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1.  Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation 
eLife  null;4:e07777.
Intrinsically disordered proteins/regions (IDPs/IDRs) are proteins or peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. In this study, we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.
DOI: http://dx.doi.org/10.7554/eLife.07777.001
eLife digest
DNA contains instructions to make all the proteins and other molecules that drive essential processes in cells. To issue such specific sets of instructions, a section of DNA—called a gene—is first copied to make molecules of messenger ribonucleic acid (or mRNA for short) in a process called transcription. This process is tightly regulated in all living organisms so that only a subset of genes are actively transcribed at any time in a given cell.
A group or ‘complex’ of proteins called TFIID plays an essential role in starting the transcription of genes that encode proteins in humans and other eukaryotic organisms. However, it is tricky to study how TFIID works because mutant cells that are missing individual components of the complex are unable to properly transcribe the required genes and soon die. Consequently, many studies of TFIID have used purified proteins in artificial systems where it is possible to examine particular aspects of TFIID activity in depth.
Here, Zhang et al. used a combination of chemistry, biochemistry, and molecular biology techniques to identify a new molecule that can selectively bind to the TFIID complex. In an artificial system containing purified proteins and other molecules, this molecule ‘locks’ TFIID onto DNA and prevents a change in shape that is required for transcription to start. The experiments show that this rearrangement is only required to make the first mRNA copy of a gene because the molecule had no effect on initiating further rounds of transcription on the same DNA.
Zhang et al.'s findings reveal that TFIID is very dynamic in controlling transcription, and that subsequent rounds of transcription follow a different path to make mRNAs. The next steps are to use new techniques such as single-molecule imaging to directly visualize the molecules involved in transcription, and to use the new molecule to block the start of transcription in living cells.
DOI: http://dx.doi.org/10.7554/eLife.07777.002
doi:10.7554/eLife.07777
PMCID: PMC4582147  PMID: 26314865
transcription factors; intrinsically disordered proteins/regions; metal oxo complexes; preinitiation complex; conformational isomerization; transcription reinitiation; D. melanogaster; human
2.  Indolinyl-Thiazole Based Inhibitors of Scavenger Receptor-BI (SR-BI)-Mediated Lipid Transport 
ACS Medicinal Chemistry Letters  2015;6(4):375-380.
A potent class of indolinyl-thiazole based inhibitors of cellular lipid uptake mediated by scavenger receptor, class B, type I (SR-BI) was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR) in an assay measuring the uptake of the fluorescent lipid DiI from HDL particles. This class of compounds is represented by ML278 (17–11), a potent (average IC50 = 6 nM) and reversible inhibitor of lipid uptake via SR-BI. ML278 is a plasma-stable, noncytotoxic probe that exhibits moderate metabolic stability, thus displaying improved properties for in vitro and in vivo studies. Strikingly, ML278 and previously described inhibitors of lipid transport share the property of increasing the binding of HDL to SR-BI, rather than blocking it, suggesting there may be similarities in their mechanisms of action.
doi:10.1021/ml500154q
PMCID: PMC4599563  PMID: 26478787
ML278; SR-BI inhibitor; HDL receptor; reverse cholesterol transport; indoline; thiazole; HTS; MLP; HCV
3.  The promise and peril of chemical probes 
Nature chemical biology  2015;11(8):536-541.
Chemical probes are powerful reagents with increasing impacts on biomedical research. However, probes of poor quality or that are used incorrectly generate misleading results. To help address these shortcomings, we will create a community-driven wiki resource to improve quality and convey current best practice.
doi:10.1038/nchembio.1867
PMCID: PMC4706458  PMID: 26196764
4.  High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission 
Cell Host & Microbe  2016;19(1):114-126.
Summary
Preventing transmission is an important element of malaria control. However, most of the current available methods to assay for malaria transmission blocking are relatively low throughput and cannot be applied to large chemical libraries. We have developed a high-throughput and cost-effective assay, the Saponin-lysis Sexual Stage Assay (SaLSSA), for identifying small molecules with transmission-blocking capacity. SaLSSA analysis of 13,983 unique compounds uncovered that >90% of well-characterized antimalarials, including endoperoxides and 4-aminoquinolines, as well as compounds active against asexual blood stages, lost most of their killing activity when parasites developed into metabolically quiescent stage V gametocytes. On the other hand, we identified compounds with consistent low nanomolar transmission-blocking activity, some of which showed cross-reactivity against asexual blood and liver stages. The data clearly emphasize substantial physiological differences between sexual and asexual parasites and provide a tool and starting points for the discovery and development of transmission-blocking drugs.
Graphical Abstract
Highlights
•Developed SaLSSA, a serum-free one-step assay for malaria transmission-blocking activity•13,983 known and new compounds analyzed by SaLSSA•>90% known antimalarial drugs do not show activity against late-stage gametocytes•Compounds with consistent low nanomolar transmission-blocking activity identified
Preventing human-mosquito transmission is important for malaria control. Plouffe et al. developed SaLSSA, a one-step high-throughput assay to screen for malaria transmission-blocking activity. A large panel of known and new small molecules was analyzed by SaLSSA. This provides starting points for the discovery and development of transmission-blocking drugs.
doi:10.1016/j.chom.2015.12.001
PMCID: PMC4723716  PMID: 26749441
transmission; malaria; chemotherapy; gametocytes; Plasmodium
5.  High-Throughput Screen in Cryptococcus neoformans Identifies a Novel Molecular Scaffold That Inhibits Cell Wall Integrity Pathway Signaling 
ACS Infectious Diseases  2015;2(1):93-102.
Cryptococcus neoformans is one of the most important human fungal pathogens; however, no new therapies have been developed in over 50 years. Fungicidal activity is crucially important for an effective anticryptococal agent and, therefore, we screened 361,675 molecules against C. neoformans using an adenylate kinase release assay that specifically detects fungicidal activity. A set of secondary assays narrowed the set of hits to molecules that interfere with fungal cell wall integrity and identified three benzothioureas with low in vitro mammalian toxicity and good in vitro anticryptococcal (minimum inhibitory concentration = 4 μg/mL). This scaffold inhibits signaling through the cell wall integrity MAP kinase cascade. Structure–activity studies indicate that the thiocarbonyl moiety is crucial for activity. Genetic and biochemical data suggest that benzothioureas inhibit signaling upstream of the kinase cascade. Thus, the benzothioureas appear to be a promising new scaffold for further exploration in the search for new anticryptococcal agents.
doi:10.1021/acsinfecdis.5b00111
PMCID: PMC4709821  PMID: 26807437
Cryptococcus neoformans; antifungal; cell wall integrity pathway; high-throughput screening; yeast cell wall; mitogen-activated protein kinase
6.  Synthesis of Oxazocenones via Gold(I)-Catalyzed 8-Endo-Dig Hydroalkoxylation of Alkynamides 
Organic Letters  2015;17(3):418-421.
Several benzoxazocenones have been found to exhibit novel cellular activities. In the present study, we report a gold(I)-catalyzed 8-endo-dig hydroalkoxylation reaction of alkynamides to access analogous oxazocenone scaffolds. This methodology provided an advanced intermediate, which was elaborated to a des-benzo analog of a bioactive benzoxazocenone.
doi:10.1021/ol503273v
PMCID: PMC4323038  PMID: 25569027
7.  HDAC6 Inhibitors Modulate Lys49 Acetylation and Membrane Localization of β-Catenin in Human iPSC-Derived Neuronal Cells 
ACS Chemical Biology  2014;10(3):883-890.
We examined the effects of isoform-specific histone deacetylase (HDAC) inhibitors on β-catenin posttranslational modifications in neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs). β-catenin is a multifunctional protein with important roles in the developing and adult central nervous system. Activation of the Wnt pathway results in stabilization and nuclear translocation of β-catenin, resulting in activation of multiple target genes. In addition, β-catenin forms a complex with cadherins at the plasma membrane as part of the adherens junctions. The N-terminus of β-catenin has phosphorylation, ubiquitination, and acetylation sites that regulate its stability and signaling. In the absence of a Wnt signal, Ser33, Ser37, and Thr41 are constitutively phosphorylated by glycogen synthase kinase 3β (GSK3β). β-Catenin phosphorylated at these sites is recognized by β-transducin repeat-containing protein (βTrCP), which results in ubiquitination and degradation by the ubiquitin-proteasome pathway. The N-terminal regulatory domain of β-catenin also includes Ser45, a phosphorylation site for Casein Kinase 1α (CK1α) and Lys49, which is acetylated by the acetyltransferase p300/CBP-associated factor (PCAF). The relevance of Lys49 acetylation and Ser45 phosphorylation to the function of β-catenin is an active area of investigation. We find that HDAC6 inhibitors increase Lys49 acetylation and Ser45 phosphorylation but do not affect Ser33, Ser37, and Thr41 phosphorylation. Lys49 acetylation results in decreased ubiquitination of β-catenin in the presence of proteasome inhibition. While increased Lys49 acetylation does not affect total levels of β-catenin, it results in increased membrane localization of β-catenin.
doi:10.1021/cb500838r
PMCID: PMC4372110  PMID: 25546293
9.  Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity 
PLoS Neglected Tropical Diseases  2015;9(9):e0004094.
Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.
Author Summary
New drugs for visceral leishmaniasis, are urgently required as existing drugs have serious shortcomings including toxicity and drug resistance. This disease is caused by parasites from the Leishmania family which live inside human cells. Screening large collections of chemicals (>100,000) to identify compounds that kill parasites has been used to identify new start points for drug discovery. It is complex and expensive to look at such numbers using intracellular parasites. To circumvent this, many groups screen using parasites adapted to grow outside human cells (axenic forms). However, the established protocols identify growth slowing compounds as well as compounds that kill parasites. Cytocidal compounds are better start points for drug discovery. Here we present a screening cascade based on a modified axenic Leishmania assay adapted to identify compounds that kill parasites. We show that these compounds have a higher probability of being active against intracellular parasites. This new screening cascade was used to screen a compound collection and led to the identification of two new chemical series with antileishmanial activity. Their activity was confirmed against intracellular parasites. They are potential candidates for further drug development. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.
doi:10.1371/journal.pntd.0004094
PMCID: PMC4583543  PMID: 26407168
10.  Diversity-Oriented Synthesis-Facilitated Medicinal Chemistry: Toward the Development of Novel Antimalarial Agents 
Journal of Medicinal Chemistry  2014;57(20):8496-8502.
Here, we describe medicinal chemistry that was accelerated by a diversity-oriented synthesis (DOS) pathway, and in vivo studies of our previously reported macrocyclic antimalarial agent that derived from the synthetic pathway. Structure–activity relationships that focused on both appendage and skeletal features yielded a nanomolar inhibitor of P. falciparum asexual blood-stage growth with improved solubility and microsomal stability and reduced hERG binding. The build/couple/pair (B/C/P) synthetic strategy, used in the preparation of the original screening library, facilitated medicinal chemistry optimization of the antimalarial lead.
doi:10.1021/jm500994n
PMCID: PMC4207553  PMID: 25211597
11.  Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells 
Cell stem cell  2009;4(4):348-358.
Summary
An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives, including lung, liver and pancreas, are of interest for regenerative medicine, but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds, two cell permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm, a higher efficiency than that achieved with Activin A or Nodal, commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers, can participate in normal development when injected into the embryonic gut tube and can form pancreatic progenitors in vitro. The application of small molecules to differentiate mouse and human ESCs into endoderm, and pancreatic progenitors represents a step toward achieving a reproducible and efficient production of desired ES cell derivatives.
doi:10.1016/j.stem.2009.01.014
PMCID: PMC4564293  PMID: 19341624
Chemical screen; embryonic stem cells; endoderm
12.  Linking tumor mutations to drug responses via a quantitative chemical-genetic interaction map 
Cancer discovery  2014;5(2):154-167.
There is an urgent need in oncology to link molecular aberrations in tumors with therapeutics that can be administered in a personalized fashion. One approach identifies synthetic-lethal genetic interactions or dependencies that cancer cells acquire in the presence of specific mutations. Using engineered isogenic cells, we generated a systematic and quantitative chemical-genetic interaction map that charts the influence of 51 aberrant cancer genes on 90 drug responses. The dataset strongly predicts drug responses found in cancer cell line collections, indicating that isogenic cells can model complex cellular contexts. Applied to triple-negative breast cancer, we report clinically actionable interactions with the MYC oncogene including resistance to AKT/PI3K pathway inhibitors and an unexpected sensitivity to dasatinib through LYN inhibition in a synthetic-lethal manner, providing new drug and biomarker pairs for clinical investigation. This scalable approach enables the prediction of drug responses from patient data and can accelerate the development of new genotype-directed therapies.
doi:10.1158/2159-8290.CD-14-0552
PMCID: PMC4407699  PMID: 25501949
systems biology; synthetic lethal; genetic interactions; networks
13.  NAMPT Is the Cellular Target of STF-31-Like Small-Molecule Probes 
ACS Chemical Biology  2014;9(10):2247-2254.
The small-molecule probes STF-31 and its analogue compound 146 were discovered while searching for compounds that kill VHL-deficient renal cell carcinoma cell lines selectively and have been reported to act via direct inhibition of the glucose transporter GLUT1. We profiled the sensitivity of 679 cancer cell lines to STF-31 and found that the pattern of response is tightly correlated with sensitivity to three different inhibitors of nicotinamide phosphoribosyltransferase (NAMPT). We also performed whole-exome next-generation sequencing of compound 146-resistant HCT116 clones and identified a recurrent NAMPT-H191R mutation. Ectopic expression of NAMPT-H191R conferred resistance to both STF-31 and compound 146 in cell lines. We further demonstrated that both STF-31 and compound 146 inhibit the enzymatic activity of NAMPT in a biochemical assay in vitro. Together, our cancer-cell profiling and genomic approaches identify NAMPT inhibition as a critical mechanism by which STF-31-like compounds inhibit cancer cells.
doi:10.1021/cb500347p
PMCID: PMC4201331  PMID: 25058389
14.  Chemical Genetics Identifies Small-Molecule Modulators of Neuritogenesis Involving Neuregulin-1/ErbB4 Signaling 
ACS Chemical Neuroscience  2010;1(4):325-342.
Genetic findings have suggested that neuregulin-1 (Nrg1) and its receptor v-erb-a erythroblastic leukemia viral oncogene homologue 4 (ErbB4) may play a role in neuropsychiatric diseases. However, the downstream signaling events and relevant phenotypic consequences of altered Nrg1 signaling in the nervous system remain poorly understood. To identify small molecules for probing Nrg1−ErbB4 signaling, a PC12-cell model was developed and used to perform a live-cell, image-based screen of the effects of small molecules on Nrg1-induced neuritogenesis. By comparison of the resulting phenotypic data to that of a similar screening performed with nerve growth factor (NGF), this multidimensional screen identified compounds that directly inhibit Nrg1−ErbB4 signaling, such as the 4-anilino-quinazoline Iressa (gefitinib), as well as compounds that potentiate Nrg1−ErbB4 signaling, such as the indolocarbazole K-252a. These findings provide new insights into the regulation of Nrg1−ErbB4 signaling events and demonstrate the feasibility of using such a multidimensional, chemical-genetic approach for discovering probes of pathways implicated in neuropsychiatric diseases.
doi:10.1021/cn900046a
PMCID: PMC2873204  PMID: 20495671
Neuregulin; ErbB4; automated imaging; neuritogenesis; quinazoline; indolocarbazole
15.  The kinase DYRK1A reciprocally regulates the differentiation of Th17 and regulatory T cells 
eLife  null;4:e05920.
The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity.
DOI: http://dx.doi.org/10.7554/eLife.05920.001
eLife digest
Inflammation is used by the immune system to protect and repair tissues after an injury or infection. However, if inflammation is too strong, or goes on for too long, it can damage tissues. This is seen in autoimmune diseases such as inflammatory bowel disease and type 1 diabetes. Therefore, precise regulation of the inflammatory response is essential for maintaining human health.
White blood cells known as T cells are central regulators of tissue inflammation. To achieve this goal, they develop into subtypes with specialized roles. For example, some T helper cells release chemical signals that trigger inflammation and other immune responses. Regulatory T (Treg) cells then shut down these immune responses once they are no longer needed. Many autoimmune and other inflammatory diseases are thought to arise—at least partially—because Treg cells fail to stop the inflammatory response. Boosting the number or the activity of Treg cells could therefore help to treat these diseases. However, technical difficulties have made it difficult to investigate the genes and molecular pathways that control how this subtype of white blood cells develops.
Khor et al. thought that discovering new chemicals that increase the number of Treg cells without harming them could help to identify the pathways that control their development. Khor et al. screened over 3000 chemicals, many of which are drugs currently approved for use in humans, for their effect on immature T cells that were taken from mice and grown in the laboratory. This ‘unbiased chemical biology’ approach identified several chemicals that both encouraged the T cells to develop into Treg cells and reduced the numbers that became inflammation-promoting T helper cells.
Khor et al. then focused on one of these chemicals, called harmine. Tests in mice showed that harmine reduces the extent of experimentally induced inflammatory reactions. Treg cells generated by treating immature T cells with harmine had the same effect. Further experiments showed that harmine exerts these effects, at least in part, by inhibiting the activity of a protein called DYRK1A. When DYRK1A was removed from maturing mouse T cells grown in the laboratory, the T cells tended to develop into anti-inflammatory Treg cells.
These findings therefore identify DYRK1A as part of a pathway that suppresses the development of Treg cells. It remains to be discovered how it does this, and whether other DYRK protein family members have similar roles.
DOI: http://dx.doi.org/10.7554/eLife.05920.002
doi:10.7554/eLife.05920
PMCID: PMC4441007  PMID: 25998054
T cell differentiation; inflammation; dual-specificity tyrosine-regulated kinase signaling; human; mouse
16.  Identification of Inhibitors of PvdQ, an Enzyme Involved in the Synthesis of the Siderophore Pyoverdine 
ACS Chemical Biology  2014;9(7):1536-1544.
Pseudomonas aeruginosa produces the peptide siderophore pyoverdine, which is used to acquire essential Fe3+ ions from the environment. PvdQ, an Ntn hydrolase, is required for the biosynthesis of pyoverdine. PvdQ knockout strains are not infectious in model systems, suggesting that disruption of siderophore production via PvdQ inhibition could be exploited as a target for novel antibacterial agents, by preventing cells from acquiring iron in the low iron environments of most biological settings. We have previously described a high-throughput screen to identify inhibitors of PvdQ that identified inhibitors with IC50 values of ∼100 μM. Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase. ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318. Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.
doi:10.1021/cb5001586
PMCID: PMC4215858  PMID: 24824984
17.  Discovery of a Small-Molecule Probe for V-ATPase Function 
Lysosomes perform a critical cellular function as a site of degradation for diverse cargoes including proteins, organelles, and pathogens delivered through distinct pathways, and defects in lysosomal function have been implicated in a number of diseases. Recent studies have elucidated roles for the lysosome in the regulation of protein synthesis, metabolism, membrane integrity, and other processes involved in homeostasis. Complex small-molecule natural products have greatly contributed to the investigation of lysosomal function in cellular physiology. Here we report the discovery of a novel, small-molecule modulator of lysosomal acidification derived from diversity-oriented synthesis through high-content screening.
doi:10.1021/jacs.5b02150
PMCID: PMC4416280  PMID: 25860544
18.  ChemBank: a small-molecule screening and cheminformatics resource database 
Nucleic Acids Research  2007;36(Database issue):D351-D359.
ChemBank (http://chembank.broad.harvard.edu/) is a public, web-based informatics environment developed through a collaboration between the Chemical Biology Program and Platform at the Broad Institute of Harvard and MIT. This knowledge environment includes freely available data derived from small molecules and small-molecule screens and resources for studying these data. ChemBank is unique among small-molecule databases in its dedication to the storage of raw screening data, its rigorous definition of screening experiments in terms of statistical hypothesis testing, and its metadata-based organization of screening experiments into projects involving collections of related assays. ChemBank stores an increasingly varied set of measurements derived from cells and other biological assay systems treated with small molecules. Analysis tools are available and are continuously being developed that allow the relationships between small molecules, cell measurements, and cell states to be studied. Currently, ChemBank stores information on hundreds of thousands of small molecules and hundreds of biomedically relevant assays that have been performed at the Broad Institute by collaborators from the worldwide research community. The goal of ChemBank is to provide life scientists unfettered access to biomedically relevant data and tools heretofore available primarily in the private sector.
doi:10.1093/nar/gkm843
PMCID: PMC2238881  PMID: 17947324
19.  Diversity-Oriented Synthesis Yields a New Drug Lead for Treatment of Chagas Disease 
ACS Medicinal Chemistry Letters  2013;5(2):149-153.
A phenotypic high-throughput screen using ∼100,000 compounds prepared using Diversity-Oriented Synthesis yielded stereoisomeric compounds with nanomolar growth-inhibition activity against the parasite Trypanosoma cruzi, the etiological agent of Chagas disease. After evaluating stereochemical dependence on solubility, plasma protein binding and microsomal stability, the SSS analogue (5) was chosen for structure–activity relationship studies. The p-phenoxy benzyl group appended to the secondary amine could be replaced with halobenzyl groups without loss in potency. The exocyclic primary alcohol is not needed for activity but the isonicotinamide substructure is required for activity. Most importantly, these compounds are trypanocidal and hence are attractive as drug leads for both acute and chronic stages of Chagas disease. Analogue (5) was nominated as the molecular libraries probe ML341 and is available through the Molecular Libraries Probe Production Centers Network.
doi:10.1021/ml400403u
PMCID: PMC4027639  PMID: 24900788
Trypanosoma cruzi; diversity-oriented synthesis; Chagas disease; neglected disease; high-throughput screening; phenotypic assay; infectious disease; Molecular Libraries Probe Production Centers Network
20.  Regulation of Ferroptotic Cancer Cell Death by GPX4 
Cell  2014;156(0):317-331.
SUMMARY
Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosisinducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.
doi:10.1016/j.cell.2013.12.010
PMCID: PMC4076414  PMID: 24439385
21.  Selective modulation of autophagy, innate immunity and adaptive immunity by small molecules 
ACS chemical biology  2013;8(12):2724-2733.
Autophagy is an evolutionarily conserved catabolic process that directs cytoplasmic proteins, organelles and microbes to lysosomes for degradation. Autophagy acts at the intersection of pathways involved in cellular stress, host defense, and modulation of inflammatory and immune responses; however, the details of how the autophagy network intersects with these processes remain largely undefined. Given the role of autophagy in several human diseases, it is important to determine the extent to which modulators of autophagy also modify inflammatory or immune pathways, and whether it is possible to modulate a subset of these pathways selectively. Here, we identify small-molecule inducers of basal autophagy (including several FDA-approved drugs) and characterize their effects on IL-1β production, autophagic engulfment and killing of intracellular bacteria, and development of Treg, TH17, and TH1 subsets from naïve T cells. Autophagy inducers with distinct, selective activity profiles were identified that reveal the functional architecture of connections between autophagy, and innate and adaptive immunity. In macrophages from mice bearing a conditional deletion of the essential autophagy gene Atg16L1, the small molecules inhibit IL-1β production to varying degrees suggesting that individual compounds may possess both autophagy-dependent and autophagy-independent activity on immune pathways. The small molecule autophagy inducers constitute useful probes to test the contributions of autophagy-related pathways in diseases marked by impaired autophagy or elevated IL-1β, and to test novel therapeutic hypotheses.
doi:10.1021/cb400352d
PMCID: PMC3951132  PMID: 24168452
22.  Atg16l1 is Required for Autophagy in Intestinal Epithelial Cells and Protection of Mice from Salmonella Infection 
Gastroenterology  2013;145(6):10.1053/j.gastro.2013.08.035.
Background & Aims
Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting anti-microbial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice.
Methods
We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1f/f x Villin-cre) or CD11c+ cells (Atg16l1f/f x CD11c-cre); these mice were used to assess cell type-specific, anti-bacterial autophagy. All responses were compared to Atg16l1f/f mice (controls). Mice were infected with Salmonella enterica serovar Typhimurium; cecum and small intestine tissues were collected for immunofluorescence, histology, and quantitative reverse transcription PCR analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on anti-bacterial responses in human epithelial cells.
Results
Autophagy was induced in small intestine and cecum following infection with S Typhimurium, and required Atg16l1. S Typhimurium colocalized with microtubule-associated protein 1 light chain 3 beta (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1f/f x Villin-cre mice. Atg16l1f/f x Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMP. Consistent with these defective immune responses, Atg16l1f/f x Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1f/f x CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1.
Conclusions
Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It is also required to prevent systemic infection of mice with enteric bacteria.
doi:10.1053/j.gastro.2013.08.035
PMCID: PMC3840157  PMID: 23973919
mouse model; autophagy; intestinal barrier; mucosa
23.  Diversity-Oriented Synthesis Probe Targets Plasmodium falciparum Cytochrome b Ubiquinone Reduction Site and Synergizes With Oxidation Site Inhibitors 
The Journal of Infectious Diseases  2014;211(7):1097-1103.
Background. The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways.
Methods. We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds.
Results. We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite.
Conclusions. The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria.
doi:10.1093/infdis/jiu565
PMCID: PMC4354981  PMID: 25336726
cytochrome b; diversity-oriented synthesis; drug development; drug resistance; malaria; target identification
24.  An interactive resource to identify cancer genetic and lineage dependencies targeted by small molecules 
Cell  2013;154(5):1151-1161.
Summary
The high rate of clinical response to protein kinase-targeting drugs matched to cancer patients with specific genomic alterations has prompted efforts to use cancer cell-line (CCL) profiling to identify additional biomarkers of small-molecule sensitivities. We have quantitatively measured the sensitivity of 242 genomically characterized CCLs to an Informer Set of 354 small molecules that target many nodes in cell circuitry, uncovering protein dependencies that: 1) associate with specific cancer-genomic alterations and 2) can be targeted by small molecules. We have created the Cancer Therapeutics Response Portal (www.broadinstitute.org/ctrp) to enable users to correlate genetic features to sensitivity in individual lineages and control for confounding factors of CCL profiling. We report a candidate dependency, associating activating mutations in the oncogene β-catenin with sensitivity to the Bcl2-family antagonist, navitoclax. The resource can be used to develop novel therapeutic hypotheses and accelerate discovery of drugs matched to patients by their cancer genotype and lineage.
doi:10.1016/j.cell.2013.08.003
PMCID: PMC3954635  PMID: 23993102
25.  Stereochemical Control of the Passerini Reaction 
Organic letters  2004;6(23):4231-4233.
A catalytic asymmetric Passerini reaction using tridentate indan (pybox) Cu(II) Lewis acid complex 4 with substrates capable of bidentate coordination has been achieved. The reaction occurs via ligand-accelerated catalysis.
doi:10.1021/ol0482893
PMCID: PMC4134661  PMID: 15524450

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