Conformational flexibility and inhibitor binding to unphosphorylated interleukin-1 receptor-associated kinase 4 (IRAK4).

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a key player in innate immune and inflammatory responses, performing a critical role in signal transduction downstream of Toll-like receptors and interleukin-1 (IL-1) receptors. Upon ligand binding and via its N-terminal death domain, IRAK4 is recruited to an oligomeric receptor that is proximal to the Myddosome signaling complex, inducing IRAK4 kinase domain dimerization, autophosphorylation, and activation. To date, all known IRAK4 structures are in the active conformation, precluding a good understanding of IRAK4's conformational dynamics. To address this issue, here we first solved three crystal structures of the IRAK4 kinase domain (at ≤2.6 Å resolution), in its unphosphorylated, inactive state bound to either the ATP analog AMP-PNP or to one of the two small-molecule inhibitors JH-I-25 and JH-I-17. The structures disclosed that although the structure in complex with AMP-PNP is in an "αC-out" inactive conformation, those in complex with type I inhibitors assume an active "Asp-Phe-Gly (DFG)-in" and "αC-in" conformation. The ability of unphosphorylated IRAK4 to take on variable conformations prompted us to screen for small-molecule inhibitors that bind preferentially to unphosphorylated IRAK4, leading to the identification of ponatinib and HG-12-6. Solving the structures of unphosphorylated IRAK4 in complex with these two inhibitors, we found that they both bind as type II inhibitors with IRAK4 in a "DFG-out" conformation. Collectively, these structures reveal conformational flexibility of unphosphorylated IRAK4 and provide unexpected insights into the potential use of small molecules to modulate IRAK4 activity in cancer, autoimmunity, and inflammation.

The small molecule CA140 inhibits the neuroinflammatory response in wild-type mice and a mouse model of AD.

BACKGROUND: Neuroinflammation is associated with neurodegenerative diseases, including Alzheimer's disease (AD). Thus, modulating the neuroinflammatory response represents a potential therapeutic strategy for treating neurodegenerative diseases. Several recent studies have shown that dopamine (DA) and its receptors are expressed in immune cells and are involved in the neuroinflammatory response. Thus, we recently developed and synthesized a non-self-polymerizing analog of DA (CA140) and examined the effect of CA140 on neuroinflammation. METHODS: To determine the effects of CA140 on the neuroinflammatory response, BV2 microglial cells were pretreated with lipopolysaccharide (LPS, 1 µg/mL), followed by treatment with CA140 (10 µM) and analysis by reverse transcription-polymerase chain reaction (RT-PCR). To examine whether CA140 alters the neuroinflammatory response in vivo, wild-type mice were injected with both LPS (10 mg/kg, intraperitoneally (i.p.)) and CA140 (30 mg/kg, i.p.), and immunohistochemistry was performed. In addition, familial AD (5xFAD) mice were injected with CA140 or vehicle daily for 2 weeks and examined for microglial and astrocyte activation. RESULTS: Pre- or post-treatment with CA140 differentially regulated proinflammatory responses in LPS-stimulated microglia and astrocytes. Interestingly, CA140 regulated D1R levels to alter LPS-induced proinflammatory responses. CA140 significantly downregulated LPS-induced phosphorylation of ERK and STAT3 in BV2 microglia cells. In addition, CA140-injected wild-type mice exhibited significantly decreased LPS-induced microglial and astrocyte activation. Moreover, CA140-injected 5xFAD mice exhibited significantly reduced microglial and astrocyte activation. CONCLUSIONS: CA140 may be beneficial for preventing and treating neuroinflammatory-related diseases, including AD.

Pathophysiological significance and therapeutic targeting of germinal center kinase in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, yet 40% to 50% of patients will eventually succumb to their disease, demonstrating a pressing need for novel therapeutic options. Gene expression profiling has identified messenger RNAs that lead to transformation, but critical events transforming cells are normally executed by kinases. Therefore, we hypothesized that previously unrecognized kinases may contribute to DLBCL pathogenesis. We performed the first comprehensive analysis of global kinase activity in DLBCL, to identify novel therapeutic targets, and discovered that germinal center kinase (GCK) was extensively activated. GCK RNA interference and small molecule inhibition induced cell-cycle arrest and apoptosis in DLBCL cell lines and primary tumors in vitro and decreased the tumor growth rate in vivo, resulting in a significantly extended lifespan of mice bearing DLBCL xenografts. GCK expression was also linked to adverse clinical outcome in a cohort of 151 primary DLBCL patients. These studies demonstrate, for the first time, that GCK is a molecular therapeutic target in DLBCL tumors and that inhibiting GCK may significantly extend DLBCL patient survival. Because the majority of DLBCL tumors (∼80%) exhibit activation of GCK, this therapy may be applicable to most patients.

Systematic identification of genomic markers of drug sensitivity in cancer cells.

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.

Discovery of a Highly Potent and Broadly Effective Epidermal Growth Factor Receptor and HER2 Exon 20 Insertion Mutant Inhibitor.

Exon 20 insertion (Ex20Ins) mutations are the third most prevalent epidermal growth factor receptor (EGFR) activating mutation and the most prevalent HER2 mutation in non-small cell lung cancer (NSCLC). Novel therapeutics for the patients with Ex20Ins mutations are urgently needed, due to their poor responses to the currently approved EGFR and HER2 inhibitors. Here we report the discovery of highly potent and broadly effective EGFR and HER2 Ex20Ins mutant inhibitors. The co-crystal structure of compound 1 b in complex with wild type EGFR clearly revealed an additional hydrophobic interaction of 4-fluorobenzene ring within a deep hydrophobic pocket, which has not been widely exploited in the development of EGFR and HER2 inhibitors. As compared with afatinib, compound 1 a exhibited superior inhibition of proliferation and signaling pathways in Ba/F3 cells harboring either EGFR or HER2 Ex20Ins mutations, and in the EGFR P772_H773insPNP patient-derived lung cancer cell line DFCI127. Our study identifies promising strategies for development of EGFR and HER2 Ex20Ins mutant inhibitors.

In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C.

Directly targeting oncogenic V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) with small-molecule inhibitors has historically been considered prohibitively challenging. Recent reports of compounds that bind directly to the K-Ras G12C mutant suggest avenues to overcome key obstacles that stand in the way of developing such compounds. We aim to target the guanine nucleotide (GN)-binding pocket because the natural contents of this pocket dictate the signaling state of K-Ras. Here, we characterize the irreversible inhibitor SML-8-73-1 (SML), which targets the GN-binding pocket of K-Ras G12C. We report a high-resolution X-ray crystal structure of G12C K-Ras bound to SML, revealing that the compound binds in a manner similar to GDP, forming a covalent linkage with Cys-12. The resulting conformation renders K-Ras in the open, inactive conformation, which is not predicted to associate productively with or activate downstream effectors. Conservation analysis of the Ras family GN-binding pocket reveals variability in the side chains surrounding the active site and adjacent regions, especially in the switch I region. This variability may enable building specificity into new iterations of Ras and other GTPase inhibitors. High-resolution in situ chemical proteomic profiling of SML confirms that SML effectively discriminates between K-Ras G12C and other cellular GTP-binding proteins. A biochemical assay provides additional evidence that SML is able to compete with millimolar concentrations of GTP and GDP for the GN-binding site.

Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells.

Genetic alterations that reduce the function of the immunoregulatory cytokine IL-10 contribute to colitis in mouse and man. Myeloid cells such as macrophages (MΦs) and dendritic cells (DCs) play an essential role in determining the relative abundance of IL-10 versus inflammatory cytokines in the gut. As such, using small molecules to boost IL-10 production by DCs-MΦs represents a promising approach to increase levels of this cytokine specifically in gut tissues. Toward this end, we screened a library of well-annotated kinase inhibitors for compounds that enhance production of IL-10 by murine bone-marrow-derived DCs stimulated with the yeast cell wall preparation zymosan. This approach identified a number of kinase inhibitors that robustly up-regulate IL-10 production including the Food and Drug Administration (FDA)-approved drugs dasatinib, bosutinib, and saracatinib that target ABL, SRC-family, and numerous other kinases. Correlating the kinase selectivity profiles of the active compounds with their effect on IL-10 production suggests that inhibition of salt-inducible kinases (SIKs) mediates the observed IL-10 increase. This was confirmed using the SIK-targeting inhibitor HG-9-91-01 and a series of structural analogs. The stimulatory effect of SIK inhibition on IL-10 is also associated with decreased production of the proinflammatory cytokines IL-1ß, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in human DCs-MΦs and anti-inflammatory CD11c(+) CX3CR1(hi) cells isolated from murine gut tissue. Collectively, these studies demonstrate that SIK inhibition promotes an anti-inflammatory phenotype in activated myeloid cells marked by robust IL-10 production and establish these effects as a previously unidentified activity associated with several FDA-approved multikinase inhibitors.

Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages.

Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation, and resolution of inflammation. Classically activated (M1) macrophages produce proinflammatory mediators to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages (M2b) produce high levels of anti-inflammatory molecules, such as IL-10, and low levels of proinflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Here, we demonstrate that the salt-inducible kinases (SIKs) restrict the formation of regulatory macrophages and that their inhibition induces striking increases in many of the characteristic markers of regulatory macrophages, greatly stimulating the production of IL-10 and other anti-inflammatory molecules. We show that SIK inhibitors elevate IL-10 production by inducing the dephosphorylation of cAMP response element-binding protein (CREB)-regulated transcriptional coactivator (CRTC) 3, its dissociation from 14-3-3 proteins and its translocation to the nucleus where it enhances a gene transcription program controlled by CREB. Importantly, the effects of SIK inhibitors on IL-10 production are lost in macrophages that express a drug-resistant mutant of SIK2. These findings identify SIKs as a key molecular switch whose inhibition reprograms macrophages to an anti-inflammatory phenotype. The remarkable effects of SIK inhibitors on macrophage function suggest that drugs that target these protein kinases may have therapeutic potential for the treatment of inflammatory and autoimmune diseases.

Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor.

We report the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggest that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. These first-in-class covalent K-Ras inhibitors demonstrate that irreversible targeting of the K-Ras guanine-nucleotide binding site is potentially a viable therapeutic strategy for inhibition of Ras signaling.

Suppression of Glioblastoma Stem Cell Potency and Tumor Growth via LRRK2 Inhibition.

Leucine-rich repeat kinase 2 (LRRK2), a large GTP-regulated serine/threonine kinase, is well-known for its mutations causing late-onset Parkinson's disease. However, the role of LRRK2 in glioblastoma (GBM) carcinogenesis has not yet been fully elucidated. Here, we discovered that LRRK2 was overexpressed in 40% of GBM patients, according to tissue microarray analysis, and high LRRK2 expression correlated with poor prognosis in GBM patients. LRRK2 and stemness factors were highly expressed in various patient-derived GBM stem cells, which are responsible for GBM initiation. Canonical serum-induced differentiation decreased the expression of both LRRK2 and stemness factors. Given that LRRK2 is a key regulator of glioma stem cell (GSC) stemness, we developed DNK72, a novel LRRK2 kinase inhibitor that penetrates the blood-brain barrier. DNK72 binds to the phosphorylation sites of active LRRK2 and dramatically reduced cell proliferation and stemness factors expression in in vitro studies. Orthotopic patient-derived xenograft mouse models demonstrated that LRRK2 inhibition with DNK72 effectively reduced tumor growth and increased survival time. We propose that LRRK2 plays a significant role in regulating the stemness of GSCs and that suppression of LRRK2 kinase activity leads to reduced GBM malignancy and proliferation. In the near future, targeting LRRK2 in patients with high LRRK2-expressing GBM could offer a superior therapeutic strategy and potentially replace current clinical treatment methods.

Essential role for IKKß in production of type 1 interferons by plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are characterized by their ability to produce high levels of type 1 interferons in response to ligands that activate TLR7 and TLR9, but the signaling pathways required for IFN production are incompletely understood. Here we exploit the human pDC cell line Gen2.2 and improved pharmacological inhibitors of protein kinases to address this issue. We demonstrate that ligands that activate TLR7 and TLR9 require the TAK1-IKKß signaling pathway to induce the production of IFNß via a pathway that is independent of the degradation of IκBα. We also show that IKKß activity, as well as the subsequent IFNß-stimulated activation of the JAK-STAT1/2 signaling pathway, are essential for the production of IFNα by TLR9 ligands. We further show that TLR7 ligands CL097 and R848 fail to produce significant amounts of IFNα because the activation of IKKß is not sustained for a sufficient length of time. The TLR7/9-stimulated production of type 1 IFNs is inhibited by much lower concentrations of IKKß inhibitors than those needed to suppress the production of NFκB-dependent proinflammatory cytokines, such as IL-6, suggesting that drugs that inhibit IKKß may have a potential for the treatment of forms of lupus that are driven by self-RNA and self-DNA-induced activation of TLR7 and TLR9, respectively.

FRTX-02, a selective and potent inhibitor of DYRK1A, modulates inflammatory pathways in mouse models of psoriasis and atopic dermatitis.

Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) has been proposed as a novel regulator of adaptive immune homeostasis through modulating T cell polarization. Thus, DYRK1A could present a potential target in autoimmune disorders. Here, we identify FRTX-02 as a novel compound exhibiting potent and selective inhibition of DYRK1A. FRTX-02 induced transcriptional activity of the DYRK1A substrate NFAT in T cell lines. Correspondingly, FRTX-02 promoted ex vivo CD4+ polarization into anti-inflammatory Tregs and reduced their polarization into pro-inflammatory Th1 or Th17 cells. We show that FRTX-02 could also limit innate immune responses through negative regulation of the MyD88/IRAK4-NF-κB axis in a mast cell line. Finally, in mouse models of psoriasis and atopic dermatitis, both oral and topical formulations of FRTX-02 reduced inflammation and disease biomarkers in a dose-dependent manner. These results support further studies of DYRK1A inhibitors, including FRTX-02, as potential therapies for chronic inflammatory and autoimmune conditions.

New scaffolds for type II JAK2 inhibitors overcome the acquired G993A resistance mutation.

Recurrent JAK2 alterations are observed in myeloproliferative neoplasms, B-cell acute lymphoblastic leukemia, and other hematologic malignancies. Currently available type I JAK2 inhibitors have limited activity in these diseases. Preclinical data support the improved efficacy of type II JAK2 inhibitors, which lock the kinase in the inactive conformation. By screening small molecule libraries, we identified a lead compound with JAK2 selectivity. We highlight analogs with on-target biochemical and cellular activity and demonstrate in vivo activity using a mouse model of polycythemia vera. We present a co-crystal structure that confirms the type II binding mode of our compounds with the "DFG-out" conformation of the JAK2 activation loop. Finally, we identify a JAK2 G993A mutation that confers resistance to the type II JAK2 inhibitor CHZ868 but not to our analogs. These data provide a template for identifying novel type II kinase inhibitors and inform further development of agents targeting JAK2 that overcome resistance.

Discovery of a small-molecule type II inhibitor of wild-type and gatekeeper mutants of BCR-ABL, PDGFRalpha, Kit, and Src kinases: novel type II inhibitor of gatekeeper mutants.

Many clinically validated kinases, such as BCR-ABL, c-Kit, PDGFR, and EGFR, become resistant to adenosine triphosphate-competitive inhibitors through mutation of the so-called gatekeeper amino acid from a threonine to a large hydrophobic amino acid, such as an isoleucine or methionine. We have developed a new class of adenosine triphosphate competitive inhibitors, exemplified by HG-7-85-01, which is capable of inhibiting T315I- BCR-ABL (clinically observed in chronic myeloid leukemia), T670I-c-Kit (clinically observed in gastrointestinal stromal tumors), and T674I/M-PDGFRalpha (clinically observed in hypereosinophilic syndrome). HG-7-85-01 is unique among all currently reported kinase inhibitors in having the ability to accommodate either a gatekeeper threonine, present in the wild-type forms of these kinases, or a large hydrophobic amino acid without becoming a promiscuous kinase inhibitor. The distinctive ability of HG-7-85-01 to simultaneously inhibit both wild-type and mutant forms of several kinases of clinical relevance is an important step in the development of the next generation of tyrosine kinase inhibitors.

Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function.

Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and for the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, the development of a potent, selective small-molecule inhibitor of Mps1 should facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 cocrystal structures of new, selective small-molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells having extra centrosomes (an abnormality found in some cancers), Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.

Development of 'DFG-out' inhibitors of gatekeeper mutant kinases.

HG-7-85-01(22) and HG-7-86-01(26) are thiazolo[5,4-b]pyridine containing type II tyrosine kinase inhibitors with potent cellular activity against both wild-type and 'gatekeeper' mutant T315I- Bcr-Abl. Here we report on the 'hybrid design' concept and subsequent structure activity guided optimization efforts that resulted in the development of these inhibitors.

Characterization of TAE684 as a potent LRRK2 kinase inhibitor.

Leucine-rich repeat kinase 2 (LRRK2) is linked to Parkinson's disease and may represent an attractive therapeutic target. Here we report a 2,4-dianilino-5-chloro-pyrimidine, TAE684, a previously reported inhibitor of anaplastic lymphoma kinase (ALK), is also a potent inhibitor of LRRK2 kinase activity (IC(50) of 7.8nM against wild-type LRRK2, 6.1nM against the G2019S mutant). TAE684 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1-0.3µM in cells and in mouse spleen and kidney, but not in brain, following oral doses of 10mg/kg.

GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor.

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for some forms of Parkinson's disease. Here we report the discovery and characterization of 2-arylmethyloxy-5-subtitutent-N-arylbenzamides with potent LRRK2 activities exemplified by GSK2578215A which exhibits biochemical IC(50)s of around 10 nM against both wild-type LRRK2 and the G2019S mutant. GSK2578215A exhibits exceptionally high selectivity for LRRK2 across the kinome, substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.3-1.0 µM in cells and in mouse spleen and kidney, but not in brain, following intraperitoneal injection of 100mg/kg.

Pan-Src kinase inhibitor treatment attenuates diabetic kidney injury via inhibition of Fyn kinase-mediated endoplasmic reticulum stress.

Src family kinases (SFKs) have been implicated in the pathogenesis of kidney fibrosis. However, the specific mechanism by which SFKs contribute to the progression of diabetic kidney disease (DKD) remains unclear. Our preliminary transcriptome analysis suggested that SFK expression was increased in diabetic kidneys and that the expression of Fyn (a member of the SFKs), along with genes related to unfolded protein responses from the endoplasmic reticulum (ER) stress signaling pathway, was upregulated in the tubules of human diabetic kidneys. Thus, we examined whether SFK-induced ER stress is associated with DKD progression. Mouse proximal tubular (mProx24) cells were transfected with Fyn or Lyn siRNA and exposed to high glucose and palmitate (HG-Pal). Streptozotocin-induced diabetic rats were treated with KF-1607, a novel pan-Src kinase inhibitor (SKI) with low toxicity. The effect of KF-1607 was compared to that of losartan, a standard treatment for patients with DKD. Among the SFK family members, the Fyn and Lyn kinases were upregulated under diabetic stress. HG-Pal induced p70S6 kinase and JNK/CHOP signaling and promoted tubular injury. Fyn knockdown but not Lyn knockdown inhibited this detrimental signaling pathway. In addition, diabetic rats treated with KF-1607 showed improved kidney function and decreased ER stress, inflammation, and fibrosis compared with those treated with losartan. Collectively, these findings indicate that Fyn kinase is a specific member of the SFKs implicated in ER stress activation leading to proximal tubular injury in the diabetic milieu and that pan-SKI treatment attenuates kidney injury in diabetic rats. These data highlight Fyn kinase as a viable target for the development of therapeutic agents for DKD.

Discovery and optimization of potent and selective benzonaphthyridinone analogs as small molecule mTOR inhibitors with improved mouse microsome stability.

Starting from small molecule mTOR inhibitor Torin1, replacement of the piperazine ring with a phenyl ring resulted in a new series of mTOR inhibitors (as exemplified by 10) that showed superior potency and selectivity for mTOR, along with significantly improved mouse liver microsome stability and a longer in vivo half-life.