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1.  Structural Mechanisms Determining Inhibition of the Collagen Receptor DDR1 by Selective and Multi-Targeted Type II Kinase Inhibitors 
Journal of Molecular Biology  2014;426(13):2457-2470.
The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9 nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a β-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.
Graphical Abstract
•DDR kinases bind potently to clinically relevant type II kinase inhibitors.•Co-crystal structures of DDR1 reveal the inhibitor binding modes.•Structural differences to other kinases allow for DDR-selective inhibitors.•Type II kinase inhibitors have potential to treat DDR-associated diseases.
PMCID: PMC4058747  PMID: 24768818
A-loop, activation loop; CML, chronic myeloid leukemia; DDR, discoidin domain receptor; IGF1R, insulin-like growth factor 1 receptor; ITC, isothermal titration calorimetry; KID, kinase insert domain; RTKs, receptor tyrosine kinases; TCEP, tris(2-carboxyethyl)phosphine; TKI, tyrosine kinase inhibitor; TrkB, tropomyosin-related kinase B; phosphorylation; crystallography; drug design; oncology; gleevec
2.  Using combination therapy to override stromal-mediated chemoresistance in mutant FLT3-positive AML: Synergism between FLT3 inhibitors, dasatinib/multi-targeted inhibitors, and JAK inhibitors 
Leukemia  2012;26(10):2233-2244.
Acute myeloid leukemia (AML) progenitors are frequently characterized by activating mutations in the receptor tyrosine kinase FLT3. Protein tyrosine kinases are integral components of signaling cascades that play a role in both FLT3-mediated transformation as well as viability pathways that are advantageous to leukemic cell survival. The bone marrow microenvironment can diminish AML sensitivity to tyrosine kinase inhibitors (TKIs). We hypothesized that inhibition of protein kinases in addition to FLT3 may be effective in overriding drug resistance in AML. We used a cell-based model mimicking stromal protection as part of an unbiased high-throughput chemical screen to identify kinase inhibitors with the potential to override microenvironment-mediated drug resistance in mutant FLT3-positive AML. Several related multi-targeted kinase inhibitors, including dasatinib, with the capability of reversing microenvironment-induced resistance to FLT3 inhibition were identified and validated. We validated synergy in vitro and demonstrated effective combination potential in vivo. In particular Janus kinase (JAK) inhibitors were effective in overriding stromal protection and potentiating FLT3 inhibition in primary AML and cell lines. These results hint at a novel concept of using combination therapy to override drug resistance in mutant FLT3-positive AML in the bone marrow niche and suppress or eradicate residual disease.
PMCID: PMC4054699  PMID: 22469781
acute myeloid leukemia; FLT3 inhibitor; multi-targeted kinase inhibitor; mutant FLT3; PKC412; AC220; stromal-mediated chemoresistance; drug resistance; synergy
3.  MELK is an oncogenic kinase essential for mitotic progression in basal-like breast cancer cells 
eLife  2014;3:e01763.
Despite marked advances in breast cancer therapy, basal-like breast cancer (BBC), an aggressive subtype of breast cancer usually lacking estrogen and progesterone receptors, remains difficult to treat. In this study, we report the identification of MELK as a novel oncogenic kinase from an in vivo tumorigenesis screen using a kinome-wide open reading frames (ORFs) library. Analysis of clinical data reveals a high level of MELK overexpression in BBC, a feature that is largely dependent on FoxM1, a master mitotic transcription factor that is also found to be highly overexpressed in BBC. Ablation of MELK selectively impairs proliferation of basal-like, but not luminal breast cancer cells both in vitro and in vivo. Mechanistically, depletion of MELK in BBC cells induces caspase-dependent cell death, preceded by defective mitosis. Finally, we find that Melk is not required for mouse development and physiology. Together, these data indicate that MELK is a normally non-essential kinase, but is critical for BBC and thus represents a promising selective therapeutic target for the most aggressive subtype of breast cancer.
eLife digest
Not all cancers are the same. There are, for example, at least five types of breast cancer. Different types of cancer can have different mutations and express different genes that determine how aggressively the tumors grow and how well they respond to different therapies. By exploiting these differences, scientists have developed therapies that target specific tumor types, and these targeted therapies have proven useful against most breast cancers.
One type of breast cancer, however, has proven hard to treat. Basal-like breast cancer grows rapidly and there are few treatment options for women with this type of cancer. One reason for this is that, unlike other forms of breast cancer, these cancers do not have the hormone receptors that are the targets of existing therapies.
Enzymes called kinases are promising alternate targets, and many kinase-inhibiting drugs can kill tumor cells in mice. Nevertheless, it has proven difficult to develop kinase inhibitors that are safe for use in humans because these drugs can also kill normal cells. To avoid this side effect, cancer researchers have been searching for a kinase that is active in cancer cells but not in normal cells.
Wang et al. tested a large collection of kinases and found that one called MELK caused tumors to grow in the mammary glands of mice. Further examination of tumor samples collected from hundreds of women in previous clinical studies revealed that MELK expression was increased in basal-like breast cancers and other breast cancer tumors that lack the usual hormone receptor targets.
When Wang et al. treated tumor cells and mice with tumors with a chemical that stops MELK working, basal-like breast cancer cells stopped multiplying and died. On the other hand, tumor cells that had the usual hormone receptors continued to multiply. To see if MELK is important in healthy mice, Wang et al. genetically engineered mice to delete the MELK gene and found that these mutant mice appear normal. The next challenge will be to test if drugs that inhibit MELK can kill basal-like breast cancer cells without having the side effect of harming normal cells.
PMCID: PMC4059381  PMID: 24844244
MELK; basal-like breast cancer; triple-negative breast cancer; FoxM1; targeted therapy; human; mouse
5.  TYK2-STAT1-BCL2 Pathway Dependence in T-Cell Acute Lymphoblastic Leukemia 
Cancer discovery  2013;3(5):564-577.
Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) screens, we identified pathway dependence on a member of the JAK tyrosine kinase family, TYK2, and its downstream effector STAT1 in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently demonstrated TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK kinase activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway i n T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of IL-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the anti-apoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway.
PMCID: PMC3651770  PMID: 23471820
Tyrosine kinase; TYK2; STAT1; BCL2; T-ALL
6.  Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC 
The Journal of Clinical Investigation  2014;124(5):2037-2049.
Genome-wide analyses determined previously that the receptor tyrosine kinase (RTK) EPHA2 is commonly overexpressed in non–small cell lung cancers (NSCLCs). EPHA2 overexpression is associated with poor clinical outcomes; therefore, EPHA2 may represent a promising therapeutic target for patients with NSCLC. In support of this hypothesis, here we have shown that targeted disruption of EphA2 in a murine model of aggressive Kras-mutant NSCLC impairs tumor growth. Knockdown of EPHA2 in human NSCLC cell lines reduced cell growth and viability, confirming the epithelial cell autonomous requirements for EPHA2 in NSCLCs. Targeting EPHA2 in NSCLCs decreased S6K1-mediated phosphorylation of cell death agonist BAD and induced apoptosis. Induction of EPHA2 knockdown within established NSCLC tumors in a subcutaneous murine model reduced tumor volume and induced tumor cell death. Furthermore, an ATP-competitive EPHA2 RTK inhibitor, ALW-II-41-27, reduced the number of viable NSCLC cells in a time-dependent and dose-dependent manner in vitro and induced tumor regression in human NSCLC xenografts in vivo. Collectively, these data demonstrate a role for EPHA2 in the maintenance and progression of NSCLCs and provide evidence that ALW-II-41-27 effectively inhibits EPHA2-mediated tumor growth in preclinical models of NSCLC.
PMCID: PMC4001547  PMID: 24713656
7.  Discovery of a Potent and Selective DDR1 Receptor Tyrosine Kinase Inhibitor 
ACS Chemical Biology  2013;8(10):2145-2150.
The DDR1 receptor tyrosine kinase is activated by matrix collagens and has been implicated in numerous cellular functions such as proliferation, differentiation, adhesion, migration, and invasion. Here we report the discovery of a potent and selective DDR1 inhibitor, DDR1-IN-1, and present the 2.2 Å DDR1 co-crystal structure. DDR1-IN-1 binds to DDR1 in the ‘DFG-out’ conformation and inhibits DDR1 autophosphorylation in cells at submicromolar concentrations with good selectivity as assessed against a panel of 451 kinases measured using the KinomeScan technology. We identified a mutation in the hinge region of DDR1, G707A, that confers >20-fold resistance to the ability of DDR1-IN-1 to inhibit DDR1 autophosphorylation and can be used to establish what pharmacology is DDR1-dependent. A combinatorial screen of DDR1-IN-1 with a library of annotated kinase inhibitors revealed that inhibitors of PI3K and mTOR such as GSK2126458 potentiate the antiproliferative activity of DDR1-IN-1 in colorectal cancer cell lines. DDR1-IN-1 provides a useful pharmacological probe for DDR1-dependent signal transduction.
PMCID: PMC3800496  PMID: 23899692
9.  Discovery and Characterization of Novel Mutant FLT3 Kinase Inhibitors 
Molecular cancer therapeutics  2010;9(9):2468-2477.
For a subpopulation of acute myeloid leukemia (AML) patients, the constitutively activated tyrosine kinase, mutant FLT3, has emerged as a promising target for therapy. The development of drug resistance, however, is a growing concern for mutant FLT3 inhibitors, such as PKC412. Potential therapeutic benefit can arise from the combination of two structurally diverse inhibitors that target- but bind differently to- the same protein or from two inhibitors with completely different mechanisms of action. Thus, there is a need for identification and development of novel FLT3 inhibitors that have the ability to positively combine with PKC412 or standard chemotherapeutic agents used to treat AML as a way to suppress the development of drug resistance and consequently prolong disease remission. Here, we report the effects of the novel type II ATP competitive inhibitors, HG-7-85-01 and HG-7-86-01, which potently and selectively target mutant FLT3 protein kinase activity, and inhibit the proliferation of cells harboring FLT3-ITD or FLT3 kinase domain point mutants via induction of apoptosis and cell cycle inhibition. Anti-leukemic activity of HG-7-85-01 was demonstrated in vivo to be comparable to that observed with PKC412 in a bioluminescence assay utilizing NCr nude mice harboring Ba/F3-FLT3-ITD-luc+ cells. HG-7-85-01 was also observed to override PKC412 resistance. Finally, HG-7-85-01 and HG-7-86-01 synergized with PKC412 and standard chemotherapeutic agents against mutant PKC412-sensitive and some PKC412-resistant, FLT3-positive cells. Thus, we present a structurally novel class of FLT3 inhibitors that warrants consideration for clinical testing against drug-resistant disease in AML patients.
PMCID: PMC3967795  PMID: 20807780
10.  Development of ATP-competitive mTOR Inhibitors 
The mTOR mediated signaling transduction pathway has been observed to be deregulated in a wide variety of cancer and metabolic diseases. Despite extensive clinical development efforts, the well-known allosteric mTOR inhibitor rapamycin and structurally related rapalogs have failed to show significant single-agent anti-tumor efficacy in most types of cancer. This limited clinical success maybe due to the inability of the rapalogs to maintain a complete blockade mTOR mediated signaling. Therefore numerous efforts have been initiated to develop ATP-competitive mTOR inhibitors that would block both mTORC1 and mTORC2 complex activity. Here we describe our experimental approaches to develop Torin1 using a medium throughput cell-based screening assay and structure-guided drug design.
PMCID: PMC3964610  PMID: 22125084
mTOR; mTORC1; mTORC2; PI3K; PIKK; Akt; Rapamycin; Torin1
11.  Interleukin-6 Secretion by Astrocytes Is Dynamically Regulated by PI3K-mTOR-Calcium Signaling 
PLoS ONE  2014;9(3):e92649.
After contusion spinal cord injury (SCI), astrocytes become reactive and form a glial scar. While this reduces spreading of the damage by containing the area of injury, it inhibits regeneration. One strategy to improve the recovery after SCI is therefore to reduce the inhibitory effect of the scar, once the acute phase of the injury has passed. The pleiotropic cytokine interleukin-6 (IL-6) is secreted immediately after injury and regulates scar formation; however, little is known about the role of IL-6 in the sub-acute phases of SCI. Interestingly, IL-6 also promotes axon regeneration, and therefore its induction in reactive astrocytes may improve regeneration after SCI. We found that IL-6 is expressed by astrocytes and neurons one week post-injury and then declines. Using primary cultures of rat astrocytes we delineated the molecular mechanisms that regulate IL-6 expression and secretion. IL-6 expression requires activation of p38 and depends on NF-κB transcriptional activity. Activation of these pathways in astrocytes occurs when the PI3K-mTOR-AKT pathway is inhibited. Furthermore, we found that an increase in cytosolic calcium concentration was necessary for IL-6 secretion. To induce IL-6 secretion in astrocytes, we used torin2 and rapamycin to block the PI3K-mTOR pathway and increase cytosolic calcium, respectively. Treating injured animals with torin2 and rapamycin for two weeks, starting two weeks after injury when the scar has been formed, lead to a modest effect on mechanical hypersensitivity, limited to the period of treatment. These data, taken together, suggest that treatment with torin2 and rapamycin induces IL-6 secretion by astrocytes and may contribute to the reduction of mechanical hypersensitivity after SCI.
PMCID: PMC3965459  PMID: 24667246
12.  Discovery of BAF312 (Siponimod), a Potent and Selective S1P Receptor Modulator 
ACS Medicinal Chemistry Letters  2013;4(3):333-337.
A novel series of alkoxyimino derivatives as S1P1 agonists were discovered through de novo design using FTY720 as the chemical starting point. Extensive structure–activity relationship studies led to the discovery of (E)-1-(4-(1-(((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (32, BAF312, Siponimod), which has recently completed phase 2 clinical trials in patients with relapsing–remitting multiple sclerosis.
PMCID: PMC4027137  PMID: 24900670
S1P receptor; S1P1 agonist; lymphocytes
13.  Developing irreversible inhibitors of the protein kinase cysteinome 
Chemistry & biology  2013;20(2):146-159.
Protein kinases are a large family of approximately 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues such as tyrosine, serine and threonine which serves as a ubiquitous mechanism for cellular signal transduction. The clinical success of a number of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large number of kinases may represent therapeutically relevant targets. To-date the majority of clinical and preclinical kinase inhibitors are ATP-competitive, non-covalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a number of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency and ability to validate pharmacological specificity through mutation of the reactive cysteine residue. Here we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biological activity profiles. In addition, we provided an informatics assessment of the potential ‘kinase-cysteinome’ and discuss strategies for the efficient development of new covalent inhibitors.
PMCID: PMC3583020  PMID: 23438744
Protein Kinases; Irreversible Kinase inhibitors
14.  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.
PMCID: PMC3929239  PMID: 21621413
mTOR; PI3K; Torin1
15.  Discovery of 1-(4-(4-propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one as a highly potent, selective Mammalian Target of Rapamycin (mTOR) inhibitor for the treatment of cancer 
Journal of medicinal chemistry  2010;53(19):7146-7155.
The mTOR protein is a master regulator of cell growth and proliferation, and inhibitors of its kinase activity have the potential to become new class of anti-cancer drugs. Starting from quinoline 1, which was identified in a biochemical mTOR assay, we developed a tricyclic benzonaphthyridinone inhibitor Torin1(26), which inhibited phosphorylation of mTORC1 and mTORC2 substrates in cells at concentrations of 2 nM and 10 nM, respectively. Moreover, Torin1 exhibits 1000-fold selectivity for mTOR over PI3K (EC50 = 1800 nM) and exhibits 100-fold binding selectivity relative to 450 other protein kinases. Torin1 was efficacious at a dose of 20 mg/kg in a U87MG xenograft model, and demonstrated good pharmacodynamic inhibition of downstream effectors of mTOR in tumor and peripheral tissues. These results demonstrate that Torin1 is a useful probe of mTOR-dependent phenomena and that benzonaphthridinones represent a promising scaffold for the further development of mTOR-specific inhibitors with the potential for clinical utility.
PMCID: PMC3893826  PMID: 20860370
17.  Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases 
Biochemical Journal  2013;457(Pt 1):215-225.
The related NUAK1 and NUAK2 are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor kinase. Recent work suggests they play important roles in regulating key biological processes including Myc-driven tumorigenesis, senescence, cell adhesion and neuronal polarity. In the present paper we describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms (IC50 for NUAK1 is 20 nM and for NUAK2 is 100 nM), whereas HTH-01-015 inhibits only NUAK1 (IC50 is 100 nM). These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases that were tested including ten AMPK family members. In all cell lines tested, WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterized substrate, MYPT1 (myosin phosphate-targeting subunit 1) that is phosphorylated by NUAK1 at Ser445. We also identify a mutation (A195T) that does not affect basal NUAK1 activity, but renders it ~50-fold resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating the phosphorylation of MYPT1 we find that in cells overexpressing drug-resistant NUAK1[A195T], but not wild-type NUAK1, phosphorylation of MYPT1 at Ser445 is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to MEFs (mouse embryonic fibroblasts) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1-knockout. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knockdown. We find that WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knockdown. The results of the present study indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAK kinases.
We describe the discovery of structurally diverse kinase inhibitors to dissect the physiological roles of the NUAK isoforms. We recommend use of an inhibitor-resistant NUAK1[A195T] mutant to verify that the physiological effects of these compounds is indeed mediated through inhibition of NUAKs
PMCID: PMC3969223  PMID: 24171924
AMP-activated protein kinase (AMPK); AMPK-related kinase 5 (ARK5); kinase inhibitor; kinase profiling; liver kinase B1 (LKB1); myosin phosphate-targeting subunit 1(MYPT1); sucrose-non-fermenting protein kinase/AMPKrelated protein kinase (SNARK); ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; BRSK, brain-specific kinase; DMEM, Dulbecco’s modified Eagle’s medium; HA, haemagglutinin; HEK, human embryonic kidney; LKB1, liver kinase B1; MARK, microtubule-affinity-regulating kinase; MEF, mouse embryonic fibroblast; MYPT1, myosin phosphate-targeting subunit 1; NF-κB, nuclear factor κB; PEI, polyethylenimine; PP1, protein phosphatase 1; SIK, salt-induced kinase
18.  Structural determinants for ERK5 (MAPK7) and leucine rich repeat kinase 2 activities of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones 
The benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-one core was discovered as a novel ERK5 (also known as MAPK7 and BMK1) inhibitor scaffold, previously. Further structure–activity relationship studies of this scaffold led to the discovery of ERK5-IN-1 (26) as the most selective and potent ERK5 inhibitor reported to date. 26 potently inhibits ERK5 biochemically with an IC50 of 0.162 ± 0.006 μM and in cells with a cellular EC50 for inhibiting epidermal growth factor induced ERK5 autophosphorylation of 0.09 ± 0.03 μM. Furthermore, 26 displays excellent selectivity over other kinases with a KINOMEscan selectivity score (S10) of 0.007, and exhibits exceptional bioavailability (F%) of 90% in mice. 26 will serve as a valuable tool compound to investigate the ERK5 signaling pathway and as a starting point for developing an ERK5 directed therapeutic agent.
Graphical abstract
•Structural determinants of benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones for ERK5.•Highly selective ERK5 inhibitor with good efficacy both in vitro and in vivo.•Represents a good template for developing ERK5 directed therapeutic agent.
PMCID: PMC3914206  PMID: 24239623
ERK5 inhibitor; Kinase selectivity; Benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-one; BMK1, big MAP kinase 1; DIEA, N,N-diisopropylethylamine; DCAMKL2, doublecortin and CaM kinase-like 2; DMA, N,N-dimethylacetamide; EGF, epidermal growth factor; ERK5, extracelluar-signal-regulated kinase 5; HCC, hepatocellular carcinoma; LRRK2, leucine rich repeat kinase 2; MAPK, mitogen-activated protein kinase; ERK5, mitogen-activated protein kinase 7; MEK5, MAP kinase kinase 5; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium-(0); PLK, polo-like kinase; PML, promyelocytic leukemia protein; RSK, ribosomal S6 kinase; SAR, structure–activity relationship; X-phos, 2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-biphenyl
19.  Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM and ATR 
Cancer research  2013;73(8):2574-2586.
mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here we report the characterization of Torin2, a second generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pM with approximately 800-fold selectivity for cellular mTOR versus PI3K. Torin2 also exhibited potent biochemical and cellular activity against PIKK family kinases including ATM (EC50 28 nM), ATR (EC50 35 nM) and DNA-PK (EC50 118 nM) (PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with MEK inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncological settings where mTOR signaling has a pathogenic role.
PMCID: PMC3760004  PMID: 23436801
mTOR; ATM; ATR; lung cancer; kinase inhibitors
20.  Reactivation of ERK Signaling causes resistance to EGFR kinase inhibitors 
Cancer discovery  2012;2(10):934-947.
The clinical efficacy of EGFR kinase inhibitors is limited by the development of drug resistance. The irreversible EGFR kinase inhibitor WZ4002 is effective against the most common mechanism of drug resistance mediated by the EGFR T790M mutation. Here we show, in multiple complementary models, that resistance to WZ4002 develops through aberrant activation of ERK signaling caused by either an amplification of MAPK1 or by downregulation of negative regulators of ERK signaling. Inhibition of MEK or ERK restores sensitivity to WZ4002 and prevents the emergence of drug resistance. We further identify MAPK1 amplification in an erlotinib resistant EGFR mutant NSCLC patient. In addition, the WZ4002 resistant MAPK1 amplified cells also demonstrate an increase both in EGFR internalization and a decrease in sensitivity to cytotoxic chemotherapy. Our findings provide insights into mechanisms of drug resistance to EGFR kinase inhibitors and highlight rationale combination therapies that should be evaluated in clinical trials.
PMCID: PMC3477553  PMID: 22961667
Drug resistance; EGFR mutation; gene amplification
21.  Leveraging kinase inhibitors to develop small molecule tools for imaging kinases by fluorescence microscopy◊ 
Molecular bioSystems  2012;8(10):2523-2526.
As the usage of fluorescence microscopy as a tool to study biological systems continues to grow, so does the need for additional tools that permit the selective detection of proteins of interest. Existing selective and well-characterized kinase inhibitors may be exploited to develop novel small molecule probes useful in imaging kinases by fluorescence microscopy.
PMCID: PMC3616611  PMID: 22673640
22.  mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin 
Science (New York, N.Y.)  2013;341(6144):1236566.
The mTOR Complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs lifespan in model organisms. A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin. We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin as well as to nutrient and growth factor starvation within cells. Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation. Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway. Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals.
PMCID: PMC3771538  PMID: 23888043
23.  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.
PMCID: PMC3415686  PMID: 22819762
T315I Bcr-Abl; Kinase inhibitor; Gatekeeper mutant; Type II inhibitor; Thiazolo[5; 4-b]pyridine
24.  Brain Penetrant LRRK2 Inhibitor 
ACS medicinal chemistry letters  2012;3(8):658-662.
Activating mutations in leucine-rich repeat kinase 2 (LRRK2) are present in a subset of Parkinson’s disease (PD) patients and may represent an attractive therapeutic target. Here we report a 2-anilino-4-methylamino-5-chloropyrimidine, HG-10-102-01(4) is a potent and selective inhibitor of wild-type LRRK2 and the G2019S mutant. Compound 4 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1–0.3 µM in cells and is the first compound reported to be capable of inhibiting Ser910 and Ser935 phosphorylation in mouse brain following intraperitoneal delivery of doses as low as 50 mg/kg.
PMCID: PMC3467149  PMID: 23066449
LRRK2; Blood-brain barrier; Brain Penetrant Inhibitor; 2,4-diaminopyrimidine
25.  Brain Penetrant LRRK2 Inhibitor 
ACS Medicinal Chemistry Letters  2012;3(8):658-662.
Activating mutations in leucine-rich repeat kinase 2 (LRRK2) are present in a subset of Parkinson's disease (PD) patients and may represent an attractive therapeutic target. Here, we report that a 2-anilino-4-methylamino-5-chloropyrimidine, HG-10-102-01 (4), is a potent and selective inhibitor of wild-type LRRK2 and the G2019S mutant. Compound 4 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1–0.3 μM in cells and is the first compound reported to be capable of inhibiting Ser910 and Ser935 phosphorylation in mouse brain following intraperitoneal delivery of doses as low as 50 mg/kg.
PMCID: PMC3467149  PMID: 23066449
LRRK2; blood−brain barrier; brain penetrant inhibitor; 2,4-diaminopyrimidine

Results 1-25 (75)