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In Alzheimer disease (AD), amyloid β peptide (Aβ) accumulates in plaques in the brain. Receptor for advanced glycation end products (RAGE) mediates Aβ-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a high-affinity RAGE-specific inhibitor (FPS-ZM1) that blocked Aβ binding to the V domain of RAGE and inhibited Aβ40- and Aβ42-induced cellular stress in RAGE-expressing cells in vitro and in the mouse brain in vivo. FPS-ZM1 was nontoxic to mice and readily crossed the blood-brain barrier (BBB). In aged APPsw/0 mice overexpressing human Aβ-precursor protein, a transgenic mouse model of AD with established Aβ pathology, FPS-ZM1 inhibited RAGE-mediated influx of circulating Aβ40 and Aβ42 into the brain. In brain, FPS-ZM1 bound exclusively to RAGE, which inhibited β-secretase activity and Aβ production and suppressed microglia activation and the neuroinflammatory response. Blockade of RAGE actions at the BBB and in the brain reduced Aβ40 and Aβ42 levels in brain markedly and normalized cognitive performance and cerebral blood flow responses in aged APPsw/0 mice. Our data suggest that FPS-ZM1 is a potent multimodal RAGE blocker that effectively controls progression of Aβ-mediated brain disorder and that it may have the potential to be a disease-modifying agent for AD. [ABSTRACT FROM AUTHOR]

Aims: Heart failure (HF) is a growing health problem, yet there are limited data on patients with HF in Malaysia. The Malaysian Heart Failure (MY-HF) Registry aims to gain insights into the epidemiology, aetiology, management, and outcome of Malaysian patients with HF and identify areas for improvement within the national HF services., Methods and Results: The MY-HF Registry is a 3-year prospective, observational study comprising 2717 Malaysian patients admitted for acute HF. We report the description of baseline data at admission and outcomes of index hospitalization of these patients. The mean age was 60.2 ± 13.6 years, 66.8% were male, and 34.3% had de novo HF. Collectively, 55.7% of patients presented with New York Heart Association (NYHA) Class III or IV; ischaemic heart disease was the most frequent aetiology (63.2%). Most admissions (87.3%) occurred via the emergency department, with 13.7% of patients requiring intensive care, and of these, 21.8% needed intubation. The proportion of patients receiving guideline-directed medical therapy increased at discharge (84.2% vs. 93.6%). The median length of stay (LOS) was 5 days, and in-hospital mortality was 2.9%. Predictors of LOS and/or in-hospital mortality were age, NYHA class, estimated glomerular filtration rate, and comorbid anaemia. LOS and in-hospital mortality were similar regardless of ejection fraction., Conclusions: The MY-HF Registry showed that the HF population in Malaysia is younger, predominantly male, and ischaemic-driven and has good prospects with hospitalization for optimization of treatment. These findings suggest a need to reassess current clinical practice and guide resource allocation to improve patient outcomes., (© 2023 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models., (© 2023. The Author(s).)

Background: The use of urinary sodium to guide diuretics in acute heart failure is recommended by experts and the most recent European Society of Cardiology guidelines. However, there are limited data to support this recommendation. The ENACT-HF study (Efficacy of a Standardized Diuretic Protocol in Acute Heart Failure) investigated the feasibility and efficacy of a standardized natriuresis-guided diuretic protocol in patients with acute heart failure and signs of volume overload., Methods: ENACT-HF was an international, multicenter, open-label, pragmatic, 2-phase study, comparing the current standard of care of each center with a standardized diuretic protocol, including urinary sodium to guide therapy. The primary end point was natriuresis after 1 day. Secondary end points included cumulative natriuresis and diuresis after 2 days of treatment, length of stay, and in-hospital mortality. All end points were adjusted for baseline differences between both treatment arms., Results: Four hundred one patients from 29 centers in 18 countries worldwide were included in the study. The natriuresis after 1 day was significantly higher in the protocol arm compared with the standard of care arm (282 versus 174 mmol; adjusted mean ratio, 1.64; P <0.001). After 2 days, the natriuresis remained higher in the protocol arm (538 versus 365 mmol; adjusted mean ratio, 1.52; P <0.001), with a significantly higher diuresis (5776 versus 4381 mL; adjusted mean ratio, 1.33; P <0.001). The protocol arm had a shorter length of stay (5.8 versus 7.0 days; adjusted mean ratio, 0.87; P =0.036). In-hospital mortality was low and did not significantly differ between the 2 arms (1.4% versus 2.0%; P =0.852)., Conclusions: A standardized natriuresis-guided diuretic protocol to guide decongestion in acute heart failure was feasible, safe, and resulted in higher natriuresis and diuresis, as well as a shorter length of stay., Competing Interests: Disclosures Dr Dauw received speaker fees from AstraZeneca, Boehringer-Ingelheim, and Bayer; Dr Lelonek received speaker and consulting fees from Novartis, Novo Nordisk, Servier, AstraZeneca, Boehringer-Ingelheim, Bausch Health, Bayer, Ewopharma, and Gedeon Richter and was involved in clinical trials from Amgen, Novartis, Novo Nordisk, and Boehringer-Ingelheim; Dr Borbély received speaker fees from Astra Zeneca, Bayer, Boehringer-Ingelheim, and Novartis; Dr Cobo-Marcos received speaker fees from Astra Zeneca, Boehringer-Ingelheim, Novartis, Vifor Pharma, Novo Nordisk, and Bayer; Dr Barge-Caballero received travel grants and speaker fees from Astra Zeneca, Boehringer-Ingelheim, Novartis, Viatris, and Pfizer and received research grants from Pfizer; Dr Barker received speaker fees from AstraZeneca, Novartis, and Medtronic; Dr Doghmi received speaker fees from Novartis, Boehringer-Ingelheim, and Pfizer; Dr Nijst received speaker fees from Novartis, Boehringer-Ingelheim, and Bayer; and Dr Martens received consultancy fees from Novartis and CLS Vifor and is supported by a research grant from the Belgian American Educational Foundation and the Frans Van de Werf Fund. The other authors report no conflicts.

Background: Bleeding outcomes are crucial primary safety endpoints in studies involving thrombolytic agents. This study aimed to determine the incidence, characteristics and mortality outcomes of bleeding following ST-elevation myocardial infarction (STEMI) thrombolysis in an Asian population., Methods: This single-centre retrospective study included all STEMI patients who received thrombolytic therapy from 2016 to 2020 in a Malaysian tertiary hospital. Total population sampling was used in this study. The primary outcome was bleeding events post-thrombolysis, categorised using the Thrombolysis in Myocardial Infarction (TIMI) bleeding criteria. Inferential statistics were used to determine the associations between relevant variables., Results: Data from 941 patients were analysed. A total of 156 (16.6%) STEMI patients bled post-thrombolysis. Major, minor, and minimal TIMI occurred in 7 (0.7%), 17 (1.8%), and 132 (14.0%) patients, respectively. Age 65 years ( P =0.031) and Malaysian Chinese ( P =0.008) were associated with a higher incidence of bleeding post-thrombolysis. Conversely, foreigners ( P =0.032) and current smoker ( P =0.007) were associated with a lower incidence of bleeding. Both TIMI major ( P <0.001) and TIMI minor ( P <0.001) were associated with a higher incidence of all-cause in-hospital mortality among STEMI patients. TIMI minor bleeding was significantly higher in the streptokinase recipients. The bleeding sites were comparable between streptokinase and tenecteplase recipients, except for a significantly higher incidence of gastrointestinal bleeding in the streptokinase recipients ( P =0.027)., Conclusion: In our Asian population, the incidence of total bleeding events following STEMI thrombolysis is comparable to that previously reported. The development of TIMI major and minor bleeding complications is associated with higher mortality., Competing Interests: Conflicts of interest: The authors declare that they have no conflicts of interest., (Copyright: © World Journal of Emergency Medicine.)

Pharmacological reperfusion remains the primary strategy for ST-elevation myocardial infarction (STEMI) in low- and medium-income countries. Literature has reported inconsistent incidences and outcomes of failed thrombolysis (FT). This study aimed to identify the incidence, mortality outcomes and predictors of FT in STEMI pharmacological reperfusion. This single-centre retrospective cohort study analyzed data on consecutive STEMI patients who received thrombolytic therapy from 2016 to 2020 in a public tertiary hospital. Total population sampling was used in this study. Logistic regression analyses were used to assess independent predictors of the mortality outcomes and FT. We analyzed 941 patients with a mean age of 53.0 ± 12.2 years who were predominantly male (n = 846, 89.9%). The in-hospital mortality was 10.3% (n = 97). FT occurred in 86 (9.1%) patients and was one of the predictors of mortality (aOR 3.847, p < 0.001). Overall, tenecteplase use (aOR 1.749, p = 0.021), pre-existing hypertension (aOR 1.730, p = 0.024), history of stroke (aOR 4.176, p = 0.004), and heart rate ≥ 100 bpm at presentation (aOR 2.333, p < 0.001) were the general predictors of FT. The predictors of FT with streptokinase were Killip class ≥ II (aOR 3.197, p = 0.004) and heart rate ≥ 100 bpm at presentation (aOR 3.536, p = 0.001). History of stroke (aOR 6.144, p = 0.004) and heart rate ≥ 100 bpm at presentation (aOR 2.216, p = 0.015) were the predictors of FT in STEMI patients who received tenecteplase. Mortality following STEMI thrombolysis remained high in our population and was attributed to FT. Identified predictors of FT enable early risk stratification to evaluate the patients' prognosis to manage them better., (© 2023. The Author(s), under exclusive licence to Società Italiana di Medicina Interna (SIMI).)

Globally, heart failure with preserved ejection fraction (HFpEF) is quickly becoming the dominant form of heart failure (HF) in ageing populations. However, there are still multiple gaps and challenges in making a firm diagnosis of HFpEF in many low-to-middle income Asian countries. In response to this unmet need, the Malaysian HFpEF Working Group (MY-HPWG) gathered and reviewed evidence surrounding the use of different diagnostic modalities indicated for patients with HFpEF to identify diagnostic tools that could be conveniently accessed across different healthcare settings. As a result, five recommendation statements were proposed and an accompanying algorithm was developed, with the aim of improving the diagnostic rate of HFpEF. The MY-HPWG recommends using more easily accessible and non-invasive tools, such as natriuretic peptide (NP) biomarkers and basic echocardiogram (ECHO), to ensure timely HFpEF diagnosis in the primary and secondary care settings, and prompt referral to a tertiary care centre for more comprehensive assessments in uncertain cases., Competing Interests: Conflict of Interest None., (© Penerbit Universiti Sains Malaysia, 2023.)

Introduction: Some guidelines had recommended "thrombolysis first" in ST-elevated myocardial infarction (STEMI) during the Coronavirus Disease 2019 (COVID-19) outbreak. The impact of COVID-19 solely on STEMI thrombolysis is lacking as most studies reported outcomes related to percutaneous coronary intervention (PCI) setting. Thus, this study aimed to assess the impact of the COVID-19 pandemic on STEMI thrombolysis outcomes and the Emergency Department's performance in a non-PCI capable centre., Methods: This single-centre retrospective study analysed data on consecutive STEMI patients who received thrombolytic therapy from May 2019 to December 2020 (20 months) in a non-PCI capable tertiary hospital. Total population sampling was used in this study. We compared all patients' characteristics and outcomes ten months before and during the pandemic. Regression models were used to assess the impact of COVID-19 pandemic on door-to-needle time (DNT), mortality, bleeding events, and the number of overnight stays., Results and Discussion: We analysed 323 patients with a mean age of 52.9 ± 12.9 years and were predominantly male (n = 280, 88.9%). There was a 12.5% reduction in thrombolysis performed during the pandemic. No significant difference in timing from symptoms onset to thrombolysis and DNT was observed. In-hospital mortality was significantly higher during the pandemic (OR 2.02, 95% CI 1.02-4.00, p = 0.044). Bleeding events post thrombolysis remained stable and there was no significant difference in the number of overnight stays during the pandemic., Conclusion: STEMI thrombolysis cases were reduced during the COVID-19 pandemic, with an inverse increase in mortality despite the preserved Emergency Department performance in timely thrombolysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Fibrinolysis remains the primary reperfusion strategy for ST-elevation myocardial infarction (STEMI) in many Asian countries. The outcomes and factors affecting mortality in STEMI fibrinolysis in the Asian population are lacking despite being widely used., Objectives: This study aimed to assess the clinical profile of patients and predictors affecting STEMI mortality in an Asian population., Methods: This single-center retrospective study analyzed data from STEMI patients who received fibrinolytic therapy from 2016 to 2020 in a tertiary hospital. Logistic regression analysis was performed to identify the significant predictors of the 30-day all-cause mortality, the primary outcome., Results: A total of 859 patients were included. Their mean age was 53.6 ±12.1 years and they were predominantly male (n=769, 89.4%). The majority of them had anterior involvement STEMI (n = 477, 55.5%) and presented with Killip ≥ II (n = 424, 49.4%). The 30-day all-cause mortality was 12.0% (n =  103). The final model found six predictors for 30-day mortality: age ≥75 (aOR 4.784, p < 0.001), female gender (aOR 2.869, p = 0.001), pre-existing hypertension (aOR 1.623, p = 0.046), anterior myocardial infarction (MI) (aOR 1.947, p < 0.001), Killip class (p < 0.001) and heart rate ≥100 at presentation (aOR 1.823, p =  0.016). Following fibrinolytic therapy, five predictors were found to affect 30-day mortality, i.e. failed fibrinolysis (aOR 2.094, p = 0.041), bleeding events, congestive heart failure (aOR 3.554, p = 0.046), ventricular fibrillation/ tachycardia (aOR 5.920, p < 0.001), and atrial fibrillation/ flutter (aOR 2.968, p = 0.016)., Conclusion: Our STEMI patients were younger and more ill at presentation. The risk predictors on 30-day all-cause mortality identified in our Asian population allow the clinicians to better triage and manage STEMI patients., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background Fibrinolysis using streptokinase or tenecteplase remains the primary reperfusion strategy for ST-elevation myocardial infarction (STEMI) in many Asian countries, including Malaysia. Comparative outcomes of these two fibrinolytic agents in the Asian population were inconclusive despite being widely used. Aim We aimed to assess and compare the outcomes of streptokinase versus tenecteplase in STEMI reperfusion of an Asian population. Method This single-centre retrospective study analysed data on STEMI patients who received fibrinolytic therapy from 2016 to 2020 in the Emergency Department of the largest tertiary hospital in Malaysia. Total population sampling was used in this study. Based on the propensity score matching, 359 patients receiving streptokinase were matched against 359 patients receiving tenecteplase by incorporating 16 variables that potentially affect mortality. 30-day mortality, stroke and major bleeding were the primary outcome measures. Results There was no significant difference in 30-day mortality between streptokinase (n = 39, 11.2%) and tenecteplase (n = 46, 13.2%) groups (p = 0.418). The rates of ischemic strokes [streptokinase (n = 1, 0.3%) versus tenecteplase (n = 3, 0.9%), p = 0.624], intracranial haemorrhage [streptokinase (n = 3, 0.9%) versus tenecteplase (n = 1, 0.3%), p = 0.624] and major bleeding [streptokinase (n = 4, 1.1%) versus tenecteplase (n = 3, 0.9%), p = 0.624], were comparable for the two groups. The incidences of failed thrombolysis were significantly higher in the tenecteplase arm. Hypotension and allergic reaction were significantly higher in the streptokinase arm. Conclusion Streptokinase and tenecteplase are fibrinolytic agents with similar efficacy and safety in STEMI reperfusion therapy in our Asian population., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Although the burden of premature myocardial infarction (MI) is high in Malaysia, direct evidence on the determinants of MI in this multi-ethnic population remains sparse., Objective: The Malaysian Acute Vascular Events Risk (MAVERIK) study is a retrospective case-control study established to investigate the genomic, lipid-related, and other determinants of acute MI in Malaysia. In this paper, we report the study protocol and early results., Methods: By June 2019, we had enrolled approximately 2500 patients with their first MI and 2500 controls without cardiovascular disease, who were frequency-matched by age, sex, and ethnicity, from 17 hospitals in Malaysia. For each participant, serum and whole blood have been collected and stored. Clinical, demographic, and behavioral information has been obtained using a 200-item questionnaire., Results: Tobacco consumption, a history of diabetes, hypertension, markers of visceral adiposity, indicators of lower socioeconomic status, and a family history of coronary disease were more prevalent in cases than in controls. Adjusted (age and sex) logistic regression models for traditional risk factors indicated that current smoking (odds ratio [OR] 4.11, 95% CI 3.56-4.75; P<.001), previous smoking (OR 1.34, 95% CI 1.12-1.60; P=.001), a history of high blood pressure (OR 2.13, 95% CI 1.86-2.44; P<.001), a history of diabetes mellitus (OR 2.72, 95% CI 2.34-3.17; P<.001), a family history of coronary heart disease (OR 1.28, 95% CI 1.07-1.55; P=.009), and obesity (BMI >30 kg/m 2 ; OR 1.19, 95% CI 1.05-1.34; P=.009) were associated with MI in age- and sex-adjusted models., Conclusions: The MAVERIK study can serve as a useful platform to investigate genetic and other risk factors for MI in an understudied Southeast Asian population. It should help to hasten the discovery of disease-causing pathways and inform regionally appropriate strategies that optimize public health action., International Registered Report Identifier (irrid): RR1-10.2196/31885., (©Rajiv Chowdhury, Mohd Fairulnizal Md Noh, Sophia Rasheeqa Ismail, Kim Robin van Daalen, Puteri Sofia Nadira Megat Kamaruddin, Siti Hafizah Zulkiply, Nur Hayati Azizul, Norhayati Mustafa Khalid, Azizan Ali, Izyan Mohd Idris, Yong Shih Mei, Shazana Rifham Abdullah, Norfashihah Faridus, Nur Azirah Md Yusof, Nur Najwa Farahin M Yusoff, Rahman Jamal, Aizai Azan Abdul Rahim, Abdul Kahar Abdul Ghapar, Ammu Kutty Radhakrishnan, Alan Yean Yip Fong, Omar Ismail, Saravanan Krishinan, Chuey Yan Lee, Liew Houng Bang, Eashwary Mageswaren, Kauthaman Mahendran, Nor Hanim Mohd Amin, Gunavathy Muthusamy, Aaron Ong Hean Jin, Ahmad Wazi Ramli, Noel Thomas Ross, Anwar Irawan Ruhani, Mansor Yahya, Yusniza Yusoff, Siti Khairani Zainal Abidin, Laryssa Amado, Thomas Bolton, Sophie Weston, Jason Crawte, Niko Ovenden, Ank Michielsen, Md Mostafa Monower, Wan Rozita Wan Mahiyuddin, Angela Wood, Emanuele Di Angelantonio, Nur Suffia Sulaiman, John Danesh, Adam S Butterworth. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 10.02.2022.)

Aims: Although acute heart failure (AHF) with volume overload is treated with loop diuretics, their dosing and type of administration are mainly based upon expert opinion. A recent position paper from the Heart Failure Association (HFA) proposed a step-wise pharmacologic diuretic strategy to increase the diuretic response and to achieve rapid decongestion. However, no study has evaluated this protocol prospectively., Methods and Results: The Efficacy of a Standardized Diuretic Protocol in Acute Heart Failure (ENACT-HF) study is an international, multicentre, non-randomized, open-label, pragmatic study in AHF patients on chronic loop diuretic therapy, admitted to the hospital for intravenous loop diuretic therapy, aiming to enrol 500 patients. Inclusion criteria are as follows: at least one sign of volume overload (oedema, ascites, or pleural effusion), use ≥ 40 mg of furosemide or equivalent for >1 month, and a BNP > 250 ng/L or an N-terminal pro-B-type natriuretic peptide > 1000 pg/L. The study is designed in two sequential phases. During Phase 1, all centres will treat consecutive patients according to the local standard of care. In the Phase 2 of the study, all centres will implement a standardized diuretic protocol in the next cohort of consecutive patients. The protocol is based upon the recently published HFA algorithm on diuretic use and starts with intravenous administration of two times the oral home dose. It includes early assessment of diuretic response with a spot urinary sodium measurement after 2 h and urine output after 6 h. Diuretics will be tailored further based upon these measurements. The study is powered for its primary endpoint of natriuresis after 1 day and will be able to detect a 15% difference with 80% power. Secondary endpoints are natriuresis and diuresis after 2 days, change in congestion score, change in weight, in-hospital mortality, and length of hospitalization., Conclusions: The ENACT-HF study will investigate whether a step-wise diuretic approach, based upon early assessment of urinary sodium and urine output as proposed by the HFA, is feasible and able to improve decongestion in AHF with volume overload., (© 2021 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas., Competing Interests: Declaration of interests All authors (except otherwise noted) are or were at the time of their involvement with the research employees of Novartis Institutes for BioMedical Research and may hold stock in Novartis. This study was funded by the Novartis Institutes for BioMedical Research and the Novartis-Berkeley Center for Proteomics and Chemistry Technologies. D.K.N. is a co-founder, shareholder, and adviser for Frontier Medicines. D.E.F. has a financial interest in Soltego, a company developing salt-inducible kinase inhibitors for topical skin-darkening treatments that might be used for a broad set of human applications. The interests of D.E.F. were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

There is a wide variation on the efficacy of three-factor Prothrombin Complex Concentrate (3F-PCC) in warfarin reversal. We aimed to determine the efficacy and safety of 3F-PCC in warfarin reversal. This multicentre prospective study analysed data from adult patients on warfarin who received 3F-PCC (Prothrombinex-VF®) for anticoagulation reversal between June 2019 to October 2020. Purposive sampling was used in this study. Study endpoints included target INR achievement, adverse drug reactions (ADRs), and in-hospital all-cause mortality. Logistic regression analyses were used to assess independent predictors of study endpoints. One-hundred thirty-seven patients with a median age of 68 (59-76) years were recruited, who were predominantly male (59.9%, n = 82). A total of 102 patients required 3F-PCC for life-threatening (40.9%, n = 56) and clinically significant bleeding (33.6%, n = 46). Initial INRs ranged from 1.55 to undetectable high (> 26). All patients had INR reduction, of which 62% (n = 85) achieved target INR, whereas 12.4% (n = 17) achieved INR below the target range. Median INR was reduced from 4.76 (3.14-8.32) to 1.54 (1.27-1.88) post-3F-PCC (p < 0.001). The use of adjunctive reversal agents and initial INR < 3.6 were the significant predictors for target INR achievement. Six (4.4%) ADRs were observed. Two (1.5%) cases with the suspected acute coronary syndrome were associated with mortality. Ischemic stroke occurred in one (0.7%) patient. The incidence of in-hospital all-cause mortality was 21.2% (n = 29). The rate of INR achievement was 62% in our study without apparent increased risk of thromboembolic events and in-hospital all-cause mortality., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Protein turnover is highly regulated by the posttranslational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. The development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is key for UPS drug discovery. Conventional approaches for studying ubiquitination either have high protein requirements or rely on exogenous or modified ubiquitin moieties, thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay that combines the NanoBiT luminescence-based technology with tandem ubiquitin binding entities (TUBEs) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied compound-induced ubiquitination of the G to S Phase Transition 1 (GSPT1) protein. Using this assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a live-cell-based approach for assaying substrate ubiquitination that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying the ubiquitination of target proteins with diverse functions.

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-messenger RNA 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 synthetic lethal node in a subset 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 an avenue to therapeutic intervention in cancer.

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.

Protein degradation is critical for proteostasis, and the addition of polyubiquitin chains to a substrate is necessary for its recognition by the 26S proteasome. Therapeutic intervention in the ubiquitin proteasome system has implications ranging from cancer to neurodegeneration. Novel screening methods and chemical biology tools for targeting E1-activating, E2-conjugating and deubiquitinating enzymes will be discussed in this review. Approaches for targeting E3 ligase-substrate interactions as well as the proteasome will also be covered, with a focus on recently described approaches., (Copyright © 2017. Published by Elsevier Ltd.)

Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products.

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

The drug discovery process classically revolves around a set of biochemical and cellular assays to drive potency optimization and structural-activity relationship models. Layered on top of these concepts is the inclusion of molecular features that affect final drug use, things like: bioavailability, toxicity, stability, solubility, formulation, route of administration, etc. Paradoxically, most drugs entering clinical trials are only tested in a handful of human genetic backgrounds before they are given to people. Here we review efforts and opine on the use of large scale in vitro cellular and in vivo models that attempt to model human disease and include diversity found in the human genetic population. Because hundreds to thousands of individual assays are needed to scratch the surface of human genetic diversity, sophisticated high throughput automation technologies or pooling and deconvolution strategies are required. Characterization of each model needs to be extensive to enable non-biased informatics based modeling. Such approaches will enable deep understanding of genetic to pharmacological response and result in new methods for patient stratification in the clinic. Oncology medicines and cancer genetics have been paving the way for these approaches and we expect to see continued expansion to other fields such as immunology and neuroscience.

Cell-based therapies hold the potential to alleviate the growing burden of liver diseases. Such therapies require human hepatocytes, which, within the stromal context of the liver, are capable of many rounds of replication. However, this ability is lost ex vivo, and human hepatocyte sourcing has limited many fields of research for decades. Here we developed a high-throughput screening platform for primary human hepatocytes to identify small molecules in two different classes that can be used to generate renewable sources of functional human hepatocytes. The first class induced functional proliferation of primary human hepatocytes in vitro. The second class enhanced hepatocyte functions and promoted the differentiation of induced pluripotent stem cell-derived hepatocytes toward a more mature phenotype than what was previously obtainable. The identification of these small molecules can help address a major challenge affecting many facets of liver research and may lead to the development of new therapeutics for liver diseases.

The KRAS oncogene is found in up to 30% of all human tumors. In 2009, RNAi experiments revealed that lowering mRNA levels of a transcript encoding the serine/threonine kinase STK33 was selectively toxic to KRAS-dependent cancer cell lines, suggesting that small-molecule inhibitors of STK33 might selectively target KRAS-dependent cancers. To test this hypothesis, we initiated a high-throughput screen using compounds in the Molecular Libraries Small Molecule Repository (MLSMR). Several hits were identified, and one of these, a quinoxalinone derivative, was optimized. Extensive SAR studies were performed and led to the chemical probe ML281 that showed low nanomolar inhibition of purified recombinant STK33 and a distinct selectivity profile as compared to other STK33 inhibitors that were reported in the course of these studies. Even at the highest concentration tested (10 μM), ML281 had no effect on the viability of KRAS-dependent cancer cells. These results are consistent with other recent reports using small-molecule STK33 inhibitors. Small molecules having different chemical structures and kinase-selectivity profiles are needed to fully understand the role of STK33 in KRAS-dependent cancers. In this regard, ML281 is a valuable addition to small-molecule probes of STK33.

Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.

An aldol-based build/couple/pair (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the build phase, a series of asymmetric syn- and anti-aldol reactions were performed to produce four stereoisomers of a Boc-protected γ-amino acid. In addition, both stereoisomers of O-PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the couple step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the pair phase, three different reactions were employed to enable intramolecular ring-forming processes: nucleophilic aromatic substitution (S(N)Ar), Huisgen [3+2] cycloaddition, and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields, providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on the solid phase to yield a 14 400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition, and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.

Artificially controlled cell recognition has potentially far-reaching applications in both the understanding and altering of biological function. The event of recognition often involves a multimeric protein binding a cellular membrane. While such an interaction is energetically favorable, it has been surprisingly underexploited in artificial control of recognition. Herein we describe how changing properties of substrate (phosphocholine, PC) self-assembly can affect both binding behavior and substrate affinity to a pentameric recognition protein (C-reactive protein, CRP). PC was modified with a short, self-assembling DNA strand to make the substrate self-assembly sensitive and responsive to ionic environment. A significant shift in CRP binding affinity was observed when substrates were assembled in the presence of Cs(+) rather than K(+). Furthermore, alteration of the linker length tethering PC to DNA showed trends similar to other multivalent systems. In optimizing these linker lengths, positive cooperativity increased and K(d) of the substrate assembly to CRP improved roughly 1000-fold. Such experiments both inform our understanding of biological, multivalent interactions in self-assembling systems and present a potential method to exogenously control events in cell recognition.

Proteins modulate the majority of all biological functions and are primarily composed of highly organized secondary structural elements such as helices, turns and sheets. Many of these functions are affected by a small number of key protein-protein contacts, often involving one or more of these well-defined structural elements. Given the ubiquitous nature of these protein recognition domains, their mimicry by peptidic and non-peptidic scaffolds has become a major focus of contemporary research. This review examines several key advances in secondary structure mimicry over the past several years, particularly focusing upon scaffolds that show not only promising projection of functional groups, but also a proven effect in biological systems.

A supramolecular assembly containing an isoguanosine pentaplex with both a "protein-binding" face and a "reporter" face has been generated. When phosphocholine is appended to the protein-binding face this supramolecular assembly binds multivalently to the pentameric human C-reactive protein, a biomolecule implicated in inflammation and heart disease.

The Bcl-x(L)/Bak protein-protein interaction has emerged as an important target for cancer therapy due to its role in apoptosis. Inhibition of this interaction by small-molecule antagonists induces apoptosis in unhealthy cells. Bak, a pro-apoptotic Bcl-2 protein, projects four hydrophobic side chains (V74, L78, I81, and I85), corresponding to the i, i+4, i+7, and i+11 positions of an alpha-helix, into a hydrophobic cleft on Bcl-x(L). Herein, we present a novel family of rationally designed alpha-helix mimetics with improved solubility and synthetic feasibility based on a benzoylurea scaffold. These benzoylurea derivatives favor a linear conformation stabilized by an intramolecular hydrogen bond, and are able to mimic the spatial projection of the i, i+4, and i+7 residues of an alpha-helix. The binding of the benzoylurea derivatives to Bcl-x(L) was assessed using fluorescence polarization competition assays, isothermal titration calorimetry, and (15)N-HSQC experiments. These experiments showed that these agents bind to and disrupt Bcl-x(L) with low micromolar inhibition and dissociation constants, with (15)N-HSQC experiments confirming binding to the hydrophobic pocket of Bcl-x(L) normally occupied by the Bak helix.

The development of small molecules that disrupt protein-protein interactions is a key goal in addressing a number of disease states. The alpha-helix is commonly found at protein interaction interfaces and has been the focus of substantial small molecule mimetic efforts. One of the primary drawbacks of many small molecule alpha-helix mimetics is their hydrophobic core structures. To address this problem we have developed a novel scaffold based on a more water soluble 5-6-5 imidazole-phenyl-thiazole core. An inhibitor of this class has been shown to disrupt the Cdc42/Dbs protein-protein interaction at micromolar concentrations and may be useful in overcoming Cdc42-induced tumor resistance to anticancer therapies.

The translocated intimin receptor (TIR) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) is required for EPEC and EHEC infections, which cause widespread illness across the globe. TIR is translocated via a type-III secretion system into the intestinal epithelial cell membrane, where it serves as an anchor for E. coli attachment via its binding partner intimin. While many aspects of EPEC and EHEC infection are now well understood, the importance of the intermolecular contacts made between intimin and TIR have not been thoroughly investigated. Herein we report site-directed mutagenesis studies on the intimin-binding domain of EPEC TIR, and how these mutations affect TIR-intimin association, as analyzed by isothermal titration calorimetry and circular dichroism. These results show how two factors govern TIR's binding to intimin: A three-residue TIR hot spot is identified that largely mediates the interaction, and mutants that alter the beta-hairpin structure of TIR severely diminish binding affinity. In addition, peptides incorporating key TIR residues identified by mutagenesis are incapable of binding intimin. These results indicate that hot spot residues and structural orientation/preorganization are required for EPEC, and likely EHEC, TIR-intimin binding.

Enteropathogenic Escherichia coli (EPEC) are Gram (-) bacteria responsible for widespread illness in the form of diarrhea. EPEC cells attach to the intestinal epithelium using a Type III secretion system common to many Gram (-) bacteria. The translocated intimin receptor (TIR) is the first protein secreted through the EPEC secretion complex, and is absolutely required for pathogenesis. It inserts into the intestinal epithelium, serving as an anchor responsible for the attachment of EPEC to the host epithelial cell. Intimin is a transmembrane protein displayed on the EPEC cell surface with an extracellular domain that binds TIR. Observation of a TIR-TIR dimer in the X-ray co-crystal structure of the extracellular domains of intimin and TIR raised the question of how these protein domains interact and function in solution. Herein we report that the extracellular domain of TIR exists in a folded and active monomeric state in solution, as confirmed by analytical ultracentrifugation, analytical size-exclusion HPLC, isothermal titration calorimetry, and surface plasmon resonance.

Serum dopamine-beta-hydroxylase (DBH) activity was measured in 44 children with psychiatric disorders and 44 controls in order to determine significant variables affecting its potential use as a marker for specific molecular pathology in the neuropsychiatric disorders of childhood. The assay procedure was reliable and the serum enzyme activity for individuals appears to be stable. There is a very broad distribution of serum DBH activity in the population, and it is similar in males and females. The genetic determination of serum DBH activity was clear in this study, and the enzyme activity increased with age through childhood and adolescence. Thyroid hormone had no effect on serum DBH activity in euthyroid subjects. No difference in mean serum DBH activity emerged across diagnostic groups. When the findings were considered in relation to the results of other studies, it was concluded that the wide range of serum DBH activities in normal and patient populations, as well as developmental effects on the activity of the enzyme, make comparison of DBH activity among diagnostic groups in childhood difficult. Large subject groups, and a consideration of genetic and developmental effects, will clarify possible syndrome-related differences in enzyme activity.