Discovery of a Type III Inhibitor of LIM Kinase 2 That Binds in a DFG-Out Conformation

The first allosteric, type III inhibitor of LIM-kinase 2 (LIMK2) is reported. A series of molecules that feature both an <i>N</i>-phenylsulfonamide and tertiary amide were not only very potent at LIMK2 but also were extremely selective against a panel of other kinases. Enzymatic kinetic studies showed these molecules to be noncompetitive with ATP, suggesting allosteric inhibition. X-ray crystallography confirmed that these sulfonamides are a rare example of a type III kinase inhibitor that binds away from the highly conserved hinge region and instead resides in the hydrophobic pocket formed in the DFG-out conformation of the kinase, thus accounting for the high level of selectivity observed

Identification and Characterization of Von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that recruit an E3 ligase to a target protein to facilitate ubiquitination and subsequent degradation of that protein. While the field of targeted degraders is still relatively young, the potential for this modality to become a differentiated and therapeutic reality is strong, such that both academic and pharmaceutical institutions are now entering this interesting area of research. In this article, we describe a broadly applicable process for identifying degrader hits based on the serine/threonine kinase TANK-binding kinase 1 (TBK1) and have generalized the key structural elements associated with degradation activities. Compound <b>3i</b> is a potent hit (TBK1 DC₅₀ = 12 nM, <i>D</i>_max = 96%) with excellent selectivity against a related kinase IKKε, which was further used as a chemical tool to assess TBK1 as a target in mutant K-Ras cancer cells

Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity

<div><p>Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4⁺ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1^−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1^−/− CD4⁺ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4⁺ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.</p></div

Discovery and Development of LX7101, a Dual LIM-Kinase and ROCK Inhibitor for the Treatment of Glaucoma

The structure of LX7101, a dual LIM-kinase and ROCK inhibitor for the treatment of ocular hypertension and associated glaucoma, is disclosed. Previously reported LIM kinase inhibitors suffered from poor aqueous stability due to solvolysis of the central urea. Replacement of the urea with a hindered amide resulted in aqueous stable compounds, and addition of solubilizing groups resulted in a set of compounds with good properties for topical dosing in the eye and good efficacy in a mouse model of ocular hypertension. LX7101 was selected as a clinical candidate from this group based on superior efficacy in lowering intraocular pressure and a good safety profile. LX7101 completed IND enabling studies and was tested in a Phase 1 clinical trial in glaucoma patients, where it showed efficacy in lowering intraocular pressure

Mst1^−/− mice exhibit decreased incidence and severity of CIA.

<p>(<b>A</b>) Mice of indicated genotype (n = 11–13) were immunized with CII in CFA and observed for clinical signs of arthritis at the time points depicted on the X axis. Histological scores of synovial inflammation and cartilage/bone erosion were analyzed on day 45 after immunization. Data are expressed as mean (± SEM); * (p<0.05) indicates significant differences in comparison to WT littermates (Mann–Whitney U test). Similar data were obtained in two additional independent experiments. (<b>B</b>) Representative pictures of histological and radiographic signs of arthritis in the same mice as in (A), obtained on day 45 after immunization with CII (left and middle panels). Arrows of the H&E-stained sections of paw joints point to severe synovial inflammation and cartilage erosion in the Mst1^−/− animals. Representative µCT images of subchondral bone changes characteristic of arthritis were taken on day 45 after immunization (right panels).</p

Analysis of T cell apoptosis and functional immune responses in Mst1^−/− mice.

<p>(<b>A</b>) Increased apoptosis of activated Mst1^−/− splenic T cells in vitro. Early apoptotic T cells were quantitated by flow cytometry in after stimulation with the indicated mAbs for 48 hrs (n = 5 per genotype). (<b>B</b>) Analysis of apoptosis in unstimulated MST^−/− T cells. Early apoptotic T cells were quantitated by flow cytometry in CD44^low and CD44^high subsets of freshly isolated splenocytes (n = 5 per genotype). (<b>C</b>) The percentages of naïve (CD62L^highCD44^low), effector memory (CD62L^lowCD44^high), and CD279-, CD25-, and CD69-positive splenic CD4⁺ T cells were examined by flow cytometry (n = 5 per genotype). (<b>D</b>) Cytokine production by splenic CD62L^highCD44⁻ CD4⁺ T cells from WT and Mst1^−/− mice (n = 5 per genotype) was examined 48 hrs after stimulation with mAbs to CD3 and CD28 (both at 1 µg/ml). (<b>E</b>) Th1 and Th2 polarized cells were generated from naïve splenic CD62L^highCD44⁻ CD4⁺ T cells after in vitro culture in polarizing conditions for 5 days. The percentages of effector memory (CD62L^lowCD44^high) CD4⁺ T cells were examined by flow cytometry (n = 5 per genotype). The percentage of sub-G0/G1 apoptotic cells was determined by the BrdU/7-AAD Flow kit and flow cytometry (n = 5 per genotype) following restimulation in vitro with plate-bound CD3 mAb (5 µg/ml) for 48 hrs. (<b>F</b>) Mst1^−/− deficiency leads to decreased Ag-specific adaptive immune responses in vivo. Mst1^−/− and WT mice (n = 6 and 9, respectively) were immunized with OVA in CFA. On day 14, serum samples were analyzed for OVA-specific IgG1 and IgG2a concentrations. Values and statistical significance are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098151#pone-0098151-g002" target="_blank">Fig. 2</a> and are representative of at least two independent experiments. Pre, preimmune serum.</p

Altered immune responses of activated Mst1^−/− T cells in vitro.

<p>(<b>A</b>) Th1 and Th2 cytokine production by splenic T cells from WT and Mst1^−/− mice was examined 48 hrs after stimulation with Con A (2.5 µg/ml) or mAbs to CD3 and CD28 (both at 1 µg/ml). (<b>B</b>) IgE concentration was measured by ELISA in serum samples of WT (n = 8) and Mst1^−/− mice (n = 10). (<b>C</b>) Deficient upregulation of lymphocyte activation markers on Mst1^−/− T cells. Splenic T cells were stimulated with plate-bound CD3 mAb (1 µg/ml) for 48 hrs, stained for CD69 and CD95 (Fas/APO-1), and analyzed by flow cytometry (n = 5 per genotype). (<b>D</b>) Suppression of Mst1^−/− T cell proliferative responses in allogeneic MLR in vitro. Splenic C57Bl/6-Albino/129SvEv (H-2b) Mst1^−/− and WT T cells (responder cells; 5 mice per genotype) were stimulated with the indicated numbers of MHC-mismatched irradiated stimulator cells obtained from Balb/c mice (H-2d). Proliferation of the responder cells was assessed by [³H]-thymidine incorporation after 90 hrs of culture. Values and statistical significance are expressed as in Fig. 2 and represent results of at least two independent experiments.</p

Deficient proliferation of Mst1^−/− T and B cells in vitro.

<p>Single cell suspensions of splenic T and B lymphocytes were stimulated with various T and B cell-specific stimuli at the indicated concentrations. Cell proliferation was assessed by [³H]-thymidine incorporation after 64–66 hrs of stimulation. Data are expressed as the mean ± SEM of counts per minute (cpm) in triplicate cultures (n = 5 per genotype). Values are expressed as mean ± SEM. * (p<0.05) and ** (p<0.01) indicate significant differences in comparison to WT animals (<i>t</i> test).</p

Delayed cell cycle progression of Mst1^−/− T cells in vitro.

<p>(<b>A</b>) Mst1^−/− and WT T cells were activated for 36 hrs with the indicated stimuli, pulsed with BrdU, and analyzed by flow cytometry (n = 5 mice per genotype). The representative dot plots and fractional values show cell subsets residing in the indicated phases of cell cycle. (<b>B</b>) Nuclear DNA content at different stages of the cell cycle was determined by FACS analysis of splenic T cells stimulated for 48 hrs with plate-bound CD3 mAb (1 µg/ml) and stained with propidium iodide. Histograms are representative of 5 samples per genotype. Values on bar graphs and statistical significance are expressed as in Fig. 2. Similar data were obtained in two additional independent experiments.</p

Mst1^−/− mice are resistant to EAE.

<p>(<b>A</b>) EAE was induced in mice of indicated genotype (n = 10) by immunization with MOGp35–55 in CFA. The mean EAE score ± SEM observed in each group is plotted against time after immunization (left panel). Representative H&E stained sections of the cervical spinal cord of WT and Mst1^−/− mice obtained on day 21 after immunization (middle panels). Higher (×40) magnification shows areas of prominent mononuclear multifocal inflammation, vacuolization and gliosis in the white matter of WT mice, which are nearly absent in Mst1^−/− mice. Aggregate histopathology scores were obtained on day 21 after immunization (right panel). Similar data were obtained in two additional independent experiments. * (p<0.05) and ** (p<0.01) indicate significant differences in comparison to WT animals (Mann–Whitney U test). (<b>B</b>) Infiltrating mononuclear cells were isolated from the spinal cord of mice with the indicated genotype (n = 5/group) on day 15 after immunization with MOGp35–55, and analyzed by flow cytometry for markers of T cell differentiation and activation. Numbers above the rectangular gates represent the percentages and absolute numbers (x10⁴/spinal cord) of infiltrating CD4⁺ and CD8+ T cells for each genotype. (<b>C</b>) Frequency of IL-2-, TNF-α-, IFN-γ-, IL-4-, IL-10, and IL-17A-producing CD4⁺ T cells infiltrating the spinal cords of indicated mice was measured by flow cytometry analysis of intracellular cytokine staining. (<b>D</b>) qPCR analysis of IL-2, TNF-α, IFN-γ, IL-4, and IL-10 mRNA in the spinal cords from mice of indicated genotype (n = 6–10) was performed on day 11 after immunization with MOGp35–55. Values are expressed relative to the expression of GAPDH. <i>Values and statistical significance are expressed as in Fig. 2 and represent at least two independent experiments.</i></p