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Usefulness of the BioGIT system in screening for differences in early exposure in the fasted state on an a priori basis
No full textThe main objective of the present study was to confirm the usefulness of BioGIT data in the evaluation of the impact of dose and/or formulation on early exposure after administration of immediate release (IR) or enabling drug products of low solubility active pharmaceutical ingredients (APIs) with a glass of water in the fasted state. BioGIT experiments were performed with four APIs: Compound Α (tablet, three dose levels), Compound E (capsule PiC1, capsule PiC2 and tablet), fenofibrate (Lipidil® capsule and Lipidil 145 ONE® tablet) and bedaquiline (HP-β-CD aqueous solution and tablet). Based on mean plasma AUC0-60min values which became available after completion of the BioGIT experiments, mean BioGIT AUC0-50min values were useful for the evaluation of the impact of dose and/or formulation on early exposure. Furthermore, the log-transformed mean BioGIT AUC0-50min ratios of two doses and/or two formulations calculated from data collected in this study and in a recent study with two diclofenac potassium products (Cataflam® tablet and Voltfast® sachet, same dose) (n=7 pairs of ratios), and the corresponding log-transformed mean plasma AUC0-60min ratios calculated from clinical data were included in a previously established correlation between log-transformed mean plasma AUC0–60min ratios with log-transformed mean BioGIT AUC0–50min ratios (n=9 pairs of ratios). The correlation between log-transformed plasma AUC0–60min ratios vs. log-transformed BioGIT AUC0–50min ratios was confirmed (n=16 pairs of ratios, R=0.90). Compared with the previously established correlation the statistical characteristics were improved. Based on this study, the BioGIT system could be useful as a screening tool for assessing the impact of dose and/or formulation differences on early exposure, after administration of immediate release or enabling drug products with a glass of water in the fasted state, on an a priori basis
SINGLE CELL TRANSCRIPTOMIC ANALYSIS OF RENAL ALLOGRAFT REJECTION REVEALS NOVEL INSIGHTS INTO INTRAGRAFT TCR CLONALITY
No full textBulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection
(ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/β sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/β cDNAs from CD8EXP into Jurkat 76 cells (TCR–/–) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in
matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection
Status check: Chemocatalysis & Biocatalysis = Chemoenzymatic Catalysis. Let’s Just Call it “Catalysis”…from the eyes of the fine chemical industry
No full textWhere is organic synthesis today? On the one hand, we have reagent-based chemistry, and in particular, chemocatalysis, done in organic solvents, capable of making virtually any target molecule. Then, there is the environmentally more attractive world of biocatalysis, where enzymes, in large measure used in water,1 whether natural or “new-to-nature” complement,2 or in an increasing number of cases even replace traditional reagents. But isn’t the ideal process one that at least has the option to use both, and even better, to merge these two areas, that today is referred to as chemoenzymatic catalysis? The state-of-affairs on this very topic is the subject of our review that recently appeared, with both approaches “meeting” in water.3 But what’s the reality here? Are industrial labs moving in this direction, embracing the obvious advantages from both the economic and sustainability perspectives? What obstacles are they facing, and how can all parties on both sides of the aisle (i.e., academic and industrial labs) gain a slice of this all-encompassing and growing chemoenzymatic catalysis “pie”, which as a topic, in time might rightly be shortened to just “catalysis”
Intrinsically disordered regions in the transcription factor MYC:MAX modulate DNA binding via intramolecular interactions
No full textThe basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor (TF) MYC is in large parts an intrinsically disordered oncoprotein. In complex with its obligate heterodimerization partner MAX, MYC preferentially binds E-Box DNA sequences (CANNTG). At promotors containing these sequence motifs, MYC controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. A vast network of proteins controls MYC function via intermolecular interactions. In this work, we establish another layer of MYC regulation by intramolecular interactions. We use Nuclear Magnetic Resonance (NMR) spectroscopy to identify and map multiple binding sites for the C-terminal MYC:MAX DNA binding domain (DBD) on the intrinsically disordered regions (IDRs) in the MYC N-terminus. We find that these binding events in trans are driven by electrostatic attraction, that they have distinct affinities, and that they are competitive with DNA binding. Thereby, we observe the strongest effects for the N-terminal MYC box 0 (Mb0), a conserved motif involved in MYC transactivation and target gene induction. We prepared recombinant full-length MYC:MAX complex and demonstrate that the interactions identified in this work are also relevant in cis, i.e. as intramolecular interactions. These findings are supported by Surface Plasmon Resonance (SPR) experiments, which revealed that intramolecular IDR:DBD interactions in MYC decelerate the association of MYC:MAX complexes to DNA. Our work offers new insights how bHLH-LZ TFs are regulated by intramolecular interactions, which opens up new possibilities for drug discover
Discovery of Amino Alcohols as Highly Potent, Selective, and Orally Efficacious Inhibitors of Leukotriene A4 Hydrolase.
No full textThe 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
Myeloid-specific deletion of activating transcription factor 6 alpha increases CD11b macrophage subpopulations and aggravates lung fibrosis.
No full textIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown etiology. The accumulation of macrophages is associated with disease pathogenesis. The unfolded protein response (UPR) has been linked to macrophage activation in pulmonary fibrosis. To date, the impact of activating transcription factor 6 alpha (ATF6α), one of the UPR mediators, on the composition and function of pulmonary macrophage subpopulations during lung injury and fibrogenesis is not fully understood. We began by examining the expression of Atf6α in IPF patients' lung single-cell RNA sequencing dataset, archived surgical lung specimens, and CD14 circulating monocytes. To assess the impact of ATF6α on pulmonary macrophage composition and pro-fibrotic function during tissue remodeling, we conducted an in vivo myeloid-specific deletion of Atf6α. Flow cytometric assessments of pulmonary macrophages were carried out in C57BL/6 and myeloid specific ATF6α-deficient mice in the context of bleomycin-induced lung injury. Our results demonstrated that Atf6α mRNA was expressed in pro-fibrotic macrophages found in the lung of a patient with IPF and in CD14 circulating monocytes obtained from blood of a patient with IPF. After bleomycin administration, the myeloid-specific deletion of Atf6α altered the pulmonary macrophage composition, expanding CD11b subpopulations with dual polarized CD38 CD206 expressing macrophages. Compositional changes were associated with an aggravation of fibrogenesis including increased myofibroblast and collagen deposition. A further mechanistic ex vivo investigation revealed that ATF6α was required for CHOP induction and the death of bone marrow-derived macrophages. Overall, our findings suggest a detrimental role for the ATF6α-deficient CD11b macrophages which had altered function during lung injury and fibrosis
Parkinson’s disease causality and heterogeneity: a proteogenomic view
No full textThe pathogenesis and clinical heterogeneity of Parkinson’s disease have been evaluated from molecular, pathophysiological, and clinical perspectives. High-throughput proteomic analysis of CSF has opened new opportunities for scrutinizing this heterogeneity. To date, this is the most comprehensive CSF-based proteomics profiling study in Parkinson’s disease with 569 patients (350 idiopathic patients, 65 GBA+ mutation carriers and 154 LRRK2+ mutation carriers), 534 controls, and 4135 proteins analyzed. Combining CSF aptamer-based proteomics with genetics we determined protein quantitative trait loci (pQTLs). Analyses of pQTLs together with summary statistics from the largest Parkinson’s disease genome wide association study (GWAS) identified 68 potential causal proteins by Mendelian randomization. The top causal protein, GPNMB was previously reported to be upregulated in the substantia nigra of Parkinson’s disease patients.
We also compared the CSF proteomes of patients and controls. The Parkinson’s disease cohort comprised not only LRRK2+ and GBA+ mutation carriers but also idiopathic patients. Proteome differences between GBA+ patients and unaffected GBA+ controls suggest degeneration of dopaminergic neurons, altered dopamine metabolism and increased brain inflammation. The proteins discriminating LRRK2+ patients from unaffected LRRK2+ controls, revealed dysregulated lysosomal degradation, altered alpha-synuclein processing, and neurotransmission. Proteome differences between idiopathic patients and controls suggest increased neuroinflammation, mitochondrial dysfunction / oxidative stress, altered iron metabolism and potential neuroprotection mediated by vasoactive substances.
Finally, we used proteomic data to stratify idiopathic patients into "endotypes". The identified endotypes show differences in cognitive and motor disease progression based on the use of previously reported protein-based risk scores.
In summary, we: i) identified causal proteins for Parkinson’s disease, ii) assessed CSF proteome differences in Parkinson’s disease patients of genetic and idiopathic etiology, and. iii) stratified idiopathic patients into robust clinically relevant subtypes. Our findings not only contribute to the identification of new therapeutic targets but also to shaping personalized medicine in CNS neurodegeneration
A chemically controlled Cas9 switch enables temporal modulation of diverse effectors.
No full textCRISPR-Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies
A Novel Autologous CAR-T Therapy, YTB323, with Preserved T-Cell Stemness Shows Enhanced CAR T-Cell Efficacy in Preclinical and Early Clinical Development.
No full textCAR T-cell product quality and stemness (Tstem) are major determinants of in vivo expansion, efficacy, and clinical response. Prolonged ex vivo culturing is known to deplete Tstem, affecting clinical outcome. YTB323, a novel autologous CD19-directed CAR T-cell therapy expressing the same validated CAR as tisagenlecleucel, is manufactured using a next-generation platform in <2 days. Here, we report preclinical development and preliminary clinical data of YTB323 in adults with r/r DLBCL (NCT03960840). In preclinical mouse models, YTB323 exhibited enhanced in vivo expansion and antitumor activity at lower doses than traditionally manufactured CAR T-cells. Clinically, at doses 25-fold lower than tisagenlecleucel, YTB323 showed 1) promising overall safety (CRS [any-grade, 35%; grade ≥3, 6%], neurotoxicity [any-grade, 25%; grade ≥3, 6%]); 2) ORR of 75% and 80% for DL1 and DL2, respectively; 3) comparable CAR T-cell expansion; and 4) preservation of T-cell phenotype. Current data support continued development of YTB323 for r/r DLBCL