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1.  Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy 
eLife  null;3:e03445.
Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a ‘synthetic lethal toxicity’ arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.
eLife digest
Major advances in cancer therapy have improved the treatment options for many patients. However, many cancer treatments are toxic or have severe side effects, making them difficult for patients to tolerate. One cause of these side effects is that many cancer therapies kill both normal cells and cancer cells. Developing cancer therapies that are more targeted is therefore a priority in cancer research.
Acute myeloid leukemia is a type of blood cancer that has proven difficult to treat without causing serious side effects. This cancer is very aggressive and only about 1 in 4 patients are successfully cured of their cancer. At present, physicians treat acute myeloid leukemia with chemotherapy, which kills both the cancer cells and some of the patient's healthy cells.
Many patients with acute myeloid leukemia have mutations in the gene encoding an enzyme called Fms-like tyrosine kinase 3 (FLT3). This mutation makes the enzyme permanently active, and patients with the mutation have a greater risk of their cancer recurring or death. Scientists have recently discovered that treatments that inhibit the FLT3 enzyme can be effective against cancer. However, the drugs investigated so far also interfere with the patient's ability to produce new blood cells, which can lead to infections or an inability to recover from bleeding. Therefore, no new drugs have yet been approved for general use.
Warkentin et al. suspected the reason for the adverse effects of FLT3 inhibitors is that these drugs also inhibit another enzyme necessary for blood cell production. Previous work showed that inhibiting one or the other of the enzymes still allows blood cells to be produced as normal: it is only when both are inhibited that production problems arise. Warkentin et al. therefore looked for a chemical that inhibits only the FLT3 enzyme and found one called Star 27. Tests revealed that this inhibits FLT3 and prevents the growth and spread of cancerous cells but does not impair blood cell production. Additionally, Star 27 continues to work even when mutations arise in the cancer cells that cause resistance to other FLT3 inhibitors.
The findings demonstrate that when it comes to drug development, it is sometimes as important to avoid certain molecular targets as it is to hit others. Understanding the network of enzymes that FLT3 works with could therefore help researchers to develop more effective and safer cancer treatments.
PMCID: PMC4307180  PMID: 25531068
staurosporine; protein kinase; leukemia; FLT3; KIT; chemical synthesis; zebrafish
2.  Chemical Genetic Approach for Kinase-Substrate Mapping by Covalent Capture of Thiophosphopeptides and Analysis by Mass Spectrometry 
Mapping kinase-substrate interactions demands robust methods to rapidly and unequivocally identify substrates from complex protein mixtures. Towards this goal we present a method in which a kinase, engineered to utilize synthetic ATPγS analogs, specifically thiophosphorylates its substrates in a complex lysate. The thiophosphate label provides a bio-orthogonal tag that can be used to affinity purify and identify labeled proteins. Following the labeling reaction proteins are digested with trypsin, thiol containing peptides are then covalently captured and non-thiol containing peptides are washed from the resin. Oxidation promoted hydrolysis, at sites of thiophosphorylation, releases phosphopeptides for analysis by tandem mass spectrometry. By incorporating two specificity gates: kinase engineering and peptide affinity purification, this method yields high confidence substrate identifications. This method gives both the identity of the substrates and phosphorylation site localization. With this information investigators can analyze the biological significance of the phosphorylation mark immediately following confirmation of the kinase-substrate relationship. Here we provide an optimized version of this technique to further enable widespread utilization of this technology.
PMCID: PMC3894912  PMID: 23836541
phosphorylation; chemical genetics; analog specific kinase; kinase substrate identification; thiophosphate labeling
3.  Discovery of dual inhibitors of the immune cell PI3Ks p110δ and p110γ: a prototype for new anti-inflammatory drugs 
Chemistry & biology  2010;17(2):123-134.
PI3Kδ and PI3Kγ regulate immune cell signaling, while the related PI3Kα and PI3Kβ regulate cell survival and metabolism. Selective inhibitors of PI3Kδ/γ represent a potential class of anti-inflammatory agents lacking the anti-proliferative effects associated with PI3Kα/β inhibition. Here we report the discovery of PI3Kδ/γ inhibitors that display up to 1,000-fold selectivity over PI3Kα/β and evaluate these compounds in a high-content inflammation assay using mixtures of primary human cells. We find selective inhibition of only PI3Kδ is weakly anti-inflammatory, but PI3Kδ/γ inhibitors show superior inflammatory marker suppression through suppression of LPS-induced TNFα production and T-cell activation. Moreover, PI3Kδ/γ inhibition yields an anti-inflammatory signature distinct from pan-PI3K inhibition and known anti-inflammatory drugs, yet bears striking similarities to glucocorticoid receptor agonists. These results highlight the potential of selectively designing drugs that target kinases with shared biological function.
PMCID: PMC2858875  PMID: 20189103
4.  Inhibitor Hijacking of Akt Activation 
Nature chemical biology  2009;5(7):484-493.
The kinase Akt plays a central role as a regulator of multiple growth factor input signals, making it an attractive anti-cancer drug target. A-443654 is an ATP-competitive Akt inhibitor. Unexpectedly, treatment of cells with A-443654 causes paradoxical hyperphosphorylation of Akt at its two regulatory sites (Thr308 and Ser473). We explore whether inhibitor-induced hyperphosphorylation of Akt by A-443654 is a consequence of disrupted feedback regulation at a pathway level or whether it is a direct consequence of inhibitor binding to the ATP binding site of Akt. Catalytically inactive mutants of Akt reveal that binding of an inhibitor to the ATP site of Akt is sufficient to directly cause hyperphosphorylation of the kinase in the absence of any pathway feedback effects. We conclude that ATP-competitive Akt inhibitors impart regulatory phosphorylation of their target kinase Akt providing new insights into both natural regulation of Akt activation and Akt inhibitors entering the clinic.
PMCID: PMC2783590  PMID: 19465931
5.  A Coupled Chemical Genetic and Bioinformatic Approach to Polo-like Kinase Pathway Exploration 
Chemistry & biology  2007;14(11):1261-1272.
Protein phosphorylation is a ubiquitous mechanism for cellular signal propagation, and signaling network complexity presents a challenge to protein kinase substrate identification. Few targets of Polo-like kinases are known, despite their significant role in coordinating cell cycle progression. Here, we combine chemical genetic, bioinformatic, and proteomic tools for Polo-like kinase substrate identification. Monospecific pharmacological inhibition of budding yeast Polo-like kinase, Cdc5, resulted in a misaligned pre-anaphase spindle and subsequently delayed anaphase nuclear migration, revealing a novel Cdc5 function. A cellular screen for Cdc5 substrates identified Spc72, a spindle pole body (SPB) component and microtubule anchor required for nuclear positioning. Spc72 bound to the Cdc5 PBD in a mitosis-specific manner, was phosphorylated by Cdc5 in vitro, and demonstrated a loss of mitotic phosphorylation in vivo upon Cdc5 inhibition. Finally, an examination of Cdc5 binding by SPB-localized proteins expanded our knowledge of Cdc5 function at the SPB.
PMCID: PMC2215327  PMID: 18022565
6.  Quantitative and Temporal Requirements Revealed for Zap-70 Catalytic Activity During T Cell Development 
Nature immunology  2014;15(7):687-694.
The catalytic activity of Zap-70 is crucial for T cell receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly inhibit Zap-70 catalytic activity in a model of synchronized thymic selection, we showed that CD4+CD8+ thymocytes integrate multiple, transient, Zap-70-dependent signals over more than 36 h to reach a cumulative threshold for positive selection, whereas one hour of signaling was sufficient for negative selection. Titration of Zap-70 activity resulted in graded reductions in positive and negative selection but did not decrease the cumulative TCR signals integrated by positively selected OT-I cells, revealing heterogeneity, even among CD4+CD8+ thymocytes expressing identical TCRs undergoing positive selection.
PMCID: PMC4095875  PMID: 24908390
7.  K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions 
Nature  2013;503(7477):548-551.
Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies1–3. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP4 and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis5,6. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP7 and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.
PMCID: PMC4274051  PMID: 24256730
8.  Staurosporine-derived inhibitors broaden the scope of analog-sensitive kinase technology 
Journal of the American Chemical Society  2013;135(48):18153-18159.
Analog-sensitive (AS) kinase technology is a powerful approach for studying phospho-signaling pathways in diverse organisms and physiological processes. The key feature of this technique is that a kinase-of-interest can be mutated to sensitize it to inhibitor analogs that do not target wild-type (WT) kinases. In theory, this enables specific inhibition of any kinase in cells and in mouse models of human disease. Typically these inhibitors are identified from a small library of molecules based on the pyrazolopyrimidine (PP) scaffold. However, we recently identified a subset of native human kinases, including the Ephrin A kinase family, that are sensitive to commonly used PP inhibitors. In an effort to develop a bioorthogonal AS-kinase inhibitor and to extend this technique to PP-sensitive kinases we sought an alternative inhibitor scaffold. Here we report the structure-based design of synthetically tractable, potent, and extremely selective AS-kinase inhibitors based on the natural product staurosporine. We demonstrate that these molecules, termed staralogs, potently target AS kinases in cells and we employ X-ray crystallography to elucidate their mechanism of efficacy. Finally, we demonstrate that staralogs target AS mutants of PP-sensitive kinases at concentrations where there is little to no inhibition of native human kinases. Thus, staralogs represent a new class of AS-kinase inhibitors and a core component of the chemical genetic tool kit for probing kinase-signaling pathways.
PMCID: PMC3938282  PMID: 24171479
9.  Structures of PI4KIIIβ complexes show simultaneous recruitment of Rab11 and its effectors 
Science (New York, N.Y.)  2014;344(6187):1035-1038.
Phosphatidylinositol 4-kinases (PI4Ks) and small guanosine triphosphatases (GTPases) are essential for processes that require expansion and remodeling of phosphatidylinositol 4-phosphate (PI4P)-containing membranes, including cytokinesis, intracellular development of malarial pathogens, and replication of a wide range of RNA viruses. However, the structural basis for coordination of PI4K, GTPases and their effectors is unknown. Here, we describe structures of PI4KB (PI4KIIIβ) bound to the small GTPase Rab11a without and with the Rab11 effector protein FIP3. The Rab11-PI4KIIIβ interface is unique compared with known structures of Rab complexes, and does not involve switch regions used by GTPase effectors. Our data provide a mechanism for how PI4KIIIβ coordinates Rab11 and its effectors on PI4P-enriched membranes, and also provide strategies for the design of specific inhibitors that could potentially target plasmodial PI4KIIIβ to combat malaria.
PMCID: PMC4046302  PMID: 24876499
10.  EGFR phosphorylates tumor-derived EGFRvIII driving STAT3/5 and progression in glioblastoma 
Cancer cell  2013;24(4):10.1016/j.ccr.2013.09.004.
EGFRvIII, a frequently occurring mutation in primary glioblastoma, results in a protein product that cannot bind ligand, but signals constitutively. Deducing how EGFRvIII causes transformation has been difficult because of autocrine and paracrine loops triggered by EGFRvIII alone or in heterodimers with wild-type EGFR. Here, we document co-expression of EGFR and EGFRvIII in primary human glioblastoma that drives transformation and tumorigenesis in a cell-intrinsic manner. We demonstrate enhancement of downstream STAT signaling triggered by EGFR-catalyzed phosphorylation of EGFRvIII, implicating EGFRvIII as a substrate for EGFR. Subsequent phosphorylation of STAT3 requires nuclear entry of EGFRvIII and formation of an EGFRvIII-STAT3 nuclear complex. Our findings clarify specific oncogenic signaling relationships between EGFR and EGFRvIII in glioblastoma.
PMCID: PMC3819146  PMID: 24135280
11.  Structure-guided Inhibitor Design Expands the Scope of Analog-Sensitive Kinase Technology 
ACS chemical biology  2013;8(9):1931-1938.
Engineered analog-sensitive (AS) protein kinases have emerged as powerful tools for dissecting phospho-signaling pathways, for elucidating the cellular function of individual kinases, and for deciphering unanticipated effects of clinical therapeutics. A crucial and necessary feature of this technology is a bioorthogonal small molecule that is innocuous towards native cellular systems but can potently inhibit the engineered kinase. In order to generalize this method we sought a molecule capable of targeting divergent AS-kinases. Here we employ X-ray crystallography and medicinal chemistry to unravel the mechanism of current inhibitors and use these insights to design the most potent, selective and general AS-kinase inhibitors reported to date. We use large-scale kinase inhibitor profiling to characterize the selectivity of these molecules as well as examine the consequences of potential off-target effects in chemical genetic experiments. The molecules reported here will serve as powerful tools in efforts to extend AS-kinase technology to the entire kinome and the principles discovered may help in the design of other engineered enzyme/ligand pairs.
PMCID: PMC3938192  PMID: 23841803
12.  Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors 
Oncogene  2013;33(12):1590-1600.
The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new ATP-competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and PIK3CA mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, while those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor 4E-BP1, but not ribosomal protein S6. In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242 induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared to KRAS WT controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.
PMCID: PMC3982880  PMID: 23542178
mTOR; colon cancer; PIK3CA; KRAS; xenograft; PP242
13.  Adipocyte ALK7 links nutrient overload to catecholamine resistance in obesity 
eLife  2014;3:e03245.
Obesity is associated with blunted β-adrenoreceptor (β-AR)-mediated lipolysis and lipid oxidation in adipose tissue, but the mechanisms linking nutrient overload to catecholamine resistance are poorly understood. We report that targeted disruption of TGF-β superfamily receptor ALK7 alleviates diet-induced catecholamine resistance in adipose tissue, thereby reducing obesity in mice. Global and fat-specific Alk7 knock-out enhanced adipose β-AR expression, β-adrenergic signaling, mitochondrial biogenesis, lipid oxidation, and lipolysis under a high fat diet, leading to elevated energy expenditure, decreased fat mass, and resistance to diet-induced obesity. Conversely, activation of ALK7 reduced β-AR-mediated signaling and lipolysis cell-autonomously in both mouse and human adipocytes. Acute inhibition of ALK7 in adult mice by a chemical-genetic approach reduced diet-induced weight gain, fat accumulation, and adipocyte size, and enhanced adipocyte lipolysis and β-adrenergic signaling. We propose that ALK7 signaling contributes to diet-induced catecholamine resistance in adipose tissue, and suggest that ALK7 inhibitors may have therapeutic value in human obesity.
eLife digest
Adrenaline and noradrenaline are two hormones that trigger the burst of energy and increase in heart rate and blood pressure that are needed for the ‘fight-or-flight’ response. Both belong to a group of chemicals called catecholamines. These chemicals bind to cells carrying proteins called adrenoceptors on their surface and stimulate the breakdown of fat, which releases energy. However, when nutrients are plentiful, fat cells become resistant to catecholamines and instead store fat so it can be used for energy if food becomes scarce. In the industrialized world where food is easily and constantly accessible, this resistance can cause an unhealthy increase in body fat and result in obesity.
Increasing fat metabolism by making fat cells more able to respond to catecholamines is an attractive strategy for combating obesity. Indeed, drugs that mimic the effect of catecholamines on an adrenoceptor found in mice reduce obesity caused by over-eating. However, these drugs are ineffective in humans and can cause harmful side effects to the cardiovascular system, including high blood pressure and an increased heart rate. Devising a strategy that specifically targets catecholamine resistance in fat cells is therefore desirable.
A protein called ALK7 is a cell surface receptor that is predominantly found in fat cells and tissues involved in controlling the metabolism. Mice with a mutation in ALK7 that stops this protein from working properly accumulate less fat than mice with a functional version of the protein, but it is not known why. To understand ALK7's involvement in fat metabolism, Guo et al. created mice whose fat cells lack ALK7, but whose other cells all produce ALK7 as normal. When fed a diet rich in fat, these mice are leaner than regular mice and they burn more energy.
The metabolic responses seen in ALK7 mutant mice are very similar to those seen in mice treated with drugs targeting adrenoceptors, suggesting that there may be a link between ALK7 and catecholamine resistance. Indeed, Guo et al. demonstrate that fat cells lacking ALK7 have an increased sensitivity to catecholamines when the mice are on a high fat diet, which decreases the amount of fat the mice accumulate. Conversely, increasing the activity of ALK7 reduces the ability of the cells to respond to catecholamines, and they accumulate more fat.
Guo et al. also generated a second line of mice carrying a mutation in ALK7 that does not affect its function, but renders it sensitive to inhibition by a custom-made chemical. When these animals were on a high-fat diet, administering the chemical made the mice leaner, suggesting that inhibiting the ALK7 receptor can prevent obesity in adult animals.
Guo et al. also performed experiments in human fat cells, which showed that the ALK7 receptor works in a similar way in human cells as it does in mice. As ALK7 is largely specific for fat cells and is not known to affect the cardiovascular system, drugs that inhibit ALK7 could potentially safely suppress catecholamine resistance and reduce human obesity.
PMCID: PMC4139062  PMID: 25161195
adipose tissue; obesity; TGF-beta; beta-adrenergic; chemical-genetic; lipolysis; human; mouse
14.  Inhibition of Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full T cell receptor signaling 
Nature immunology  2013;15(2):186-194.
T cell receptor (TCR) signaling is initiated by Src-family kinases (SFKs). To understand how C-terminal Src kinase (Csk), the negative regulator of SFKs, controls the basal state and the initiation of TCR signaling, we generated mice expressing a PP1-analog inhibitor-sensitive Csk variant (CskAS). Inhibition of CskAS in thymocytes, without TCR engagement, induced potent SFK activation and proximal TCR signaling up to phospholipase C-γ1 (PLC-γ1). Surprisingly, increases in inositol phosphates (InsP), intracellular calcium and Erk phosphorylation were impaired. Altering the actin cytoskeleton pharmacologically or providing CD28 costimulation rescued these defects. Thus, Csk plays a critical role in preventing TCR signaling. However, our studies also revealed a requirement for actin remodeling, initiated by costimulation, for full TCR signaling.
PMCID: PMC3946925  PMID: 24317039
15.  Feedback circuits monitor and adjust basal Lck-dependent events in T cell antigen receptor signaling 
Science signaling  2011;4(190):ra59.
The Src family kinase Lck is crucial for initiation of T cell antigen receptor (TCR) signaling. Lck is tightly controlled to prevent erroneous immune activation, yet allows rapid responses over a range of sensitivities to antigens. Here, using an analog-sensitive Csk we report that Lck is dynamically controlled by a Csk:CD45-controlled equilibrium in T cells. By rapidly inhibiting Csk, we show that changes in this equilibrium are sufficient for activation of the canonical TCR signaling pathways independent of TCR stimulation. The activated signaling pathways show sustained and marked hyperphosphorylation, revealing a feedback circuit that is sensitive to basal signaling activity and is capable of adapting to changes in basal signal transduction machinery. We identify the inhibitory adaptor molecule Dok-1 as a candidate in the adaptive response to alterations in basal signaling activity. Our results also suggest a novel role for Csk in terminating or dampening of TCR signals.
PMCID: PMC4080844  PMID: 21917715
16.  Chemical-Genetics of Rapamycin-Insensitive TORC2 in S. cerevisiae 
Cell reports  2013;5(6):1725-1736.
Current approaches for identifying synergistic targets use cell culture models with combinations of clinically available drugs to see if the combined effect of the combination is better than predicted by their individual efficacy. New techniques are needed to systematically and rationally identify targets and pathways that have a high potential as synergistic targets. In this study, we create a tool to screen and identify molecular targets that may synergize with new inhibitors of TOR (Target of Rapamycin), a conserved protein that is a major integrator of cell proliferation signals in the nutrient-signaling pathway. While clinical results from TORC1 inhibition using rapamycin analogs (that only inhibit TORC1) have been disappointing, trials using inhibitors that also target TORC2 have been promising. To understand the molecular basis for this increased therapeutic efficacy and to discover secondary targets that may have potential in targeted combination therapy, we engineered TOR2 in S. cerevisiae to accept an orthogonal inhibitor in order to create the first chemical tool to selectively inhibit TORC2. We used this tool to create a Chemical Epistasis Mini-Array Profile, or ChE-MAP, by measuring interactions between the chemically inhibited TOR2 kinase and a diverse library of deletion mutants. The ChE-MAP identified known TOR components and distinguished between TORC1 (assessed using rapamycin) and TORC2 dependent functions. Results showed a novel TORC2-specific interaction with the pentose phosphate pathway (PPP). We used global metabolic profiling to show that that TORC2 inhibition led to decreases in metabolites specific to the PPP and confirmed that TOR2 was regulating this process using metabolic flux analysis. Regulation of the PPP is a previously unappreciated role for TORC2 that may suggest a role for the complex in balancing the high energy demand required for ribosome biogenesis.
PMCID: PMC4007695  PMID: 24360963
17.  A Cdk7-Cdk4 T-Loop Phosphorylation Cascade Promotes G1 Progression 
Molecular cell  2013;50(2):250-260.
Eukaryotic cell division is controlled by cyclin-dependent kinases (CDKs), which require phosphorylation by a CDK-activating kinase (CAK) for full activity. Chemical genetics uncovered requirements for the metazoan CAK Cdk7 in determining cyclin-specificity and activation order of Cdk2 and Cdk1 during S and G2 phases. It was unknown if Cdk7 also activates Cdk4 and Cdk6 to promote passage of the Restriction (R) Point, when continued cell-cycle progression becomes mitogen-independent; or if CDK-activating phosphorylation regulates G1 progression. Here we show that Cdk7 is a Cdk4- and Cdk6-activating kinase in human cells, required to maintain activity, not just to establish the active state, as is the case for Cdk1 and Cdk2. Activating phosphorylation of Cdk7 rises concurrently with that of Cdk4 as cells exit quiescence, and accelerates Cdk4 activation in vitro. Therefore, mitogen signaling drives a CDK-activation cascade during G1 progression, and CAK might be rate-limiting for R-point passage.
PMCID: PMC3677717  PMID: 23622515
18.  Optimizing small molecule inhibitors of calcium-dependent protein kinase 1 to prevent infection by Toxoplasma gondii 
Journal of medicinal chemistry  2013;56(7):3068-3077.
Toxoplasma gondii is sensitive to bulky pyrazolo [3,4-d] pyrimidine (PP) inhibitors due to the presence of a Gly gatekeeper in the essential calcium dependent protein kinase 1 (CDPK1). Here we synthesized a number of new derivatives of 3-methyl-benzyl-PP (3-MB-PP, or 1). The potency of PP analogs in inhibiting CDPK1 enzyme activity in vitro (low nM IC50 values) and blocking parasite growth in host cell monolayers in vitro (low μM EC50 values) were highly correlated and occurred in a CDPK1-specific manner. Chemical modification of the PP scaffold to increase half-life in the presence of microsomes in vitro led to identification of compounds with enhanced stability while retaining activity. Several of these more potent compounds were able to prevent lethal infection with T. gondii in the mouse model. Collectively the strategies outlined here provide a route for development of more effective compounds for treatment of toxoplasmosis, and perhaps related parasitic diseases.
PMCID: PMC3625458  PMID: 23470217
Serine/threonine protein kinase; gatekeeper; calcium signaling; toxoplasmosis; chemotherapy
19.  A neo-substrate that amplifies catalytic activity of Parkinson’s disease related kinase PINK1 
Cell  2013;154(4):737-747.
Mitochondria have long been implicated in the pathogenesis of Parkinson’s disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal recessive form of early onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered since no allosteric regulatory sites for PINK1 are known. Here we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD related mutant PINK1G309D and PINK1wt. Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.
PMCID: PMC3950538  PMID: 23953109
20.  Glucose Sensor O-GlcNAcylation Coordinates with Phosphorylation to Regulate Circadian Clock 
Cell metabolism  2013;17(2):291-302.
Post-translational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3β dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3β. Interestingly, OGT activity is regulated by GSK3β, hence OGT and GSK3β exhibit reciprocal regulation. Modulating OGlcNAcylation levels alter circadian period length in both mice and Drosophila, and conversely protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662–S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine tune circadian clock.
PMCID: PMC3597447  PMID: 23395175
21.  The In Vivo Response of Stem and Other Undifferentiated Spermatogonia to the Reversible Inhibition of GDNF Signaling in the Adult 
Stem cells (Dayton, Ohio)  2012;30(4):732-740.
Maintaining adequate numbers of spermatogonial stem cells is required for the production of the millions of sperm required for male fertility. To date, however, the mechanisms that regulate the size of this pool in the adult are poorly defined. Glial cell line-derived neurotrophic factor (GDNF) is required for establishing this pool in the prepubertal animal, but its in vivo function in the normal adult testis has never been examined directly. We used a chemical-genetic approach to address this issue. We generated mice carrying a single amino acid mutation (V805A) in Ret, the kinase subunit of the GDNF receptor. This mutation does not affect normal GDNF signaling, but renders it susceptible to inhibition by the ATP competitive inhibitor, NA-PP1. When GDNF signaling was blocked in adults for 11 days, only a few cells remained that expressed the stem spermatogonial markers, Gfrα1 and Zbtb16 and testicular Ret mRNA content was reduced markedly. These decreases were associated with depletion of functional stem spermatogonia; some were lost when GDNF signaling was inhibited for only 2 days while others survived for up to 11 days. However, when signaling was restored, the remaining stem cells proliferated, initiating tissue restoration. In conclusion, these results provide the first direct proof that GDNF acutely regulates the numbers of spermatogonial stem cells in the normal adult testis. Additionally, these results demonstrate different sensitivities among subpopulations of these stem cells to inhibition of GDNF signaling.
PMCID: PMC3894909  PMID: 22232066
stem spermatogonia; GDNF; Sertoli cell; Ret; spermatogenesis
22.  Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II 
Nature structural & molecular biology  2012;19(11):1108-1115.
Promoter-proximal pausing by RNA polymerase II (Pol II) ensures both gene-specific regulation and RNA quality control. Structural considerations suggested initiation factor eviction would be required for elongation factor engagement and pausing of transcription complexes. Here we show that selective inhibition of Cdk7—part of TFIIH—increases TFIIE retention, prevents DRB-sensitivity inducing factor (DSIF) recruitment and attenuates pausing in human cells. Pause release depends on Cdk9—cyclin T1 (P-TEFb); Cdk7 is also required for Cdk9-activating phosphorylation and Cdk9-dependent downstream events—Pol II carboxyl-terminal domain Ser2 phosphorylation and histone H2B ubiquitylation—in vivo. Cdk7 inhibition, moreover, impairs Pol II transcript 3′-end formation. Cdk7 thus acts through TFIIE and DSIF to establish and through P-TEFb to relieve barriers to elongation: incoherent feedforward that might create a window to recruit RNA-processing machinery. Therefore, cyclin-dependent kinases govern Pol II handoff from initiation to elongation factors and co-transcriptional RNA maturation.
PMCID: PMC3746743  PMID: 23064645
23.  PTEN expression is consistent in colorectal cancer primaries and metastases and associates with patient survival 
Cancer Medicine  2013;2(4):496-506.
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates the phosphoinositide-3-kinase (PI3K) signaling pathway. In colorectal cancer (CRC), observed frequencies of loss of PTEN expression, concordant expression in primary tumors and metastases, and the association of PTEN status with outcome vary markedly by detection method. We determined the degree to which PTEN expression is consistent in 70 matched human CRC primaries and liver metastases using a validated immunohistochemistry assay. We found loss of PTEN expression in 12.3% of assessable CRC primaries and 10.3% of assessable liver metastases. PTEN expression (positive or negative) was concordant in 98% of matched colorectal primaries and liver metastases. Next we related PTEN status to mutations in RAS and PI3K pathway genes (KRAS, NRAS, BRAF, and PIK3CA) and to overall survival (OS). PTEN expression was not significantly associated with the presence or absence of mutations in RAS or PI3K pathway genes. The median OS of patients whose tumors did not express PTEN was 9 months, compared to 49 months for patients whose tumors did express PTEN (HR = 6.25, 95% confidence intervals (CI) (1.98, 15.42), P = 0.0017). The association of absent PTEN expression with increased risk of death remained significant in multivariate analysis (HR = 6.31, 95% CI (2.03, 17.93), P = 0.0023). In summary, PTEN expression was consistent in matched CRC primaries and in liver metastases. Therefore, future investigations of PTEN in metastatic CRC can use primary tumor tissue. In patients with liver-only metastases, loss of PTEN expression predicted poor OS.
We observed concordant PTEN expression in 98% of colorectal cancer (CRC) primary and liver metastasis pairs using a validated immunohistochemistry assay. Consistent PTEN expression at both disease sites is significant because tumor tissue is usually available from CRC primaries but not metastases. Loss of PTEN expression associated with poor survival of CRC patients with liver-only metastases.
PMCID: PMC3799284  PMID: 24156022
Biomarker; colorectal cancer; concordance; PTEN; survival
24.  A genetically selective inhibitor reveals a ZAP-70 catalytic-independent function in TREG cells 
Nature immunology  2010;11(12):1085-1092.
To investigate the role of the kinase zeta-associated protein of 70 kDa (ZAP-70) in T cells, we generated mice expressing a ZAP-70 mutant whose catalytic activity can be selectively blocked by a small molecule inhibitor. Conventional naïve, effector and memory T cells were dependent on ZAP-70 kinase activity for their activation, demonstrating a non-redundant role for ZAP-70 in TCR-induced signals. In contrast, ZAP-70 catalytic activity was not required for activation of the GTPase Rap1 and inside-out signals that promote integrin adhesion. This ZAP-70 kinase-independent pathway is sufficient for regulatory T (TREG) cell suppressive activity, which was unperturbed by ZAP-70 catalytic inhibition. Our results implicate ZAP-70 as an attractive therapeutic target.
PMCID: PMC3711183  PMID: 21037577
25.  Chemical genetic discovery of targets and anti-targets for cancer polypharmacology 
Nature  2012;486(7401):80-84.
The complexity of cancer has led to recent interest in polypharmacological approaches for developing kinase-inhibitor drugs; however, optimal kinase-inhibition profiles remain difficult to predict. Using a Ret-kinase-driven Drosophila model of multiple endocrine neoplasia type 2 and kinome-wide drug profiling, here we identify that AD57 rescues oncogenic Ret-induced lethality, whereas related Ret inhibitors imparted reduced efficacy and enhanced toxicity. Drosophila genetics and compound profiling defined three pathways accounting for the mechanistic basis of efficacy and dose-limiting toxicity. Inhibition of Ret plus Raf, Src and S6K was required for optimal animal survival, whereas inhibition of the ‘anti-target’ Tor led to toxicity owing to release of negative feedback. Rational synthetic tailoring to eliminate Tor binding afforded AD80 and AD81, compounds featuring balanced pathway inhibition, improved efficacy and low toxicity in Drosophila and mammalian multiple endocrine neoplasia type 2 models. Combining kinase-focused chemistry, kinome-wide profiling and Drosophila genetics provides a powerful systems pharmacology approach towards developing compounds with a maximal therapeutic index.
PMCID: PMC3703503  PMID: 22678283

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