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Size matters – and how you measure it: a gram-negative antibacterial example exceeding typical molecular weight limits
No full textFrom a porin permeation perspective, we characterized a monobactam antibiotic (compound 1) which has a higher molecular weight (MW) than the upper limit of 600 Da typically applied in designing such compounds, yet the compound is active against Gram-negative bacteria. Despite a MW of 692 Da, the compound is able to adopt a compact conformation based on 2D NMR data. The dimensions, projection area and dipole moment derived from this conformation are compatible with porin permeation, as are locations of polar groups upon superimposition to the crystal structure of a known antibiotic, ampicillin, bound to E. coli OmpF porin crystal structure. MIC shifts in a porin knockout strain are also consistent with compound 1 predominately permeating through porins. In conclusion, we describe a carefully characterized case of a molecule outside default design parameters where MW does not adequately characterize the 3D shape which is more directly related to permeability. Leveraging 3D design criteria would open up additional chemical space currently underutilized due to limitations perceived in 2D
Looking Forward: Cutting-Edge Technologies and Skills for Pathologists in the Future
No full textToxicologic pathology is one of the most valuable fields contributing to the advancement of animal and human health. With the ever-changing technological and economic environment, the basic skill set that pathologists are equipped with may require refinement to address the current and future needs. Periodically, pathologists must add relevant, new skills to their toolbox. The Career Development and Outreach Committee of the Society of Toxicologic Pathology (STP) sponsored a career development workshop entitled “Looking Forward: Cutting-edge Technologies and Skills for Pathologists in the Future” in conjunction with the STP 38th Annual Symposium. Experts were chosen to speak on artificial intelligence, clustered regularly interspaced short palindromic repeats technology, microRNAs, and next-generation sequencing. This article provides a summary of the talks presented at the workshop
Discovery of Potent and Selective Antibody-Drug Conjugates with Eg5 Inhibitors through Linker and Payload Optimization
No full textTargeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody-drug conjugate (ADC). Preliminary study were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbonate, or MC-VC-PABC cleavable linker. However, the resulting ADCs lacked antigen-specificity in-vivo, probably from premature release of the payload. Second-generation ADCs were then developed, using non-cleavable linkers, and the resulting conjugates (ADC-4, ADC-11 and ADC-12) led to in-vivo efficacy in a HER-2 expressing (SK-OV-3ip) mouse xenograft model in a target-dependent manner
Palladium-catalyzed C‒O coupling of amino alcohols for the synthesis of aryl ethers
No full textAmine containing aryl ethers are common pharmacophore motifs that continue to emerge from drug discovery efforts. As amino alcohols are readily available building blocks, practical methodologies for incorporating them into more complex structures are highly desireable. We report our efforts to explore the application of Pd-catalyzed C-O coupling methods to the arylation of 1,2- and 1,3-amino alcohols. We established reliable conditions, under which we explored the scope and limitations of the transformation. The insights gained have been valuable in employing this methodology within a fast-moving drug discovery environment, which we anticipate will be of general interest to the synthesis and catalysis communities
YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.
No full textBiliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/β-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/β-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells
Insights on Bimetallic Micellar Nanocatalysis for Buchwald-Hartwig Aminations
No full textA nanocatalyst for micellar Buchwald-Hartwig aminations is developed, thoroughly characterized, and applied on a variety of substrates. The catalyst is stable under ambient conditions for at least six months. The catalyst retained its activity after several cycles, and its structure remained intact as confirmed by NMR spectroscopy. Association of Pd nanoparticles with Cu by a phosphine ligand is revealed by 31P NMR spectroscopy and their linkage with the activated carbon surface is re-vealed by XAS analysis. Control NMR experiments revealed the binding of the ligand with both the Cu and Pd, and all phosphine molecules are under the same environment. In addition to NMR and XAS analysis, the catalyst is characterized by SEM, HRTEM, XPS, and TGA. Reactions are highly reproducible at variable scales. Environmentally benign, proline-based amphiphile PS-750-M is critical for catalytic activity, which is achieved under mild conditions in water as the reaction medium. The inherent sustainability of these conditions coupled with a low E factor achievable through robust recycling of catalyst and reaction medium demonstrates the significant utility of this technology
Detection and kinetics of persistent neutralizing anti-interferon-beta antibodies in patients with multiple sclerosis. Results from the ABIRISK prospective cohort study
No full textTwo validated assays, a bridging ELISA and a luciferase-based bioassay, were compared for detection of anti-drug antibodies (ADA) against interferon-beta (IFN-β) in patients with multiple sclerosis. Serum samples were tested from patients enrolled in a prospective study of 18 months. In contrast to the ELISA, when IFN-β-specific rabbit polyclonal and human monoclonal antibodies were tested, the bioassay was the more sensitive to detect IFN-β ADA in patients' sera. For clinical samples, selection of method of ELISA should be evaluated prior to the use of a multi-tiered approach. A titer threshold value is reported that may be used as a predictor for persistently positive neutralizing ADA
Opportunities and Challenges Using Artificial Intelligence (AI) in ADME/Tox
No full textA recent conference entitled Artificial Intelligence (AI) Applications in Biopharma Summit meeting organized a panel of scientists who work at the interface of machine learning and absorption, distribution, metabolism, excretion, and toxicology (ADME/Tox). This group represented small and big pharma companies with a combined total of 80 years of experience in the field. The questions generated and the discussion topic was felt to be of broader interest. With the recent rebirth of AI related to pharma, it is timely to present this collaborative commentary to capture the diverging opinions on the past present and future role of AI for ADME/Tox
Imaging Mass Cytometry and Single-cell Genomics Reveals Differential Depletion and Repletion of B cell Populations Following Ofatumumab Treatment in Cynomolgus Monkeys
No full textOfatumumab is the first, fully human, anti-CD20 monoclonal antibody in Phase 3 development for multiple sclerosis (MS). The study focused on changes in lymphocyte subsets in blood and lymphoid tissues and on potential novel biomarkers as a result of anti-CD20 antibody action in Cynomolgus monkeys treated with human equivalent doses of subcutaneous (s.c.) ofatumumab on Days 0, 7, and 14. Axillary lymph nodes (LNs) and blood samples were collected at various time points until Day 90. Lymphocyte subsets were quantified by flow cytometry, while morphological and immune cell changes were assessed by imaging mass cytometry (IMC), immunohistochemistry (IHC), and transcriptome analyses using single-cell methodology. Ofatumumab treatment resulted in a potent and rapid reduction of B cells along with a simultaneous drop in CD20+ T cell counts. The absolute numbers of marginal zone B cells in axillary LNs increased with treatment based on FACS analysis time. At Day 21, IHC revealed B-cell depletion in the perifollicular and interfollicular area of axillary LNs, while only the core of the germinal center was depleted of CD20+CD21+ cells. By Day 62, the perifollicular and interfollicular areas were abundantly infiltrated by CD21+ B cells and this distribution returned to the baseline cytoarchitecture by Day 90. By IMC CD20+CD3+CD8+ cells could be identified at the margin of the follicles, with a similar pattern of distribution at Day 21 and 90. Single-cell transcriptomics analysis showed that ofatumumab induced reversible changes in t-distributed stochastic neighbor embedding (t-SNE) defined B-cell subsets that may serve as biomarkers for drug action. In summary, low dose s.c. ofatumumab potently depletes both B cells and CD20+ T cells but apparently spares marginal zone (MZ) B cells in the spleen and LN. These findings add to our molecular and tissue-architectural understanding of ofatumumab treatment effects on B-cell subsets
Joints in the appendicular skeleton: Developmental mechanisms and evolutionary influences
No full textThe joints are a diverse group of skeletal structures, and their genesis, morphogenesis, and acquisition of specialized tissues have intrigued biologists for decades. Here we review past and recent studies on important aspects of joint development, including the roles of the interzone and morphogenesis of articular cartilage. Studies have documented the requirement of interzone cells in limb joint initiation and formation of most, if not all, joint tissues. We highlight these studies and also report more detailed interzone dissection experiments in chick embryos. Articular cartilage has always received special attention owing to its complex architecture and phenotype and its importance in long-term joint function. We pay particular attention to mechanisms by which neonatal articular cartilage grows and thickens over time and eventually acquires its multi-zone structure and becomes mechanically fit in adults. These and other studies are placed in the context of evolutionary biology, specifically regarding the dramatic changes in limb joint organization during transition from aquatic to land life. We describe previous studies, and include new data, on the knee joints of aquatic axolotls that unlike those in higher vertebrates, are not cavitated, are filled with rigid fibrous tissues and resemble amphiarthroses. We show that when axolotls metamorph to life on land, their intra-knee fibrous tissue becomes sparse and seemingly more flexible and the articular cartilage becomes distinct and acquires a tidemark. In sum, there have been considerable advances toward a better understanding of limb joint development, biological responsiveness, and evolutionary influences, though much remains unclear. Future progress in these fields should also lead to creation of new developmental biology-based tools to repair and regenerate joint tissues in acute and chronic conditions