Christine Everett

Structure-based design of substituted hexafluoroisopropanol-arylsulfonamides as modulators of RORc.

The structure-activity relationships of T0901317 analogs were explored as RORc inverse agonists using the principles of property- and structure-based drug design. An X-ray co-crystal structure of T0901317 and RORc was obtained and provided molecular insight into why T0901317 functioned as an inverse agonist of RORc; whereas, the same ligand functioned as an agonist of FXR, LXR, and PXR. The structural data was also used to design inhibitors with improved RORc biochemical and cellular activities. The improved inhibitors possessed enhanced selectivity profiles (rationalized using the X-ray crystallographic data) against other nuclear receptors.

Minor Structural Change to Tertiary Sulfonamide RORc Ligands Led to Opposite Mechanisms of Action.

A minor structural change to tertiary sulfonamide RORc ligands led to distinct mechanisms of action. Co-crystal structures of two compounds revealed mechanistically consistent protein conformational changes. Optimized phenylsulfonamides were identified as RORc agonists while benzylsulfonamides exhibited potent inverse agonist activity. Compounds behaving as agonists in our biochemical assay also gave rise to an increased production of IL-17 in human PBMCs whereas inverse agonists led to significant suppression of IL-17 under the same assay conditions. The most potent inverse agonist compound showed >180-fold selectivity over the ROR isoforms as well as all other nuclear receptors that were profiled.

The crystal structure of the catalytic domain of the NF-κB inducing kinase reveals a narrow but flexible active site.

The NF-κB inducing kinase (NIK) regulates the non-canonical NF-κB pathway downstream of important clinical targets including BAFF, RANKL, and LTβ. Despite numerous genetic studies associating dysregulation of this pathway with autoimmune diseases and hematological cancers, detailed molecular characterization of this central signaling node has been lacking. We undertook a systematic cloning and expression effort to generate soluble, well-behaved proteins encompassing the kinase domains of human and murine NIK. Structures of the apo NIK kinase domain from both species reveal an active-like conformation in the absence of phosphorylation. ATP consumption and peptide phosphorylation assays confirm that phosphorylation of NIK does not increase enzymatic activity. Structures of murine NIK bound to inhibitors possessing two different chemotypes reveal conformational flexibility in the gatekeeper residue controlling access to a hydrophobic pocket. Finally, a single amino acid difference affects the ability of some inhibitors to bind murine and human NIK with the same affinity.

Reduction in lipophilicity improved the solubility, plasma-protein binding, and permeability of tertiary sulfonamide RORc inverse agonists.

Using structure-based drug design principles, we identified opportunities to reduce the lipophilicity of our tertiary sulfonamide RORc inverse agonists. The new analogs possessed improved RORc cellular potencies with >77-fold selectivity for RORc over other nuclear receptors in our cell assay suite. The reduction in lipophilicity also led to an increased plasma-protein unbound fraction and improvements in cellular permeability and aqueous solubility.

Discovery of imidazo[1,5-a]pyridines and -pyrimidines as potent and selective RORc inverse agonists.

The nuclear receptor (NR) retinoic acid receptor-related orphan receptor gamma (RORγ, RORc, or NR1F3) is a promising target for the treatment of autoimmune diseases. RORc is a critical regulator in the production of the pro-inflammatory cytokine interleukin-17. We discovered a series of potent and selective imidazo[1,5-a]pyridine and -pyrimidine RORc inverse agonists. The most potent compounds displayed >300-fold selectivity for RORc over the other ROR family members, PPARγ, and NRs in our cellular selectivity panel. The favorable potency, selectivity, and physiochemical properties of GNE-0946 (9) and GNE-6468 (28), in addition to their potent suppression of IL-17 production in human primary cells, support their use as chemical biology tools to further explore the role of RORc in human biology.

Discovery of 1-{4-[3-Fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (GNE-3500): a Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C (RORc or RORγ) Inverse Agonist

Retinoic acid receptor-related orphan receptor C (RORc, RORγ, or NR1F3) is a nuclear receptor that plays a major role in the production of interleukin (IL)-17. Considerable efforts have been directed toward the discovery of selective RORc inverse agonists as potential treatments of inflammatory diseases such as psoriasis and rheumatoid arthritis. Using the previously reported tertiary sulfonamide 1 as a starting point, we engineered structural modifications that significantly improved human and rat metabolic stabilities while maintaining a potent and highly selective RORc inverse agonist profile. The most advanced δ-sultam compound, GNE-3500 (27, 1-{4-[3-fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone), possessed favorable RORc cellular potency with 75-fold selectivity for RORc over other ROR family members and >200-fold selectivity over 25 additional nuclear receptors in a cell assay panel. The favorable potency, selectivity, in vitro ADME properties, in vivo PK, and dose-dependent inhibition of IL-17 in a PK/PD model support the evaluation of 27 in preclinical studies.

Identification of tertiary sulfonamides as RORc inverse agonists.

Screening a nuclear receptor compound subset in a RORc biochemical binding assay revealed a benzylic tertiary sulfonamide hit. Herein, we describe the identification of compounds with improved RORc biochemical inverse agonist activity and cellular potencies. These improved compounds also possessed appreciable selectivity for RORc over other nuclear receptors.

Resolving TYK2 locus genotype-to-phenotype differences in autoimmunity

Resolving TYK2 locus genotype-to-phenotype differences reveals an immune signaling optimum that may be exploited therapeutically for treating autoimmune diseases. TYK2’s balancing act Determining the biological consequences of the thousands of genetic variants that contribute to common diseases for the purpose of improving health care is challenging. Genetic variants that influence autoimmune diseases have been identified in the tyrosine kinase 2 (TYK2) gene, but conflicting evidence regarding their biological impact obscures the therapeutic potential of TYK2. By resolving this conflict, Dendrou et al. have revealed a genetic effect that drives an optimal degree of immune signaling: low enough to be protective against autoimmunity but high enough to prevent immunodeficiency. These findings indicate that TYK2 may be a potential drug target in a number of autoimmune conditions. Thousands of genetic variants have been identified, which contribute to the development of complex diseases, but determining how to elucidate their biological consequences for translation into clinical benefit is challenging. Conflicting evidence regarding the functional impact of genetic variants in the tyrosine kinase 2 (TYK2) gene, which is differentially associated with common autoimmune diseases, currently obscures the potential of TYK2 as a therapeutic target. We aimed to resolve this conflict by performing genetic meta-analysis across disorders; subsequent molecular, cellular, in vivo, and structural functional follow-up; and epidemiological studies. Our data revealed a protective homozygous effect that defined a signaling optimum between autoimmunity and immunodeficiency and identified TYK2 as a potential drug target for certain common autoimmune disorders.

A reversed sulfonamide series of selective RORc inverse agonists.

The identification of a new series of RORc inverse agonists is described. Comprehensive structure-activity relationship studies of this reversed sulfonamide series identified potent RORc inverse agonists in biochemical and cellular assays which were also selective against a panel of nuclear receptors. Our work has contributed a compound that may serve as a useful in vitro tool to delineate the complex biological pathways involved in signalling through RORc. An X-ray co-crystal structure of an analogue with RORc has also provided useful insights into the binding interactions of the new series.

Structure-Based Design of Tricyclic NF-κB Inducing Kinase (NIK) Inhibitors That Have High Selectivity over Phosphoinositide-3-kinase (PI3K)

We report here structure-guided optimization of a novel series of NF-κB inducing kinase (NIK) inhibitors. Starting from a modestly potent, low molecular weight lead, activity was improved by designing a type 11/2 binding mode that accessed a back pocket past the methionine-471 gatekeeper. Divergent binding modes in NIK and PI3K were exploited to dampen PI3K inhibition while maintaining NIK inhibition within these series. Potent compounds were discovered that selectively inhibit the nuclear translocation of NF-κB2 (p52/REL-B) but not canonical NF-κB1 (REL-A/p50).