Structure-Guided Elaboration of a Fragment-Like Hit into an Orally Efficacious Leukotriene A4 Hydrolase Inhibitor.

Leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have provided strong evidence that LTA4H is an attractive drug target for the treatment of chronic inflammatory diseases. Here, we describe the transformation of compound 2, a fragment-like hit, into the potent inhibitor of LTA4H 3. Our strategy involved two key steps. First, we aimed to increase the polarity of fragment 2 to improve its drug-likeness, particularly its solubility, while preserving both its promising potency and low molecular weight. Second, we utilized structural information and incorporated a basic amino function, which allowed for the formation of an essential hydrogen bond with Q136 of LTA4H and consequently enhanced the potency. Compound 3 exhibited exceptional selectivity and showed oral efficacy in a KRN passive serum-induced arthritis model in mice. The anticipated human dose to achieve 90% target engagement at the trough concentration was determined to be 40 mg administered once daily.

Racial Identity Attitudes and Vicarious Traumatization from Undue Police Violence on Anticipatory Traumatic Reaction Among Black Americans.

Although the viral nature of videos that capture violent and racialized policing of Black Americans in the United States can increase awareness, exposure to race-based violence can result in vicarious traumatization, particularly among Black Americans. The relationship between anticipatory traumatic reactions (ATRs) and racial identity attitudes is not clearly addressed in the extant body of literature. The current study addresses this research disparity by first analyzing group mean differences among Black Americans (N = 138) who were assigned to audiovisual, written, and imaginal exposure groups. The current study also used a cluster analysis of Black Americans to examine the differences between racial identity attitudes and ATRs following media exposure to undue police violence. Results from the study indicated that no differences in ATRs existed based on types of media exposure. Significant differences across three racial identity clusters existed between ATR in association with attitudes of assimilation, miseducation, self-hatred, anti-dominance, and ethnic-racial salience. Findings from the study suggest that mental health professionals should attend to racial identity attitudes as a relevant factor in how Black American clients experience the psychological impact of media exposure to undue police violence.

Risks of obstructive genitourinary birth defects in relation to trihalomethane and haloacetic acid exposures: expanding disinfection byproduct mixtures analyses using relative potency factors.

BACKGROUND: Some disinfection byproducts (DBPs) are teratogens based on toxicological evidence. Conventional use of predominant DBPs as proxies for complex mixtures may result in decreased ability to detect associations in epidemiological studies. OBJECTIVE: We assessed risks of obstructive genitourinary birth defects (OGDs) in relation to 12 DBP mixtures and 13 individual component DBPs. METHODS: We designed a nested registry-based case-control study (210 OGD cases; 2100 controls) in Massachusetts towns with complete quarterly 1999-2004 data on four trihalomethanes (THMs) and five haloacetic acids (HAAs). We estimated temporally-weighted average DBP exposures for the first trimester of pregnancy. We estimated adjusted odds ratios (aORs) and 95% confidence intervals (CIs) for OGD in relation to individual DBPs, unweighted mixtures, and weighted mixtures based on THM/HAA relative potency factors (RPF) from animal toxicology data for full-litter resorption, eye defects, and neural tube defects. RESULTS: We detected elevated aORs for OGDs for the highest of bromodichloromethane (aOR = 1.75; 95% CI: 1.15-2.65), dibromochloromethane (aOR = 1.71; 95% CI: 1.15-2.54), bromodichloroacetic acid (aOR = 1.56; 95%CI: 0.97-2.51), chlorodibromoacetic acid (aOR = 1.97, 95% CI: 1.23-3.15), and tribromoacetic acid (aOR = 1.90; 95%CI: 1.20-3.03). Across unweighted mixture sums, the highest aORs were for the sum of three brominated THMs (aOR = 1.74; 95% CI: 1.15-2.64), the sum of six brominated HAAs (aOR = 1.43; 95% CI: 0.89-2.31), and the sum of nine brominated DBPs (aOR = 1.80; 95% CI: 1.05-3.10). Comparing eight RPF-weighted to unweighted mixtures, the largest aOR differences were for two HAA metrics, which both were higher with RPF weighting; other metrics had reduced or minimally changed ORs in RPF-weighted models.

Discovery of Amino Alcohols as Highly Potent, Selective, and Orally Efficacious Inhibitors of Leukotriene A4 Hydrolase.

The discovery of chiral amino alcohols derived from our previously disclosed clinical LTA4H inhibitor LYS006 is described. In a biochemical assay, their optical antipodes showed similar potencies, which could be rationalized by the cocrystal structures of these compounds bound to LTA4H. Despite comparable stabilities in liver microsomes, they showed distinct in vivo PK properties. Selective O-phosphorylation of the (R)-enantiomers in blood led to clearance values above the hepatic blood flow, whereas the (S)-enantiomers were unaffected and exhibited satisfactory metabolic stabilities in vivo. Introduction of two pyrazole rings led to compound (S)-2 with a more balanced distribution of polarity across the molecule, exhibiting high selectivity and excellent potency in vitro and in vivo. Furthermore, compound (S)-2 showed favorable profiles in 16-week IND-enabling toxicology studies in dogs and rats. Based on allometric scaling and potency in whole blood, compound (S)-2 has the potential for a low oral efficacious dose administered once daily.

Anti-brolucizumab immune response as one prerequisite for rare retinal vasculitis/retinal vascular occlusion adverse events.

In October 2019, Novartis launched brolucizumab, a single-chain variable fragment molecule targeting vascular endothelial growth factor A, for the treatment of neovascular age-related macular degeneration. In 2020, rare cases of retinal vasculitis and/or retinal vascular occlusion (RV/RO) were reported, often during the first few months after treatment initiation, consistent with a possible immunologic pathobiology. This finding was inconsistent with preclinical studies in cynomolgus monkeys that demonstrated no drug-related intraocular inflammation, or RV/RO, despite the presence of preexisting and treatment-emergent antidrug antibodies (ADAs) in some animals. In this study, the immune response against brolucizumab in humans was assessed using samples from clinical trials and clinical practice. In the brolucizumab-naïve population, anti-brolucizumab ADA responses were detected before any treatment, which was supported by the finding that healthy donors can harbor brolucizumab-specific B cells. This suggested prior exposure of the immune system to proteins with structural similarity. Experiments on samples showed that naïve and brolucizumab-treated ADA-positive patients developed a class-switched, high-affinity immune response, with several linear epitopes being recognized by ADAs. Only patients with RV/RO showed a meaningful T cell response upon recall with brolucizumab. Further studies in cynomolgus monkeys preimmunized against brolucizumab with adjuvant followed by intravitreal brolucizumab challenge demonstrated that high ADA titers were required to generate ocular inflammation and vasculitis/vascular thrombosis, comparable to RV/RO in humans. Immunogenicity therefore seems to be a prerequisite to develop RV/RO. However, because only 2.1% of patients with ADA develop RV/RO, additional factors must play a role in the development of RV/RO.

A root cause analysis to identify the mechanistic drivers of immunogenicity against the anti-VEGF biotherapeutic brolucizumab.

Immunogenicity against intravitreally administered brolucizumab has been previously described and associated with cases of severe intraocular inflammation, including retinal vasculitis/retinal vascular occlusion (RV/RO). The presence of antidrug antibodies (ADAs) in these patients led to the initial hypothesis that immune complexes could be key mediators. Although the formation of ADAs and immune complexes may be a prerequisite, other factors likely contribute to some patients having RV/RO, whereas the vast majority do not. To identify and characterize the mechanistic drivers underlying the immunogenicity of brolucizumab and the consequence of subsequent ADA-induced immune complex formation, a translational approach was performed to bridge physicochemical characterization, structural modeling, sequence analysis, immunological assays, and a quantitative systems pharmacology model that mimics physiological conditions within the eye. This approach revealed that multiple factors contributed to the increased immunogenic potential of brolucizumab, including a linear epitope shared with bacteria, non-natural surfaces due to the single-chain variable fragment format, and non-native drug species that may form over prolonged time in the eye. Consideration of intraocular drug pharmacology and disease state in a quantitative systems pharmacology model suggested that immune complexes could form at immunologically relevant concentrations modulated by dose intensity. Assays using circulating immune cells from treated patients or treatment-naïve healthy volunteers revealed the capacity of immune complexes to trigger cellular responses such as enhanced antigen presentation, platelet aggregation, endothelial cell activation, and cytokine release. Together, these studies informed a mechanistic understanding of the clinically observed immunogenicity of brolucizumab and associated cases of RV/RO.

Nonhematopoietic IRAK1 drives arthritis via neutrophil chemoattractants.

IL-1 receptor-activated kinase 1 (IRAK1) is involved in signal transduction downstream of many TLRs and the IL-1R. Its potential as a drug target for chronic inflammatory diseases is underappreciated. To study its functional role in joint inflammation, we generated a mouse model expressing a functionally inactive IRAK1 (IRAK1 kinase deficient, IRAK1KD), which also displayed reduced IRAK1 protein expression and cell type-specific deficiencies of TLR signaling. The serum transfer model of arthritis revealed a potentially novel role of IRAK1 for disease development and neutrophil chemoattraction exclusively via its activity in nonhematopoietic cells. Consistently, IRAK1KD synovial fibroblasts showed reduced secretion of neutrophil chemoattractant chemokines following stimulation with IL-1ß or human synovial fluids from patients with rheumatoid arthritis (RA) and gout. Together with patients with RA showing prominent IRAK1 expression in fibroblasts of the synovial lining, these data suggest that targeting IRAK1 may be therapeutically beneficial. As pharmacological inhibition of IRAK1 kinase activity had only mild effects on synovial fibroblasts from mice and patients with RA, targeted degradation of IRAK1 may be the preferred pharmacologic modality. Collectively, these data position IRAK1 as a central regulator of the IL-1ß-dependent local inflammatory milieu of the joints and a potential therapeutic target for inflammatory arthritis.

CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

Induced Chirality in Halide Perovskite Clusters through Surface Chemistry.

Chiroptical properties are of interest for various applications, including structure determination, polarized photodetectors, and spintronics. Inducing chiroptical activity into semiconductors is challenging because of difficulties in creating asymmetric crystal structures. One promising method is to use chirality transfer by deploying chiral organic molecules as capping ligands for nanocrystals. Experimentally, chiral-capped nanocrystals show emergent chiroptical signatures, but the mechanisms for chirality transfer remain unclear. Here we utilize atomistic modeling using time-dependent density functional theory calculations to explore chirality transfer in CsPbX3 (X = Cl, I) clusters capped with chiral diaminocyclohexane (DACH) enantiomers. When DACH enantiomers are bound to the cluster surface, the perovskite optical transitions gain chiral signatures. This observed chirality transfer is best rationalized by chiral molecular dipole-cluster transition dipole coupling. With multiple DACH molecules bound to the cluster surface, anisotropy factors are found to increase proportionally to the surface ligand density, providing mechanistic insight toward improving chiroptical functionality in semiconductor nanomaterials.

The effect of tactile and verbal guidance during scapulothoracic exercises: An EMG and kinematic investigation.

BACKGROUND: Clinician-led training through tactile and verbal guidance to improve muscle activity and joint motion are a common but understudied focus of therapeutic interventions for shoulder pain. The purpose of this study was to determine if clinician guidance changes scapulothoracic muscle activity and kinematics compared to unguided shoulder exercises. METHODS: Eleven participants with shoulder pain were studied. Electromyographic (EMG) sensors were placed on the serratus anterior and upper and lower trapezii. Scapulothoracic and sternoclavicular kinematics were collected using electromagnetic sensors. Five common resisted shoulder exercises were performed with the following guidance: unguided, combined (verbal and tactile cues), and verbal guidance only. One-way repeated measures ANOVAs determined the effect of guidance versus unguided conditions for each exercise. RESULTS: Nine of ten combinations of exercise and guidance techniques demonstrated a significant effect of guidance for either muscle activity or joint kinematics. The guidance condition with the most frequent significant improvements across all variables was the combined condition. The exercises with the most frequent significant improvements across all variables were the external rotation exercises. Variables improved most frequently were: upper:lower trapezius EMG ratio (up to 11%), sternoclavicular elevation (up to 6°) and scapulothoracic internal rotation positioning (up to 8°), and sternoclavicular retraction displacement (up to 5°). CONCLUSION: Shoulder muscle activity and kinematics during exercises can be modified by tactile and verbal guidance. Most improvements in muscle activity occurred with verbal guidance during external rotation exercises. Most improvements in joint positioning and movement occurred with combined guidance during external rotation exercises.

TNF leads to mtDNA release and cGAS/STING-dependent interferon responses that support inflammatory arthritis.

Tumor necrosis factor (TNF) is a key driver of several inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, in which affected tissues show an interferon-stimulated gene signature. Here, we demonstrate that TNF triggers a type-I interferon response that is dependent on the cyclic guanosine monophosphate-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. We show that TNF inhibits PINK1-mediated mitophagy and leads to altered mitochondrial function and to an increase in cytosolic mtDNA levels. Using cGAS-chromatin immunoprecipitation (ChIP), we demonstrate that cytosolic mtDNA binds to cGAS after TNF treatment. Furthermore, TNF induces a cGAS-STING-dependent transcriptional response that mimics that of macrophages from rheumatoid arthritis patients. Finally, in an inflammatory arthritis mouse model, cGAS deficiency blocked interferon responses and reduced inflammatory cell infiltration and joint swelling. These findings elucidate a molecular mechanism linking TNF to type-I interferon signaling and suggest a potential benefit for therapeutic targeting of cGAS/STING in TNF-driven diseases.

Process analysis of pluripotent stem cell differentiation to megakaryocytes to make platelets applying European GMP.

Quality, traceability and reproducibility are crucial factors in the reliable manufacture of cellular therapeutics, as part of the overall framework of Good Manufacturing Practice (GMP). As more and more cellular therapeutics progress towards the clinic and research protocols are adapted to comply with GMP standards, guidelines for safe and efficient adaptation have become increasingly relevant. In this paper, we describe the process analysis of megakaryocyte manufacture from induced pluripotent stem cells with a view to manufacturing in vitro platelets to European GMP for transfusion. This process analysis has allowed us an overview of the entire manufacturing process, enabling us to pinpoint the cause and severity of critical risks. Risk mitigations were then proposed for each risk, designed to be GMP compliant. These mitigations will be key in advancing this iPS-derived therapy towards the clinic and have broad applicability to other iPS-derived cellular therapeutics, many of which are currently advancing towards GMP-compliance. Taking these factors into account during protocol design could potentially save time and money, expediting the advent of safe, novel therapeutics from stem cells.

Audit of Psychosocial and Palliative Care Support for Children Having Allogeneic Stem Cell Transplants at the New Zealand National Allogeneic Transplant Centre.

Psychosocial and palliative care support during stem cell transplants (SCT) is known to improve outcomes. AIM: evaluate the support provided to children and families at the New Zealand National Allogeneic Stem Cell Transplant unit (NATC). METHOD: the psychosocial and palliative care support for children who received SCT between December 2012 and April 2018 was audited. RESULTS: of the 101 children who received SCT, 97% were reviewed by the social work team (SW) and 82% by the psychiatric consult liaison team (CLT) at least once during their illness. However, pre-transplant psychological assessment only occurred in 16%, and during the SCT admission, only 55% received SW support, and 67% received CLT support. Eight out of eighty-five families (9%) were offered support for siblings. Eight of the sixteen children who died were referred for pediatric palliative care (PPC) with all supported and half the families who experienced a death (n = 8; 50%) received bereavement follow up. CONCLUSION: although the majority received some social work and psychological support, auditing against the standards suggests the consistency of involvement could be improved. Referrals for PPC were inadequate and largely for end-of-life phase. Sibling support, in particular donor siblings, had insufficient psychological assessment and support. Key recommendations are provided to address this underperformance.

Transfer to the clinic: refining forward programming of hPSCs to megakaryocytes for platelet production in bioreactors.

The production of in vitro-derived platelets has great potential for transfusion medicine. Here, we build on our experience in the forward programming (FoP) of human pluripotent stem cells (hPSCs) to megakaryocytes (MKs) and address several aspects of the complex challenges to bring this technology to the bedside. We first identify clinical-grade hPSC lines that generate MKs efficiently. We design a bespoke media to maximize both production and maturity of MKs and improve platelet output. Crucially, we transition the lentiviral-based FoP of hPSCs to a nonviral inducible system. We also show how small molecules promote a definitive hematopoiesis phenotype during the differentiation process, thereby increasing the quality of the final product. Finally, we generate platelets using a bioreactor designed to reproduce the physical cues that promote platelet production in the bone marrow. We show that these platelets are able to contribute to both thrombus formation in vitro and have a hemostatic effect in thrombocytopenic mice in vivo.

Discovery of LYS006, a Potent and Highly Selective Inhibitor of Leukotriene A4 Hydrolase.

The cytosolic metalloenzyme leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB4 generation at low exposures in vivo, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.

An unexpected COVID-19 diagnosis during emergency surgery in a neonate.

A 4-day-old, 3.3 kg infant presented with suspected intestinal malrotation, necessitating emergent diagnostic laparoscopy. Intra-operatively, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) came back positive. This is the first case report of emergency surgery and anesthesia in a positive SARS-CoV-2 newborn. This report highlights a neonate with an incidental positive SARS-CoV-2 test, no known exposure history, negative polymerase chain reaction maternal testing, and absence of respiratory symptoms who required modified pressure control ventilation settings to adequately ventilate with the high-efficiency particulate air filter in situ.

IVF or ICSI for fertility preservation?

In IVF, eggs and sperm are added together for fertilisation to occur whereas ICSI involves injecting a single sperm into each egg. ICSI is very effective where sperm count or swimming is poor (male infertility) but is slightly riskier than IVF in terms of health problems in children, although these risks are small. However, the risk of no eggs fertilising is higher for IVF compared to ICSI and couples undertaking fertility preservation, for example, before cancer treatment, usually only have time for one attempt. Using fertility preservation treatment cycle data reported to Human Fertilisation and Embryology Authority (HFEA), this study shows that ICSI results in higher number of fertilised eggs and embryos for storage or treatment compared to IVF. However, 19% of eggs are not used in ICSI treatment, so IVF appears to be better overall. Clinics should choose IVF or ICSI for fertility preservation depending on sperm characteristics rather than using ICSI for all.

Chiroptical characterization tools for asymmetric small molecules - experimental and computational approaches for electronic circular dichroism (ECD) and anisotropy spectroscopy.

Synchrotron radiation electronic circular dichroism (SRECD) and anisotropy spectroscopy for both enantiomers of a group of small non-planar chiral molecules are reported here. The experimental SRECD spectra are compared to computational ECD spectra generated using time-dependent density functional theory and a thermal averaging over relevant molecular configurations. The combination of these experimental and computational characterization methodologies for such molecules enables the prediction and understanding of the spectral behavior of other small molecules, in addition to chiroptically characterizing members of the mandelic acid family substructure. Enantiomeric purity of samples can be evaluated in comparison with these spectra and the extent of photolytic enantioinduction can also be predicted using these experimental/calculated SRECD and anisotropy spectra.

Missed Opportunity Encounters for Early Diagnosis of HIV Infection in Adolescents.

BACKGROUND: Early diagnosis of HIV infection improves patient outcomes and reduces transmission. Adolescents make up one-fifth of new HIV diagnoses in the United States. We sought to quantify the number of missed opportunity encounters (MOEs) before HIV diagnosis for adolescents at a pediatric hospital (PediHosp) and a proximate adult hospital which employs universal HIV screening in its emergency department (ED) (CountyHosp). METHODS: An observational study at 2 academic tertiary care hospitals in the United States that included all adolescents 13-20 years old with a new diagnosis of behaviorally-acquired HIV infection from 2006 to 2017. MOE were defined as any encounter at PediHosp or CountyHosp after the latter of the individual's 13th birthday or the date 3 months after the individual's most recent negative HIV screen, and before the encounter of HIV diagnosis. Comparisons were made by site of diagnosis and location of MOE. RESULTS: Two-hundred five subjects met inclusion criteria: 68% male, 76% Black and 81% men who have sex with men. There were 264 MOE, the proportion of adolescent ED encounters that were MOE at the PediHosp ED was 8.3 MOE per 10,000 encounters and the proportion at the CountyHosp ED was 1.2 (relative risk = 6.7; 95% CI: 4.1-11.0; P < 0.001). CONCLUSIONS: MOE for HIV diagnosis in adolescents occur frequently and are greater in number at a PediHosp as compared with a similar adult setting with universal screening. Universal HIV screening protocols at PediHosp may identify HIV-positive adolescents earlier.

Interferon-γ signaling in human iPSC-derived neurons recapitulates neurodevelopmental disorder phenotypes.

Maternal immune activation increases the risk of neurodevelopmental disorders. Elevated cytokines, such as interferon-γ (IFN-γ), in offspring's brains play a central role. IFN-γ activates an antiviral cellular state, limiting viral entry and replication. Moreover, IFN-γ is implicated in brain development. We tested the hypothesis that IFN-γ signaling contributes to molecular and cellular phenotypes associated with neurodevelopmental disorders. Transient IFN-γ treatment of neural progenitors derived from human induced pluripotent stem cells increased neurite outgrowth. RNA sequencing analysis revealed that major histocompatibility complex class I (MHCI) genes were persistently up-regulated through neuronal differentiation-an effect that was mediated by IFN-γ-induced promyelocytic leukemia protein (PML) nuclear bodies. Critically, IFN-γ-induced neurite outgrowth required both PML and MHCI. We also found evidence that IFN-γ disproportionately altered the expression of genes associated with schizophrenia and autism, suggesting convergence between genetic and environmental risk factors. Together, these data implicate IFN-γ signaling in neurodevelopmental disorder etiology.