Douglas Marcotte

Small molecules inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism.

Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

Background: Janus kinase 3 (Jak3) inhibitors hold promise for treatment of autoimmunity, but developing selective inhibitors is challenging. Results: We designed Jak3 inhibitors that avoid inhibition of the other JAKs. Conclusion: Our inhibitors possess high selectivity against other kinases and can potently inhibit Jak3 activity in cell-based assays. Significance: This class of irreversible inhibitors may be useful as selective agents of Jak3 inhibition. The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.

Discovery of biaryl carboxylamides as potent RORγ inverse agonists

RORγt is a pivotal regulator of a pro-inflammatory gene expression program implicated in the pathology of several major human immune-mediated diseases. Evidence from mouse models demonstrates that genetic or pharmacological inhibition of RORγ activity can block the production of pathogenic cytokines, including IL-17, and convey therapeutic benefit. We have identified and developed a biaryl-carboxylamide series of RORγ inverse agonists via a structure based design approach. Co-crystal structures of compounds 16 and 48 supported the design approach and confirmed the key interactions with RORγ protein; the hydrogen bonding with His479 was key to the significant improvement in inverse agonist effect. The results have shown this is a class of potent and selective RORγ inverse agonists, with demonstrated oral bioavailability in rodents.

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode.

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.

Structure-based design of 2,6,7-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines as Aurora kinases inhibitors.

This Letter reports the optimization of a pyrrolopyrimidine series as dual inhibitors of Aurora A/B kinases. This series derived from a pyrazolopyrimidine series previously reported as inhibitors of aurora kinases and CDKs. In an effort to improve the selectivity of this chemotype, we switched to the pyrrolopyrimidine core which allowed functionalization on C-2. In addition, the modeling rationale was based on superimposing the structures of Aurora-A kinase and CDK2 which revealed enough differences leading to a path for selectivity improvement. The synthesis of the new series of pyrrolopyrimidine analogs relied on the development of a different route for the two key intermediates 7 and 19 which led to analogs with both tunable activity against CDK1 and maintained cell potency.

Discovery of novel pyrazole-containing benzamides as potent RORγ inverse agonists

The nuclear receptor RORγ plays a central role in controlling a pro-inflammatory gene expression program in several lymphocyte lineages including TH17 cells. RORγ-dependent inflammation has been implicated in the pathogenesis of several major autoimmune diseases and thus RORγ is an attractive target for therapeutic intervention in these diseases. Starting from a lead biaryl compound 4a, replacement of the head phenyl moiety with a substituted aminopyrazole group resulted in a series with improved physical properties. Further SAR exploration led to analogues (e.g., 4j and 5m) as potent RORγ inverse agonists.

Rational mutagenesis to support structure-based drug design: MAPKAP kinase 2 as a case study.

BackgroundStructure-based drug design (SBDD) can provide valuable guidance to drug discovery programs. Robust construct design and expression, protein purification and characterization, protein crystallization, and high-resolution diffraction are all needed for rapid, iterative inhibitor design. We describe here robust methods to support SBDD on an oral anti-cytokine drug target, human MAPKAP kinase 2 (MK2). Our goal was to obtain useful diffraction data with a large number of chemically diverse lead compounds. Although MK2 structures and structural methods have been reported previously, reproducibility was low and improved methods were needed.ResultsOur construct design strategy had four tactics: N- and C-terminal variations; entropy-reducing surface mutations; activation loop deletions; and pseudoactivation mutations. Generic, high-throughput methods for cloning and expression were coupled with automated liquid dispensing for the rapid testing of crystallization conditions with minimal sample requirements. Initial results led to development of a novel, customized robotic crystallization screen that yielded MK2/inhibitor complex crystals under many conditions in seven crystal forms. In all, 44 MK2 constructs were generated, ~500 crystals were tested for diffraction, and ~30 structures were determined, delivering high-impact structural data to support our MK2 drug design effort.ConclusionKey lessons included setting reasonable criteria for construct performance and prioritization, a willingness to design and use customized crystallization screens, and, crucially, initiation of high-throughput construct exploration very early in the drug discovery process.

Discovery of biaryls as RORγ inverse agonists by using structure-based design.

RORγ plays a critical role in controlling a pro-inflammatory gene expression program in several lymphocyte lineages including T cells, γδ T cells, and innate lymphoid cells. RORγ-mediated inflammation has been linked to susceptibility to Crohns disease, arthritis, and psoriasis. Thus inverse agonists of RORγ have the potential of modulating inflammation. Our goal was to optimize two RORγ inverse agonists: T0901317 from literature and 1 that we obtained from internal screening. We used information from internal X-ray structures to design two libraries that led to a new biaryl series.

Structural determinant for inducing RORgamma specific inverse agonism triggered by a synthetic benzoxazinone ligand

BackgroundThe nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. Our goal was to develop a RORγ specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent.ResultsWe identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β.ConclusionThe synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism.

Germinal-center kinase-like kinase co-crystal structure reveals a swapped activation loop and C-terminal extension.

Germinal‐center kinase‐like kinase (GLK, Map4k3), a GCK‐I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation‐loop swapped dimer with more than 20 amino acids of ordered density at the carboxy‐terminus. This C‐terminal PEST region binds intermolecularly to the hydrophobic groove of the N‐terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an “active” kinase, including the salt bridge between the C‐helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P‐loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.