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Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

By analyzing data sets of replicate DNA-Encoded Library (DEL) selections, an approach for estimating the noise level of the experiment has been developed. Using a logarithm transformation of the number of counts associated with each compound and a subset of compounds with the highest number of counts, it is possible to assess the quality of the data through normalizing the replicates and use this same data to estimate the noise in the experiment. The noise level is seen to be dependent on sequencing depth as well as specific selection conditions. The noise estimation is independent of any cutoff used to remove low frequency compounds from the data analysis. The removal of compounds with only 1-5 read counts greatly reduces some of the challenges encountered in DEL data analysis as it can reduce the data set by greater than 100-fold without impacting the interpretation of the results.

The ability to predict chemical structure from DNA sequence has to date been a necessary cornerstone of DNA-encoded library technology. DNA-encoded libraries (DELs) are typically screened by immobilized affinity selection and enriched library members are identified by counting the number of times an individual compound's sequence is observed in the resultant dataset. Those with high signal reads (DEL hits) are subsequently followed up through off-DNA synthesis of the predicted small molecule structures. However, hits followed-up in this manner often fail to translate to confirmed ligands. To address this low conversion rate of DEL hits to off-DNA ligands, we have developed an approach that eliminates the reliance on chemical structure prediction from DNA sequence. Here we describe our method of combining non-combinatorial resynthesis on-DNA following library procedures as a rapid means to assess the probable molecules attached to the DNA barcode. Furthermore, we apply our Bead-Assisted Ligand Isolation Mass Spectrometry (BALI-MS) technique to identify the true binders found within the mixtures of on-DNA synthesis products. Finally, we describe a Normalized Enrichment (NE) metric that allows for the quantitative assessment of affinity selection in these studies. We exemplify how this combined approach enables the identification of putative hit matter against a clinically relevant therapeutic target bisphosphoglycerate mutase, BPGM., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Herein, we report a general and simplified synthesis of fluorophosphonates directly from p-nitrophenylphosphonates. This FP on-demand reaction is mediated by a commercially available polymer-supported fluoride reagent that produces a variety (25 examples) of fluorophosphonates in high yields while only requiring reagent filtration for pure fluorophosphonate isolation. This reaction protocol facilitates the rapid profiling of serine hydrolases with diverse and novel sets of activated phosphonates with differential proteome reactivity. Moreover, slight modification of the procedure into a reaction-to-assay format has enabled additional screening efficiency., (© 2021 Wiley-VCH GmbH.)

Over the past 20 years, the toolbox for discovering small-molecule therapeutic starting points has expanded considerably. Pharmaceutical researchers can now choose from technologies that, in addition to traditional high-throughput knowledge-based and diversity screening, now include the screening of fragment and fragment-like libraries, affinity selection mass spectrometry, and selection against DNA-encoded libraries (DELs). Each of these techniques has its own unique combination of advantages and limitations that makes them more, or less, suitable for different target classes or discovery objectives, such as desired mechanism of action. Layered on top of this are the constraints of the drug-hunters themselves, including budgets, timelines, and available platform capacity; each of these can play a part in dictating the hit identification strategy for a discovery program. In this article, we discuss some of the factors that we use to govern our building of a hit identification roadmap for a program and describe the increasing role that DELs are playing in our discovery strategy. Furthermore, we share our learning during our initial exploration of DEL and highlight the approaches we have evolved to maximize the value returned from DEL selections. Topics addressed include the optimization of library design and production, reagent validation, data analysis, and hit confirmation. We describe how our thinking in these areas has led us to build a DEL platform that has begun to deliver tractable matter to our global discovery portfolio.

DEL selections are binding assays conducted with mixtures of chemically diverse DNA-tagged ligands and a screening target. DEL selections use DNA sequence counts to measure target binding, where ideally higher affinity ligands will have higher counts than weaker affinity ligands. However, there is not always a clear relationship between DNA sequence count (assay signal) and binding affinity. This disconnect may be due to the fidelity of library chemistry, where reactions often do not go to completion, and also to repetitive rounds of binding and elution that are standard practice in most DEL selection experiments. We describe here a strategy that addresses both of these issues and provides a means to calculate ligand affinity from primary selection data. The reaction yields of selected compounds during DEL library synthesis can also be predicted with this method., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020. Published by Elsevier Inc.)

DNA-encoded libraries (DELs) can contain billions of unique chemical species; selecting against such large inputs, it is typical to find more candidate binders than is reasonable to pursue for follow-up synthesis and testing. Given this wealth of choices, common practice is to limit synthesis to only those compounds estimated to have the greatest chance of being high-affinity binders; of the many potential factors contributing to this estimation, the strength of the selection signal of a candidate binder is always important. We define here methods and equations which relate the theoretical selection signal of a compound to its affinity and chemical yield. Tests using known binders of BRD4 and ROCK2 support the theory backing these equations and suggest they should be of use for prospectively determining affinity and chemical yield from primary DEL selection data., Competing Interests: Declaration of competing interest The author declares no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA-encoded chemical library (DECL) synthesis must occur in aqueous media under conditions that preserve the integrity of the DNA encoding tag. While the identification of "DNA-compatible" reaction conditions is critical for the development of DECL designs that explore previously inaccessible chemical space, reports measuring such compatibility have been largely restricted to methods that do not faithfully capture the impact of reaction conditions on DNA fidelity in solution phase. Here we report a comprehensive methodology that uses soluble DNA substrates that exactly recapitulate DNA's exposure to the chemically reactive species of DECL synthesis. This approach includes the assessment of chemical fidelity (reaction yield and purity), encoding fidelity (ligation efficiency), and readability (DNA compatibility), revealing the fate of the DNA tag during DECL chemistry from a single platform.

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists., (© 2018 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society of Clinical Pharmacology and Therapeutics.)

Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log  D, improving solubility, and minimizing chemical lability. Compound 15 achieves excellent CNS exposure, extended 2-AG elevation effect in vivo, and decreased brain inflammatory markers in response to an inflammatory challenge.

Monoacylglycerol lipase (MAGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the CNS. MAGL catalyzes the conversion of 2-AG to arachidonic acid (AA), a precursor to the proinflammatory eicosannoids such as prostaglandins. Herein we describe highly efficient MAGL inhibitors, identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of azetidine and piperidine-derived carbamates. The discovery and optimization of 3-substituted azetidine carbamate irreversible inhibitors of MAGL were aided by the generation of inhibitor-bound MAGL crystal structures. Compound 6, a highly efficient and selective MAGL inhibitor against recombinant enzyme and in a cellular context, was tested in vivo and shown to elevate central 2-AG levels at a 10 mg/kg dose.

C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.

The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is a potent inhibitor of the mRNA decapping scavenger enzyme (DcpS), but the mechanism whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic lipophilic molecule that is predicted to accumulate in lysosomes. To understand if the in vivo efficacy is due to DcpS inhibition or other effects resulting from the physicochemical properties of the chemotype, we undertook structure based molecular design to identify DcpS inhibitors with improved physicochemical properties. Herein we describe the design, synthesis, and in vitro pharmacological characterization of these DcpS inhibitors along with the in vivo mouse CNS PK profile of PF-DcpSi (compound 24), one of the analogs found to be efficacious in SMA mouse model.

We describe a polyacrylamide gel casting cassette that overcomes limitations of commercially available gel electrophoresis equipment. This apparatus molds a single polyacrylamide gel that can evaluate more than 200 samples in parallel, is loaded with a multichannel pipettor, and is flexible with respect to composition of the separating matrix. We demonstrate its use to characterize inhibitors of enzymes that modify protein and nucleic acid substrates. Throughputs of greater than 1000 samples per day were achieved when this system was paired with a quantitative laser-based imaging system, yielding data of remarkable quality.

Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca(2+)-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNA(S100B)) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B-p53 complex and restore active p53 in this deadly cancer. Using a structure-activity relationship by nuclear magnetic resonance approach (SAR by NMR), three persistent binding pockets are found on S100B, termed sites 1-3. While inhibitors that simultaneously bind sites 2 and 3 are in place, no molecules that simultaneously bind all three persistent sites are available. For this purpose, Cys84 was used in this study as a potential means to bridge sites 1 and 2 because it is located in a small crevice between these two deeper pockets on the protein. Using a fluorescence polarization competition assay, several Cys84-modified S100B complexes were identified and examined further. For five such SBiX-S100B complexes, crystallographic structures confirmed their covalent binding to Cys84 near site 2 and thus present straightforward chemical biology strategies for bridging sites 1 and 3. Importantly, one such compound, SC1982, showed an S100B-dependent death response in assays with WM115 malignant melanoma cells, so it will be particularly useful for the design of SBiX molecules with improved affinity and specificity.

4'-Phosphopantetheinyl transferases (PPTases) catalyze a post-translational modification essential to bacterial cell viability and virulence. We present the discovery and medicinal chemistry optimization of 2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides, which exhibit submicromolar inhibition of bacterial Sfp-PPTase with no activity toward the human orthologue. Moreover, compounds within this class possess antibacterial activity in the absence of a rapid cytotoxic response in human cells. An advanced analogue of this series, ML267 (55), was found to attenuate production of an Sfp-PPTase-dependent metabolite when applied to Bacillus subtilis at sublethal doses. Additional testing revealed antibacterial activity against methicillin-resistant Staphylococcus aureus , and chemical genetic studies implicated efflux as a mechanism for resistance in Escherichia coli . Additionally, we highlight the in vitro absorption, distribution, metabolism, and excretion and in vivo pharmacokinetic profiles of compound 55 to further demonstrate the potential utility of this small-molecule inhibitor.

Phosphopantetheinyl transferase (PPTase; E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low-molecular weight CoA substrate to higher-molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of PPTase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human PPTase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict the therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity.

Introduction: Since the first application of antibiotics to treat bacterial infections, the development and spread of resistance has been a persistent threat. An ever evolving pipeline of next-generation therapeutics is required for modern medicine to remain one step ahead of pathogens., Areas Covered: This review describes recent efforts to develop drugs that interrupt the assimilation of iron by bacteria: a process that is vital to cellular homeostasis and is not currently targeted by antibiotics used in the clinic. This review also covers the mechanisms by which bacteria acquire iron for their environment, and details efforts to intervene in these processes, using small molecule inhibitors that target key steps in these pathways, with a special emphasis on recent advances published during the 2010 - 2012 period., Expert Opinion: For decades, the routes used by bacteria to assimilate iron from host and environmental settings have been the subject of intense study. While numerous investigations have identified inhibitors of these pathways, many have stopped short of translating the in vitro results to in vivo proof of concept experiments. The extension of preliminary findings in this manner will significantly increase the impact of the field.

Understanding the mode of action of small molecules is an integral facet of drug discovery. We report an optimized immunoaffinity fluorescent method that allows one to conduct parallel studies at both the cellular and molecular level using a single probe construct. Viability of the method has been evaluated analytically and applied using glycyrrhetic acid as a model.

In high throughput screening (HTS) campaigns, the quality and cost of commercial reagents suitable for pilot studies often create obstacles upon scale-up to a full screen. We faced such challenges in our efforts to implement an HTS for inhibitors of the phosphopantetheinyl transferase Sfp using an assay that had been validated using commercially available reagents. Here we demonstrate a facile route to the synthetic preparation of reactive tetraethylrhodamine and quencher probes, and their application to economically produce fluorescent and quencher-modified substrates. These probes were prepared on a scale that would allow a full, quantitative HTS of more than 350,000 compounds.

Surfactin-type phosphopantetheinyl transferases (Sfp-PPTases) are responsible for modifying type I polyketide and non-ribosomal peptide synthases of prokaryotes and have been implicated in the activation of a variety of pathogen-associated virulence factors. As such, inhibitors of this enzyme class represent enticing leads for antibiotic development and can serve as tools in studies of bacterial metabolism. Currently, no small molecule inhibitors of Sfp-PPTase are known, highlighting the need for efficient methods for PPTase inhibitor identification and development. Herein, we present the design and implementation of a robust and miniaturized high-throughput kinetic assay for inhibitors of Sfp-PPTase using the substrate combination of rhodamine-labeled coenzyme A and Black Hole Quencher-2 labeled consensus acceptor peptide YbbR. Upon PPTase-catalyzed transfer of the rhodamine-labeled phosphopantetheinyl arm onto the acceptor peptide, the fluorescent donor and quencher are covalently joined and the fluorescence signal is reduced. This assay was miniaturized to a low 4 microL volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC(1280)). Top inhibitors identified by the screen were further characterized in secondary assays, including protein phosphopantetheinylation detected by gel electrophoresis. The present assay enables the screening of large compound libraries against Sfp-PPTase in a robust and automated fashion and is applicable to designing assays for related transferase enzymes.

Efforts to isolate carrier protein-mediated synthases from natural product-producing organisms using reporter-linked post-translational modification have been complicated by the efficiency of the endogenous process. To address this issue, we chose to target endogenous phosphopantetheinyl transferases (PPTases) for inhibitor design to facilitate natural product synthase isolation through a chemical genetics approach. Herein we validate secondary metabolism-associated PPTase for chemical probe development. We synthesized and evaluated a panel of compounds based on the anthranilate 4H-oxazol-5-one pharmacophore previously described to attenuate PPTase activity within bacterial cultures. Through the use of a new high-throughput Förster resonance energy transfer assay, we demonstrated that these compounds exclusively inhibit fatty acid synthase-specific PPTases. In vivo, a lead compound within this panel demonstrated selective antibiotic activity in a Bacillus subtilis model. Further evaluation demonstrated that the compound enhances actinorhodin production in Streptomyces coelicolor, revealing the ability of this class of molecules to stimulate precocious secondary metabolite production.

A significant gap exists between genetics-based investigations of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways and our understanding of their regulation, interaction, and activity in living systems. To help bridge this gap, here we present an orthogonal active site identification system (OASIS) for the proteomic identification and analysis of PKS/NRPS biosynthetic enzymes. OASIS probes target conserved features of PKS/NRPS active sites to provide activity-based enrichment of modular synthases, followed by analysis through multidimensional protein identification technology (MudPIT) LC-MS/MS analysis. When applied to the model bacterium Bacillus subtilis, this functional proteomics method detects and quantifies all four modular synthases in the organism. Furthermore, tandem application of multiple OASIS probes enhances identification of specific PKS/NRPS modules from complex proteomic mixtures. By expanding the dynamic range of proteomic analysis for PKS/NRPS enzymes, OASIS offers a valuable tool for strain comparison, culture condition optimization, and enzyme discovery.

(+/-)-Laetirobin (1) was isolated as a cytostatic lead from Laetiporus sulphureus growing parasitically on the black locust tree, Robinia pseudoacacia, by virtue of a reverse-immunoaffinity system. Using an LC/MS procedure, milligram quantities of (+/-)-laetirobin (1) were obtained, and the structure of 1 was elucidated by X-ray crystallography and confirmed by NMR spectroscopy. Preliminary cellular studies indicated that (+/-)-laetirobin (1) rapidly enters in tumor cells, blocks cell division at a late stage of mitosis, and invokes apoptosis.

Phosphopantetheinyl transferase plays an essential role in activating fatty acid, polyketide, and nonribosomal peptide biosynthetic pathways, catalyzing covalent attachment of a 4'-phosphopantetheinyl group to a conserved residue within carrier protein domains. This enzyme has been validated as an essential gene to primary metabolism and presents a target for the identification of antibiotics with a new mode of action. Here we report the development of a homogeneous resonance energy transfer assay using fluorescent coenzyme A derivatives and a surrogate peptide substrate that can serve to identify inhibitors of this enzyme class. This assay lays a blueprint for translation of these techniques to other transferase enzymes that accept fluorescent substrate analogues.

Mycolactone is a polyketide natural product secreted by Mycobacterium ulcerans, the organism responsible for the tropical skin disease Buruli ulcer. The finding that this small molecule virulence factor is sufficient to reconstitute the necrotic pathology associated with Buruli ulcer suggests that a better understanding of mycolactone biosynthesis, particularly the processes which are distinct from those in human metabolism, may provide a unique avenue for the development of selective therapeutics. In the present study we have cloned, expressed, and biochemically characterized the putative macrocycle forming thioesterase for mycolactone, MLSA2 TE. We have evaluated the enzyme both as the truncated thioesterase domain and as a carrier protein-linked didomain construct. The results of these analyses distinguish MLSA2 TE from traditional fatty acid and polyketide synthase TE-domains in terms of its sequence, kinetic parameters, and susceptibility to traditional active-site directed inhibitors. These findings suggest that MLSA2 TE utilizes a unique biochemical mechanism for macrocycle formation.

Although chemical methods to modify proteins in a sequence-specific manner have yet to be developed, site-specific post-translational modification of proteins has recently emerged as a major focus in biological chemistry. Post-translational modification with functionalized substrate analogues opens up several unique avenues to induce selective reactivity into proteins in a sequence-specific manner, and can be applied to protein identification and manipulation in both in vitro and in vivo contexts. Further in vivo applications of this method will enable the imaging of cellular processes, avoiding nonspecific labeling and probe scattering, major complications observed in nonenzymatic methods. Additionally, new tools for in vitro protein modification have been developed that offer more versatile ways to study protein structure and function.

Engineering biosynthetic pathways into suitable host organisms has become an attractive venue for the design, evaluation, and production of small molecule therapeutics. Polyketide (PK) and nonribosomal peptide (NRP) synthases have been of particular interest due to their modular structure, yet routine cloning and expression of these enzymes remains challenging. Here we describe a method to covalently label carrier proteins from PK and NRP synthases using the enzymatic transfer of a modified coenzyme A analog by a 4'-phosphopantetheinyltransferase. Using this method, carrier proteins can be loaded with single fluorescent or affinity reporters, providing novel entry for protein visualization, Western blot identification, and affinity purification. Application of these methods provides an ideal tool to track and quantify metabolically engineered pathways. Such techniques are valuable to measure protein expression, solubility, activity, and native posttranslational modification events in heterologous systems.

Food and fluid deprived and nondeprived male rats showed 36% and 46% decreases, respectively, in sucrose consumption 15-min after injection with 2 mg/kg of naloxone in one hr tests. The magnitude of this decrease was not correlated with an index of naloxone's ability to produce a sickness, as measured by the conditioned taste aversion test. Tests with animals scheduled to drink water in a 15-min daily session showed naloxone had similar effects in reducing water intake in 23-hr and 47-hr water deprived rats. Morphine, when self-administered, produced an increase in water intake during 6-hr sessions. The data support the idea that naloxone disrupts a component of normal regulation of ingestion.

Opiate antagonists were tested for their effects upon either drinking or eating in eight experiments. Naloxone, nalorphine, and the active isomer of WIN 44,441 all reduce drinking. Neither an analog of nalorphine that does not cross the blood-brain barrier, nor the inactive isomer of WIN 44,441 is effective in reducing water intake. These data provide support for the conclusion that these antagonists ahve stereospecific effects within the central nervous system. Naloxone suppresses drinking following procedures inducing osmotic, volemic, or hormonal thirst. Naloxone suppresses eating following procedures inducing glucoprivation but does not alter eating elicited by tail-pressure. Collectively, these data lead to the conclusion that endorphins play a role in the organization of ingestive behavior following challenges to homeostasis.