Structure guided design of a series of sphingosine kinase (SphK) inhibitors.

Sphingosine-1-phosphate (S1P) signaling plays a vital role in mitogenesis, cell migration and angiogenesis. Sphingosine kinases (SphKs) catalyze a key step in sphingomyelin metabolism that leads to the production of S1P. There are two isoforms of SphK and observations made with SphK deficient mice show the two isoforms can compensate for each other's loss. Thus, inhibition of both isoforms is likely required to block SphK dependent angiogenesis. A structure based approach was used to design and synthesize a series of SphK inhibitors resulting in the identification of the first potent inhibitors of both isoforms of human SphK. Additionally, to our knowledge, this series of inhibitors contains the only sufficiently potent inhibitors of murine SphK1 with suitable physico-chemical properties to pharmacologically interrogate the role of SphK1 in rodent models and to reproduce the phenotype of SphK1 (-/-) mice.

Nonclinical Efficacy and Safety of CX-2029, an Anti-CD71 Probody-Drug Conjugate.

Probody therapeutics (Pb-Txs) are conditionally activated antibody-drug conjugates (ADCs) designed to remain inactive until proteolytically activated in the tumor microenvironment, enabling safer targeting of antigens expressed in both tumor and normal tissue. Previous attempts to target CD71, a highly expressed tumor antigen, have failed to establish an acceptable therapeutic window due to widespread normal tissue expression. This study evaluated whether a probody-drug conjugate targeting CD71 can demonstrate a favorable efficacy and tolerability profile in preclinical studies for the treatment of cancer. CX-2029, a Pb-Tx conjugated to maleimido-caproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E, was developed as a novel cancer therapeutic targeting CD71. Preclinical studies were performed to evaluate the efficacy and safety of this anti-CD71 PDC in patient-derived xenograft (PDX) mouse models and cynomolgus monkeys, respectively. CD71 expression was detected at high levels by IHC across a broad range of tumor and normal tissues. In vitro, the masked Pb-Tx form of the anti-CD71 PDC displayed a >50-fold reduced affinity for binding to CD71 on cells compared with protease-activated, unmasked anti-CD71 PDC. Potent in vivo tumor growth inhibition (stasis or regression) was observed in >80% of PDX models (28/34) at 3 or 6 mg/kg. Anti-CD71 PDC remained mostly masked (>80%) in circulation throughout dosing in cynomolgus monkeys at 2, 6, and 12 mg/kg and displayed a 10-fold improvement in tolerability compared with an anti-CD71 ADC, which was lethal. Preclinically, anti-CD71 PDC exhibits a highly efficacious and acceptable safety profile that demonstrates the utility of the Pb-Tx platform to target CD71, an otherwise undruggable target. These data support further clinical development of the anti-CD71 PDC CX-2029 as a novel cancer therapeutic.

The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism.

Glucose homeostasis is regulated systemically by hormones such as insulin and glucagon, and at the cellular level by energy status. Glucagon enhances glucose output from the liver during fasting by stimulating the transcription of gluconeogenic genes via the cyclic AMP-inducible factor CREB (CRE binding protein). When cellular ATP levels are low, however, the energy-sensing kinase AMPK inhibits hepatic gluconeogenesis through an unknown mechanism. Here we show that hormonal and energy-sensing pathways converge on the coactivator TORC2 (transducer of regulated CREB activity 2) to modulate glucose output. Sequestered in the cytoplasm under feeding conditions, TORC2 is dephosphorylated and transported to the nucleus where it enhances CREB-dependent transcription in response to fasting stimuli. Conversely, signals that activate AMPK attenuate the gluconeogenic programme by promoting TORC2 phosphorylation and blocking its nuclear accumulation. Individuals with type 2 diabetes often exhibit fasting hyperglycaemia due to elevated gluconeogenesis; compounds that enhance TORC2 phosphorylation may offer therapeutic benefits in this setting.

Thermodynamic analysis of mRNA cap binding by the human initiation factor eIF4E via free energy perturbations.

Eukaryotic mRNAs are appended at the 5' end, with the 7-methylguanosine cap linked by a 5'-5'-triphosphate bridge to the first transcribed nucleoside (m7GpppX). Initiation of cap-dependent translation of mRNA requires direct interaction between the cap structure and the eukaryotic translation initiation factor eIF4E. Biophysical studies of the association between eIF4E and various cap analogs have demonstrated that m(7)GTP binds to the protein ca. -5.0 kcal/mol more favorably than unmethylated GTP. In this work, a thermodynamic analysis of the binding process between eIF4E and several cap analogs has been conducted using Monte Carlo (MC) simulations in conjunction with free energy perturbation (FEP) calculations. To address the role of the 7-methyl group in the eIF4E/m7GpppX cap interaction, binding free energies have been computed for m(7)GTP, GTP, protonated GTP at N(7), the 7-methyldeazaguanosine 5'-triphosphate (m(7)DTP), and 7-deazaguanosine 5'-triphosphate (DTP) cap analogs. The MC/FEP simulations for the GTP-->m(7)DTP transformation demonstrate that half of the binding free energy gain of m(7)GTP with respect to GTP can be attributed to favorable van der Waals interactions with Trp166 and reduced desolvation penalty due to the N(7) methyl group. The methyl group both eliminates the desolvation penalty of the N(7) atom upon binding and creates a larger cavity within the solvent that further facilitates the desolvation step. Analysis of the pair m(7)GTP-m(7)DTP suggests that the remaining gain in affinity is related to the positive charge created on the guanine moiety due to the N(7) methylation. The charge provides favorable cation-pi interactions with Trp56 and Trp102 and decreases the negative molecular charge, which helps the transfer from the solvent, a more polar environment, to the protein.

Understanding smoking cessation in rural communities.

CONTEXT: Rural communities are adversely impacted by increased rates of tobacco use. Rural residents may be exposed to unique communal norms and other factors that influence smoking cessation. PURPOSE: This study explored facilitating factors and barriers to cessation and the role of rural health care systems in the smoking-cessation process. METHODS: Focus groups were conducted with smokers (N = 63) in 7 Midwestern rural communities. Qualitative analysis and thematic coding of transcripts was conducted. FINDINGS: Three levels of pertinent themes--intrinsic, health-system resource, and community/social factors--were identified. Intrinsic factors facilitating cessation included willingness to try various cessation methods, beliefs about consequences of continuing smoking (eg, smoking-related illnesses), and benefits of quitting (eg, saving money). Intrinsic barriers included skepticism about resources, low self-efficacy and motivation for smoking cessation, concern about negative consequences of quitting (eg, weight gain), and perceived benefits of continued smoking (eg, enjoyment). Key health-system resource facilitators were pharmacotherapy use and physician visits. Resource barriers included infrequent physician visits, lack of medical/financial resources, limited local smoking-cessation programs, and lack of knowledge of existing resources. In terms of community/social factors, participants acknowledged the negative social impact/image of smoking, but also cited a lack of alternative activities, few public restrictions, stressors, and exposure to other smokers as barriers to cessation. CONCLUSIONS: Smokers in rural communities face significant challenges that must be addressed. A multilevel model centered on improving access to health care system resources while addressing intrinsic and community/social factors might enhance smoking-cessation interventions and programs in rural communities.

Kansas primary care weighs in: a pilot randomized trial of a chronic care model program for obesity in 3 rural Kansas primary care practices.

CONTEXT: Obesity is a chronic disease of epidemic proportions in the United States. Primary care providers are critical to timely diagnosis and treatment of obesity, and need better tools to deliver effective obesity care. PURPOSE: To conduct a pilot randomized trial of a chronic care model (CCM) program for obesity care in rural Kansas primary care. METHODS: We enrolled 107 participants to a 6-month, 2-armed, randomized trial comparing a CCM for obesity with usual care. The primary outcome was weight change at 90 days. The usual care arm received educational weight loss materials and outcome assessments at day 0, 90, and 180. The active arm received the same elements as the usual care arm plus a multicomponent obesity CCM. FINDINGS: The Day 90 mean +/- SD weight change for the active arm (n = 34) and control arm (n = 33), respectively, was -4.5 +/- 7.7 pounds and -2.4 +/- 8.1 pounds (P = .27 for difference). The Day 180 mean +/- SD weight change for the active (n = 27) and control (n = 27) arms, respectively, was -9.4 +/- 10.3 pounds and -2.1 +/- 10.7 pounds (P = .01 for difference). There was no significant change in physical activity, or fruit and vegetable intake at day 90 or day 180. CONCLUSIONS: Improving the recognition and treatment of obesity in primary care settings is a critical initiative. Rural populations suffer disproportionately with obesity, and better methods of delivering obesity care are needed for this population. Further research is needed to establish the effectiveness of a CCM approach for obesity care.

Characterization of a high-molecular-weight Notch complex in the nucleus of Notch(ic)-transformed RKE cells and in a human T-cell leukemia cell line.

Notch genes encode a family of transmembrane proteins that are involved in many cellular processes, such as differentiation, proliferation, and apoptosis. It is well established that all four Notch genes can act as oncogenes; however, the mechanism by which Notch proteins transform cells remains unknown. Previously, we reported that both nuclear localization and transcriptional activation are required for neoplastic transformation of RKE cells. Furthermore, we identified cyclin D1 as a direct transcriptional target of constitutively active Notch molecules. In an effort to understand the mechanism by which Notch functions in the nucleus, we sought to determine if Notch formed stable complexes using size exclusion chromatography. Herein, we report that the Notch intracellular domain (N(ic)) forms distinct high-molecular-weight complexes in the nuclei of transformed RKE cells. The largest complex is approximately 1.5 MDa and contains both endogenous CSL (for CBF1, Suppressor of Hairless, and Lag-1) and Mastermind-Like-1 (Maml). N(ic) molecules that do not have the high-affinity binding site for CSL (RAM) retain the ability to associate with CSL in a stable complex through interactions involving Maml. However, Maml does not directly bind to CSL. Furthermore, Maml can rescue Delta RAM transcriptional activity on a CSL-dependent promoter. These results indicate that deletion of the RAM domain does not equate to CSL-independent signaling. Moreover, in SUP-T1 cells, N(ic) exists exclusively in the largest N(ic)-containing complex. SUP-T1 cells are derived from a T-cell leukemia that harbors the t(7;9)(q34;q34.3) translocation and constitutively express N(ic). Taken together, our data indicate that complex formation is likely required for neoplastic transformation by Notch(ic).

Acetylation of Mastermind-like 1 by p300 Drives the Recruitment of NACK to Initiate Notch-Dependent Transcription.

Although it has long been appreciated that p300 acts as a critical Notch coactivator, the mechanistic details of p300 in Notch-mediated transcription remain unclear. We previously demonstrated that PEAK1-related kinase activating pseudokinase 1 (NACK), also known as SGK223, is a critical coactivator of Notch signaling and binds to the Notch1 ternary complex. Herein we report that p300 and CBP acetylate Mastermind-like 1 (Maml1) on amino acid residues K188 and K189 to recruit NACK to the Notch1 ternary complex, which results in the recruitment of RNA polymerase II to initiate transcription. NACK is recruited to the ternary complexes containing Maml1 and Maml3, but not Maml2. Simultaneous inhibition of p300/CBP and Notch has a synergistic effect in esophageal adenocarcinoma. In summary, this study provides a deeper mechanistic understanding of the assembly of the Notch transcriptional complex and provides rationale and proof of concept for a combinatorial therapeutic attack on Notch-dependent cancers. Cancer Res; 77(16); 4228-37. ©2017 AACR.

Strategies for recruiting African-American residents of public housing developments into a randomized controlled trial.

OBJECTIVES: Two community-based strategies used to implement a clinical trial within public housing developments are discussed: 1) hiring and training community outreach residents (CORE) team members to recruit and retain primarily African-American participants; and 2) conducting health fairs to recruit participants into a trial examining the effects of nicotine gum and motivational interviewing on smoking cessation rates. DESIGN: A cluster randomized, community-based clinical trial. SETTING: This trial was conducted in housing developments within a metropolitan area in the Midwest. PARTICIPANTS: Over a period of 20 months, the research team recruited 813 residents, 80% of whom were African-American, to attend health fairs. Of this number, 273 (33%) smokers were identified, and 173 were ultimately enrolled into the study. RESULTS: Attendance at health fairs of public housing development residents ranged from 8%-66% across the housing developments, with an average of 21%. A brief survey was conducted at the health fair to assess smoking status, fruit/vegetable consumption, and physical activity. CONCLUSIONS: A number of possible explanations for the relatively high participation rates among a community-based trial include engaging the community in the research process, offering free health screening services, building recruitment incentives for the CORE, and tailoring health education/promotion materials according to the demographic make-up of the developments. Details regarding the development of recruitment strategies that may boost recruitment rates in community-based clinical trials with predominantly ethnic minorities are provided.

Stage of change movement across three health behaviors: the role of self-efficacy.

PURPOSE: In this study, we examined the influence of self-efficacy in predicting stage of change (SOC) movement, without intervention, over a 1-month period for smoking cessation, exercise adoption, and dietary fat reduction. DESIGN: The design of this study was longitudinal. Patients' stage of change and self-efficacy were assessed at baseline, and stage of change was reassessed at a 1-month follow-up. Patients were categorized as (1) Regressors (moved backward at least one stage), (2) Stables (no change), or (3) Progressors (moved forward at least one stage). Chi-square analyses were used to determine the ability of self-efficacy to predict stage movement at 1-month follow-up. SETTING: The data were collected at a large, inner city, academic hospital in the southeastern United States. Patients were attending primary care clinics. SUBJECTS: Five hundred fifty-four low income, predominantly African-American, individuals attending primary care clinics were participants in the study. MEASURES: Previously validated scales of stage of change and self-efficacy from Prochaska's laboratory were used in this study. RESULTS: Results showed statistically significant differences between predicted and actual SOC movement for smoking cessation, exercise adoption, and dietary fat intake reduction. Baseline self-efficacy ratings were significantly related to stage progression, regression, and stability of stage of change for all three health behaviors. Thirty-seven percent of smokers who were predicted to progress on the basis of their self-efficacy scores progressed. For exercise adoption and dietary fat reduction, 50% and 44%, respectively, of individuals expected to progress at least one stage on the basis of self-efficacy scores progressed. CONCLUSION: Self-efficacy influences SOC movement for smoking cessation, dietary fat reduction, and exercise adoption.

Use of a brief Smoking Consequences Questionnaire for Adults (SCQ-A) in African American smokers.

Purposes of the present study were to (a) examine psychometric properties of a brief Smoking Consequences Questionnaire-Adult (SCQ-A) among an African American sample and (b) explore differences in smoking expectancies across levels of smoking-nicotine dependence. Four hundred eighty-four smokers attending an urban health clinic completed the brief SCQ-A. Maximum likelihood factor extraction with a varimax rotation specifying 9 factors replicated 9 factors of the original SCQ-A. Evidence for the brief SCQ-A's reliability and validity was found. Heavier and/or more dependent smokers had significantly higher scores than lighter and/or less dependent smokers on positive expectancies SCQ-A subscales. Results suggest the brief SCQ-A may be a useful alternative to the full scale SCQ-A. Results also provide evidence for the SCQ-A's validity with African American smokers.

Gender, smoking expectancies, and readiness to quit among urban African American smokers.

The relationship between smoking expectancies and readiness to quit as well as gender differences in expectancies and readiness to quit was examined among 484 urban African American smokers. Univariate analyses revealed that higher positive expectancies were generally associated with less readiness to quit and higher negative expectancies were associated with greater readiness to quit. A multivariable model indicated that stimulation/state enhancement, taste/sensorimotor manipulation, and weight control were most strongly related to intention to quit. Although men and women did not differ on readiness to quit, women reported higher scores on the negative affect reduction subscale than did men.

Applying exercise stage of change to a low-income underserved population.

OBJECTIVES: To validate the transtheoretical model for exercise behavior and the constructs of decisional balance and self-efficacy for exercise in a low-income, poorly educated primary care sample. METHODS: Patients attending public primary-care clinics from 4 separate sites in Louisiana were interviewed regarding their health behaviors. RESULTS: The data provide equivocal support for applying the transtheoretical model for exercise and integrating it with other models of behavior change within this population. CONCLUSIONS: Further studies modifying the decisional balance measures are necessary before definitive statements regarding the applicability of these models to exercise within this specialized population can be made.

Sphingosine kinase activity is not required for tumor cell viability.

Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.

Structure-guided design, synthesis, and evaluation of guanine-derived inhibitors of the eIF4E mRNA-cap interaction.

The eukaryotic initiation factor 4E (eIF4E) plays a central role in the initiation of gene translation and subsequent protein synthesis by binding the 5' terminal mRNA cap structure. We designed and synthesized a series of novel compounds that display potent binding affinity against eIF4E despite their lack of a ribose moiety, phosphate, and positive charge as present in m7-GMP. The biochemical activity of compound 33 is 95 nM for eIF4E in an SPA binding assay. More importantly, the compound has an IC(50) of 2.5 µM for inhibiting cap-dependent mRNA translation in a rabbit reticular cell extract assay (RRL-IVT). This series of potent, truncated analogues could serve as a promising new starting point toward the design of neutral eIF4E inhibitors with physicochemical properties suitable for cellular activity assessment.

Psychometric properties of a Brief Smoking Consequences Questionnaire for Adults (SCQ-A) among African American light smokers.

Despite a decline in cigarette smoking over the past few decades, rates remain unacceptably high for certain segments of the population, such as urban African Americans (AAs). AA smokers, on average, smoke fewer cigarettes per day than European American samples; however, AA smokers are less likely to achieve abstinence during a quit attempt. Outcome expectancies have previously been association with cessation outcomes, but prior research has not examined expectancies among treatment-seeking AA light smokers. The 33-item Smoking Consequences Questionnaire-Adult (SCQ-A) was evaluated among 751 AA light smokers (i.e.,

Seeking help for disaster services after a flood.

OBJECTIVES: This article describes how the frequency of exposure to a flood is associated with the probability of seeking help from agencies (eg, Federal Emergency Management Agency, Red Cross) that provide disaster-related services. The article also describes the population characteristics for the people who are most likely to seek help for disaster services. METHODS: Prospective cohort data from 1735 respondents of the Iowa Health Poll were used. Multivariate logistic regression was used to model the odds of seeking help from any agency for flood-related problems. RESULTS: Overall, most people, regardless of flood exposure, did not seek help from disaster service agencies. Disaster services were sought by 23% of respondents who experienced 1 flood, 31% who have experienced 2 floods, and 26% who have experienced 3 or more floods. Multivariate adjusted odds of seeking help were associated with number of flood experiences (odds ratio [OR] 1.58), white race (OR 0.24), economic hardship (OR 1.43), urban residence (OR 0.43), and social support (OR 0.55). CONCLUSIONS: On average, the probability of seeking disaster relief services increases with the number of flood experiences. Racial/ethnic minorities, rural residents, economically challenged individuals, and people with low levels of perceived social support may be more likely than people without these characteristics to seek services.

Smoking cessation in homeless populations: a pilot clinical trial.

This study, which tested two motivational interviewing treatment approaches, assessed the feasibility of conducting a community-based smoking cessation intervention among homeless smokers. Participants (N = 46) were recruited from multiple facilities in the Kansas City area and were randomized to two counseling conditions in which they received five individual motivational interviewing sessions, six group meetings, and their choice of 8 weeks of 21-mg nicotine patch or 4-mg nicotine lozenge. The two counseling conditions consisted of motivational interviewing targeted either to smoking behaviors exclusively (smoking only) or to smoking and other addictions or life events that could affect ability to quit (smoking plus). Group meetings were designed to provide educational information and social support. Measures of feasibility assessed included the proportion of participants who returned for randomization among those eligible, adherence to prescribed nicotine replacement therapies, retention rates at the week 26 final study visit, and biochemically verified 7-day abstinence at week 26. Most participants (69.6%) chose nicotine patches, and 32% of those participants reported using at least four patches per week. Carbon monoxide verified 7-day abstinence rates in the smoking-only and smoking-plus groups were 13.04% and 17.39% (ns), respectively, at week 8 and 8.70% and 17.39% (ns), respectively, at week 26. Participants who used at least four patches per week were more likely to have quit at 8 weeks than were those who used fewer patches (33.3% vs. 10.5%, p = .30). Results support the feasibility of conducting a smoking cessation intervention among homeless smokers. Findings also show promising effects for nicotine replacement therapy and counseling in this population. Developing programs to improve smoking cessation outcomes in underserved populations is an essential step toward achieving national health objectives and for ultimately reducing tobacco-related health disparities.

Repression of activator protein-1-mediated transcriptional activation by the Notch-1 intracellular domain.

Developmental decisions that control cell fate are commonly regulated by the Notch signaling pathway. Activation of transmembrane Notch receptors results in proteolytic liberation of the intracellular domain of Notch, which translocates into the nucleus, binds a repressor (C promoter binding factor 1/RBP-Jkappa, Su(H), and Lag-1 (CSL)), and induces target genes. We found that the intracellular domain of human Notch-1 (NIC-1) represses activator protein-1 (AP-1)-mediated transactivation. Because numerous genes that control immune and inflammatory responses are AP-1-dependent and Notch regulates immune cell function, we investigated the underlying molecular mechanisms. Repression of AP-1 by NIC-1 did not represent a general inhibitory effect on transcription because nuclear factor kappaB-dependent transcription and transcription driven by a constitutive promoter and enhancer were not affected by NIC-1. The physiological relevance of the repression was supported by the facts that repression was apparent in multiple cell lines, endogenous AP-1 target genes were repressed, and similar concentrations of NIC-1 were required for CSL-dependent activation and AP-1 repression. The RBP-Jkappa-associated molecule domain of NIC-1 that mediates CSL binding and distinct sequences necessary for transactivation were required for repression. However, there was not a strict correlation between the sequence requirements for CSL-dependent activation and AP-1 repression. Repression correlated with predominant nuclear localization of NIC-1 and was not accompanied by disruption of c-Jun amino-terminal kinase-dependent signaling events required for AP-1 activation or by defective AP-1 DNA binding activity. These results provide evidence for negative cross-talk between Notch and AP-1, which may have important consequences for controlling diverse biological processes.

Development of a culturally relevant body image instrument among urban African Americans.

OBJECTIVE: To validate a culturally relevant body image instrument among urban African Americans through three distinct studies. RESEARCH METHODS AND PROCEDURES: In Study 1, 38 medical practitioners performed content validity tests on the instrument. In Study 2, three research staff rated the body image of 283 African-American public housing residents (75% women, mean age = 44 years), with the residents completing body image, BMI, and percentage body fat measures. In Study 3, 35 African Americans (57% men, mean age = 42) completed body image measures and evaluated their cultural relevance. RESULTS: In Study 1, 97% to 100% of practitioners sorted the jumbled figures into the correct ascending order. The correlation between the body image figures and the practitioners' weight classifications of the figures was high (r = 0.91). In Study 2, observers arrived at similar ratings of body size with excellent consistency (alpha = 0.95). Ratings of body image were strongly correlated with participant BMI (r = 0.89 to 0.93 across observers and 0.81 for all participants) and percentage of body fat (r = 0.77 to 0.89 across observers and 0.76 for all participants). In Study 3, body image ratings with the new scale were positively correlated with other validated figural scales. The majority of participants reported that figures in the new body image scale looked most like themselves and other African Americans and were easiest to identify themselves with. DISCUSSION: The instrument displayed strong psychometric performance and cultural relevance, suggesting that the scale is a promising tool for examining body image and obesity among African Americans.