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Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside homology model guided design, to develop chemical biology approaches for the study of BRD8 inhibition, and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.

Bromodomain‐containing proteins are epigenetic modulators involved in a wide range of cellular processes, from recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl‐lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule‐based approach to covalently modify recombinant and endogenous bromodomain‐containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag allowed in‐gel visualization and pull‐down of the desired bromodomains.

Using a comprehensive chemical genetics approach, we identified a member of the lignan natural product family, HTP-013, which exhibited significant cytotoxicity across various cancer cell lines. Correlation of compound activity across a panel of reporter gene assays suggested the vacuolar-type ATPase (v-ATPase) as a potential target for this compound. Additional cellular studies and a yeast haploinsufficiency screen strongly supported this finding. Competitive photoaffinity labeling experiments demonstrated that the ATP6V0A2 subunit of the v-ATPase complex binds directly to HTP-013, and further mutagenesis library screening identified resistance-conferring mutations in ATP6V0A2. The positions of these mutations suggest the molecule binds a novel pocket within the domain of the v-ATPase complex responsible for proton translocation. While other mechanisms of v-ATPase regulation have been described, such as dissociation of the complex or inhibition by natural products including bafilomycin A1 and concanamycin, this work provides detailed insight into a distinct binding pocket within the v-ATPase complex.

We screened a library of bioactive small molecules for activators and inhibitors of innate immune signaling through IRF3 and NFkB pathways with the goals of advancing pathway understanding and discovering probes for immunology research. We used high content screening to measure the translocation from the cytoplasm to nucleus of IRF3 and NFkB in primary human macrophages; these transcription factors play a critical role in the activation of STING and other pro-inflammatory pathways. Our pathway activator screen yielded a diverse set of hits that promoted nuclear translocation of IRF3 and/or NFkB, but the majority of these compounds did not cause activation of downstream pathways. Screening for antagonists of the STING pathway yielded multiple kinase inhibitors, some of which inhibit kinases not previously known to regulate the activity of this pathway. Structure-activity relationships (SARs) and subsequent chemical proteomics experiments suggested that MAPKAPK5 (PRAK) is a kinase that regulates IRF3 translocation in human macrophages. Our work establishes a high content screening approach for measuring pro-inflammatory pathways in human macrophages and identifies novel ways to inhibit such pathways; among the targets of the screen are several molecules that may merit further development as anti-inflammatory drugs.

The microbial metabolite Chivosazole F has been described to affect the cytoskeleton and to inhibit actin polymerization in vitro. Applying orthogonal genomic and proteomics approaches, we now show for the first time that Chivosazole F exerts its effect by directly interacting with actin and demonstrate the cellular impact of Chivosazole F in an unbiased, genome-wide context in yeast and in mammalian cells. Furthermore, mutation-based resistance mapping identifies two SNPs located in the putative Chivosazole F binding site of actin. Comparing chemogenomic profiles and responses to the Chivosazole F-resistant SNPs shows a partially conserved mechanism of action for Chivosazole F and Latrunculin A, but clear divergence from Chondramide. In addition, C14orf80 is an evolutionarily highly conserved ORF, lacking any functional annotation. As editing of C14orf80 leads to Chivosazole F hyper-resistance, we propose a function for this gene product in counteracting perturbation of actin filaments.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only a few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.

Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity across many types of human cancers; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114 substrate recognition, leading to inhibition of ubiquitination and degradation of the tumor-suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the utility of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-mRNA endonuclease Cleavage and Polyadenylation Specificity Factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a novel synthetic lethal node in a sub-set of acute myeloid leukemia (AML) and Ewing’s sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing’s sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing a new avenue to therapeutic intervention in cancer.

Everyday discrimination is a potent source of stress for racial minorities, and is associated with a wide range of negative health outcomes, spanning both mental and physical health. Few studies have examined the relationships linking race and discrimination to mortality in later life. We examined the longitudinal association among race, everyday discrimination, and all-cause mortality in 12,081 respondents participating in the Health and Retirement Study. Cox proportional hazards models showed that everyday discrimination, but not race, was positively associated with mortality; depressive symptoms and lifestyle factors partially accounted for the relationship between everyday discrimination and mortality; and race did not moderate the association between everyday discrimination and mortality. These findings contribute to a growing body of evidence on the role that discrimination plays in shaping the life chances, resources, and health of people, and, in particular, minority members, who are continuously exposed to unfair treatment in their everyday lives.

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.

This chapter investigates the linkage between race/ethnicity and social relationships. The topic is discussed within the framework of the “racialized life course,” a perspective that argues that personal life pathways are ordered differently across the racial and ethnic groups. The chapter reviews several strands of scholarly literature on the topics of social participation, social capital, and social support that contribute to a theoretical understanding of race/ethnicity and social relationships. The chapter brings data to bear on these issues using nationally-representative survey data from the General Social Survey using measures of social network characteristics, associational memberships, and social participation. Consistent with prior research, the study finds a great deal of racial homophily in social network choices, suggesting that African-Americans are more socially isolated. At the same time, results presented in this chapter with respect to social participation (contact with relatives and members of one’s neighborhood) and associational ties (church membership and attendance) indicate that African-Americans are more likely to activate their social networks. The results indicate that there are racial-ethnic differences in the nature of social relations, and the chapter concludes with a discussion of their significance in understanding the nature of racial inequalities in contemporary American society.

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.

Targeted degradation of proteins through the ubiquitin-proteasome system (UPS) via the activities of E3 ubiquitin ligases regulates diverse cellular processes, and misregulation of these enzymes contributes to the pathogenesis of human diseases. One of the challenges facing the UPS field is to delineate the complete cohort of substrates for a particular E3 ligase. Advances in mass spectrometry and the development of antibodies recognizing the Lys-ϵ-Gly-Gly (diGly) remnant from ubiquitinated proteins following trypsinolysis have provided a tool to address this question. We implemented an inducible loss of function approach in combination with quantitative diGly proteomics to find novel substrates of HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase), an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DNA damage-inducible transcript 4 (DDIT4) as a putative HUWE1 substrate. Cell-based assays demonstrated that HUWE1 interacts with and regulates ubiquitination and stability of DDIT4. Together these data suggest a model in which HUWE1 mediates DDIT4 proteasomal degradation. Our results demonstrate proof of concept that inducible knockdown of an E3 ligase in combination with diGly proteomics provides a potentially advantageous method for identifying novel E3 substrates that may help to identify candidates for therapeutic modulation in the UPS.

The combination of photoaffinity labeling (PAL) and quantitative chemoproteomics enables the comprehensive, unbiased determination of protein interaction profiles to support target identification of bioactive small molecules. This approach is amenable to cells in culture and compatible with pharmacologically relevant transmembrane target classes like G-protein coupled receptors and ions channels which have been notoriously hard to access by conventional chemoproteomics approaches. Here, we describe a strategy that combines PAL probe titration and competition with excess parental compounds with the goal of enabling the identification of specific interactors as well as assessing the functional relevance of a binding event for the phenotype under investigation.

Background Identifying selective kinase inhibitors remains a major challenge. The design of bivalent inhibitors provides a rational strategy for accessing potent and selective inhibitors. While bivalent kinase inhibitors have been successfully designed, no comprehensive assessment of affinity and selectivity for a series of bivalent inhibitors has been performed. Here, we present an evaluation of the structure activity relationship for bivalent kinase inhibitors targeting ABL1. Methods Various SNAPtag constructs bearing different specificity ligands were expressed in vitro. Bivalent inhibitor formation was accomplished by synthesizing individual ATP-competitive kinase inhibitors containing a SNAPtag targeting moiety, enabling the spontaneous self-assembly of the bivalent inhibitor. Assembled bivalent inhibitors were incubated with K562 lysates, and then subjected to affinity enrichment using various ATP-competitive inhibitors immobilized to sepharose beads. Resulting eluents were analyzed using Tandem Mass Tag (TMT) labeling and two-dimensional liquid chromatography-tandem mass spectrometry (2D–LC-MS/MS). Relative binding affinity of the bivalent inhibitor was determined by calculating the concentration at which 50% of a given kinase remained bound to the affinity matrix. Results The profiling of three parental ATP-competitive inhibitors and nine SNAPtag conjugates led to the identification of 349 kinase proteins. In all cases, the bivalent inhibitors exhibited enhanced binding affinity and selectivity for ABL1 when compared to the parental compound conjugated to SNAPtag alone. While the rank order of binding affinity could be predicted by considering the binding affinities of the individual specificity ligands, the resulting affinity of the assembled bivalent inhibitor was not predictable. The results from this study suggest that as the potency of the ATP-competitive ligand increases, the contribution of the specificity ligand towards the overall binding affinity of the bivalent inhibitor decreases. However, the affinity of the specificity components in its interaction with the target is essential for achieving selectivity. Conclusion Through comprehensive chemical proteomic profiling, this work provides the first insight into the influence of ATP-competitive and specificity ligands binding to their intended target on a proteome-wide scale. The resulting data suggest a subtle interplay between the ATP-competitive and specificity ligands that cannot be accounted for by considering the specificity or affinity of the individual components alone. Electronic supplementary material The online version of this article (doi:10.1186/s12953-017-0125-1) contains supplementary material, which is available to authorized users.

Scientific Reports, 7, ISSN:2045-2322

The combination of photoaffinity labeling (PAL) and quantitative chemoproteomics enables the comprehensive, unbiased determination of protein interaction profiles to support target identification of bioactive small molecules. This approach is amenable to cells in culture and compatible with pharmacologically relevant transmembrane target classes like G-protein coupled receptors and ions channels which have been notoriously hard to access by conventional chemoproteomics approaches. Here, we describe a strategy that combines PAL probe titration and competition with excess parental compounds with the goal of enabling the identification of specific interactors as well as assessing the functional relevance of a binding event for the phenotype under investigation.

In recent years, the number of drug candidates with a covalent mechanism of action progressing through clinical trials or being approved by the FDA has increased significantly. And as interest in covalent inhibitors has increased, the technical challenges for characterizing and optimizing these inhibitors have become evident. A number of new tools have been developed to aid this process, but these have not gained wide-spread use. This review will highlight a number of methods and tools useful for prosecuting covalent inhibitor drug discovery programs.

The assessment of the impact that socioeconomic determinants have on the prevalence of certain chronic conditions reported by respondents in population surveys must confront two problems. First, the self-reports could be in error (false positives and false negatives). Second, those reporting are a selected sample of those who ever experience the problem, and this selection is heavily influenced by excess mortality attributable to the condition being reported. In this article, we use a combination of empirical data and microsimulation to (a) assess the magnitude of the bias attributable to the selection problem, and (b) suggest an adjustment procedure that corrects for this bias. We find that the proposed adjustment procedure considerably reduces the bias arising from differential mortality.

The evolution of an HIV epidemic involves many population dynamics that are closely linked with age. Understanding these dynamics can help policy makers and program planners design interventions, measure progress in the fight against HIV, and prepare for the future impact of the disease. To this end, we propose a model for sex- and age-specific projections of HIV prevalence that uses B-splines to model changes in HIV incidence. We are able to combine HIV prevalence data from antenatal clinics with prevalence data from nationally representative surveys to estimate the model parameters. Several approaches are also proposed for generating forecasts from our relatively parsimonious B-spline model. We apply these techniques to investigate the dynamics of HIV incidence in Tanzania. The results support a rapid increase in HIV incidence from the early 1980s to the early 1990s, followed by a decline up through 2003. We also generate forecasts of HIV prevalence for 2007 and 2011, and compare them to nationally representative data from these years. Our assessment of the predictive performance of the approaches suggests that additional information is needed to produce accurate forecasts of sex- and age-specific prevalence, but that B-splines provide a potentially useful approach for achieving this goal.

This chapter explores the integration of life course and developmental approaches to the understanding of cognitive growth, maintenance, and decline over the life span. We employ the idea of event-centered growth models as an essential contribution that at once brings more contextual factors into developmental models of cognitive function (CF) and shows how the life course approach can contribute to an understanding of a phenomenon that is otherwise viewed as a developmental (or aging) process. After introducing the concepts, theories, and methods necessary for understanding the main focus of our attention—namely within-person change in CF—we apply this framework to four major areas where our approach can potentially lead to new insights and understandings: (1) early child cognitive development and the transition to school; (2) the transition to adulthood and midlife, in which CF in adolescence leads to major influences on educational achievement, occupational success and CF in adulthood; (3) the potential for changes in CF during midlife; and (4) transitions in later life (e.g. health events and labor force transitions),and how theoretically CF has a role to play as both consequence and cause of these transitions. The approach we propose can be applied to the study of a wide array of developmental phenomena where life course events and transitions play a role.

BACKGROUND Much of our knowledge of the epidemiology and demography of HIV epidemics in Africais derived from models fit to sparse, non-representative data. These often average over ageand other important dimensions, rarely quantify uncertainty, and typically do not imposeconsistency on the epidemiology and the demography of the population. OBJECTIVE This work conducts an empirical investigation of the history of the HIV epidemic inUganda and Tanzania through the late 1990s, focusing on sex-age-specific incidence,uses those results to produce probabilistic forecasts of HIV prevalence ten years later,and compares those to measures of HIV prevalence at the later time to describe the sexagepattern of changes in prevalence over the intervening period. METHODS We adapt an epidemographic model of a population affected by HIV so that its parameterscan be estimated using both the Bayesian melding with IMIS estimation methodand maximum likelihood methods. Using the Bayesian version of the model we produceprobabilistic forecasts of the population with HIV. RESULTS We produce estimates of sex-age-specific HIV incidence in Uganda and Tanzania in thelate 1990s, produce probabilistic forecasts of the HIV epidemics in Uganda and Tanzaniaduring the early 2000s, describe the sex-age pattern of changes in HIV prevalence inUganda during the early 2000s, and compare the performance and results of the Bayesianand maximum likelihood estimation procedures. CONCLUSIONS We demonstrate that: (1) it is possible to model HIV epidemics in Africa taking accountof sex and age, (2) there are important advantages to the Bayesian estimation method,including rigorous quantification of uncertainty and the ability to make probabilistic forecasts,and (3) that there were important age-specific changes in HIV incidence in Ugandaduring the early 2000s.

The association between childhood family structure and offspring well-being is well-documented. Recent research shows that adult children of divorced parents will likely marry someone whose parents’ divorced (i.e., family structure homogamy) and are subsequently likely to divorce themselves. This literature has focused primarily on marital unions, despite the rise in cohabitation and nonmarital childbearing. Research suggests that marriage and cohabitation are different types of unions and have different implications for the well-being of children. Therefore, we extend the literature by examining the role of family structure homogamy in matching patterns and union stability among unmarried, cohabiting couples. Data from the Fragile Families and Child Wellbeing Study suggest that unmarried, cohabiting mothers and fathers are both more likely to be from nonintact childhood family structures and are significantly more likely to dissolve their unions compared to married parents who both tend to be from intact childhood family structures.

Loss-of-function studies are valuable for elucidating kinase function and the validation of new drug targets. While genetic techniques, such as RNAi and genetic knockouts, are highly specific and easy to implement, in many cases post-translational perturbation of kinase activity, specifically pharmacological inhibition, is preferable. However, due to the high degree of structural similarity between kinase active sites and the large size of the kinome, identification of pharmacological agents that are sufficiently selective to probe the function of a specific kinase of interest is challenging, and there is currently no systematic method for accomplishing this goal. Here, we present a modular chemical genetic strategy that uses antibody mimetics as highly selective targeting components of bivalent kinase inhibitors. We demonstrate that it is possible to confer high kinase selectivity to a promiscuous ATP-competitive inhibitor by tethering it to an antibody mimetic fused to the self-labeling protein SNAPtag. With this approach, a potent bivalent inhibitor of the tyrosine kinase Abl was generated. Profiling in complex cell lysates, with competition-based quantitative chemical proteomics, revealed that this bivalent inhibitor possesses greatly enhanced selectivity for its target, BCR-Abl, in K562 cells. Importantly, we show that both components of the bivalent inhibitor can be assembled in K562 cells to block the ability of BCR-Abl to phosphorylate a direct cellular substrate. Finally, we demonstrate the generality of using antibody mimetics as components of bivalent inhibitors by generating a reagent that is selective for the activated state of the serine/threonine kinase ERK2.

This article presents an extension of the cohort-component model of population projection (CCMPP) first formulated by Heuveline (2003) that is capable of modeling a population affected by HIV. Heuveline proposes a maximum likelihood approach to estimate the age profile of HIV incidence that produced the HIV epidemics in East Africa during the 1990s. We extend this work by developing the Leslie matrix representation of the CCMPP, which greatly facilitates the implementation of the model for parameter estimation and projection. The Leslie matrix also contains information about the stable tendencies of the corresponding population, such as the stable age distribution and time to stability. Another contribution of this work is that we update the sources of data used to estimate the parameters, and use these data to estimate a modified version of the CCMPP that includes (estimated) parameters governing the survival experience of the infected population. A further application of the model to a small population with high HIV prevalence in rural South Africa is presented as an additional demonstration. This work lays the foundation for development of more robust and flexible Bayesian estimation methods that will greatly enhance the utility of this and similar models.

SummaryGDE1 is a mammalian glycerophosphodiesterase (GDE) implicated by in vitro studies in the regulation of glycerophophoinositol (GroPIns) and possibly other glycerophospho (GroP) metabolites. Here, we show using untargeted metabolomics that GroPIns is profoundly (>20-fold) elevated in brain tissue from GDE1(−/−) mice. Furthermore, two additional GroP metabolites not previously identified in eukaryotic cells, glycerophosphoserine (GroPSer) and glycerophosphoglycerate (GroPGate), were also highly elevated in GDE1(−/−) brains. Enzyme assays with synthetic GroP metabolites confirmed that GroPSer and GroPGate are direct substrates of GDE1. Interestingly, our metabolomic profiles also revealed that serine (both L-and D-) levels were significantly reduced in brains of GDE1(−/−) mice. These findings designate GroPSer as a previously unappreciated reservoir for free serine in the nervous system and suggest that GDE1, through recycling serine from GroPSer, may impact D-serine-dependent neural signaling processes in vivo.PaperClip

Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarin A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarin A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarin A binds a dimeric PfAp3Aase and prevents the formation of the enzyme⋅substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarin A in Mycobacterium tuberculosis. Thus, cyclomarin A is a rare example of a natural product with two distinct and specific modes of action.

Decades of research have produced evidence that parental divorce is negatively associated with offspring outcomes from early childhood, through adolescence, and into the adult years. This study adds to the literature on the effects of parental divorce by examining how the timing of a parental divorce influences the total effect on adult health. Furthermore, we look at how this long-term effect of parental divorce depends on mediators such as the family's socioeconomic status, parental involvement, cognitive test scores, behavioural problems, smoking, and the offspring's own experience with divorce. The analyses use data from the National Child Development Study, which includes nine waves of data beginning at birth in 1958 and continuing through age 50. Results from a structural equation model suggest that a parental divorce experienced before age 7 does influence adult health by operating primarily through family socioeconomic status and smoking in adulthood.

Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1.

Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means are often required to determine the biologically relevant target (or targets) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.

A major mechanism for bacterial resistance to antibiotics is through the acquisition of a plasmid coding for resistance-mediating proteins. Described herein is a strategy to eliminate these plasmids from bacteria, thus resensitizing the bacteria to antibiotics. This approach involves mimicking a natural mechanism for plasmid elimination, known as plasmid incompatibility. The compound apramycin was identified as a tight binder to SLI RNA (Kd = 93 nM), the in vivo target of the plasmid incompatibility determinate RNA I, and footprinting/mutagenesis studies indicate apramycin binds SLI in the important regulatory region that dictates plasmid replication control and incompatibility. In vivo studies demonstrate that this compound causes significant plasmid loss and resensitizes bacteria to conventional antibiotics. The demonstration that a small molecule can mimic incompatibility, cause plasmid elimination, and resensitize bacteria to antibiotics opens up new targets for antibacterial research.

Nat. Chem. Biol. 11, 511–517 (2015); published online 1 June 2015; corrected online 15 July 2015 and 11 February 2016 In the version of this article originally published online, the schematic for the construct in Figure 4a was incorrect. A corrected figure has been provided in the HTML and PDF versions of the article.

Elucidation of the molecular forces governing small molecule-RNA binding is paramount to the progress of rational design strategies. The extensive characterization of the aminoglycoside-16S rRNA A-site interaction has deepened our understanding of how aminoglycosides bind to their target and exert their antimicrobial effects. However, to date no other RNA binding compounds have undergone such rigorous evaluation, and in general the origins of small molecule-RNA binding remain a mystery. We recently reported the identification of small molecules, dimers of 2-deoxystreptamine, which are able to bind selectively to RNA tetraloops and octaloops, respectively [Thomas, Liu, and Hergenrother (2005) J. Am. Chem. Soc. 127, 12434-12435]. Described herein is the biochemical and biophysical characterization of the RNA binding properties of the most selective compound, B-12, as well as closely related analogues. These studies further substantiate that B-12 is indeed selective for RNA octaloop sequences and indicate that the origin of this selectivity may lie in B-12's unusual binding mode, in which entropic factors are major contributors to the overall binding energy. In fact, isothermal titration calorimetry (ITC) experiments indicate that the binding of B-12 and most of its analogues is associated with a strong entropic contribution to the total binding energy. This is in stark contrast to the aminoglycosides, for which favorable enthalpy typically provides the driving force for binding. These studies are the first to examine small molecule-RNA hairpin loop binding in detail and are a necessary step toward the design of compounds that are specific binders for a given RNA sequence.

The targeting of one mRNA in the transcriptome requires small molecules that bind with substantial affinity and specificity. As such, compounds with specificity for individual RNA secondary structural motifs could be useful for targeting RNA. Described herein is the synthesis of a combinatorial library of 105 dimers of deoxystreptamine and the subsequent identification of compounds with specificity for specific RNA hairpin loop sizes, including tetraloops and octaloops. Such compounds will be useful for the perturbation of RNA function in vivo.

Bacteria routinely become resistant to antibiotics through the uptake of plasmids that encode resistance-mediating proteins. Such plasmid-based resistance is seen extensively in clinical settings and has been documented for a wide variety of bacterial infections from both Gram-positive and Gram-negative organisms. We recently reported that a small molecule could be used to mimic a natural process of plasmid elimination, called plasmid incompatibility, and that the addition of this compound causes elimination of an IncB plasmid from E. coli and a subsequent resensitization to antibiotics [DeNap, Thomas, Musk, and Hergenrother (2004) J. Am. Chem. Soc. 126, 15402-15404]. Described herein is a further substantiation and validation of the notion that plasmid incompatibility can be mimicked with small molecules that bind to important RNA targets controlling plasmid replication. In this study, the dissociation constant and stoichiometry of RNA binding are determined for 12 aminoglycosides with stem-loop I (SLI) of the IncB replication machinery. Importantly, it is found that compounds that do not bind to this RNA replication control element fail to induce plasmid loss in vivo, whereas those that do bind to the RNA typically cause measurable plasmid loss. These results highlight the potential for targeting key RNA regions for induction of plasmid loss and the subsequent resensitization of bacteria to antibiotics.

RNA is known to have multiple roles in critical cellular functions. Thus, there is great potential for RNA-binding small molecules as both therapeutic agents and cellular probes. Unfortunately, the multiple secondary structures that RNA can adopt have caused difficulty in the development of a general paradigm for RNA-small molecule binding. In particular, the standard RNA-binding compounds such as aminoglycosides do not generally bind RNA hairpin loops, a widespread and vitally important secondary structural motif. In this manuscript we report that dimers of deoxystreptamine bind to RNA hairpin loops with affinities rivaling that of RNA-aminoglycoside interactions.