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.

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.

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.

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.

Identification of Thieno[3,2-d]pyrimidine Derivatives as Dual Inhibitors of Focal Adhesion Kinase and FMS-like Tyrosine Kinase 3.

Focal adhesion kinase (FAK) is overexpressed in highly invasive and metastatic cancers. To identify novel FAK inhibitors, we designed and synthesized various thieno[3,2-d]pyrimidine derivatives. An intensive structure-activity relationship (SAR) study led to the identification of 26 as a lead. Moreover, 26, a multitargeted kinase inhibitor, possesses excellent potencies against FLT3 mutants as well as FAK. Gratifyingly, 26 remarkably inhibits recalcitrant FLT3 mutants, including F691L, that cause drug resistance. Importantly, 26 is superior to PF-562271 in terms of apoptosis induction, anchorage-independent growth inhibition, and tumor burden reduction in the MDA-MB-231 xenograft mouse model. Also, 26 causes regression of tumor growth in the MV4-11 xenograft mouse model, indicating that it could be effective against acute myeloid leukemia (AML). Finally, in an orthotopic mouse model using MDA-MB-231, 26 remarkably prevents metastasis of orthotopic tumors to lymph nodes. Taken together, the results indicate that 26 possesses potential therapeutic value against highly invasive cancers and relapsed AML.

X-ray Crystal Structure-Guided Design and Optimization of 7H-Pyrrolo[2,3-d]pyrimidine-5-carbonitrile Scaffold as a Potent and Orally Active Monopolar Spindle 1 Inhibitor.

Triple-negative breast cancer (TNBC) is an aggressive breast-cancer subtype associated with poor prognosis and high relapse rates. Monopolar spindle 1 kinase (MPS1) is an apical dual-specificity protein kinase that is over-expressed in TNBC. We herein report a highly selective MPS1 inhibitor based on a 7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile scaffold. Our lead optimization was guided by key X-ray crystal structure analysis. In vivo evaluation of candidate (9) is shown to effectively mitigate human TNBC cell proliferation.

KF-1607, a Novel Pan Src Kinase Inhibitor, Attenuates Obstruction-Induced Tubulointerstitial Fibrosis in Mice.

Src family kinases (SFKs), an important group of non-receptor tyrosine kinases, are suggested to be excessively activated during various types of tissue fibrosis. The present study investigated the effect of KF-1607, an orally active and a newly synthesized Src kinase inhibitor (SKI) with proposed low toxicity, in preventing the progression of renal interstitial fibrosis. Unilateral ureteral obstruction (UUO) surgery was performed in 6-week-old male C57BL/6 mice to induce renal interstitial fibrosis. Either KF-1607 (30 mg/kg, oral gavage) or PP2 (2 mg/kg, intraperitoneal injection), a common experimental SKI, was administered to mice for seven days, started one day prior to surgery. UUO injury-induced SFK expression, including Src, Fyn, and Lyn kinase. SFK inhibition by KF-1607 prevented the progression of tubular injury in UUO mice, as indicated by decreases in albuminuria, urinary KIM-1 excretion, and kidney NGAL protein expression. Renal tubulointerstitial fibrosis was attenuated in response to KF-1607, as shown by decreases in α-SMA, collagen I and IV protein expression, along with reduced Masson's trichrome and collagen-I staining in kidneys. KF-1607 also inhibited inflammation in the UUO kidney, as exhibited by reductions in F4/80 positive-staining and protein expression of p-NFκB and ICAM. Importantly, the observed effects of KF-1607 were similar to those of PP2. A new pan Src kinase inhibitor, KF-1607, is a potential pharmaceutical agent to prevent the progression of renal interstitial fibrosis.

Structural Basis of AZD9291 Selectivity for EGFR T790M.

AZD9291 (Osimertinib) is highly effective in treating EGFR-mutated non-small-cell lung cancers (NSCLCs) with T790M-mediated drug resistance. Despite the remarkable success of AZD9291, its binding pose with EGFR T790M remains unclear. Here, we report unbiased, atomic-level molecular dynamics (MD) simulations in which spontaneous association of AZD9291 with EGFR kinases having WT and T790M mutant gatekeepers was observed. Simulation-generated structural models suggest that the binding pose of AZD9291 with T790M differs from its binding pose with the WT, and that AZD9291 interacts extensively with the gatekeeper residue (Met 790) in T790M but not with Thr 790 in the WT, which explains why AZD9291 binds T790M with higher affinity. The MD simulation-generated models were confirmed by experimentally determined EGFR/T790M complex crystal structures. This work may facilitate the rational design of drugs that can overcome resistance mutations to AZD9291, and more generally it suggests the potential of using unbiased MD simulation to elucidate small-molecule binding poses.

Identification of a Unique Resorcylic Acid Lactone Derivative That Targets Both Lymphangiogenesis and Angiogenesis.

We synthesized 11 novel L-783277 derivatives, in which a structure rigidifying phenyl ring is incorporated into the 14-membered chiral resorcylic acid lactone system. The SAR study with these substances demonstrated that 17 possesses excellent kinase selectivity against a panel of 335 kinases in contrast to L-783277 and inhibits VEGFR3, VEGFR2, and FLT3 with single-digit nanomolar IC50 values. Also, we found that 21, a stereoisomer of 17, has excellent potency (IC50 = 9 nM) against VEGFR3 and selectivity over VEGFR2 and FLT3. 17, a potent dual VEGFR3 and VEGFR2 inhibitor, effectively suppresses both lymphangiogenesis and angiogenesis in a 3D-microfluidic tumor lymphangiogenesis assay and in vivo corneal assay while SAR131675 blocks only lymphangiogenesis. In addition, 17 blocks the endothelial tube formation and suppresses proliferation of PHE tumor vascular model. 17 will be a valuable templatefor developing therapeutically active and selective substances that target both lymphangiogenesis and angiogenesis.

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.

Brain injury induces HIF-1α-dependent transcriptional activation of LRRK2 that exacerbates brain damage.

Leucine-rich repeat kinase 2 (LRRK2), originally identified as a causative genetic factor in Parkinson's disease, is now associated with a number of pathologies. Here, we show that brain injury induces a robust expression of endogenous LRRK2 and suggest a role of LRRK2 after injury. We found that various in vitro and in vivo models of traumatic brain injury (TBI) markedly enhanced LRRK2 expression in neurons and also increased the level of hypoxia-inducible factor (HIF)-1α. Luciferase reporter assay and chromatin immunoprecipitation revealed direct binding of HIF-1α in LRRK2 proximal promoter. We also found that HIF-1α-dependent transcriptional induction of LRRK2 exacerbated neuronal cell death following injury. Furthermore, application of G1023, a specific, brain-permeable inhibitor of LRRK2, substantially prevented brain tissue damage, cell death, and inflammatory response and alleviated motor and cognitive defects induced by controlled cortical impact injury. Together, these results suggest HIF-1α-LRRK2 axis as a potential therapeutic target for brain injury.

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.

Novel Scaffolds for Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase (DYRK1A) Inhibitors.

DYRK1A is one of five members of the dual-specificity tyrosine (Y) phosphorylation-regulated kinase (DYRK) family. The DYRK1A gene is located in the Down syndrome critical region and regulates cellular processes related to proliferation and differentiation of neuronal progenitor cells during early development. This has focused research on its role in neuronal degenerative diseases, including Alzheimer's and Down syndrome. Recent studies have also shown a possible role of DYRK1A in diabetes. Here we report a variety of scaffolds not generally known for DYRK1A inhibition, demonstrating their effects in in vitro assays and also in cell cultures. These inhibitors effectively block the tau phosphorylation that is a hallmark of Alzheimer's disease. The crystal structures of these inhibitors support the design of optimized and novel therapeutics.

First SAR Study for Overriding NRAS Mutant Driven Acute Myeloid Leukemia.

GNF-7, a multitargeted kinase inhibitor, served as a dual kinase inhibitor of ACK1 and GCK, which provided a novel therapeutic strategy for overriding AML expressing NRAS mutation. This SAR study with GNF-7 derivatives, designed to target NRAS mutant-driven AML, led to identification of the extremely potent inhibitors, 10d, 10g, and 11i, which possess single-digit nanomolar inhibitory activity against both ACK1 and GCK. These substances strongly suppress proliferation of mutant NRAS expressing AML cells via apoptosis and AKT/mTOR signaling blockade. Compound 11i is superior to GNF-7 in terms of kinase inhibitory activity, cellular activity, and differential cytotoxicity. Moreover, 10k possessing a favorable mouse pharmacokinetic profile prolonged life-span of Ba/F3-NRAS-G12D injected mice and significantly delayed tumor growth of OCI-AML3 xenograft model without causing the prominent level of toxicity found with GNF-7. Taken together, this study provides insight into the design of novel ACK1 and GCK dual inhibitors for overriding NRAS mutant-driven AML.

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.

A Chemoproteomic Approach to Query the Degradable Kinome Using a Multi-kinase Degrader.

Heterobifunctional molecules that recruit E3 ubiquitin ligases, such as cereblon, for targeted protein degradation represent an emerging pharmacological strategy. A major unanswered question is how generally applicable this strategy is to all protein targets. In this study, we designed a multi-kinase degrader by conjugating a highly promiscuous kinase inhibitor with a cereblon-binding ligand, and used quantitative proteomics to discover 28 kinases, including BTK, PTK2, PTK2B, FLT3, AURKA, AURKB, TEC, ULK1, ITK, and nine members of the CDK family, as degradable. This set of kinases is only a fraction of the intracellular targets bound by the degrader, demonstrating that successful degradation requires more than target engagement. The results guided us to develop selective degraders for FLT3 and BTK, with potentials to improve disease treatment. Together, this study demonstrates an efficient approach to triage a gene family of interest to identify readily degradable targets for further studies and pre-clinical developments.

Structural basis of mutant-selectivity and drug-resistance related to CO-1686.

Non-small-cell lung cancers (NSCLCs) caused by activating mutations in the kinase domain of epidermal growth factor receptor (EGFR) initially respond to first-generation reversible drugs gefitinib and erlotinib. However, clinical efficacy is limited due to the development of drug-resistance that in more than half of the cases are driven by the secondary T790M mutation. CO-1686 is one of the third generation irreversible inhibitors that inhibits EGFR activating mutants, including those with concurrent T790M, while avoiding the off-target toxicity owing to inhibition of wild-type EGFR in treating EGFR mutation-positive NSCLCs. Despite the remarkable success, the experimentally determined structure of this agent in complex with EGFR T790M remains unknown. In this study, we determined crystal structures of EGFR T790M or L858R mutants covalently bound by CO-1686. Based on these structural data, we can explain why CO-1686 irreversibly inhibits EGFR and selectively prefers T790M, which may help improving this or similar compounds, and explain why EGFR L718Q and L844V mutations incur resistance to this agent.

A UV-Independent Topical Small-Molecule Approach for Melanin Production in Human Skin.

The presence of dark melanin (eumelanin) within human epidermis represents one of the strongest predictors of low skin cancer risk. Topical rescue of eumelanin synthesis, previously achieved in "redhaired" Mc1r-deficient mice, demonstrated significant protection against UV damage. However, application of a topical strategy for human skin pigmentation has not been achieved, largely due to the greater barrier function of human epidermis. Salt-inducible kinase (SIK) has been demonstrated to regulate MITF, the master regulator of pigment gene expression, through its effects on CRTC and CREB activity. Here, we describe the development of small-molecule SIK inhibitors that were optimized for human skin penetration, resulting in MITF upregulation and induction of melanogenesis. When topically applied, pigment production was induced in Mc1r-deficient mice and normal human skin. These findings demonstrate a realistic pathway toward UV-independent topical modulation of human skin pigmentation, potentially impacting UV protection and skin cancer risk.

Discovery of a potent dual ALK and EGFR T790M inhibitor.

The mutational activations of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR) are validated oncogenic events and the targets of approved drugs to treat non-small cell lung cancer (NSCLC). Here we report highly potent dual small molecule inhibitors of both ALK and EGFR, particularly the T790M mutant which confers resistance to first generation EGFR inhibitors. Dual ALK/EGFR inhibitors may provide an efficient approach to prevent resistance that arises as a consequence of clinically reported reciprocal activation mechanisms. Our lead compound 7c displayed remarkable inhibitory activities against both ALK and EGFR in enzymatic and cellular assays. We demonstrate that 7c is capable of recapitulating the signaling effects and antiproliferative activity of combined treatment with the approved ALK inhibitor ceritinib and T790M EGFR inhibitor osimertinib against patient-derived non-small cell lung cancer cell line, DFCI032 which harbors both EML4-ALK and activated EGFR.

Small-Molecule Inhibitors of LRRK2.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) protein have been genetically and functionally linked to Parkinson's disease (PD). The kinase activity of LRRK2 is increased by pathogenic mutations; therefore, modulation of LRRK2 kinase activity by a selective small-molecule inhibitor has been proposed as a potentially viable treatment for Parkinson's disease. This chapter presents a historical overview of the development and bioactivity of several small-molecule LRRK2 inhibitors that have been used to inhibit LRRK2 kinase activity in vitro or in vivo. These compounds are important tools for understanding the cellular biology of LRRK2 and for evaluating the potential of LRRK2 inhibitors as disease-modifying PD therapies.