Virus-like particles displaying the mature C-terminal domain of filamentous hemagglutinin are immunogenic and protective against Bordetella pertussis respiratory infection in mice.

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.

Evaluating the efficacy of a premedication regimen including high-dose cetirizine in reduction of hypersensitivity reactions to paclitaxel: A retrospective cohort study.

INTRODUCTION: Hypersensitivity reactions (HSR) are a known adverse effect of paclitaxel, occurring in approximately 10% of patients, typically during the first or second infusion of the medication. Corticosteroids, histamine-1 and histamine-2 receptor antagonists are given prior to paclitaxel infusions to reduce the incidence of HSR. There are limited data that suggest administration of cetirizine given prior to a platinum infusion as secondary prophylaxis may reduce HSR rates. METHODS: The objective of this study was to assess the impact of a novel paclitaxel hypersensitivity prevention protocol including high-dose cetirizine administered 12 and 6 h prior to paclitaxel infusion on the rate of HSR compared to a historical control. The primary objective was the rate of HSR of any grade after the first cycle of paclitaxel. Secondary outcomes included grade of infusion reaction and incidence of severe HSR. RESULTS: A total of 104 patients were included for analysis in the cetirizine group and 124 in the control group. Hypersensitivity reactions occurred in 37 (16.2%) patients in the overall population, and no statistical difference was observed between groups. (13.46% vs 18.55%; p = 0.23). Numerically more grade 3-4 HSRs occurred in the control group than the treatment group (30.77% vs 69.23; p = 0.51). CONCLUSIONS: The addition of cetirizine to paclitaxel infusions resulted in numerically lower rates of HSR and a reduction in severity of grade 3-4 HSRs. Future studies with more robust compliance data and a larger patient population would be needed to appropriately assess the efficacy of our novel treatment regimen.

Short-term exposure of female BALB/cJ mice to e-cigarette aerosol promotes neutrophil recruitment and enhances neutrophil-platelet aggregation in pulmonary microvasculature.

Despite the perception that e-cigarettes are safer than conventional cigarettes, numerous findings demonstrated that e-cigarette aerosol (EC) exposure induced compromised immune functionality, vascular changes even after acute exposure, and lung injury. Notably, altered neutrophil functionality and platelet hemodynamics have been observed post-EC exposure. It was hypothesized that EC exposure initiates an inflammatory response resulting in altered neutrophil behavior and increased neutrophil-platelet interaction in the pulmonary microvasculature. Neutrophil and platelet responses were examined up to 48 hrs following whole-body, short-term EC exposure without flavorants or nicotine in a murine model, which most closely modeled secondhand exposure. This study is the first to investigate the impact of EC exposure through lung intravital imaging. Compared to room air-exposed mice, EC-exposed mice displayed significantly increased 1.7‒1.9-fold number of neutrophils in the pulmonary microvasculature associated with no marked change in neutrophils within whole blood or bronchoalveolar lavage fluid (BALF). Neutrophil-platelet interactions were also significantly elevated 1.9‒2.5-fold in exposed mice. Plasma concentration of myeloperoxidase was markedly reduced 1.5-fold 48 hr following exposure cessation, suggesting suppressed neutrophil antimicrobial activity. Cytokine expression exhibited changes indicating vascular damage. Effects persisted for 48 hr post-EC exposure. Data demonstrated that EC exposure repeated for 3 consecutive days in 2.5 hr intervals in the absence of flavorants or nicotine resulted in modified pulmonary vasculature hemodynamics, altered immune functionality, and a pro-inflammatory state in female BALB/cJ mice.

Defining the Mechanistic Correlates of Protection Conferred by Whole-Cell Vaccination against Pseudomonas aeruginosa Acute Murine Pneumonia.

Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe pulmonary infections associated with high morbidity and mortality in immunocompromised patients. The development of a vaccine against P. aeruginosa could help prevent infections caused by this highly antibiotic-resistant microorganism. We propose that identifying the vaccine-induced correlates of protection against P. aeruginosa will facilitate the development of a vaccine against this pathogen. In this study, we investigated the mechanistic correlates of protection of a curdlan-adjuvanted P. aeruginosa whole-cell vaccine (WCV) delivered intranasally. The WCV significantly decreased bacterial loads in the respiratory tract after intranasal P. aeruginosa challenge and raised antigen-specific antibody titers. To study the role of B and T cells during vaccination, anti-CD4, -CD8, and -CD20 depletions were performed prior to WCV vaccination and boosting. The depletion of CD4+, CD8+, or CD20+ cells had no impact on the bacterial burden in mock-vaccinated animals. However, depletion of CD20+ B cells, but not CD8+ or CD4+ T cells, led to the loss of vaccine-mediated bacterial clearance. Also, passive immunization with serum from WCV group mice alone protected naive mice against P. aeruginosa, supporting the role of antibodies in clearing P. aeruginosa We observed that in the absence of T cell-dependent antibody production, mice vaccinated with the WCV were still able to reduce bacterial loads. Our results collectively highlight the importance of the humoral immune response for protection against P. aeruginosa and suggest that the production of T cell-independent antibodies may be sufficient for bacterial clearance induced by whole-cell P. aeruginosa vaccination.

Intranasal Immunization with Acellular Pertussis Vaccines Results in Long-Term Immunity to Bordetella pertussis in Mice.

Bordetella pertussis colonizes the respiratory mucosa of humans, inducing an immune response seeded in the respiratory tract. An individual, once convalescent, exhibits long-term immunity to the pathogen. Current acellular pertussis (aP) vaccines do not induce the long-term immune response observed after natural infection in humans. In this study, we evaluated the durability of protection from intranasal (i.n.) pertussis vaccines in mice. Mice that convalesced from B. pertussis infection served as a control group. Mice were immunized with a mock vaccine (phosphate-buffered saline [PBS]), aP only, or an aP base vaccine combined with one of the following adjuvants: alum, curdlan, or purified whole glucan particles (IRI-1501). We utilized two study designs: short term (challenged 35 days after priming vaccination) and long term (challenged 6 months after boost). The short-term study demonstrated that immunization with i.n. vaccine candidates decreased the bacterial burden in the respiratory tract, reduced markers of inflammation, and induced significant serum and lung antibody titers. In the long-term study, protection from bacterial challenge mirrored the results observed in the short-term challenge study. Immunization with pertussis antigens alone was surprisingly protective in both models; however, the alum and IRI-1501 adjuvants induced significant B. pertussis-specific IgG antibodies in both the serum and lung and increased numbers of anti-B. pertussis IgG-secreting plasma cells in the bone marrow. Our data indicate that humoral responses induced by the i.n. vaccines correlated with protection, suggesting that long-term antibody responses can be protective.

Antibody-mediated immunological memory correlates with long-term Lyme veterinary vaccine protection in mice.

Lyme disease, caused by the bacterium Borrelia burgdorferi, is the most common tick-borne illness in the United States. Despite the rise in Lyme disease incidence, there is no vaccine against B. burgdorferi approved for human use. Little is known about the immune correlates of protection needed to prevent Lyme disease. In this work, a mouse model was used to characterize the immune response and compare the protection provided by two USDA-approved vaccines for use in canines: Duramune (bacterin vaccine) and Vanguard crLyme (subunit vaccine composed of two outer surface proteins, OspA and OspC). C3H/HeNCrl mice were immunized with two doses of either Duramune or Vanguard, and immune responses and protection against B. burgdorferi were assessed in short (35 days) and long-term (120 days) studies. Flow cytometry, ELISPOT detection of antibody-producing cells, and antibody affinity studies were performed to identify correlates of vaccine-mediated protection. Both vaccines induced humoral responses, with high IgG titers against B. burgdorferi. However, the levels of anti-B. burgdorferi antibodies decayed over time in Vanguard-vaccinated mice. While both vaccines triggered the production of antibodies against both OspA and OspC, antibody levels against these proteins were also lower in Vanguard-vaccinated mice 120 days post-vaccination. Both vaccines only provided partial protection against B. burgdorferi at the dose used in this model. The protection provided by Duramune was superior to Vanguard 120 days post-vaccination, and was characterized by higher antibody titers, higher abundance of long-lived plasma cells, and higher avidity antibodies than Vanguard. Overall, these studies provide insights into the importance of the humoral memory response to veterinary vaccines against Lyme disease and will help inform the development of future human vaccines.

Impact of botanical oils on polyunsaturated fatty acid metabolism and leukotriene generation in mild asthmatics.

BACKGROUND: Dietary supplementation with botanical oils that contain n-6 and n-3 eighteen carbon chain (18C)-PUFA such as γ linolenic acid (GLA, 18:3n-6), stearidonic acid (SDA, 18:4n-3) and α linolenic acid (ALA, 18:3n-3) have been shown to impact PUFA metabolism, alter inflammatory processes including arachidonic acid (AA) metabolism and improve inflammatory disorders. METHODS: The diet of mild asthmatics patients was supplemented for three weeks with varying doses of two botanical seed oils (borage oil [Borago officinalis, BO] and echium seed oil [Echium plantagineum; EO]) that contain SDA, ALA and GLA. A three week wash out period followed. The impact of these dietary manipulations was evaluated for several biochemical endpoints, including in vivo PUFA metabolism and ex vivo leukotriene generation from stimulated leukocytes. RESULTS: Supplementation with several EO/BO combinations increased circulating 20-22 carbon (20-22C) PUFAs, including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and dihommo-gammalinolenic acid (DGLA), which have been shown to inhibit AA metabolism and inflammation without impacting circulating AA levels. BO/EO combinations also inhibited ex vivo leukotriene generation with some combinations attenuating cysteinyl leukotriene generation in stimulated basophils by >50% and in stimulated neutrophils by >35%. CONCLUSIONS: This study shows that dietary supplementation with BO/EO alters 20-22C PUFA levels and attenuates leukotriene production in a manner consistent with a reduction in inflammation.

Protocol for aerosolization challenge of mice with Bordetella pertussis.

Bordetella pertussis causes whooping cough and is transmitted via respiratory droplets. Here, we present a protocol to challenge mice with Bordetella pertussis. We describe bacteria preparation and long-term storage, followed by manufacturing a challenge dose for use in a commercial exposure chamber with controlled nebulization of B. pertussis into aerosols. We then detail the aerosol challenge of mice through a more natural administration than intranasal instillation and post-challenge data collection. This protocol allows for better comparisons between preclinical pertussis studies.

Intranasal COVID-19 vaccine induces respiratory memory T cells and protects K18-hACE mice against SARS-CoV-2 infection.

Current COVID-19 vaccines prevent severe disease, but do not induce mucosal immunity or prevent infection with SARS-CoV-2, especially with recent variants. Furthermore, serum antibody responses wane soon after immunization. We assessed the immunogenicity and protective efficacy of an experimental COVID-19 vaccine based on the SARS-CoV-2 Spike trimer formulated with a novel adjuvant LP-GMP, comprising TLR2 and STING agonists. We demonstrated that immunization of mice twice by the intranasal (i.n.) route or by heterologous intramuscular (i.m.) prime and i.n. boost with the Spike-LP-GMP vaccine generated potent Spike-specific IgG, IgA and tissue-resident memory (TRM) T cells in the lungs and nasal mucosa that persisted for at least 3 months. Furthermore, Spike-LP-GMP vaccine delivered by i.n./i.n., i.m./i.n., or i.m./i.m. routes protected human ACE-2 transgenic mice against respiratory infection and COVID-19-like disease following lethal challenge with ancestral or Delta strains of SARS-CoV-2. Our findings underscore the potential for nasal vaccines in preventing infection with SARS-CoV-2 and other respiratory pathogen.

Optimization of Aspergillus versicolor Culture and Aerosolization in a Murine Model of Inhalational Fungal Exposure.

Aspergillus versicolor is ubiquitous in the environment and is particularly abundant in damp indoor spaces. Exposure to Aspergillus species, as well as other environmental fungi, has been linked to respiratory health outcomes, including asthma, allergy, and even local or disseminated infection. However, the pulmonary immunological mechanisms associated with repeated exposure to A. versicolor have remained relatively uncharacterized. Here, A. versicolor was cultured and desiccated on rice then placed in an acoustical generator system to achieve aerosolization. Mice were challenged with titrated doses of aerosolized conidia to examine deposition, lymphoproliferative properties, and immunotoxicological response to repeated inhalation exposures. The necessary dose to induce lymphoproliferation was identified, but not infection-like pathology. Further, it was determined that the dose was able to initiate localized immune responses. The data presented in this study demonstrate an optimized and reproducible method for delivering A. versicolor conidia to rodents via nose-only inhalation. Additionally, the feasibility of a long-term repeated exposure study was established. This experimental protocol can be used in future studies to investigate the physiological effects of repeated pulmonary exposure to fungal conidia utilizing a practical and relevant mode of delivery. In total, these data constitute an important foundation for subsequent research in the field.

Intranasal challenge with B. pertussis leads to more severe disease manifestations in mice than aerosol challenge.

The murine Bordetella pertussis challenge model has been utilized in preclinical research for decades. Currently, inconsistent methodologies are employed by researchers across the globe, making it difficult to compare findings. The objective of this work was to utilize the CD-1 mouse model with two routes of challenge, intranasal and aerosol administration of B. pertussis, to understand the differences in disease manifestation elicited via each route. We observed that both routes of B. pertussis challenge result in dose-dependent colonization of the respiratory tract, but overall, intranasal challenge led to higher bacterial burden in the nasal lavage, trachea, and lung. Furthermore, high dose intranasal challenge results in induction of leukocytosis and pro-inflammatory cytokine responses compared to aerosol challenge. These data highlight crucial differences in B. pertussis challenge routes that should be considered during experimental design.

Development of an anti-Pseudomonas aeruginosa therapeutic monoclonal antibody WVDC-5244.

The rise of antimicrobial-resistant bacterial infections is a crucial health concern in the 21st century. In particular, antibiotic-resistant Pseudomonas aeruginosa causes difficult-to-treat infections associated with high morbidity and mortality. Unfortunately, the number of effective therapeutic interventions against antimicrobial-resistant P. aeruginosa infections continues to decline. Therefore, discovery and development of alternative treatments are necessary. Here, we present pre-clinical efficacy studies on an anti-P. aeruginosa therapeutic monoclonal antibody. Using hybridoma technology, we generated a monoclonal antibody and characterized its binding to P. aeruginosa in vitro using ELISA and fluorescence correlation spectroscopy. We also characterized its function in vitro and in vivo against P. aeruginosa. The anti-P. aeruginosa antibody (WVDC-5244) bound P. aeruginosa clinical strains of various serotypes in vitro, even in the presence of alginate exopolysaccharide. In addition, WVDC-5244 induced opsonophagocytic killing of P. aeruginosa in vitro in J774.1 murine macrophage, and complement-mediated killing. In a mouse model of acute pneumonia, prophylactic administration of WVDC-5244 resulted in an improvement of clinical disease manifestations and reduction of P. aeruginosa burden in the respiratory tract compared to the control groups. This study provides promising pre-clinical efficacy data on a new monoclonal antibody with therapeutic potential for P. aeruginosa infections.

Long-Term Analysis of Pertussis Vaccine Immunity to Identify Potential Markers of Vaccine-Induced Memory Associated With Whole Cell But Not Acellular Pertussis Immunization in Mice.

Over two decades ago acellular pertussis vaccines (aP) replaced whole cell pertussis vaccines (wP) in several countries. Since then, a resurgence in pertussis has been observed, which is hypothesized to be linked, in part, to waning immunity. To better understand why waning immunity occurs, we developed a long-term outbred CD1 mouse model to conduct the longest murine pertussis vaccine studies to date, spanning out to 532 days post primary immunization. Vaccine-induced memory results from follicular responses and germinal center formation; therefore, cell populations and cytokines involved with memory were measured alongside protection from challenge. Both aP and wP immunization elicit protection from intranasal challenge by decreasing bacterial burden in both the upper and lower airways, and by generation of pertussis specific antibody responses in mice. Responses to wP vaccination were characterized by a significant increase in T follicular helper cells in the draining lymph nodes and CXCL13 levels in sera compared to aP mice. In addition, a population of B. pertussis+ memory B cells was found to be unique to wP vaccinated mice. This population peaked post-boost, and was measurable out to day 365 post-vaccination. Anti-B. pertussis and anti-pertussis toxoid antibody secreting cells increased one day after boost and remained high at day 532. The data suggest that follicular responses, and in particular CXCL13 levels in sera, could be monitored in pre-clinical and clinical studies for the development of the next-generation pertussis vaccines.

Bordetella pertussis whole cell immunization protects against Pseudomonas aeruginosa infections.

Whole cell vaccines are complex mixtures of antigens, immunogens, and sometimes adjuvants that can trigger potent and protective immune responses. In some instances, such as whole cell Bordetella pertussis vaccination, the immune response to vaccination extends beyond the pathogen the vaccine was intended for and contributes to protection against other clinically significant pathogens. In this study, we describe how B. pertussis whole cell vaccination protects mice against acute pneumonia caused by Pseudomonas aeruginosa. Using ELISA and western blot, we identified that B. pertussis whole cell vaccination induces production of antibodies that bind to lab-adapted and clinical strains of P. aeruginosa, regardless of immunization route or adjuvant used. The cross-reactive antigens were identified using immunoprecipitation, mass spectrometry, and subsequent immunoblotting. We determined that B. pertussis GroEL and OmpA present in the B. pertussis whole cell vaccine led to production of antibodies against P. aeruginosa GroEL and OprF, respectively. Finally, we showed that recombinant B. pertussis OmpA was sufficient to induce protection against P. aeruginosa acute murine pneumonia. This study highlights the potential for use of B. pertussis OmpA as a vaccine antigen for prevention of P. aeruginosa infection, and the potential of broadly protective antigens for vaccine development.

Variability of Dosing and Number of Medications Needed to Achieve Adequate Sedation in Mechanically Ventilated Pediatric Intensive Care Patients.

Children admitted to the pediatric intensive care unit (PICU) often require multiple medications to achieve comfort and sedation. Although starting doses are available, these medications are typically titrated to the desired effect. Both oversedation and undersedation are associated with adverse events. The aim of this retrospective study was to evaluate cumulative medication burden necessary to achieve comfort in patients in the PICU and determine relevant predictors of medication needs. In order to account for all of the sedative medications, z-scores were used to assess the population average dose of each medication and compare each patient day to this population average. Sedation regimens for 130 patients in the PICU were evaluated. Mean overall infusion rates of fentanyl, morphine, and hydromorphone were 1.67 ± 0.81 µg/kg/hour, 0.12 ± 0.08 mg/kg/hour, and 17.84 ± 13.4 µg/kg/hour, respectively. The mean infusion rate of dexmedetomidine was 0.59 ± 0.28 µg/kg/hour, and midazolam was 0.14 ± 0.1 mg/kg/hour. Summation z-sores were used to rank the amount of sedation medication needed to achieve comfort for each individual patient for his/her PICU stay in relation to the entire sample. Patient age, weight, and length of mechanical ventilation were all significant predictors of sedation requirement. This study will provide data necessary to develop a model of cumulative medication burden needed to achieve appropriate sedation in this population. This descriptive model in appropriately ranking patients based on sedative needs is the first step in exploring potential genetic factors that may provide an insight into homing in on the appropriate sedation regimen.

Effect of a multifaceted, church-based wellness program on metabolic syndrome in 41 overweight or obese congregants.

INTRODUCTION: A rise in obesity, poor-quality diets, and low physical activity has led to a dramatic increase in the number of Americans with metabolic syndrome and diabetes. Our objective was to determine the effect of a short-term, multifaceted wellness program carried out in a church setting on weight, metabolic syndrome, and self-reported wellness. METHODS: Forty-one overweight or obese adults in a church congregation provided fasting blood samples and answered a wellness questionnaire before and after completing an 8-week diet and exercise program. We also measured weight, body fat, body mass index, and waist and hip circumference. RESULTS: The intervention decreased weight, body fat, and central adiposity; improved indexes of metabolic syndrome; and increased self-reported wellness. CONCLUSION: A multifaceted wellness intervention that emphasizes diet and exercise can rapidly influence weight, insulin resistance, metabolic syndrome, and self-reported wellness.

The content of favorable and unfavorable polyunsaturated fatty acids found in commonly eaten fish.

Changes in diet during the past century have caused a marked increase in consumption of saturated fatty acids and n-6 polyunsaturated fatty acids (PUFAs) with a concomitant decrease in the intake of n-3 PUFAs. Increased fish consumption has been shown to be the only realistic way to increase dietary quantities of beneficial long-chain n-3 PUFAs such as eicosapentaenoic acid and docosahexaenoic acid and re-establish more balanced n-6:n-3 ratios in the diets of human beings. Our objective in this research was to characterize some of the relevant fatty acid chemistry of commonly consumed fish, with a particular focus on the four most commonly consumed farmed fish. To do this, 30 commonly consumed farmed and wild fish were collected from supermarkets and wholesalers throughout the United States. Fatty acid composition of samples from these fish was determined using gas chromatography. The 30 samples studied contained n-3 PUFAs ranging from fish having almost undetectable levels to fish having nearly 4.0 g n-3 PUFA per 100 g fish. The four most commonly farmed fish, Atlantic salmon, trout, tilapia, and catfish, were more closely examined. This analysis revealed that trout and Atlantic salmon contained relatively high concentrations of n-3 PUFA, low n-6:n-3 ratios, and favorable saturated fatty acid plus monounsaturated fatty acid to PUFA ratios. In contrast, tilapia (the fastest growing and most widely farmed fish) and catfish have much lower concentrations of n-3 PUFA, very high ratios of long chain n-6 to long chain n-3 PUFAs, and high saturated fatty acid plus monounsaturated fatty acid to PUFA ratios. Taken together, these data reveal that marked changes in the fishing industry during the past decade have produced widely eaten fish that have fatty acid characteristics that are generally accepted to be inflammatory by the health care community.

Notch Represses Transcription by PRC2 Recruitment to the Ternary Complex.

It is well established that Notch functions as a transcriptional activator through the formation of a ternary complex that comprises Notch, Maml, and CSL. This ternary complex then serves to recruit additional transcriptional cofactors that link to higher order transcriptional complexes. The mechanistic details of these events remain unclear. This report reveals that the Notch ternary complex can direct the formation of a repressor complex to terminate gene expression of select target genes. Herein, it is demonstrated that p19Arf and Klf4 are transcriptionally repressed in a Notch-dependent manner. Furthermore, results indicate that Notch recruits Polycomb Repressor Complex 2 (PRC2) and Lysine Demethylase 1 (KDM1A/LSD1) to these promoters, which leads to changes in the epigenetic landscape and repression of transcription. The demethylase activity of LSD1 is a prerequisite for Notch-mediated transcriptional repression. In addition, a stable Notch transcriptional repressor complex was identified containing LSD1, PRC2, and the Notch ternary complex. These findings demonstrate a novel function of Notch and provide further insight into the mechanisms of Notch-mediated tumorigenesis.Implications: This study provides rationale for the targeting of epigenetic enzymes to inhibit Notch activity or use in combinatorial therapy to provide a more profound therapeutic response. Mol Cancer Res; 15(9); 1173-83. ©2017 AACR.

The Small Molecule IMR-1 Inhibits the Notch Transcriptional Activation Complex to Suppress Tumorigenesis.

In many cancers, aberrant Notch activity has been demonstrated to play a role in the initiation and maintenance of the neoplastic phenotype and in cancer stem cells, which may allude to its additional involvement in metastasis and resistance to therapy. Therefore, Notch is an exceedingly attractive therapeutic target in cancer, but the full range of potential targets within the pathway has been underexplored. To date, there are no small-molecule inhibitors that directly target the intracellular Notch pathway or the assembly of the transcriptional activation complex. Here, we describe an in vitro assay that quantitatively measures the assembly of the Notch transcriptional complex on DNA. Integrating this approach with computer-aided drug design, we explored potential ligand-binding sites and screened for compounds that could disrupt the assembly of the Notch transcriptional activation complex. We identified a small-molecule inhibitor, termed Inhibitor of Mastermind Recruitment-1 (IMR-1), that disrupted the recruitment of Mastermind-like 1 to the Notch transcriptional activation complex on chromatin, thereby attenuating Notch target gene transcription. Furthermore, IMR-1 inhibited the growth of Notch-dependent cell lines and significantly abrogated the growth of patient-derived tumor xenografts. Taken together, our findings suggest that a novel class of Notch inhibitors targeting the transcriptional activation complex may represent a new paradigm for Notch-based anticancer therapeutics, warranting further preclinical characterization. Cancer Res; 76(12); 3593-603. ©2016 AACR.