RpoN-Based stapled peptides with improved DNA binding suppress Pseudomonas aeruginosa virulence.

Stapled peptides have the ability to mimic α-helices involved in protein binding and have proved to be effective pharmacological agents for disrupting protein-protein interactions. DNA-binding proteins such as transcription factors bind their cognate DNA sequences via an α-helix interacting with the major groove of DNA. We previously developed a stapled peptide based on the bacterial alternative sigma factor RpoN capable of binding the RpoN DNA promoter sequence and inhibiting RpoN-mediated expression in Escherichia coli. We have elucidated a structure-activity relationship for DNA binding by this stapled peptide, improving DNA binding affinity constants in the high nM range. Lead peptides were shown to have low toxicity as determined by their low hemolytic activity at 100 µM and were shown to have anti-virulence activity in a Galleria mellonella model of Pseudomonas aeruginosa infection. These findings support further preclinical development of stapled peptides as antivirulence agents targeting P. aeruginosa.

Transcranial Direct Current Stimulation to the Left Dorsolateral Prefrontal Cortex Improves Cognitive Control in Patients With Attention-Deficit/Hyperactivity Disorder: A Randomized Behavioral and Neurophysiological Study.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder associated with significant morbidity and mortality that may affect over 5% of children and approximately 2.8% of adults worldwide. Pharmacological and behavioral therapies for ADHD exist, but critical symptoms such as dysexecutive deficits remain unaffected. In a randomized, sham-controlled, double-blind, crossover mechanistic study, we assessed the cognitive and physiological effects of transcranial direct current stimulation (tDCS) in 40 adult patients with ADHD in order to identify diagnostic (cross-sectional) and treatment biomarkers (targets). METHODS: Patients performed three experimental sessions in which they received 30 minutes of 2 mA anodal tDCS targeting the left dorsolateral prefrontal cortex, 30 minutes of 2 mA anodal tDCS targeting the right dorsolateral prefrontal cortex, and 30 minutes of sham. Before and after each session, half the patients completed the Eriksen flanker task and the other half completed the stop signal task while we assessed behavior (reaction time, accuracy) and neurophysiology (event-related potentials). RESULTS: Anodal tDCS to the left dorsolateral prefrontal cortex modulated cognitive (reaction time) and physiological (P300 amplitude) measures in the Eriksen flanker task in a state-dependent manner, but no effects were found in the stop signal reaction time of the stop signal task. CONCLUSIONS: These findings show procognitive effects in ADHD associated with the modulation of event-related potential signatures of cognitive control, linking target engagement with cognitive benefit, proving the value of event-related potentials as cross-sectional biomarkers of executive performance, and mechanistically supporting the state-dependent nature of tDCS. We interpret these results as an improvement in cognitive control but not action cancellation, supporting the existence of different impulsivity constructs with overlapping but distinct anatomical substrates, and highlighting the implications for the development of individualized therapeutics.

A positive psychology intervention for patients with bipolar depression: a randomized pilot trial.

Background: There are few effective treatments for bipolar depression, a common and debilitating illness.Aims: We aimed to examine the feasibility and preliminary efficacy of a four-week, telephone-delivered positive psychology (PP) intervention for patients with bipolar depression.Methods: Twenty-five patients hospitalized for bipolar depression were randomized to receive a PP (n = 14) or control condition (CC; n = 11) intervention. Following discharge, participants completed weekly exercises and phone calls with a study trainer. PP intervention feasibility was assessed by the number of exercises completed, and acceptability was examined on five-point Likert-type scales of ease and utility. Between-group differences on psychological constructs at 4 and 8 weeks post-enrollment were assessed using mixed effects regression models.Results: Participants in the PP group completed an average of three out of four PP exercises and found PP exercises to be subjectively helpful, though neither easy nor difficult. Compared to CC, the PP intervention led to trends towards greater improvements in positive affect and optimism at follow-up, with large effect sizes (modified Cohen's d = 0.95-1.24). PP had variable, non-significant effects on negative psychological constructs.Conclusions: Larger, randomized trials are needed to further evaluate the efficacy of this intervention in this high-risk population.

Relationships Between Life Stressors, Health Behaviors, and Chronic Medical Conditions in Mid-Life Adults: A Narrative Review.

OBJECTIVES: (1) To explore stressors affecting midlife adults and understand their impact on health behaviors and the development of chronic medical conditions; (2) To identify midlife-specific interventions that mitigate the impact of stressors on the health of this population. METHODS: We searched the PubMed database from inception to December 2017 using the search terms [mid-life] and [midlife] paired with related behaviors and conditions. Eligible articles provided data on: (1) sources of stress and effects on health behaviors, (2) development of chronic medical conditions, or (3) midlife interventions targeting health-related stressors or behaviors. We also reviewed the references of articles found in the initial search to identify additional articles. We included studies not focused on midlife for comparison. RESULTS: This review revealed that interpersonal stress (e.g., caregiving and loneliness), occupational stress, and financial stress are highly prevalent in midlife and have a substantial impact on the health and health behaviors of this population. Many of these stressors converge, intensifying associated distress and health impact. Although not always targeted specifically to this population, interventions focused on diminishing these stressors have showed promising results, particularly group interventions and those focused on positive psychological well-being and mindfulness. CONCLUSION: Midlife is a large and growing population at high risk for chronic medical conditions. Specific stressors during this period are negatively associated with health outcomes. Further research is needed on midlife-specific interventions focused on mitigating these stressors, as such interventions have the potential to improve quality of life and promote health in this significant and vulnerable segment of society.

Does timeframe adjustment of the Life Orientation Test-Revised assess optimism as a state?: Data from the PEACE-III trial in patients with heart disease.

Optimism is prospectively associated with superior health outcomes in cardiac patients, making it an attractive target for well-being interventions in this population. However, optimism measured by the Life Orientation Test-Revised (LOT-R) has largely been considered a static, dispositional construct. Among 125 patients with a recent acute coronary syndrome who received a positive psychology intervention, we assessed the properties of a modified LOT-R that changed the timeframe of items from general dispositional statements to queries about 'right now.' We aimed to learn whether this modified LOT-R was more dynamic than the original LOT-R via administration of both instruments at three timepoints over the 16-week study period. Contrary to our hypothesis, this modified LOT-R showed no greater change in mean score or intra-individual variance than the original LOT-R over 16 weeks. This suggests that simply changing the timeframe of the LOT-R may not facilitate assessment of more state-like optimism in medical patients.

A Positive Psychology Intervention to Promote Health Behaviors in Heart Failure: A Proof-of-Concept Trial.

Patients with heart failure (HF) frequently struggle to adhere to health behaviors, and psychological factors may contribute to nonadherence. We examined the feasibility and acceptability of a 10-week, positive psychology (PP)-based intervention to promote health behavior adherence in patients (N = 10) with mild to moderate HF and suboptimal health behavior adherence. Participants engaged in weekly phone sessions, completed PP exercises (e.g., writing a gratitude letter, using a personal strength), and set goals related to diet, medication adherence, and physical activity. Feasibility was assessed by the number of sessions completed, and acceptability by participant ratings of ease and utility. Preliminary efficacy was measured by changes in psychological and adherence outcomes. The intervention was feasible (87% of exercises completed) and acceptable. Furthermore, in exploratory analyses, the intervention was associated with improvements in psychological and health behavior adherence outcomes. Larger, randomized trials are needed to further investigate the utility of this intervention. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02938052.

Inhibition of Bacterial Gene Transcription with an RpoN-Based Stapled Peptide.

In response to environmental and other stresses, the σ54 subunit of bacterial RNA polymerase (RNAP) controls expression of several genes that play a significant role in the virulence of both plant and animal pathogens. Recruitment of σ54 to RNAP initiates promoter-specific transcription via the double-stranded DNA denaturation mechanism of the cofactor. The RpoN box, a recognition helix found in the C-terminal region of σ54, has been identified as the component necessary for major groove insertion at the -24 position of the promoter. We employed the hydrocarbon stapled peptide methodology to design and synthesize stapled σ54 peptides capable of penetrating Gram-negative bacteria, binding the σ54 promoter, and blocking the interaction between endogenous σ54 and its target DNA sequence, thereby reducing transcription and activation of σ54 response genes.

Cooperative Domain Formation by Homologous Motifs in HOIL-1L and SHARPIN Plays A Crucial Role in LUBAC Stabilization.

The linear ubiquitin chain assembly complex (LUBAC) participates in inflammatory and oncogenic signaling by conjugating linear ubiquitin chains to target proteins. LUBAC consists of the catalytic HOIP subunit and two accessory subunits, HOIL-1L and SHARPIN. Interactions between the ubiquitin-associated (UBA) domains of HOIP and the ubiquitin-like (UBL) domains of two accessory subunits are involved in LUBAC stabilization, but the precise molecular mechanisms underlying the formation of stable trimeric LUBAC remain elusive. We solved the co-crystal structure of the binding regions of the trimeric LUBAC complex and found that LUBAC-tethering motifs (LTMs) located N terminally to the UBL domains of HOIL-1L and SHARPIN heterodimerize and fold into a single globular domain. This interaction is resistant to dissociation and plays a critical role in stabilizing trimeric LUBAC. Inhibition of LTM-mediated HOIL-1L/SHARPIN dimerization profoundly attenuated the function of LUBAC, suggesting LTM as a superior target of LUBAC destabilization for anticancer therapeutics.

Magnitude and sources of distress in mid-life adults with chronic medical illness: an exploratory mixed-methods analysis.

Most mid-life adults have at least one chronic medical condition (CMC) and are at risk for developing additional CMCs. Stressors specific to this life stage may contribute to CMC development by hindering healthy behaviors. The goal of this study was to compare sources and intensity of distress, as they relate to health behaviors, between mid-life and non-mid-life adults with CMCs. We utilized a mixed-methods approach by analyzing quantitative self-report measures of psychiatric symptoms and psychological well-being, with in-depth, semi-structured qualitative interviews to identify sources of stress in three cohorts of patients with CMCs (heart failure, type 2 diabetes, and coronary artery disease). Between-group differences on self-report measures were compared via independent samples t-tests and relevant themes from interview transcripts were compared via chi-square analysis. We found that mid-life participants (n = 30) reported greater psychological distress (depression/anxiety) than non-mid-life (n = 62) participants (Hospital Anxiety and Depression Scale scores 13.8 [SD 7.3] vs. 10.6 [SD 6.6]; t(90)=2.13; p = .035), and qualitative analysis revealed several specific sources of stress significantly more common (p < .001) in mid-life adults. Interventions targeting the needs of this population could reduce distress, improve health behaviors, and have a major impact on public health.

Biophysical and biological evaluation of optimized stapled peptide inhibitors of the linear ubiquitin chain assembly complex (LUBAC).

Linear ubiquitylation, in which ubiquitin units are covalently linked through N- and C-terminal amino acids, is a unique cellular signaling mechanism. This process is controlled by a single E3 ubiquitin ligase, the linear ubiquitin chain assembly complex (LUBAC), which is composed of three proteins - HOIL-1L, HOIP and SHARPIN. LUBAC is involved in the activation of the canonical NF-κB pathway and has been linked to NF-κB dependent malignancies. In this work, we present HOIP-based stapled alpha-helical peptides designed to inhibit LUBAC through the disruption of the HOIL-1L-HOIP interaction and loss of the functional complex. We find our HOIP peptides to be active LUBAC ubiquitylation inhibitors in vitro, though through interaction with HOIP rather than HOIL. Active peptides were shown to have inhibitory effects on cell viability, reduced NF-κB activity and decreased production of NF-κB related gene products. This work further demonstrates the potential of LUBAC as a therapeutic target and of the use of stapled peptides as inhibitors of protein-protein interactions.

Alternative treatment strategies for catatonia: A systematic review.

BACKGROUND: Catatonia is a commonly encountered syndrome, affecting 10-20% of various psychiatric populations and carrying significant medical co-morbidities. However, there are few established alternative treatment strategies when benzodiazepines are ineffective and electroconvulsive therapy is unavailable. OBJECTIVE: The authors systematically review evidence for alternative treatment strategies for catatonia using Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. METHOD: The authors conducted a search of PubMed database from 1983 to August 2016 to identify articles. Eligible reports presented cases involving treatment of catatonia using modalities other than benzodiazepines or electroconvulsive therapy. RESULTS: The authors identified 72 articles, comprising 98 individual cases. N-methyl-d-aspartate-receptor antagonists, anti-epileptic drugs, and atypical antipsychotic agents appeared to have the largest number of reports supporting their effectiveness and safety in treating catatonia patients. CONCLUSIONS: Based on the case report literature, the authors propose an updated algorithm for catatonia treatment in cases where benzodiazepines fail and electroconvulsive therapy is not available.

Challenges in Targeting a Basic Helix-Loop-Helix Transcription Factor with Hydrocarbon-Stapled Peptides.

Basic helix-loop-helix (bHLH) transcription factors play critical roles in organism development and disease by regulating cell proliferation and differentiation. Transcriptional activity, whether by bHLH homo- or heterodimerization, is dependent on protein-protein and protein-DNA interactions mediated by α-helices. Thus, α-helical decoys have been proposed as potential targeted therapies for pathologic bHLH transcription. Here, we developed a library of stabilized α-helices of OLIG2 (SAH-OLIG2) to test the capacity of hydrocarbon-stapled peptides to disrupt OLIG2 homodimerization, which drives the development and chemoresistance of glioblastoma multiforme, one of the deadliest forms of human brain cancer. Although stapling successfully reinforced the α-helical structure of bHLH constructs of varying length, sequence-specific dissociation of OLIG2 dimers from DNA was not achieved. Re-evaluation of the binding determinants for OLIG2 self-association and stability revealed an unanticipated role of the C-terminal domain. These data highlight potential pitfalls in peptide-based targeting of bHLH transcription factors given the liabilities of their positively charged amino acid sequences and multifactorial binding determinants.

Synthesis of stabilized alpha-helical peptides.

Stabilized alpha-helical (SAH) peptides are valuable laboratory tools to explore important protein-protein interactions. Whereas most peptides lose their secondary structure when isolated from the host protein, stapled peptides incorporate an all-hydrocarbon "staple" that reinforces their natural alpha-helical structure. Thus, stapled peptides retain their functional ability to bind their native protein targets and serve multiple experimental uses. First, they are useful for structural studies such as NMR or crystal structures that map and better define binding sites. Second, they can be used to identify small molecules that specifically target that interaction site. Third, stapled peptides can be used to test the importance of specific amino acid residues or posttranslational modifications to the binding. Fourth, they can serve as structurally competent bait to identify novel binding partners to specific alpha-helical motifs. In addition to markedly improved alpha-helicity, stapled peptides also display resistance to protease cleavage and enhanced cell permeability. Most importantly, they are useful for intracellular experiments that explore the functional consequences of blocking particular protein interactions. Because of their remarkable stability, stapled peptides can be applied to whole-animal, in vivo studies. Here we describe a protocol for the synthesis of a peptide that incorporates an all-hydrocarbon "staple" employing a ring-closing olefin metathesis reaction. With proper optimization, stapled peptides can be a fundamental, accurate laboratory tool in the modern chemical biologist's armory.

Essential role of the linear ubiquitin chain assembly complex in lymphoma revealed by rare germline polymorphisms.

UNLABELLED: Constitutive activation of NF-κB is a hallmark of the activated B cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), owing to upstream signals from the B-cell receptor (BCR) and MYD88 pathways. The linear polyubiquitin chain assembly complex (LUBAC) attaches linear polyubiquitin chains to IκB kinase-γ, a necessary event in some pathways that engage NF-κB. Two germline polymorphisms affecting the LUBAC subunit RNF31 are rare among healthy individuals (∼1%) but enriched in ABC DLBCL (7.8%). These polymorphisms alter RNF31 α-helices that mediate binding to the LUBAC subunit RBCK1, thereby increasing RNF31-RBCK1 association, LUBAC enzymatic activity, and NF-κB engagement. In the BCR pathway, LUBAC associates with the CARD11-MALT1-BCL10 adapter complex and is required for ABC DLBCL viability. A stapled RNF31 α-helical peptide based on the ABC DLBCL-associated Q622L polymorphism inhibited RNF31-RBCK1 binding, decreased NF-κB activation, and killed ABC DLBCL cells, credentialing this protein-protein interface as a therapeutic target. SIGNIFICANCE: We provide genetic, biochemical, and functional evidence that the LUBAC ubiquitin ligase is a therapeutic target in ABC DLBCL, the DLBCL subtype that is most refractory to current therapy. More generally, our findings highlight the role of rare germline-encoded protein variants in cancer pathogenesis.

High levels of Hdmx promote cell growth in a subset of uveal melanomas.

The p53 tumor suppressor pathway is inactivated in cancer either via direct mutation or via deregulation of upstream regulators or downstream effectors. P53 mutations are rare in uveal melanoma. Here we investigated the role of the p53 inhibitor Hdmx in uveal melanoma. We found Hdmx over-expression in a subset of uveal melanoma cell lines and fresh-frozen tumor samples. Hdmx depletion resulted in cell-line dependent growth inhibition, apparently correlating with differential Hdm2 levels. Surprisingly, p53 knockdown hardly rescued cell cycle arrest and apoptosis induction upon Hdmx knockdown, whereas it effectively prevented growth suppression induced by the potent p53 activator Nutlin-3. In addition, two compounds inhibiting Hdmx function or expression, SAH-p53-8 and XI-011, also elicited a growth inhibitory effect in a partly p53-independent manner. These findings suggest a novel, growth-promoting function of Hdmx that does not rely on its ability to inhibit p53. We provide evidence for a contribution of p27 protein induction to the observed p53-independent G1 arrest in response to Hdmx knockdown. In conclusion, our study establishes the importance of Hdmx as an oncogene in a subset of uveal melanomas and widens the spectrum of its function beyond p53 inhibition.

MDM4 is a key therapeutic target in cutaneous melanoma.

The inactivation of the p53 tumor suppressor pathway, which often occurs through mutations in TP53 (encoding tumor protein 53) is a common step in human cancer. However, in melanoma-a highly chemotherapy-resistant disease-TP53 mutations are rare, raising the possibility that this cancer uses alternative ways to overcome p53-mediated tumor suppression. Here we show that Mdm4 p53 binding protein homolog (MDM4), a negative regulator of p53, is upregulated in a substantial proportion (∼65%) of stage I-IV human melanomas and that melanocyte-specific Mdm4 overexpression enhanced tumorigenesis in a mouse model of melanoma induced by the oncogene Nras. MDM4 promotes the survival of human metastatic melanoma by antagonizing p53 proapoptotic function. Notably, inhibition of the MDM4-p53 interaction restored p53 function in melanoma cells, resulting in increased sensitivity to cytotoxic chemotherapy and to inhibitors of the BRAF (V600E) oncogene. Our results identify MDM4 as a key determinant of impaired p53 function in human melanoma and designate MDM4 as a promising target for antimelanoma combination therapy.

A stapled p53 helix overcomes HDMX-mediated suppression of p53.

Cancer cells neutralize p53 by deletion, mutation, proteasomal degradation, or sequestration to achieve a pathologic survival advantage. Targeting the E3 ubiquitin ligase HDM2 can lead to a therapeutic surge in p53 levels. However, the efficacy of HDM2 inhibition can be compromised by overexpression of HDMX, an HDM2 homolog that binds and sequesters p53. Here, we report that a stapled p53 helix preferentially targets HDMX, blocks the formation of inhibitory p53-HDMX complexes, induces p53-dependent transcriptional upregulation, and thereby overcomes HDMX-mediated cancer resistance in vitro and in vivo. Importantly, our analysis of p53 interaction dynamics provides a blueprint for reactivating the p53 pathway in cancer by matching HDM2, HDMX, or dual inhibitors to the appropriate cellular context.

Synthesis and biophysical characterization of stabilized alpha-helices of BCL-2 domains.

Rational design of compounds to mimic the functional domains of BCL-2 family proteins requires chemical reproduction of the biologic complexity afforded by the relatively large and folded surfaces of BCL-2 homology (BH) domain peptide alpha-helices. Because the intermolecular handshakes of BCL-2 proteins are so critical to controlling cellular fate, we undertook the development of a toolbox of peptidic ligands that harness the natural potency and specificity of BH alpha-helices to interrogate and potentially medicate the deregulated apoptotic pathways of human disease. To overcome the classic deficiencies of peptide reagents, including loss of bioactive structure in solution, rapid proteolytic degradation in vivo, and cellular impermeability, we developed a new class of compounds based on hydrocarbon stapling of BH3 death domain peptides. Here we describe the chemical synthesis of Stabilized Alpha-Helices of BCL-2 domains or SAHBs, and the analytical methods used to characterize their secondary structure, proteolytic stability, and cellular penetrance.

Dissection of the BCL-2 family signaling network with stabilized alpha-helices of BCL-2 domains.

The BCL-2 family of apoptotic proteins regulates the critical balance between cellular life and death and, thus, has become the focus of intensive basic science inquiry and a fundamental target for therapeutic development in oncology and other diseases. Classified based on the presence of conserved alpha-helical motifs and pro- and anti-apoptotic functionalities, BCL-2 proteins participate in a complex interaction network that determines cellular fate. The identification of BCL-2 homology domain 3 (BH3) as a critical death helix that engages and regulates BCL-2 family proteins has inspired the development of molecular tools to decode and drug the interaction network. Stabilized Alpha-Helices of BCL-2 domains (SAHBs) are structurally reinforced, protease-resistant, and cell-permeable compounds that retain the specificity of native BH3 death ligands and, therefore, serve as ideal reagents to dissect BCL-2 family interactions in vitro and in vivo. Here, we describe the in vitro and cell-based methods that exploit SAHB compounds to determine the functional consequences of BH3 interactions in regulating apoptosis.

Exploration of treatment options for the management of stage I testicular seminoma.

Seminoma is the epitome of a highly treatable neoplastic malignancy. Approximately 80% of patients presenting with seminomatous germ cell tumors are diagnosed with stage I disease. Men with clinical stage I seminomas have an excellent chance of achieving a cure irrespective of the treatment option selected. With such high disease-specific survival rates, attention has turned to reducing treatment-related morbidity. Adjuvant radiotherapy to the para-aortic retroperitoneal lymph nodes in conjunction with orchidectomy has been the standard treatment since the mid-1990s. There is some evidence, however, suggesting potential deleterious long-term sequelae from radiation treatment. Adjuvant chemotherapy has gained support as an acceptable adjuvant treatment strategy for stage I seminoma. While long-term studies are limited, short-term data regarding the therapeutic efficacy of single-agent carboplatin are promising. Finally, surveillance following orchidectomy is an attractive option for motivated patients interested in avoiding immediate adjuvant therapy. It may be the optimal choice for compliant men who are able to handle the mental burden of not receiving active treatment.