Binding site elucidation and structure guided design of macrocyclic IL-17A antagonists

Abstract

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.

Figure 1. Chemical structures of example IL-17A inhibitors used in this study.

Compound 1: example of a lead IL-17A antagonist with a linear peptide motif. Compounds 2 and 3: macrocyclic IL-17A antagonists designed on basis of the structure of compound 1 complexed with IL-17A.

Figure 2. Characterization of the central binding pocket of the IL-17A dimer (surface presentation with the two polypeptide chains colored in ice blue and gold, respectively) probed using the VISM algorithm (red balls represent the probes used).

The high druggability of the pocket is manifested by the large hydrophobic cavity and the favorable druggability score (∆G) which assesses the optimal binding affinity of the binding site.

Figure 3. Binding of compound 1 in a central pocket of IL-17A.

(A) Overall structure of the Fab/HAP/IL-17A/compound 1 complex. Component polypeptides appear as ribbons, with two HAP molecules in blue and red, respectively, and the two chains of IL-17A in ice blue and gold, respectively. Atoms of compound 1 appear as spheres, with carbons in green, nitrogens in blue, and oxygens in red. Hydrogen bonds are shown as dashes. Picture prepared using program CCP4MG. (B) Close view of compound 1 bound in the central pocket at the IL-17A dimer interface and interacting with both IL-17A monomers (surface representation). Dashed lines are hydrogen bonds. (C) Compound 1 binding enlarges the central pocket and makes the N-terminal half of the IL-17A dimer much less compact. (D) For comparison, the central pocket is much smaller in the apo IL-17A dimer and completely enclosed. Compound 1 would clash with the closed binding site.

Figure 4. A covalent probe targeting the compound binding site in IL-17A.

(A) Structure of compound 4, an analog of compound 1 designed as a covalent probe of the IL-17A binding site with a FRET IC₅₀ of 0.2 μM. (B) Compound 4 specifically and covalently labeled full length IL-17A. IL-17A labeled by compound 4 was biotinylated by click chemistry, and pulled down by streptavidin. Eluates were immunoblotted. (C) In this model of compound 4 in the IL-17A binding pocket, the reactive sulfonyl fluoride is close to Tyr85. (D) Mass spectra of IL-17A (a) before and (b) after treatment with compound 4. The predicted mass shift resulting from a single modification of the protein was 795.3 Da.

Figure 5. Mechanism of inhibition of the formation of the IL-17A/IL-17RA complex by compound 1.

(A) IL-17A/IL-17RA binary complex (PDB accession 4HSA) is incompatible with compound 1 binding. Compound 1 (stick model) was superimposed into its IL-17A binding site in IL-17A/IL-17RA complex (surface representation, with IL-17RA colored in coral). (B) IL-17A/compound 1 binary complex is incompatible with IL-17RA binding. IL-17RA (coral surface) was superimposed onto IL-17A/compound 1 binary complex.

Figure 6. Cyclization strategy inspired by the bound bioactive conformation of compound 1.

(A) In solution, lack of an NOE signal from the cyclopentyl and phenyl linker of compound 1 (left) indicated that the bound conformation was not highly populated in solution. Cyclization between the L-phenylalanine side chain and the N,N-dimethylamide (right) may reinforce the bound bioactive conformation (right). (B) Use of MD/MM-GBSA to predict binding affinities in design of macrocyclic compounds. Examples are for the discussed compounds. Box regions correspond to 50% of the distribution, lines extend to max 1.5 times of this interval, and averages are denoted by dashed lines in the boxes. (C) Compound 2 bound to IL-17A. D. Compound 3 bound to IL-17A.