The clinical success of multitargeted kinase inhibitors has stimulated efforts to identify promiscuous drugs with optimal selectivity profiles. It remains unclear to what extent such drugs can be rationally designed, particularly for combinations of targets that are structurally divergent. Here we report the systematic discovery of molecules that potently inhibit both tyrosine kinases and phosphatidylinositol-3-OH kinases, two protein families that are among the most intensely pursued cancer drug targets. Through iterative chemical synthesis, X-ray crystallography and kinome-level biochemical profiling, we identified compounds that inhibit a spectrum of new target combinations in these two families. Crystal structures revealed that the dual selectivity of these molecules is controlled by a hydrophobic pocket conserved in both enzyme classes and accessible through a rotatable bond in the drug skeleton. We show that one compound, PP121, blocks the proliferation of tumor cells by direct inhibition of oncogenic tyrosine kinases and phosphatidylinositol-3-OH kinases. These molecules demonstrate the feasibility of accessing a chemical space that intersects two families of oncogenes.

Red blood cells (RBC) must coordinate their rate of growth and proliferation with the availability of nutrients, such as iron, but the signaling mechanisms that link the nutritional state to RBC growth are incompletely understood. We performed a screen for cell types that have high levels of signaling through mTORC1, a protein kinase that couples nutrient availability to cell growth. This screen revealed that reticulocytes show high levels of phosphorylated ribosomal protein S6, a downstream target of mTORC1. We found that mTORC1 activity in RBCs is regulated by dietary iron and that genetic activation or inhibition of mTORC1 results in macrocytic or microcytic anemia, respectively. Finally, ATP competitive mTOR inhibitors reduced RBC proliferation and were lethal after treatment with phenylhydrazine, an inducer of hemolysis. These results identify the mTORC1 pathway as a critical regulator of RBC growth and proliferation and establish that perturbations in this pathway result in anemia.

While the SCN controls the circadian clock, further evidence suggests the existence of a food-entrainable oscillator (FEO) that links behavior to changes in food availability such as during restricted feeding (RF). We found that the activity of AgRP/NPY neurons changed rhythmically during RF suggesting that these neurons are a component of the FEO. We next ablated AgRP/NPY neurons in neonates with diphtheria toxin resulting in the loss of ∼50% of AgRP/NPY neurons. Body weight and food intake were unchanged in adult animals after neonatal ablation, as were the responses to leptin treatment, leptin withdrawal, food deprivation and ghrelin treatment. However, ablated animals showed 30% mortality within 4 days of RF. Moreover, the recovery of body weight and food intake in surviving animals lagged behind controls with an absence of food anticipatory activity even after three days. These findings identify AgRP/NPY neurons as a key cellular component of the food-entrained oscillator.

Animals cannot synthesize nine essential amino acids (EAAs) and must therefore obtain them from food. Mice reportedly reject food lacking a single EAA within the first hour of feeding. This remarkable phenomenon is proposed to involve post-ingestive sensing of amino acid imbalance by the protein kinase GCN2 in the brain. Here, we systematically re-examine dietary amino acid sensing in mice. In contrast to previous results, we find that mice cannot rapidly identify threonine- or leucine-deficient food in common feeding paradigms. However, mice attain the ability to identify EAA-deficient food following 2 days of EAA deprivation, suggesting a requirement for physiologic need. In addition, we report that mice can rapidly identify lysine-deficient food without prior EAA deficit, revealing a distinct sensing mechanism for this amino acid. These behaviors are independent of the proposed amino acid sensor GCN2, pointing to the existence of an undescribed mechanism for rapid sensing of dietary EAAs.

Artificial stimulation of Agouti-Related Peptide (AgRP) neurons promotes intense food consumption, yet paradoxically during natural behavior these cells are inhibited before feeding begins. Previously, to reconcile these observations, we showed that brief stimulation of AgRP neurons can generate hunger that persists for tens of minutes, but the mechanisms underlying this sustained hunger drive remain unknown (Chen et al., 2016). Here we show that Neuropeptide Y (NPY) is uniquely required for the long-lasting effects of AgRP neurons on feeding behavior. We blocked the ability of AgRP neurons to signal through AgRP, NPY, or GABA, and then stimulated these cells using a paradigm that mimics their natural regulation. Deletion of NPY, but not AgRP or GABA, abolished optically-stimulated feeding, and this was rescued by NPY re-expression selectively in AgRP neurons. These findings reveal a unique role for NPY in sustaining hunger in the interval between food discovery and consumption.

Energy homeostasis requires precise measurement of the quantity and quality of ingested food. The vagus nerve innervates the gut and can detect diverse interoceptive cues, but the identity of the key sensory neurons and corresponding signals that regulate food intake remains unknown. Here, we use an approach for target-specific, single-cell RNA sequencing to generate a map of the vagal cell types that innervate the gastrointestinal tract. We show that unique molecular markers identify vagal neurons with distinct innervation patterns, sensory endings, and function. Surprisingly, we find that food intake is most sensitive to stimulation of mechanoreceptors in the intestine, whereas nutrient-activated mucosal afferents have no effect. Peripheral manipulations combined with central recordings reveal that intestinal mechanoreceptors, but not other cell types, potently and durably inhibit hunger-promoting AgRP neurons in the hypothalamus. These findings identify a key role for intestinal mechanoreceptors in the regulation of feeding.

Body weight is regulated by interoceptive neural circuits that track energy need, but how the activity of these circuits is altered in obesity remains poorly understood. Here we describe the in vivo dynamics of hunger-promoting AgRP neurons during the development of diet-induced obesity in mice. We show that high-fat diet attenuates the response of AgRP neurons to an array of nutritionally-relevant stimuli including food cues, intragastric nutrients, cholecystokinin and ghrelin. These alterations are specific to dietary fat but not carbohydrate or protein. Subsequent weight loss restores the responsiveness of AgRP neurons to exterosensory cues but fails to rescue their sensitivity to gastrointestinal hormones or nutrients. These findings reveal that obesity triggers broad dysregulation of hypothalamic hunger neurons that is incompletely reversed by weight loss and may contribute to the difficulty of maintaining a reduced weight.

Animals must learn through experience which foods are nutritious and should be consumed, and which are toxic and should be avoided. Enteroendocrine cells (EECs) are the principal chemosensors in the GI tract, but investigation of their role in behavior has been limited by the difficulty of selectively targeting these cells in vivo. Here, we describe an intersectional genetic approach for manipulating EEC subtypes in behaving mice. We show that multiple EEC subtypes inhibit food intake but have different effects on learning. Conditioned flavor preference is driven by release of cholecystokinin whereas conditioned taste aversion is mediated by serotonin and substance P. These positive and negative valence signals are transmitted by vagal and spinal afferents, respectively. These findings establish a cellular basis for how chemosensing in the gut drives learning about food.

p110 alpha (PIK3CA) is the most frequently mutated kinase in human cancer, and numerous drugs targeting this kinase are currently in preclinical development or early-stage clinical trials. Clinical resistance to protein kinase inhibitors frequently results from point mutations that block drug binding; similar mutations in p110 alpha are likely, but currently none have been reported. Using a S. cerevisiae screen against a structurally diverse panel of PI3K inhibitors, we have identified a potential hotspot for resistance mutations (I800), a drug-sensitizing mutation (L814C), and a surprising lack of resistance mutations at the "gatekeeper" residue. Our analysis further reveals that clinical resistance to these drugs may be attenuated by using multitargeted inhibitors that simultaneously inhibit additional PI3K pathway members.

Chemoattractants like f-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent signals that promote the formation of protrusive filamentous actin (F-actin; frontness) and RhoA-dependent actomyosin contraction (backness). Frontness locally inhibits backness and vice versa. In neutrophil-like HL60 cells, blocking phosphatidylinositol-3,4,5-tris-phosphate (PIP3) accumulation with selective inhibitors of PIP3 synthesis completely prevents fMLP from activating a PIP3-dependent kinase and Cdc42 but not from stimulating F-actin accumulation. PIP3-deficient cells show reduced fMLP-dependent Rac activity and unstable pseudopods, which is consistent with the established role of PIP3 as a mediator of positive feedback pathways that augment Rac activation at the front. Surprisingly, such cells also show reduced RhoA activation and RhoA-dependent contraction at the trailing edge, leading to the formation of multiple lateral pseudopods. Cdc42 mediates PIP3's positive effect on RhoA activity. Thus, PIP3 and Cdc42 maintain stable polarity with a single front and a single back not only by strengthening pseudopods but also, at longer range, by promoting RhoA-dependent actomyosin contraction at the trailing edge.

HIV-1 Nef, which is required for the efficient onset of AIDS, enhances viral replication and infectivity by exerting multiple effects on infected cells. Nef downregulates cell-surface MHC-I molecules by an uncharacterized PI3K pathway requiring the actions of two Nef motifs-EEEE(65) and PXXP(75). We report that the Nef EEEE(65) targeting motif enables Nef PXXP(75) to bind and activate a trans-Golgi network-localized Src family tyrosine kinase (SFK). The Nef/SFK complex then recruits and phosphorylates the tyrosine kinase ZAP-70, which binds class I PI3K to trigger MHC-I downregulation in primary CD4+ T cells. In promonocytic cells, Nef/SFK recruits the ZAP-70 homolog Syk to downregulate MHC-I, implicating this PI3K pathway in multiple HIV-1 reservoirs. Isoform-specific PI3K inhibitors repress MHC-I downregulation, identifying them as potential therapeutic agents to combat HIV-1. The discovery of this Nef-SFK-ZAP-70/Syk-PI3K signaling pathway explains the hierarchal role of the Nef motifs in effecting immunoevasion.

Neuroscience relies on techniques for imaging the structure and dynamics of neural circuits, but the cell bodies of individual neurons are often obscured by overlapping fluorescence from axons and dendrites in surrounding neuropil. Here, we describe two strategies for using the ribosome to restrict the expression of fluorescent proteins to the neuronal soma. We show first that a ribosome-tethered nanobody can be used to trap GFP in the cell body, thereby enabling direct visualization of previously undetectable GFP fluorescence. We then design a ribosome-tethered GCaMP for imaging calcium dynamics. We show that this reporter faithfully tracks somatic calcium dynamics in the mouse brain while eliminating cross-talk between neurons caused by contaminating neuropil. In worms, this reporter enables whole-brain imaging with faster kinetics and brighter fluorescence than commonly used nuclear GCaMPs. These two approaches provide a general way to enhance the specificity of imaging in neurobiology.

In humans and other mammalian species, lesions in the preoptic area of the hypothalamus cause profound sleep impairment, indicating a crucial role of the preoptic area in sleep generation. However, the underlying circuit mechanism remains poorly understood. Electrophysiological recordings and c-Fos immunohistochemistry have shown the existence of sleep-active neurons in the preoptic area, especially in the ventrolateral preoptic area and median preoptic nucleus. Pharmacogenetic activation of c-Fos-labelled sleep-active neurons has been shown to induce sleep. However, the sleep-active neurons are spatially intermingled with wake-active neurons, making it difficult to target the sleep neurons specifically for circuit analysis. Here we identify a population of preoptic area sleep neurons on the basis of their projection target and discover their molecular markers. Using a lentivirus expressing channelrhodopsin-2 or a light-activated chloride channel for retrograde labelling, bidirectional optogenetic manipulation, and optrode recording, we show that the preoptic area GABAergic neurons projecting to the tuberomammillary nucleus are both sleep active and sleep promoting. Furthermore, translating ribosome affinity purification and single-cell RNA sequencing identify candidate markers for these neurons, and optogenetic and pharmacogenetic manipulations demonstrate that several peptide markers (cholecystokinin, corticotropin-releasing hormone, and tachykinin 1) label sleep-promoting neurons. Together, these findings provide easy genetic access to sleep-promoting preoptic area neurons and a valuable entry point for dissecting the sleep control circuit.

The brain transforms the need for water into the desire to drink, but how this transformation is performed remains unknown. Here we describe the motivational mechanism by which the forebrain thirst circuit drives drinking. We show that thirst-promoting subfornical organ neurons are negatively reinforcing and that this negative-valence signal is transmitted along projections to the organum vasculosum of the lamina terminalis (OVLT) and median preoptic nucleus (MnPO). We then identify molecularly defined cell types within the OVLT and MnPO that are activated by fluid imbalance and show that stimulation of these neurons is sufficient to drive drinking, cardiovascular responses, and negative reinforcement. Finally, we demonstrate that the thirst signal exits these regions through at least three parallel pathways and show that these projections dissociate the cardiovascular and behavioral responses to fluid imbalance. These findings reveal a distributed thirst circuit that motivates drinking by the common mechanism of drive reduction.

Food and water are rewarding in part because they satisfy our internal needs1,2. Dopaminergic neurons in the ventral tegmental area (VTA) are activated by gustatory rewards3-5, but how animals learn to associate these oral cues with the delayed physiological effects of ingestion is unknown. Here we show that individual dopaminergic neurons in the VTA respond to detection of nutrients or water at specific stages of ingestion. A major subset of dopaminergic neurons tracks changes in systemic hydration that occur tens of minutes after thirsty mice drink water, whereas different dopaminergic neurons respond to nutrients in the gastrointestinal tract. We show that information about fluid balance is transmitted to the VTA by a hypothalamic pathway and then re-routed to downstream circuits that track the oral, gastrointestinal and post-absorptive stages of ingestion. To investigate the function of these signals, we used a paradigm in which a fluid's oral and post-absorptive effects can be independently manipulated and temporally separated. We show that mice rapidly learn to prefer one fluid over another based solely on its rehydrating ability and that this post-ingestive learning is prevented if dopaminergic neurons in the VTA are selectively silenced after consumption. These findings reveal that the midbrain dopamine system contains subsystems that track different modalities and stages of ingestion, on timescales from seconds to tens of minutes, and that this information is used to drive learning about the consequences of ingestion.

Leptin regulates body weight by signaling to the brain the availability of energy stored as fat. This negative feedback loop becomes disrupted in most obese individuals, resulting in a state known as leptin resistance. The physiological causes of leptin resistance remain poorly understood. Here we test the hypothesis that hyperleptinemia is required for the development of leptin resistance in diet-induced obese mice. We show that mice whose plasma leptin has been clamped to lean levels develop obesity in response to a high-fat diet, and the magnitude of this obesity is indistinguishable from wild-type controls. Yet these obese animals with constant low levels of plasma leptin remain highly sensitive to exogenous leptin even after long-term exposure to a high fat diet. This shows that dietary fats alone are insufficient to block the response to leptin. The data also suggest that hyperleptinemia itself can contribute to leptin resistance by downregulating cellular response to leptin as has been shown for other hormones.

The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue.

Thirst motivates animals to drink in order to maintain fluid balance. Thirst has conventionally been viewed as a homeostatic response to changes in blood volume or tonicity. However, most drinking behaviour is regulated too rapidly to be controlled by blood composition directly, and instead seems to anticipate homeostatic imbalances before they arise. How this is achieved remains unknown. Here we reveal an unexpected role for the subfornical organ (SFO) in the anticipatory regulation of thirst in mice. By monitoring deep-brain calcium dynamics, we show that thirst-promoting SFO neurons respond to inputs from the oral cavity during eating and drinking and then integrate these inputs with information about the composition of the blood. This integration allows SFO neurons to predict how ongoing food and water consumption will alter fluid balance in the future and then to adjust behaviour pre-emptively. Complementary optogenetic manipulations show that this anticipatory modulation is necessary for drinking in several contexts. These findings provide a neural mechanism to explain longstanding behavioural observations, including the prevalence of drinking during meals, the rapid satiation of thirst, and the fact that oral cooling is thirst-quenching.

The rewarding taste of food is critical for motivating animals to eat, but whether taste has a parallel function in promoting meal termination is not well understood. Here we show that hunger-promoting AgRP neurons are rapidly inhibited during each bout of ingestion by a signal linked to the taste of food. Blocking these transient dips in activity via closed-loop optogenetic stimulation increases food intake by selectively delaying the onset of satiety. We show that upstream leptin receptor-expressing neurons in the dorsomedial hypothalamus (DMHLepR) are tuned to respond to sweet or fatty tastes and exhibit time-locked activation during feeding that is the mirror image of downstream AgRP cells. These findings reveal an unexpected role for taste in the negative feedback control of ingestion. They also reveal a mechanism by which AgRP neurons, which are the primary cells that drive hunger, are able to influence the moment-by-moment dynamics of food consumption.

The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)-->Akt-->mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.