Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

Identification of epidermal growth factor receptor-tyrosine kinase inhibitor targeting the VP1 pocket of human rhinovirus.

Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.

Genetically Encoded Whole Cell Biosensor for Drug Discovery of HIF-1 Interaction Inhibitors.

The heterodimeric transcription factor, hypoxia inducible factor-1 (HIF-1), is an important anticancer target as it supports the adaptation and response of tumors to hypoxia. Here, we optimized the repressed transactivator yeast two-hybrid system to further develop it as part of a versatile yeast-based drug discovery platform and validated it using HIF-1. We demonstrate both fluorescence-based and auxotrophy-based selections that could detect HIF-1α/HIF-1ß dimerization inhibition. The engineered genetic selection is tunable and able to differentiate between strong and weak interactions, shows a large dynamic range, and is stable over different growth phases. Furthermore, we engineered mechanisms to control for cellular activity and off-target drug effects. We thoroughly characterized all parts of the biosensor system and argue this tool will be generally applicable to a wide array of protein-protein interaction targets. We anticipate this biosensor will be useful as part of a drug discovery platform, particularly when screening DNA-encoded new modality drugs.

Pharmacological characterization of a novel peptide inhibitor of the Keap1-Nrf2 protein-protein interaction.

LAS200813 is a novel bicyclic lipopeptide that activates Nrf2 by binding to Keap1, thereby antagonising the Keap1-Nrf2 protein-protein interaction. In this work we report the pharmacological characterization of LAS200813 in Nrf2-dependent translational preclinical models. LAS200813 binds to Keap1 with high affinity (IC50: 0.73 nM) and is able to induce the translocation of Nrf2 to the nucleus. Furthermore, LAS200813 increases the expression of Nrf2 target genes in human bronchial epithelial cells (EC50 of 96 and 70 nM for srxn1 and nqo1, respectively). Similarly, the intratracheal administration of LAS200813 to rats increases the expression of Nrf2-dependent genes in lung tissue, an effect that lasts for a few hours. Moreover, in cells exposed to cigarette smoke, LAS200813 shows an antioxidant effect by increasing the production of glutathione and prevents cellular apoptosis. In conclusion, the results described herein demonstrate that LAS200813 is a potent non-electrophilic Nrf2-activating peptide designed to be administered by inhaled route which may be a potential therapeutic strategy for respiratory diseases driven by oxidative stress.

ACR Appropriateness Criteria® Chronic Chest Pain-High Probability of Coronary Artery Disease: 2021 Update.

Management of patients with chronic chest pain in the setting of high probability of coronary artery disease (CAD) relies heavily on imaging for determining or excluding presence and severity of myocardial ischemia, hibernation, scarring, and/or the presence, site, and severity of obstructive coronary lesions, as well as course of management and long-term prognosis. In patients with no known ischemic heart disease, imaging is valuable in determining and documenting the presence, extent, and severity of obstructive coronary narrowing and presence of myocardial ischemia. In patients with known ischemic heart disease, imaging findings are important in determining the management of patients with chronic myocardial ischemia and can serve as a decision-making tool for medical therapy, angioplasty, stenting, or surgery. This document summarizes the recent growing body of evidence on various imaging tests and makes recommendations for imaging based on the available data and expert opinion. This document is focused on epicardial CAD and does not discuss the microvascular disease as the cause for CAD. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment.

Public-Private Partnerships: Compound and Data Sharing in Drug Discovery and Development.

Collaborative efforts between public and private entities such as academic institutions, governments, and pharmaceutical companies form an integral part of scientific research, and notable instances of such initiatives have been created within the life science community. Several examples of alliances exist with the broad goal of collaborating toward scientific advancement and improved public welfare. Such collaborations can be essential in catalyzing breaking areas of science within high-risk or global public health strategies that may have otherwise not progressed. A common term used to describe these alliances is public-private partnership (PPP). This review discusses different aspects of such partnerships in drug discovery/development and provides example applications as well as successful case studies. Specific areas that are covered include PPPs for sharing compounds at various phases of the drug discovery process-from compound collections for hit identification to sharing clinical candidates. Instances of PPPs to support better data integration and build better machine learning models are also discussed. The review also provides examples of PPPs that address the gap in knowledge or resources among involved parties and advance drug discovery, especially in disease areas with unfulfilled and/or social needs, like neurological disorders, cancer, and neglected and rare diseases.

A MALT1 inhibitor suppresses human myeloid DC, effector T-cell and B-cell responses and retains Th1/regulatory T-cell homeostasis.

The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) regulates nuclear-factor-kappa-B (NF-κB) activation downstream of surface receptors with immunoreceptor tyrosine-based activation motifs (ITAMs), such as the B-cell or T-cell receptor and has thus emerged as a therapeutic target for autoimmune diseases. However, recent reports demonstrate the development of lethal autoimmune inflammation due to the excessive production of interferon gamma (IFN-É£) and defective differentiation of regulatory T-cells in genetically modified mice deficient in MALT1 paracaspase activity. To address this issue, we explored the effects of pharmacological MALT1 inhibition on the balance between T-effector and regulatory T-cells. Here we demonstrate that allosteric inhibition of MALT1 suppressed Th1, Th17 and Th1/Th17 effector responses, and inhibited T-cell dependent B-cell proliferation and antibody production. Allosteric MALT1 inhibition did not interfere with the suppressive function of human T-regulatory cells, although it impaired de novo differentiation of regulatory T-cells from naïve T-cells. Treatment with an allosteric MALT1 inhibitor alleviated the cytokine storm, including IFN-É£, in a mouse model of acute T-cell activation, and long-term treatment did not lead to an increase in IFN-É£ producing CD4 cells or tissue inflammation. Together, our data demonstrate that the effects of allosteric inhibition of MALT1 differ from those seen in mice with proteolytically inactive MALT1, and thus we believe that MALT1 is a viable target for B and T-cell driven autoimmune diseases.

Cardiac phenotype in ATP1A3-related syndromes: A multicenter cohort study.

OBJECTIVE: To define the risks and consequences of cardiac abnormalities in ATP1A3-related syndromes. METHODS: Patients meeting clinical diagnostic criteria for rapid-onset dystonia-parkinsonism (RDP), alternating hemiplegia of childhood (AHC), and cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) with ATP1A3 genetic analysis and at least 1 cardiac assessment were included. We evaluated the cardiac phenotype in an Atp1a3 knock-in mouse (Mashl+/-) to determine the sequence of events in seizure-related cardiac death. RESULTS: Ninety-eight patients with AHC, 9 with RDP, and 3 with CAPOS (63 female, mean age 17 years) were included. Resting ECG abnormalities were found in 52 of 87 (60%) with AHC, 2 of 3 (67%) with CAPOS, and 6 of 9 (67%) with RDP. Serial ECGs showed dynamic changes in 10 of 18 patients with AHC. The first Holter ECG was abnormal in 24 of 65 (37%) cases with AHC and RDP with either repolarization or conduction abnormalities. Echocardiography was normal. Cardiac intervention was required in 3 of 98 (≈3%) patients with AHC. In the mouse model, resting ECGs showed intracardiac conduction delay; during induced seizures, heart block or complete sinus arrest led to death. CONCLUSIONS: We found increased prevalence of ECG dynamic abnormalities in all ATP1A3-related syndromes, with a risk of life-threatening cardiac rhythm abnormalities equivalent to that in established cardiac channelopathies (≈3%). Sudden cardiac death due to conduction abnormality emerged as a seizure-related outcome in murine Atp1a3-related disease. ATP1A3-related syndromes are cardiac diseases and neurologic diseases. We provide guidance to identify patients potentially at higher risk of sudden cardiac death who may benefit from insertion of a pacemaker or implantable cardioverter-defibrillator.

Discovery of the Oral Leukotriene C4 Synthase Inhibitor (1S,2S)-2-({5-[(5-Chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic Acid (AZD9898) as a New Treatment for Asthma.

While bronchodilators and inhaled corticosteroids are the mainstay of asthma treatment, up to 50% of asthmatics remain uncontrolled. Many studies show that the cysteinyl leukotriene cascade remains highly activated in some asthmatics, even those on high-dose inhaled or oral corticosteroids. Hence, inhibition of the leukotriene C4 synthase (LTC4S) enzyme could provide a new and differentiated core treatment for patients with a highly activated cysteinyl leukotriene cascade. Starting from a screening hit (3), a program to discover oral inhibitors of LTC4S led to (1S,2S)-2-({5-[(5-chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic acid (AZD9898) (36), a picomolar LTC4S inhibitor (IC50 = 0.28 nM) with high lipophilic ligand efficiency (LLE = 8.5), which displays nanomolar potency in cells (peripheral blood mononuclear cell, IC50,free = 6.2 nM) and good in vivo pharmacodynamics in a calcium ionophore-stimulated rat model after oral dosing (in vivo, IC50,free = 34 nM). Compound 36 mitigates the GABA binding, hepatic toxicity signal, and in vivo toxicology findings of an early lead compound 7 with a human dose predicted to be 30 mg once daily.

Collision-avoiding imaging trajectories for linac mounted cone-beam CT.

BACKGROUND: Some patients cannot be imaged with cone-beam CT for image-guided radiation therapy because their size, pose, or fixation devices cause collisions with the machine. OBJECTIVE: To investigate imaging trajectories that avoid such collisions by using virtual isocenter and variable magnification during acquisition while yielding comparable image quality. METHODS: The machine components most likely to collide are the gantry and kV detector. A virtual isocenter trajectory continuously moves the patient during gantry rotation to maintain an increased separation between the two. With dynamic magnification, the kV detector is dynamically moved to increase clearance for an angular range around the potential collision point while acquiring sufficient data to maintain the field-of-view. Both strategies were used independently and jointly with the resultant image quality evaluated against the standard circular acquisition. RESULTS: Collision avoiding trajectories show comparable contrast and resolution to standard techniques. For an anthropomorphic phantom, the RMSE is <7×10- 4, multi-scale structural similarity index is >0.97, and visual image fidelity is >0.96 for all trajectories when compared to a standard circular scan. CONCLUSIONS: The proposed trajectories avoid machine-patient collisions while providing comparable image quality to the current standard thereby enabling CBCT imaging for patients that could not otherwise be scanned.

Long-Term Outcomes of Hypertrophic Cardiomyopathy Diagnosed During Childhood: Results From a National Population-Based Study.

BACKGROUND: Late survival and symptomatic status of children with hypertrophic cardiomyopathy have not been well defined. We examined long-term outcomes for pediatric hypertrophic cardiomyopathy. METHODS: The National Australian Childhood Cardiomyopathy Study is a longitudinal population-based cohort study of children (0-10 years of age) diagnosed with cardiomyopathy between 1987 and 1996. The primary study end point was time to death or cardiac transplantation. RESULTS: There were 80 patients with hypertrophic cardiomyopathy, with a median age at diagnosis of 0.48 (interquartile range, 0.1, 2.5) years. Freedom from death/transplantation was 86% (95% confidence interval [CI], 77.0-92.0) 1 year after presentation, 80% (95% CI, 69.0-87.0) at 10 years, and 78% (95% CI, 67.0-86.0) at 20 years. From multivariable analyses, risk factors for death/transplantation included symmetrical left ventricular hypertrophy at the time of diagnosis (hazard ratio, 4.20; 95% CI, 1.60-11.05; P=0.004), Noonan syndrome (hazard ratio, 2.88; 95% CI, 1.02-8.08; P=0.045), higher posterior wall thickness z score (hazard ratio, 1.45; 95% CI, 1.22-1.73; P<0.001), and lower fractional shortening z score (hazard ratio, 0.84; 95% CI, 0.74-0.95; P=0.005) during follow-up. Nineteen (23%) subjects underwent left ventricular myectomy. At a median of 15.7 years of follow-up, 27 (42%) of 63 survivors were treated with ß-blocker, and 13 (21%) had an implantable cardioverter-defibrillator. CONCLUSIONS: The highest risk of death or transplantation for children with hypertrophic cardiomyopathy is within 1 year after diagnosis, with low attrition rates thereafter. Many subjects receive medical, surgical, or device therapy.