Brian Nicholas Cook

5-Aminomethylbenzimidazoles as potent ITK antagonists

Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.

Discovery of potent inhibitors of interleukin-2 inducible T-cell kinase (ITK) through structure-based drug design

Interleukin-2 inducible T-cell kinase (ITK) is a member of the Tec kinase family and is involved with T-cell activation and proliferation. Due to its critical role in acting as a modulator of T-cells, ITK inhibitors could provide a novel route to anti-inflammatory therapy. This work describes the discovery of ITK inhibitors through structure-based design where high-resolution crystal structural information was used to optimize interactions within the kinase specificity pocket of the enzyme to improve both potency and selectivity.

Itk kinase inhibitors: initial efforts to improve the metabolical stability and the cell activity of the benzimidazole lead.

Previously, we reported a series of novel benzimidazole based Itk inhibitors that exhibited excellent enzymatic potency and selectivity but low microsomal stability. Employing a structure based approach a new series of inhibitors with comparable potency and selectivity to the original series and with a potential for improved microsome stability was identified.

Discovery, SAR and X-ray structure of 1H-benzimidazole-5-carboxylic acid cyclohexyl-methyl-amides as inhibitors of inducible T-cell kinase (Itk)

A series of novel potent benzimidazole based inhibitors of interleukin-2 T-cell kinase (Itk) were prepared. In this report, we discuss the structure-activity relationship (SAR), selectivity, and cell-based activity for the series. We also discuss the SAR associated with an X-ray structure of one of the small-molecule inhibitors bound to ITK.

Discovery of 1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide analogs as new RORC modulators.

Structure-based and pharmacophore-based virtual screening in combination with combinatorial chemistry and X-ray crystallography led to the discovery of a new class of benzothiadiazole dioxide analogs with functional activity as RORC inverse agonists. The early RORC SAR compound 14 exhibited RORC inhibition in a cell based reporter gene assay of 5.7 μM and bound to RORC with an affinity of 1.6 μM in a fluorescence polarization assay displacing a ligand binding site probe. Crystallography confirmed the binding mode of the compound in the ligand binding domain displaying the engagement of a novel sub pocket close to Ser404. Subsequent optimization yielded compounds with enhanced RORC inverse agonist activity. The most active compound 19 showed an IC50 of 440 nM in a human PBMC assay.

Piperazinyl-oxadiazoles as selective sphingosine-1-phosphate receptor agonists.

The discovery of a new series of selective S1P1 agonists is described. This series of piperazinyl-oxadiazole derivatives was rapidly optimized starting from high-throughput screening hit 1 to afford potent and selective lead compound 10d. Further SAR studies showed that 10d was converted to the active phosphate metabolite 29 in vivo. Oral administration of compound 10d to rats was shown to induce lymphopenia at 3 mg/kg.

Structural studies unravel the active conformation of apo RORγt nuclear receptor and a common inverse agonism of two diverse classes of RORγt inhibitors

The nuclear receptor retinoid acid receptor-related orphan receptor γt (RORγt) is a master regulator of the Th17/IL-17 pathway that plays crucial roles in the pathogenesis of autoimmunity. RORγt has recently emerged as a highly promising target for treatment of a number of autoimmune diseases. Through high-throughput screening, we previously identified several classes of inverse agonists for RORγt. Here, we report the crystal structures for the ligand-binding domain of RORγt in both apo and ligand-bound states. We show that apo RORγt adopts an active conformation capable of recruiting coactivator peptides and present a detailed analysis of the structural determinants that stabilize helix 12 (H12) of RORγt in the active state in the absence of a ligand. The structures of ligand-bound RORγt reveal that binding of the inverse agonists disrupts critical interactions that stabilize H12. This destabilizing effect is supported by ab initio calculations and experimentally by a normalized crystallographic B-factor analysis. Of note, the H12 destabilization in the active state shifts the conformational equilibrium of RORγt toward an inactive state, which underlies the molecular mechanism of action for the inverse agonists reported here. Our findings highlight that nuclear receptor structure and function are dictated by a dynamic conformational equilibrium and that subtle changes in ligand structures can shift this equilibrium in opposite directions, leading to a functional switch from agonists to inverse agonists.

CCR1 Plays a Critical Role in Modulating Pain through Hematopoietic and Non-Hematopoietic Cells

Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1−/− animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freunds adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1−/− mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.