The constituent polypeptides of the interleukin-17 family form six different homodimeric cytokines (IL-17A-F) and the heterodimeric IL-17A/F. Their interactions with IL-17 receptors A-E (IL-17RA-E) mediate host defenses while also contributing to inflammatory and autoimmune responses. IL-17A and IL-17F both preferentially engage a receptor complex containing one molecule of IL-17RA and one molecule of IL-17RC. More generally, IL-17RA appears to be a shared receptor that pairs with other members of its family to allow signaling of different IL-17 cytokines. Here we report crystal structures of homodimeric IL-17A and its complex with IL-17RA. Binding to IL-17RA at one side of the IL-17A molecule induces a conformational change in the second, symmetry-related receptor site of IL-17A. This change favors, and is sufficient to account for, the selection of a different receptor polypeptide to complete the cytokine-receptor complex. The structural results are supported by biophysical studies with IL-17A variants produced by site-directed mutagenesis.

Auristatins, synthetic analogues of the antineoplastic natural product Dolastatin 10, are ultrapotent cytotoxic microtubule inhibitors that are clinically used as payloads in antibody-drug conjugates (ADCs). The design and synthesis of several new auristatin analogues with N-terminal modifications that include amino acids with α,α-disubstituted carbon atoms are described, including the discovery of our lead auristatin, PF-06380101. This modification of the peptide structure is unprecedented and led to analogues with excellent potencies in tumor cell proliferation assays and differential ADME properties when compared to other synthetic auristatin analogues that are used in the preparation of ADCs. In addition, auristatin cocrystal structures with tubulin are being presented that allow for the detailed examination of their binding modes. A surprising finding is that all analyzed analogues have a cis-configuration at the Val-Dil amide bond in their functionally relevant tubulin bound state, whereas in solution this bond is exclusively in the trans-configuration. This remarkable observation shines light onto the preferred binding mode of auristatins and serves as a valuable tool for structure-based drug design.

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2′, 3′ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2′-5′ and 3′-5′ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

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.

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RAInterleukin-17 (IL-17) cytokines are homo or heterodimeric proteins formed by combinations of six distinct polypeptides designated IL17A-F 1 . They are essential to a fully functional immune system 2,3 , but dysregulated expression of IL-17A is implicated in autoimmune disorders such as psoriasis, psoriatic arthritis, rheumatoid arthritis and multiple sclerosis 4-6 . The IL-17A covalent homodimer's significance in psoriasis is evidenced by the recent success of anti-IL-17A biologics as therapeutics. Secukinumab (Costentyx TM ), a monoclonal antibody targeting IL-17A, was recently approved for the treatment of moderate to severe plaque psoriasis 7,8 and is being investigated in other IL-17A-driven immunological diseases 9 . Additionally, two other biologics, ixekizumab (anti-IL17A) 10,11 and brodalumab (an antibody to the IL-17 receptor, IL-17RA) 12,13 , have shown efficacy in psoriasis in late stage clinical trials.IL-17A signaling occurs through its membrane-bound receptors, IL-17RA and IL-17RC, and elicits multiple inflammatory and immune responses [14][15][16] . The cytokine binds to IL-17RA with low single-digit nanomolar affinity 14,15,17,18 . and the structure of their complex is known 17 . The emerging biologics block this interaction by binding to one or other of the partners, but our goal was to determine whether it could be blocked or modulated with a small molecule as this could afford orally active agents.Small-molecule inhibition of a protein-protein interaction (PPI) is invariably challenging 19 . Even the discovery of early lead matter tends to be difficult because corporate compound collections are largely designed to target the active centers of enzymes, and are deficient in compounds suitable to the longer and shallower binding sites on which PPIs tend to depend. As the industry expands the "druggable genome", continued efforts at small molecule inhibition of PPIs will be required 20 . ResultsLead small molecule IL-17A antagonists. Our effort to discover small-molecule antagonists of IL-17A was initiated from disclosed inhibitors 21,22 exemplified by compound 1 (Fig. 1), a polyamide with clear structure-activity relationships (SAR) representative of the series. For example, the amide bonds, correct chiral center and cyclopentyl group were all required for activity. Surface plasmon resonance (SPR) measurements showed