets region I of IL-17A, an area that has the least sequence conservation in IL-17 cytokines. This lack of sequence conservation in the HAP binding site explains the observed specificity of HAP binding to human IL-17A. For example, inspection of the published IL-17F crystal structure (PDB code 1JPY) revealed a pocket of IL-17F similar to that of IL-17A for W12 of HAP binding, but it is occupied by a Phe-Phe motif at the N-terminal peptide of IL-17F. This Phe-Phe motif is missing in IL-17A. Sequence alignments between human and mouse IL-17A indicated that among IL-17A residues that interacting with HAP, majority differences occur in strand 0 of IL-17A which interacts with the N-terminal β-strand of HAP. In human IL-17A the sequences are 21TVMVNLNI, and in mouse they are 21NVKVNLKV. 0.9994549 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.99918175 structure_element cleaner0 2023-07-20T08:54:15Z SO: region I 0.99885875 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9995997 protein_type cleaner0 2023-07-20T08:20:46Z MESH: IL-17 cytokines 0.9996042 site cleaner0 2023-07-20T09:03:35Z SO: HAP binding site 0.9994111 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9990902 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.9993593 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9993753 protein cleaner0 2023-07-20T09:24:38Z PR: IL-17F 0.99959993 evidence cleaner0 2023-07-20T09:43:11Z DUMMY: crystal structure 0.99950004 site cleaner0 2023-07-20T09:39:56Z SO: pocket 0.99945974 protein cleaner0 2023-07-20T09:24:38Z PR: IL-17F 0.99938995 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9996131 residue_name_number cleaner0 2023-07-20T09:21:05Z DUMMY: W12 0.9995919 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9995097 structure_element cleaner0 2023-07-20T09:03:47Z SO: Phe-Phe motif 0.9994707 protein cleaner0 2023-07-20T09:24:38Z PR: IL-17F 0.99954486 structure_element cleaner0 2023-07-20T09:03:48Z SO: Phe-Phe motif 0.99655885 protein_state cleaner0 2023-07-20T09:56:43Z DUMMY: missing 0.99916726 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.99954736 experimental_method cleaner0 2023-07-20T09:38:53Z MESH: Sequence alignments 0.9992693 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.9207432 taxonomy_domain cleaner0 2023-07-20T08:42:57Z DUMMY: mouse 0.9992469 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9975853 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9996598 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9996507 structure_element cleaner0 2023-07-20T09:47:34Z SO: strand 0 0.9992854 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.99962634 structure_element cleaner0 2023-07-20T09:47:37Z SO: β-strand 0.9995598 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.99916685 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.99937075 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.6848422 chemical cleaner0 2023-07-20T08:55:01Z CHEBI: 21TVMVNLNI 0.99622476 taxonomy_domain cleaner0 2023-07-20T08:42:58Z DUMMY: mouse 0.67692673 chemical cleaner0 2023-07-20T09:03:58Z CHEBI: 21NVKVNLKV DISCUSS title_1 23082 Discussion DISCUSS paragraph 23093 Using a combination of phage display and SAR we have discovered novel peptides that are IL-17A antagonists. One of those peptides, HAP, also shows activity in inhibiting the production of multiple inflammatory cytokines by primary human keratinocytes stimulated by IL-17A and TNF-α, a disease relevant-model. We have also determined the complex structure of IL-17A/HAP, which provides the structural basis for HAP’s antagonism to IL-17A signaling. 0.99951345 experimental_method cleaner0 2023-07-20T09:04:21Z MESH: phage display 0.99884343 experimental_method cleaner0 2023-07-20T09:04:27Z MESH: SAR 0.94560504 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9995239 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP protein_type MESH: cleaner0 2023-07-20T08:45:34Z cytokines 0.99935263 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.9938674 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.99192315 protein cleaner0 2023-07-20T08:44:23Z PR: TNF-α 0.9757987 experimental_method cleaner0 2023-07-20T09:39:01Z MESH: determined evidence DUMMY: cleaner0 2023-07-20T09:42:22Z complex structure complex_assembly GO: cleaner0 2023-07-20T08:46:26Z IL-17A/HAP 0.999521 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9747037 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A DISCUSS paragraph 23545 During IL-17A signaling, IL-17A binds to one copy of IL-17RA and one copy of IL-17RC. Since apo IL-17A is a homodimer with 2 fold symmetry, IL-17RA potentially can bind to either face of the IL-17A dimer. With two HAP molecules covering both faces of the IL-17A dimer, HAP can block IL-17RA approaching from either face. To form the 1:2 complex observed in crystal structure, it is important that there is no strong negative cooperativity in the binding of two HAP molecules. In fact, in native electrospray ionization mass spectrometry analysis only 1:2 IL-17A/HAP complex was observed even when IL-17A was in excess (Supplementary Figure S8), indicating a positive binding cooperativity that favors inhibition of IL-17RA binding by HAP. 0.99425274 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.99880075 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.99937516 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99941367 protein cleaner0 2023-07-20T08:20:07Z PR: IL-17RC 0.9996289 protein_state cleaner0 2023-07-20T08:21:59Z DUMMY: apo 0.99945265 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.99932516 oligomeric_state cleaner0 2023-07-20T09:24:11Z DUMMY: homodimer 0.9992736 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99930763 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.999328 oligomeric_state cleaner0 2023-07-20T09:18:27Z DUMMY: dimer 0.99969614 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.999307 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9993388 oligomeric_state cleaner0 2023-07-20T09:18:27Z DUMMY: dimer 0.9996923 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9994337 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.9996326 evidence cleaner0 2023-07-20T09:43:22Z DUMMY: crystal structure 0.99968565 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9995424 experimental_method cleaner0 2023-07-20T09:39:09Z MESH: native electrospray ionization mass spectrometry 0.9995264 complex_assembly cleaner0 2023-07-20T08:46:26Z GO: IL-17A/HAP 0.99953824 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9984725 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.9996768 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP DISCUSS paragraph 24284 HAP, with only 15 residues, can achieve almost the same binding affinity as the much larger IL-17RA molecule, indicating a more efficient way of binding to IL-17A. The interaction of IL-17A with IL-17RA has an extensive interface, covering ~2,200 Å2 surface area of IL-17A. Due to the discontinuous nature of the IL-17A/IL-17RA binding interface, it is classified as having tertiary structural epitopes on both binding partners, and is therefore hard to target using small molecules. Our studies of HAP demonstrated an uncommon mode of action for a peptide in inhibiting such a difficult protein-protein interaction target, and suggest further possible improvements in its binding potency. 0.99971944 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.93260455 residue_range cleaner0 2023-07-20T09:27:23Z DUMMY: 15 residues 0.9994731 evidence cleaner0 2023-07-20T09:43:25Z DUMMY: binding affinity 0.9992774 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.9994075 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9994039 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.99942493 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.9994136 site cleaner0 2023-07-20T09:40:05Z SO: interface 0.9994745 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9989624 site cleaner0 2023-07-20T09:05:20Z SO: IL-17A/IL-17RA binding interface 0.99971825 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP DISCUSS paragraph 24977 One way of further improving HAP’s potency is by dimerization. Homo-dimerization of HAP (45) achieved sub-nanomolar potency against human IL-17A in cell assay. In the crystal structure, the distance between the carbonyl of Asn14 of one HAP molecule and the N-terminus of the second is only 15.7 Å, suggesting the potential for more potent dimeric peptides to be designed by using linkers of different lengths at different positions. 0.9989919 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9997008 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP chemical CHEBI: cleaner0 2023-07-20T09:05:36Z 45 0.9993666 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.8267777 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9995927 evidence cleaner0 2023-07-20T09:43:30Z DUMMY: crystal structure 0.9998982 residue_name_number cleaner0 2023-07-20T09:21:10Z DUMMY: Asn14 0.99961996 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9993316 oligomeric_state cleaner0 2023-07-20T08:33:39Z DUMMY: dimeric 0.99874264 chemical cleaner0 2023-07-20T09:59:17Z CHEBI: peptides DISCUSS paragraph 25415 Another direction of improving HAP is by reducing its size. As demonstrated by the crystal structure, binding of the α-helix of HAP should be sufficient for preventing IL-17RA binding to IL-17A. Theoretically, it is possible to design chemicals such as stapled α-helical peptides to block α-helix-mediated IL-17A/IL-17RA interactions. Such peptides may have smaller sizes with more favorable physical properties (proteolytic resistance, serum half-life, permeability, etc). 0.9902175 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9996195 evidence cleaner0 2023-07-20T09:43:34Z DUMMY: crystal structure 0.99971706 structure_element cleaner0 2023-07-20T09:47:54Z SO: α-helix 0.9993011 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9988205 protein cleaner0 2023-07-20T08:17:00Z PR: IL-17RA 0.99886304 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.9997104 structure_element cleaner0 2023-07-20T09:47:57Z SO: α-helix complex_assembly GO: cleaner0 2023-07-20T08:28:05Z IL-17A/IL-17RA DISCUSS paragraph 25901 In summary, these peptide-based anti-IL-17A modalities could be further developed as alternative therapeutic options to the reported monoclonal antibodies. We are also very interested in finding non-peptidic small molecule IL-17A antagonists, and HAP can be used as an excellent tool peptide. The strategy utilized in generating the complex structures of HAP may also be useful for enabling structure based design of some known small molecule IL-17A antagonists. 0.8363242 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.92506737 protein_type cleaner0 2023-07-20T09:25:45Z MESH: antibodies 0.9988014 protein cleaner0 2023-07-20T08:16:52Z PR: IL-17A 0.99968266 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9987901 evidence cleaner0 2023-07-20T09:43:37Z DUMMY: structures 0.9997285 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP METHODS title_1 26364 Methods METHODS title_2 26372 Peptide synthesis METHODS paragraph 26390 Peptides were synthesized either on a PTI Symphony or Biotage Syro II synthesizer employing standard Fmoc chemistry on Rink resin with N-terminal amine capped with acetyl group unless noted otherwise. Resin, amino acids and solvents were purchased from EMD Chemicals. Peptides were then removed from polymer support using the cleavage cocktail (88:5:5:2, TFA:PhOH:H2O:TIPS) and precipitated with cold diethyl ether. For the dimers, the native peptides incorporating a primary amine (N-terminal amine or lysine at specific positions in the peptide sequence) were mixed with the bis-pentafluoroesters of the desired PEG (purchased from Quanta BioDesign) in the presence of DIEA in DMF. Peptides were purified by reversed phase HPLC on Phenomenex preparative Luna C18 columns using water:acetonitrile gradients and lyophilized. METHODS title_2 27215 HPLC-MS conditions for peptide purity analysis METHODS paragraph 27262 All peptides were assessed for purity by analytical C18 RP-HP-LCMS prior to use in biological assays. Buffers used were 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B). The standard method (1) consisted of a linear gradient of 5% to 95% B over 10 minutes on Agilent 1100 series HPLC-MSD and method (2) consisted of a linear gradient of 5% to 95% B over 8 minutes on Waters 2767 series HPLC-MSD. The C18 column (Phenomenex, Luna C18, 4.6 × 150mm) effluent was immediately mass analyzed in electrospray positive mode. Accurate mass measurements of final peptides were performed using C18 reversed-phase chromatography mass spectrometry (RPHPLCMS) and mass detected on a Waters Synapt G2 Q-Tof mass spectrometer tuned to a resolution (FWHM) of 25,000. Exact intact masses were calculated based on the monoisotopic m/z value of the base peak charge state. All peptides were analyzed using these methods. Supporting data of peptide 45 is shown (Supplementary Figure S2) as representative data sets of the all the molecules investigated (characterization of all peptides presented on Table 1S). METHODS title_2 28401 ELISA assay METHODS paragraph 28413 rhIL-17 (#317-ILB), IL-17R (#2269-IL) were from R&D Systems. ELISA wash buffer (50-63-01) and TMB SureBlue Microwell Peroxidase substrate (#50-63-01 or #52-00-00) were from KPL. Half-well high-binding ELISA plates (#3690) and full-well plates (#9018) were from Costar. Superblock (#2011 or AAA500) was from ScyTek. No-Weigh NSH-PEG4-Biotin (21329) and Zeba Desalt Spin Columns (89882) were from Pierce. METHODS paragraph 28816 ELISAs were as follows in duplicate unless otherwise specified. Plates were coated in PBS overnight at 4 °C and blocked using Superblock for one hour at RT. Subsequent incubations were for 1 hr at room temperature with dilutions in Superblock. Plates were washed 3X between steps using a Biotek ELx405 plate washer and developed using TMB substrate. Reactions were stopped in 2 M H2SO4 and OD450 values were read on a Molecular Devices SpectraMax Plus plate reader. METHODS paragraph 29289 IL-17 RA competition: half-well plates were coated with 0.5 μg/mL IL-17R-Fc and blocked. Peptides were titrated in Superblock containing 0.4 μg/mL Biotinylated IL-17A. Biotinylated IL-17A was detected by adding tetramethylbenzidine (TMB). Reactions were stopped in 2 M H2SO4 and OD450 values were read on a Molecular Devices SpectraMax Plus plate reader. METHODS title_2 29652 Human BJ fibroblast and murine MLE-12 cell assays for inhibitor screen METHODS paragraph 29723 For inhibitor screen, human BJ fibroblast cells (ATCC CRL2522) (American Type Culture Collection, VA) were used. For mouse cell based assay MLE-12 mouse epithelial cells (ATCC CRL2110) were used. Both cell lines were maintained in ATCC recommended media. Cells were seeded at 5 × 103 cells/well into 96-well flat-bottom microtiter plates in which peptides that had been pre-diluted with cytokines (1 ng/mL for human IL-17A or 15 ng/mL for mouse IL-17A) in culture medium. Cells were incubated at 37 °C for 16–24 hrs, and supernatants were collected and analyzed by ELISA for either human CXCL1/GRO-α (R&D Systems DY275) or mouse CXCL1/KC (R&D Systems DY453). METHODS title_2 30399 Primary human keratinocytes cell assay METHODS paragraph 30438 Primary human keratinocytes were cultured in Epilife medium with EDGS (Life Technology, Cascade Biologics) following product instructions. 5 days after establishing the culture from frozen vials, cells were plated at 10,000/well (80 μl) in culture media in 384 well plate. 4 hours after plating the cells, 10 μl of 10X peptide stocks were added. Final DMSO concentration was 1%. Immediately after peptide addition, 10 μl of a mixture of recombinant human IL-17A (endotoxin Level <0.10 EU per 1 μg of the protein, E.coli expression, >97% pure judged by SDS/page, R&D System, Minneapolis, MN) 100 ng /ml (final) and TNF-α (Sigma-Aldrich, St Louis, MO) 10 ng/ ml (final), or TNF-α 10 ng/ml only were added to the cells. Cell assay plates were incubated for 48 hours at 37 °C in a tissue culture incubator before harvesting culture supernatants for analysis of IL-6, IL-8 and CCL-20 production using kits K211AKB-2, K211ANB-2, and K211BEB-2, respectively (Meso Scale Discovery, Rockville, MD). METHODS title_2 31455 Protein production METHODS paragraph 31474 Details of non-commercial protein constructs used in this study and their purifications are in the Supplementary Material. METHODS title_2 31597 SPR binding assays METHODS paragraph 31616 The IL-17 SPR binding assay was run on a Biacore 3000 SPR instrument (GE Healthcare). Biotinylated human IL-17A, or IL-17A/F heteromer, or IL-17F, or mouse IL-17A (Cell Signaling) was captured on a Biacore Streptavidin chip to achieve protein density of about 2500 to 3500 RUs on the surface. The SPR running buffer was 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% P20 with 3% DMSO. Peptide samples were injected at a flow rate of 50 μl/min for 180 seconds association and at least 600 seconds dissociation using a 2–3 fold dilution series. Multiple blank injections were run before and after each peptide series for references. The data were processed and analyzed with Scrubber 2.0 (BioLogical Software) and Biaeval software (GE Healthcare) to calculate binding constants and on and off rates. METHODS paragraph 32416 The IL-17A SPR competition assay was run on a BioRAD ProteON instrument (BioRAD Laboratories). IL-17RA-Fc fusion protein (R&D Systems) was captured on a GLM chip using the standard amine coupling reaction. The chip surface activated using a mixture of sulfo-NHS and EDC, was exposed to the receptor dissolved in acetate buffer pH 5.0 at 0.01 mg/ml concentration. The surface was deactivated using 1 M ethanolamine HCl. An adjacent flow cell on the chip was treated identically without the protein and was used as a reference surface for subsequent data analysis. Three-fold dilution series of peptides mixed with 5 nM IL-17A were injected at a flow rate of 50 μl/min for 120 seconds, followed by 300 seconds of dissociation. The receptor on the surface was regenerated using a 30 second injection of 3 M MgCl2. Observed signal between 180 and 200 seconds was averaged for each sample. Average response observed for multiple 5 nM IL-17A samples without any peptide was used as 100% signal for calculating % inhibition for each concentration of compound. The data were fit with Microcal Origin software (OriginLab, MA) to calculate IC50 for peptides. METHODS title_2 33578 FRET Assay METHODS paragraph 33589 The FRET signal of a Eu3+-labeled IL-17A donor and an Alexa Fluor 647 labeled IL-17RA acceptor was measured to monitor the interaction of IL-17A and IL-17RA. Maximal FRET was observed when IL-17A was bound to IL-17RA and diminished FRET was observed when IL-17A was separated from IL-17RA. The excitation of the donor at a wavelength of 320 nm triggers fluorescence at 615 nM and this in turn serves to excite the acceptor, which then fluoresces at a wavelength of 665 nm. The fluorescence at both 615 nm and 665 nm were measured and the ratio of 665 nm/615 nm was used to monitor the IL-17A/IL-17RA binding. Final assay concentrations were 1 nM biotinylated IL-17A labeled with 0.67 nM Europium-Streptavidin (Invitrogen), 6 nM IL-17RA-Fc fusion protein (R&D Systems) labeled with 1 nM Alexa Fluor 647 antibody (BioLegand) in a buffer containing 10 mM HEPES pH 7.4, 150 mM NaCl, 0.02% BSA, and 0.01% Tween 20. Peptides were tested using a half log dilution series of 11 concentrations. The IL-17A was incubated with the Europium-Streptavidin to 1.5X final assay concentration for one hour at room temperature. Peptides were prepared at 50X final concentration in 100% DMSO and 300 nl were added to a 384-well white assay plate (Greiner). 10 μl of the Eu3+-labeled IL-17A was added to the peptides and incubated at room temperature for one hour. During this pre-incubation of peptide and IL-17A, IL-17RA was incubated with Alexa Fluor 647 antibody to 3X final assay concentration at room temperature for one hour and then 5 μl of the 3X Alexa Fluor 647 labeled IL-17RA was added to the assay for a total volume of 15.3 μl and a final DMSO concentration of 2%. The plates were covered and incubated at room temperature for 3 hours. The FRET signal of the IL-17A/IL-17RA interaction was measured using an EnVision Multilabel plate reader (PerkinElmer). The peptide data was converted into % inhibition, using 0% (no HAP) and 100% inhibition (100 nM HAP) as controls. A four parameter logistic nonlinear regression model using the percent inhibition at each concentration was used to calculate an IC50 for each peptide. METHODS title_2 35746 Protein crystallization and data collection METHODS paragraph 35790 To crystallize Fab/IL-17A/HAP complex, 30 mM HAP in DMSO stock was added to the Fab/IL-17A complex to a final concentration of 1 mM. The complex was then screened for crystals using commercial screen kits using a sitting drop vapor diffusion format. Crystals of Fab/truncated IL-17A/HAP complex were obtained under condition of 0.02 mM CdCl2, 0.02 M MgCl2, 0.02 M NiCl2, 0.1 M NaOAc pH = 4.2–4.9, and 24–28% PEG MME 2000. Crystals of Fab/full length IL-17A covalent dimer/HAP complex were obtained under conditions of 0.1 M NaOAc, pH = 4.5 and 30% PEG MME 5000. Crystals were soaked briefly in cryo solutions of the mother liquor supplemented with 25% glycerol before flash cooled in liquid nitrogen. Crystal data sets were collected at APS IMCA 17ID beamline (Chicago, IL), processed with autoPROC. Data collection statistics are listed in Supplementary Table 2. METHODS title_2 36680 Structure determination and refinement METHODS paragraph 36719 Fab/IL-17A/HAP complex structures were solved with molecular replacement method using the published FAN/IL-17A crystal structure (pdb code 2VXS), using program Phaser. Structure refinements was carried out using program Buster and manual model building using program COOT. Final refinement statistics are listed in Supplementary Table 2. METHODS title_1 37057 Additional Information METHODS paragraph 37080 How to cite this article: Liu, S. et al. Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide. Sci. Rep. 6, 26071; doi: 10.1038/srep26071 (2016). SUPPL title_1 37246 Supplementary Material 556 567 surname:Gaffen;given-names:S. L. 10.1038/nri2586 19575028 REF Nat Rev Immunol ref 9 2009 37269 Structure and signalling in the IL-17 receptor family 479 489 surname:Cua;given-names:D. J. surname:Tato;given-names:C. M. 20559326 REF Nat Rev Immunol ref 10 2010 37323 Innate IL-17-producing cells: the sentinels of the immune system 36 39 surname:Toy;given-names:D. 10.4049/jimmunol.177.1.36 16785495 REF J Immunol. ref 177 2006 37388 Cutting Edge: Interleukin 17 Signals through a Heteromeric Receptor Complex 311 321 surname:Onishi;given-names:R. M. surname:Gaffen;given-names:S. L. 10.1111/j.1365-2567.2009.03240.x 20409152 REF Immunology ref 129 2010 37464 Interleukin-17 and its target genes: mechanisms of interleukin-17 function in disease R120 128 surname:Hwang;given-names:S. Y. 10.1186/ar1038 15059275 REF Arthritis Res Ther ref 6 2004 37550 IL-17 induces production of IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts via NF-kappaB- and PI3-kinase/Akt-dependent pathways 2636 2649 surname:Mills;given-names:K. H. 10.1002/eji.200838535 18958872 REF Eur J Immunol ref 38 2008 37690 Induction, function and regulation of IL-17-producing T cells 549 553 surname:van den Berg;given-names:W. B. surname:Miossec;given-names:P. 10.1038/nrrheum.2009.179 19798029 REF Nat Rev Rheumatol ref 5 2009 37752 IL-17 as a future therapeutic target for rheumatoid arthritis 291 297 surname:Puel;given-names:A. 10.1084/jem.20091983 20123958 REF J Exp Med. ref 207 2010 37814 Autoantibodies against IL-17A, IL-17F, and IL-22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type I 65 68 surname:Puel;given-names:A. 10.1126/science.1200439 21350122 REF Science ref 332 2011 37959 Chronic Mucocutaneous Candidiasis in Humans with Inborn Errors of Interleukin-17 Immunity surname:Griffiths;given-names:C. E. M. 10.1016/S0140-6736(15)60125-8 REF Lancet ref 2015 38049 Comparison of ixekizumab with etanercept or placebo in moderate-to-severe psoriasis (UNCOVER-2 and UNCOVER-3): results from two phase 3 randomised trials 326 338 surname:Langley;given-names:R. G. 10.1056/NEJMoa1314258 25007392 REF N Engl J Med. ref 371 2014 38203 Secukinumab in Plaque Psoriasis — Results of Two Phase 3 Trials 1137 1146 surname:McInnes;given-names:I. B. 10.1016/S0140-6736(15)61134-5 26135703 REF Lancet ref 386 2015 38269 Secukinumab, a human anti-interleukin-17A monoclonal antibody, in patients with psoriatic arthritis (FUTURE 2): a randomised, double-blind, placebo-controlled, phase 3 trial surname:Papp;given-names:K. 10.1016/j.jaad.2014.08.039 REF J Am Acad Dermatol. ref 71 2014 38443 Safety and efficacy of brodalumab for psoriasis after 120 weeks of treatment REF ref 38520 U.S. Food and Drug Administration. FDA approves new psoriasis drug Cosentyx. U.S. FDA press release (2015). 1345 1352 surname:Kotake;given-names:S. 10.1172/JCI5703 10225978 REF J Clin Invest ref 103 1999 38628 IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis 963 970 surname:Chabaud;given-names:M. 10.1002/1529-0131(199905)42:5 10323452 REF Arthritis Rheum ref 42 1999 38738 Human interleukin-17: A T cell-derived proinflammatory cytokine produced by the rheumatoid synovium 2832 2838 surname:Ziolkowska;given-names:M. 10679127 REF J Immunol ref 164 2000 38838 High levels of IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin A-sensitive mechanism R93 surname:Shahrara;given-names:S. surname:Huang;given-names:Q. surname:Mandelin;given-names:A. M.;suffix:2nd surname:Pope;given-names:R. M. 10.1186/ar2477 18710567 REF Arthritis Res Ther ref 10 2008 38972 TH-17 cells in rheumatoid arthritis 1004 1013 surname:Lubberts;given-names:E. 11441109 REF J Immunol ref 167 2001 39008 IL-1-independent role of IL-17 in synovial inflammation and joint destruction during collagen-induced arthritis 650 659 surname:Lubberts;given-names:E. 10.1002/art.20001 14872510 REF Arthritis Rheum ref 50 2004 39120 Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion 2799 2805 surname:Wright;given-names:J. F. 18684971 REF J Immunol ref 181 2008 39300 The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL-17RC receptor complex 5332 5341 surname:Hymowitz;given-names:S. G. 10.1093/emboj/20.19.5332 11574464 REF EMBO J. ref 20 2001 39400 IL‐17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL‐17F, and implications for receptor binding 1245 1251 surname:Ely;given-names:L. K. surname:Fischer;given-names:S. surname:Garcia;given-names:K. C. 10.1038/ni.1813 19838198 REF Nat. Immunol. ref 10 2009 39528 Structural basis of receptor sharing by interleukin 17 cytokines 1888 surname:Liu;given-names:S. 10.1038/ncomms2880 23695682 REF Nat Commun ref 4 2013 39593 Crystal structures of interleukin 17A and its complex with IL-17 receptor A 905 921 surname:Gerhardt;given-names:S. 10.1016/j.jmb.2009.10.008 19835883 REF J Mol Biol. ref 394 2009 39669 Structure of IL-17A in Complex with a Potent, Fully Human Neutralizing Antibody 4299 4310 surname:Rickel;given-names:E. A. 10.4049/jimmunol.181.6.4299 18768888 REF J Immunol. ref 181 2008 39749 Identification of Functional Roles for Both IL-17RB and IL-17RA in Mediating IL-25-Induced Activities 1409 1419 surname:Zhong;given-names:X. 10.1074/jbc.M112.417717 23184956 REF J Biol Chem. ref 288 2013 39851 Pyroglutamate and O-Linked Glycan Determine Functional Production of Anti-IL17A and Anti-IL22 Peptide-Antibody Bispecific Genetic Fusions 225 227 surname:Vugmeyster;given-names:Y. surname:Zhang;given-names:Y. surname:Zhong;given-names:X. surname:Wright;given-names:J. surname:Leung;given-names:S. S. 10.1016/j.intimp.2013.11.013 24295652 REF Int Immunopharmacol. ref 18 2014 39989 Pharmacokinetics of anti-IL17A and anti-IL22 peptide–antibody bispecific genetic fusions in mice 87 97 surname:Zwick;given-names:M. B. 9784192 REF Anal Biochem ref 264 1998 40088 The maltose-binding protein as a scaffold for monovalent display of peptides derived from phage libraries 435 440 surname:Chang;given-names:S. H. surname:Dong;given-names:C. 10.1038/cr.2007.35 17452998 REF Cell Res ref 17 2007 40194 A novel heterodimeric cytokine consisting of IL-17 and IL-17F regulates inflammatory responses 87 102 surname:Fouser;given-names:L. A. surname:Wright;given-names:J. F. surname:Dunussi-Joannopoulos;given-names:K. surname:Collins;given-names:M. 19161418 REF Immunol Rev. ref 226 2008 40289 Th17 cytokines and their emerging roles in inflammation and autoimmunity 677 687 surname:Chiricozzi;given-names:A. 10.1038/jid.2010.340 21085185 REF J Invest Dermatol. ref 131 2011 40362 Integrative responses to IL-17 and TNF-alpha in human keratinocytes account for key inflammatory pathogenic circuits in psoriasis AB177 surname:Loesche;given-names:C. surname:Polus;given-names:F. surname:Sinner;given-names:F. surname:Gerard;given-names:B. surname:Valentin;given-names:M. A. 10.1016/j.jaad.2014.01.735 REF J Am Acad Dermatol. ref 70 2015 40492 Novel microperfusion method confirms that psoriasis lesional skin contains higher protein levels of IL-17A and β-defensin-2 compared to nonlesional skin 285 291 surname:Griffin;given-names:L. surname:Lawson;given-names:A. 10.1111/j.1365-2249.2011.04427.x 21649648 REF Clin. Exp. Immunol ref 165 2011 40648 Antibody fragments as tools in crystallography surname:Guettner;given-names:A. surname:Machacek;given-names:M. surname:Papavassilis;given-names:C. surname:Sander;given-names:O. REF ref 40695 surname:Allan;given-names:B. surname:Chow;given-names:C. K. surname:Liu;given-names:L. surname:Lu;given-names:J. surname:Tetreault;given-names:J. W. REF ref 40696 1102 1114 surname:Arkin;given-names:Michelle R. surname:Tang;given-names:Y. surname:Wells;given-names:James A. 10.1016/j.chembiol.2014.09.001 25237857 REF Chem. Biol. ref 21 2014 40697 Small-Molecule Inhibitors of Protein-Protein Interactions: Progressing toward the Reality 3 33 surname:Verdine;given-names:G. L. surname:Hilinski;given-names:G. J. surname:Wittrup;given-names:K. Dane surname:Verdine Gregory;given-names:L. REF Methods in Enzymology Vol. Volume 503 ref 2012 40787 161 173 surname:Azzarito;given-names:V. surname:Long;given-names:K. surname:Murphy;given-names:N. S. surname:Wilson;given-names:A. J. 23422557 REF Nat Chem ref 5 2013 40788 Inhibition of [alpha]-helix-mediated protein-protein interactions using designed molecules 315 323 surname:Boersma;given-names:M. D. 10.1021/ja207148m 22040025 REF J. Am. Chem. Soc. ref 134 2012 40879 Evaluation of Diverse α/β-Backbone Patterns for Functional α-Helix Mimicry: Analogues of the Bim BH3 Domain 1810 surname:Livingston;given-names:D. 10.1002/art.39542 REF Arthritis Rheum ref 64 2012 40998 Identification and Characterization of Synthetic Small Molecule Macrocycle Antagonists of Human IL17A 293 302 surname:Vonrhein;given-names:C. 10.1107/S0907444911007773 21460447 REF Acta Crystallogr D Biol Crystallogr ref 67 2011 41100 Data processing and analysis with the autoPROC toolbox 658 674 surname:McCoy;given-names:A. J. 19461840 REF J Appl Crystallogr ref 40 2007 41155 Phaser crystallographic software 2210 2221 surname:Blanc;given-names:E. 15572774 REF Acta Crystallogr D Biol Crystallogr ref 60 2004 41188 Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT 486 501 surname:Emsley;given-names:P. surname:Lohkamp;given-names:B. surname:Scott;given-names:W. G. surname:Cowtan;given-names:K. 20383002 REF Acta Crystallogr D Biol Crystallogr ref 66 2010 41271 Features and development of Coot 386 394 surname:McNicholas;given-names:S. surname:Potterton;given-names:E. surname:Wilson;given-names:K. S. surname:Noble;given-names:M. E. 10.1107/S0907444911007281 21460457 REF Acta Crystallogr D Biol Crystallogr ref 67 2011 41304 Presenting your structures: the CCP4mg molecular-graphics software SUPPL footnote 41371 Author Contributions P.J., L.D.W., J.J. and G.C. designed and performed phage display and mutagenesis optimization experiments. J.D. and B.O. designed and synthesized peptides. S.S., X.S. and M.G. created protein constructs and purified in house protein reagents. S.L. designed and performed crystallographic studies and analyzed the structures, made Figures 2 and 3, Supplementary Table S3 and Supplementary Figure S7. M.E.B., P.V.S. and W.L. performed FRET, SPR and cell assay, respectively, and made Table 3 and Figure 1. W.L. made Supplementary Figures S2 and S5. J.D., B.D.O., D.T., A.B. and C.W.B. contributed to peptide SAR. J.D., L.D.W. and P.J. prepared Tables 1 and 2, Supplementary Schemes S1 and S2, Supplementary Figures S1, S3 and S4 and Supplementary Tables S1 and S2. P.V.S. and B.A.C. performed Fab/HAP co-binding SPR and made Supplementary Figure S6. X.F. performed mass spectral analysis of the stoichiometry of HAP binding to IL-17A and made Supplementary Figure S8. L.D.W., G.C., D.T., A.B., C.W.B., G.W., R.W.L., A.T., J.M.W. and X.Q. supervised the study. S.L., J.D., J.M.W. and L.D.W. wrote the main text. All authors reviewed the manuscript. srep26071-f1.jpg f1 FIG fig_title_caption 42538 Binding of HAP to IL-17A and inhibition of IL-17A/IL-17RA are measured by SPR, FRET and cell-based assays. 0.9997749 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9971237 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A complex_assembly GO: cleaner0 2023-07-20T08:28:05Z IL-17A/IL-17RA 0.99964595 experimental_method cleaner0 2023-07-20T08:43:35Z MESH: SPR 0.99958235 experimental_method cleaner0 2023-07-20T09:39:17Z MESH: FRET 0.9995019 experimental_method cleaner0 2023-07-20T09:39:20Z MESH: cell-based assays srep26071-f1.jpg f1 FIG fig_caption 42645 (A) Typical SPR sensorgrams (black) of HAP at indicated concentrations binding to biotinylated human IL-17A immobilized on a streptavidin chip surface, fitted with single site binding model curves (red). Kinetic parameters (ka, kd) were obtained by a global fit using three concentrations in triplicate. KD determined by the standard equation, KD = kd/ka. (B) HAP inhibits SPR signaling of IL-17A binding to immobilized IL-17RA. Data are mean and error bars of +/− standard deviation of three measurements. (C) Inhibition of IL-17A and IL-17RA binding by HAP measured by FRET assay. Data are mean and error bars of +/− standard deviation from 299 experiments, each performed in duplicate. (D) Example of HAP selective inhibition of the production of IL-8 (triangles), IL-6 (squares) and CCL-20 (circles) by primary human keratinocyte cells synergistically stimulated by 100 ng/ml IL-17A and 10 ng/ml TNF-α. HAP does not inhibit the baseline production of IL-6, IL-8 and CCL-20 stimulated by 10 ng/ml TNF-α alone (gray lines and symbols). Data are mean and error bars of +/− standard deviation of duplicated experiments. 0.9996934 experimental_method cleaner0 2023-07-20T08:43:35Z MESH: SPR 0.9996536 evidence cleaner0 2023-07-20T09:14:36Z DUMMY: sensorgrams 0.9997179 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9992107 protein_state cleaner0 2023-07-20T09:15:19Z DUMMY: biotinylated 0.9993474 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.9994132 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A evidence DUMMY: cleaner0 2023-07-20T09:14:28Z single site binding model curves 0.99936134 evidence cleaner0 2023-07-20T09:15:12Z DUMMY: ka 0.998982 evidence cleaner0 2023-07-20T09:15:04Z DUMMY: kd 0.9994641 evidence cleaner0 2023-07-20T09:15:03Z DUMMY: KD 0.9994006 evidence cleaner0 2023-07-20T09:15:04Z DUMMY: KD 0.9980882 evidence cleaner0 2023-07-20T09:15:04Z DUMMY: kd 0.9948657 evidence cleaner0 2023-07-20T09:15:12Z DUMMY: ka 0.9997172 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9996642 experimental_method cleaner0 2023-07-20T08:43:35Z MESH: SPR 0.9989503 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.8585394 protein_state cleaner0 2023-07-20T09:56:49Z DUMMY: immobilized 0.9993916 protein cleaner0 2023-07-20T08:17:01Z PR: IL-17RA 0.99624085 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.9984536 protein cleaner0 2023-07-20T08:17:01Z PR: IL-17RA 0.99971646 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.99960434 experimental_method cleaner0 2023-07-20T09:39:26Z MESH: FRET assay 0.9997013 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9994752 protein_type cleaner0 2023-07-20T08:44:32Z MESH: IL-8 0.99951357 protein_type cleaner0 2023-07-20T08:44:37Z MESH: IL-6 protein_type MESH: cleaner0 2023-07-20T08:44:55Z CCL-20 0.9990013 species cleaner0 2023-07-20T08:25:04Z MESH: human 0.9985098 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.93264896 protein cleaner0 2023-07-20T08:44:23Z PR: TNF-α 0.9997168 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9995456 protein_type cleaner0 2023-07-20T08:44:37Z MESH: IL-6 0.9994187 protein_type cleaner0 2023-07-20T08:44:32Z MESH: IL-8 protein_type MESH: cleaner0 2023-07-20T08:44:55Z CCL-20 0.99070376 protein cleaner0 2023-07-20T08:44:23Z PR: TNF-α srep26071-f2.jpg f2 FIG fig_title_caption 43783 Overall structure of the Fab/IL-17A/HAP complex in ribbon presentation. 0.99678874 evidence cleaner0 2023-07-20T09:43:41Z DUMMY: structure complex_assembly GO: cleaner0 2023-07-20T08:47:07Z Fab/IL-17A/HAP srep26071-f2.jpg f2 FIG fig_caption 43855 For clarity, different molecules are colored differently. Two HAP molecules are colored blue and red, and IL-17A monomers are colored ice blue and pink, respectively. Picture prepared using program CCP4MG. (A) Overview of the distinct binding sites of Fab and HAP to IL-17A. (B) Close-in view of the IL-17A/HAP structure. IL-17A β-strands are labelled. Each of the two bound HAP interacts with both monomers of the IL-17A dimer. (C) As a comparison, the IL-17A/IL-17RA complex was shown with IL-17A in the same orientation. Three distinct areas IL-17A/IL-17RA interface are labeled. 0.999759 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.99955463 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.9991665 oligomeric_state cleaner0 2023-07-20T08:23:13Z DUMMY: monomers 0.99946296 site cleaner0 2023-07-20T09:40:12Z SO: binding sites 0.9997955 structure_element cleaner0 2023-07-20T08:47:40Z SO: Fab 0.9996724 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9994636 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.9994233 complex_assembly cleaner0 2023-07-20T08:46:26Z GO: IL-17A/HAP 0.9993218 evidence cleaner0 2023-07-20T09:43:45Z DUMMY: structure 0.9994388 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.9996136 structure_element cleaner0 2023-07-20T09:48:08Z SO: β-strands 0.99904794 protein_state cleaner0 2023-07-20T09:56:54Z DUMMY: bound 0.99970585 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9992748 oligomeric_state cleaner0 2023-07-20T08:23:13Z DUMMY: monomers 0.9994292 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.99929965 oligomeric_state cleaner0 2023-07-20T09:18:27Z DUMMY: dimer 0.9996103 complex_assembly cleaner0 2023-07-20T08:28:05Z GO: IL-17A/IL-17RA 0.9995683 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.9995848 site cleaner0 2023-07-20T09:15:57Z SO: IL-17A/IL-17RA interface srep26071-f3.jpg f3 FIG fig_title_caption 44441 Mechanism of the inhibition of the IL-17A/IL-17RA interaction by HAP. complex_assembly GO: cleaner0 2023-07-20T08:28:06Z IL-17A/IL-17RA 0.9997354 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP srep26071-f3.jpg f3 FIG fig_caption 44511 (A) HAP binds at region I of IL-17A. IL-17A dimer is in surface presentation (β-strands 0 shown as ribbons for clarity). Polar interactions are shown in dashes. HAP residues as well as key IL-17A residues are labeled. For clarity, a few HAP residues are also shown in stick model with carbon atoms colored green, oxygen in red and nitrogen in blue. (B) I-17RA (ribbon in gold) peptide Leu27-Ile32 binds to the same area as the HAP α-helix. Trp31 of IL-17RA binds to the same pocket in IL-17A as Trp12 of HAP. (C) As illustrated by overlay a single HAP molecule and β-strands 0 (grey) of the IL-17A/HAP complex in the apo IL-17A structure, conformational changes in region I of IL-17A are needed for binding of both the β-stand and α-helix of the HAP. Notice that the Trp binding pocket for W12 of HAP or W31 of IL-17RA is missing in the apo structure. 0.99956113 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.99846333 structure_element cleaner0 2023-07-20T08:54:15Z SO: region I 0.99849606 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.99864507 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.9993493 oligomeric_state cleaner0 2023-07-20T09:18:27Z DUMMY: dimer 0.99935997 structure_element cleaner0 2023-07-20T09:48:13Z SO: β-strands 0 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:18:31Z Polar interactions 0.9993606 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.99775904 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.9994431 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.9994113 protein cleaner0 2023-07-20T09:25:04Z PR: I-17RA 0.9966951 residue_range cleaner0 2023-07-20T09:16:28Z DUMMY: Leu27-Ile32 0.9990828 chemical cleaner0 2023-07-20T08:18:37Z CHEBI: HAP 0.99966097 structure_element cleaner0 2023-07-20T09:48:20Z SO: α-helix 0.9998932 residue_name_number cleaner0 2023-07-20T08:55:25Z DUMMY: Trp31 0.9993536 protein cleaner0 2023-07-20T08:17:01Z PR: IL-17RA 0.9973183 site cleaner0 2023-07-20T09:40:19Z SO: pocket 0.99910945 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.9998946 residue_name_number cleaner0 2023-07-20T08:53:17Z DUMMY: Trp12 0.99966085 chemical cleaner0 2023-07-20T08:18:38Z CHEBI: HAP 0.99909353 experimental_method cleaner0 2023-07-20T09:39:33Z MESH: overlay 0.9996959 chemical cleaner0 2023-07-20T08:18:38Z CHEBI: HAP 0.99939364 structure_element cleaner0 2023-07-20T09:48:25Z SO: β-strands 0 0.996832 complex_assembly cleaner0 2023-07-20T08:46:26Z GO: IL-17A/HAP 0.9996488 protein_state cleaner0 2023-07-20T08:21:59Z DUMMY: apo 0.9991872 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.99958926 evidence cleaner0 2023-07-20T09:43:55Z DUMMY: structure 0.999281 structure_element cleaner0 2023-07-20T08:54:15Z SO: region I 0.99816847 protein cleaner0 2023-07-20T08:16:53Z PR: IL-17A 0.99966127 structure_element cleaner0 2023-07-20T09:48:29Z SO: β-stand 0.99964935 structure_element cleaner0 2023-07-20T09:48:32Z SO: α-helix 0.9995005 chemical cleaner0 2023-07-20T08:18:38Z CHEBI: HAP 0.9996323 site cleaner0 2023-07-20T09:16:41Z SO: Trp binding pocket 0.99988747 residue_name_number cleaner0 2023-07-20T09:21:15Z DUMMY: W12 0.9996706 chemical cleaner0 2023-07-20T08:18:38Z CHEBI: HAP 0.9998939 residue_name_number cleaner0 2023-07-20T09:21:18Z DUMMY: W31 0.99928457 protein cleaner0 2023-07-20T08:17:01Z PR: IL-17RA 0.9996501 protein_state cleaner0 2023-07-20T08:21:59Z DUMMY: apo 0.99953055 evidence cleaner0 2023-07-20T09:43:59Z DUMMY: structure t1.xml t1 TABLE table_title_caption 45380 ELISA competition activity of peptide analogues of 1. 0.9044505 experimental_method cleaner0 2023-07-20T09:16:56Z MESH: ELISA competition activity t1.xml t1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50">Compound #</th><th align="center" valign="top" charoff="50">Competition IC₅₀(nM)</th><th align="center" valign="top" charoff="50">1</th><th align="center" valign="top" charoff="50">2</th><th align="center" valign="top" charoff="50">3</th><th align="center" valign="top" charoff="50">4</th><th align="center" valign="top" charoff="50">5</th><th align="center" valign="top" charoff="50">6</th><th align="center" valign="top" charoff="50">7</th><th align="center" valign="top" charoff="50">8</th><th align="center" valign="top" charoff="50">9</th><th align="center" valign="top" charoff="50">10</th><th align="center" valign="top" charoff="50">11</th><th align="center" valign="top" charoff="50">12</th><th align="center" valign="top" charoff="50">13</th><th align="center" valign="top" charoff="50">14</th><th align="center" valign="top" charoff="50">15</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><bold>1</bold></td><td align="center" valign="top" charoff="50">80</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">K</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>2</bold></td><td align="center" valign="top" charoff="50">80</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>3</bold></td><td align="center" valign="top" charoff="50">66</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50"><bold>K</bold></td></tr><tr><td align="left" valign="top" charoff="50"><bold>4</bold></td><td align="center" valign="top" charoff="50">172</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>5</bold></td><td align="center" valign="top" charoff="50">402</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>6</bold></td><td align="center" valign="top" charoff="50">7451</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>7</bold></td><td align="center" valign="top" charoff="50">474</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>8</bold></td><td align="center" valign="top" charoff="50">524</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>9</bold></td><td align="center" valign="top" charoff="50">640</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>10</bold></td><td align="center" valign="top" charoff="50">4016</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>11</bold></td><td align="center" valign="top" charoff="50">84</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>12</bold></td><td align="center" valign="top" charoff="50">2651</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>13</bold></td><td align="center" valign="top" charoff="50">11</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>14</bold></td><td align="center" valign="top" charoff="50">195</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>15</bold></td><td align="center" valign="top" charoff="50">5717</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>16</bold></td><td align="center" valign="top" charoff="50">149</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>17</bold></td><td align="center" valign="top" charoff="50">167</td><td align="center" valign="top" charoff="50"><bold>A</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>18</bold></td><td align="center" valign="top" charoff="50">45</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>H</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>19</bold></td><td align="center" valign="top" charoff="50">71</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>Q</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>20</bold></td><td align="center" valign="top" charoff="50">117</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>R</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>21</bold></td><td align="center" valign="top" charoff="50">31</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>T</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>22</bold></td><td align="center" valign="top" charoff="50">522</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>W</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>23</bold></td><td align="center" valign="top" charoff="50">64</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>Y</bold></td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>24</bold></td><td align="center" valign="top" charoff="50">3</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50"><bold>I</bold></td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>25</bold></td><td align="center" valign="top" charoff="50">33</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50"><bold>L</bold></td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>26</bold></td><td align="center" valign="top" charoff="50">6</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50"><bold>V</bold></td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>27</bold></td><td align="center" valign="top" charoff="50">31</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>M</bold></td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>28</bold></td><td align="center" valign="top" charoff="50">26</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>N</bold></td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>29</bold></td><td align="center" valign="top" charoff="50">33</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"><bold>Q</bold></td><td align="center" valign="top" charoff="50">A</td></tr></tbody></table> 45434 Compound # Competition IC50(nM) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 80 I V V T M P A D L W D W I K A 2 80 I V V T M P A D L W D W I R A 3 66 I V V T M P A D L W D W I R K 4 172 I V V T M P A D L W D W I A A 5 402 I V V T M P A D L W D W A R A 6 7451 I V V T M P A D L W D A I R A 7 474 I V V T M P A D L W A W I R A 8 524 I V V T M P A D L A D W I R A 9 640 I V V T M P A D A W D W I R A 10 4016 I V V T M P A A L W D W I R A 11 84 I V V T M P A D L W D W I R A 12 2651 I V V T M A A D L W D W I R A 13 11 I V V T A P A D L W D W I R A 14 195 I V V A M P A D L W D W I R A 15 5717 I V A T M P A D L W D W I R A 16 149 I A V T M P A D L W D W I R A 17 167 A V V T M P A D L W D W I R A 18 45 I H V T M P A D L W D W I R A 19 71 I Q V T M P A D L W D W I R A 20 117 I R V T M P A D L W D W I R A 21 31 I T V T M P A D L W D W I R A 22 522 I W V T M P A D L W D W I R A 23 64 I Y V T M P A D L W D W I R A 24 3 I V V T I P A D L W D W I R A 25 33 I V V T L P A D L W D W I R A 26 6 I V V T V P A D L W D W I R A 27 31 I V V T M P A D L W D W I M A 28 26 I V V T M P A D L W D W I N A 29 33 I V V T M P A D L W D W I Q A t1.xml t1 TABLE table_footnote 46614 All peptides are acetylated at their N-termini. t2.xml t2 TABLE table_title_caption 46662 Cell-based activity of amino acid deletions of 30. t2.xml t2 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50">Compound #</th><th align="center" valign="top" charoff="50">Cell IC₅₀(nM)</th><th align="center" valign="top" charoff="50">1</th><th align="center" valign="top" charoff="50">2</th><th align="center" valign="top" charoff="50">3</th><th align="center" valign="top" charoff="50">4</th><th align="center" valign="top" charoff="50">5</th><th align="center" valign="top" charoff="50">6</th><th align="center" valign="top" charoff="50">7</th><th align="center" valign="top" charoff="50">8</th><th align="center" valign="top" charoff="50">9</th><th align="center" valign="top" charoff="50">10</th><th align="center" valign="top" charoff="50">11</th><th align="center" valign="top" charoff="50">12</th><th align="center" valign="top" charoff="50">13</th><th align="center" valign="top" charoff="50">14</th><th align="center" valign="top" charoff="50">15</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><bold>1</bold></td><td align="center" valign="top" charoff="50">370</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">M</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">K</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>24</bold></td><td align="center" valign="top" charoff="50">18</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>26</bold></td><td align="center" valign="top" charoff="50">137</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">R</td><td align="center" valign="top" charoff="50">A</td></tr><tr><td align="left" valign="top" charoff="50"><bold>30</bold></td><td align="center" valign="top" charoff="50">17</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">H</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50">K</td></tr><tr><td align="left" valign="top" charoff="50"><bold>32</bold></td><td align="center" valign="top" charoff="50">&gt;1000</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">H</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50">K</td></tr><tr><td align="left" valign="top" charoff="50"><bold>33</bold></td><td align="center" valign="top" charoff="50">&gt;1000</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50">K</td></tr><tr><td align="left" valign="top" charoff="50"><bold>34</bold></td><td align="center" valign="top" charoff="50">&gt;1000</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50">K</td></tr><tr><td align="left" valign="top" charoff="50"><bold>35</bold></td><td align="center" valign="top" charoff="50">12</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">H</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">N</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"><bold>36</bold></td><td align="center" valign="top" charoff="50">183</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">H</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50"><bold>37</bold></td><td align="center" valign="top" charoff="50">&gt;1000</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">H</td><td align="center" valign="top" charoff="50">V</td><td align="center" valign="top" charoff="50">T</td><td align="center" valign="top" charoff="50">I</td><td align="center" valign="top" charoff="50">P</td><td align="center" valign="top" charoff="50">A</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">L</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50">D</td><td align="center" valign="top" charoff="50">W</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr></tbody></table> 46713 Compound # Cell IC50(nM) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 370 I V V T M P A D L W D W I K A 24 18 I V V T I P A D L W D W I R A 26 137 I V V T L P A D L W D W I R A 30 17 I H V T I P A D L W D W I N K 32 >1000   H V T I P A D L W D W I N K 33 >1000     V T I P A D L W D W I N K 34 >1000       T I P A D L W D W I N K 35 12 I H V T I P A D L W D W I N   36 183 I H V T I P A D L W D W I     37 >1000 I H V T I P A D L W D W       t3.xml t3 TABLE table_title_caption 47183 Binding affinity/IC50 of HAP in orthogonal in vitro assays/primary keratinocyte cell assay. t3.xml t3 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50">Assay</th><th align="center" valign="top" charoff="50">K_D/IC₅₀ (nM)</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">SPR binding (N = 3)<xref ref-type="fn" rid="t3-fn1">*</xref>: hIL-17A, hIL-17A/F, mIL-17A</td><td align="center" valign="top" charoff="50">2.6(±0.5), 4.6(±0.7), 30.1(±0.1)</td></tr><tr><td align="left" valign="top" charoff="50">SPR competition</td><td align="center" valign="top" charoff="50">0.9(±0.3)</td></tr><tr><td align="left" valign="top" charoff="50">FRET IL-17RA competition (N = 598)</td><td align="center" valign="top" charoff="50">0.8 ± 0.2</td></tr><tr><td align="left" valign="top" charoff="50">Human primary keratinocytes IC-8 (N = 9), IC-6 (N = 4), CCL-20 (N = 4)</td><td align="center" valign="top" charoff="50">151(±49), 136(±37), 253(±64)</td></tr></tbody></table> 47275 Assay KD/IC50 (nM) SPR binding (N = 3)*: hIL-17A, hIL-17A/F, mIL-17A 2.6(±0.5), 4.6(±0.7), 30.1(±0.1) SPR competition 0.9(±0.3) FRET IL-17RA competition (N = 598) 0.8 ± 0.2 Human primary keratinocytes IC-8 (N = 9), IC-6 (N = 4), CCL-20 (N = 4) 151(±49), 136(±37), 253(±64) t3.xml t3 TABLE table_footnote 47591 *KD = kd/ka. ka (M−1s−1)and kd (s−1): 2(±1) × 105, 6(±5) × 10−4 (hIL-17A); 4.6(±0.7) × 105, 2.1(±0.2) × 10−3 (hIL-17A/F); 2.7(±0.2) × 105, 8.2(±0.8) × 10−3 (mIL-17A).