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Complement factor B inhibition or deletion is not sufficient to prevent neurodegeneration in a murine model of glaucoma.
No full textPurpose: Activation of the classical complement pathway is thought to contribute to the development and progression of glaucoma. The role of alternative complement or amplification of common complement pathways in glaucoma is not well understood. We evaluated complement factor B expression in post-mortem human ocular tissues with or without glaucoma, and the effect of FB inhibition and deletion in a mouse ocular hypertensive model of glaucoma induced by photopolymerized hyaluronic acid glycidyl methacrylate (HAGM).
Methods: Human CFB mRNA in postmortem human eyes was assessed by RNAscope and TaqMan. The HAGM model was performed on C57BL6/J mice. The effect of FB in the HAGM model was evaluated with an oral FB small molecule inhibitor and Cfb-/- mice. Complement mRNA and proteins in mouse eyes were assessed by TaqMan and Western blot, respectively.
Results: CFB mRNA in human glaucomatous macular neural retina and optic nerve head was upregulated. Cfb mRNA is also upregulated in the HAGM model. Oral FB inhibitor, ED-79-GX17, dosed daily at 200 mg/kg for 3 days post IOP induction in WT mice showed complement inhibition in ocular tissues and significantly inhibited systemic complement levels. Daily dosing of ED-79-GX17 for 30 days or Cfb deletion was also unable to prevent RGC or axon loss 30 days post IOP induction in mice.
Conclusion: The essential alternative complement component FB may not substantially contribute to RGC loss in glaucoma in the mouse model of ocular hypertension despite upregulation of Cfb expression and activation of the alternative pathway. The relevance of these findings to human glaucoma remains to be determined
Use of machine learning for quantification of retinal pigment epithelium tight junctions improves assay sensitivity
No full textThe retinal pigment epithelium (RPE) is critical for maintaining outer retinal barrier homeostasis. In age-related macular degeneration (AMD), the RPE can undergo a dedifferentiation process that includes tight junction (TJ) loss and displacement of zonula occludens-1 (ZO-1), which may impair structural and functional integrity of the RPE barrier and contribute to disease pathogenesis. Our objective was to develop an automated and sensitive quantification method for TJ aberrations in an RPE immunofluorescence imaging assay, following treatment with TNFα or TGFβ2. However, quantifying ZO-1 morphological changes in the RPE using standard image analysis methods did not provide a satisfactory assay window. To address this challenge, we developed an imaging assay to quantify ZO-1 changes using a machine learning approach, enabling enhanced phenotypic characterization of the ZO-1 changes in RPE cells and improved assay sensitivity. We were also able to capture and quantify the reversal of these changes using the TNFα inhibitor, etanercept with this imaging assay. Our findings indicated that this machine learning ZO-1 quantification assay could serve as a potential phenotypic readout for RPE dedifferentiation and enabling large-scale mechanistic studies
Ophiobolin A Covalently Targets Mitochondrial Complex IV Leading to Metabolic Collapse in Cancer Cells.
No full textOphiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anticancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of the OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets: lysine-72 of cytochrome c oxidase subunit 5A (COX5A) and cysteine-53 of mitochondrial hypoxia induced gene 1 domain family member 2A (HIGD2A). These two subunit proteins are part of complex IV (cytochrome C oxidase) within the electron transport chain and contributed significantly to the antiproliferative activity of OPA. OPA activated mitochondrial respiration in a COX5A- and HIGD2A-dependent manner, leading to an initial spike in mitochondrial ATP and heightened mitochondrial oxidative stress. OPA compromised mitochondrial membrane potential, ultimately leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anticancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death
Contributions of Hyperactive Mutations in Mpro from SARS-CoV-2 to Drug Resistance.
No full textThe appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by the SARS-CoV-2 virus, are major concerns for human health. Many drugs target enzymes, and resistance-conferring mutations impact inhibitor binding or enzyme activity. Nirmatrelvir, the most widely used inhibitor currently used to treat SARS-CoV-2 infections, targets the main protease (Mpro) preventing it from processing the viral polyprotein into active subunits. Our previous work systematically analyzed resistance mutations in Mpro that reduce binding to inhibitors; here, we investigate mutations that affect enzyme function. Hyperactive mutations that increase Mpro activity can contribute to drug resistance but have not been thoroughly studied. To explore how hyperactive mutations contribute to resistance, we comprehensively assessed how all possible individual mutations in Mpro affect enzyme function using a mutational scanning approach with a fluorescence resonance energy transfer (FRET)-based yeast readout. We identified hundreds of mutations that significantly increased the Mpro activity. Hyperactive mutations occurred both proximal and distal to the active site, consistent with protein stability and/or dynamics impacting activity. Hyperactive mutations were observed 3 times more than mutations which reduced apparent binding to nirmatrelvir in recent studies of laboratory-grown viruses selected for drug resistance. Hyperactive mutations were also about three times more prevalent than nirmatrelvir binding mutations in sequenced isolates from circulating SARS-CoV-2. Our findings indicate that hyperactive mutations are likely to contribute to the natural evolution of drug resistance in Mpro and provide a comprehensive list for future surveillance efforts
Graphitic Carbon Nitride as a Photocatalyst for Decarboxylative C(sp2)-C(sp3) Couplings via Nickel Catalysis.
No full textThe development of robust and reliable methods for the construction of C(sp2)-C(sp3) bonds is vital for accessing an increased array of structurally diverse scaffolds in drug discovery and development campaigns. While significant advances towards this goal have been achieved using metallaphotoredox chemistry, many of these methods utilise photocatalysts based on precious-metals due to their efficient redox processes and tuneable properties. However, due to the cost, scarcity, and toxicity of these metals, the search for suitable replacements should be a priority. Here, we show the use of commercially available heterogeneous semiconductor graphitic carbon nitride (gCN) as a photocatalyst, combined with nickel catalysis, for the cross-coupling between aryl halide and carboxylic acid coupling partners. gCN has been shown to engage in single-electron-transfer (SET) and energy-transfer (EnT) processes for the formation of C-X bonds, and in this manuscript we overcome previous limitations to furnish C-C over C-O bonds using carboxylic acids. A broad scope of both aryl halides and carboxylic acids is presented, and recycling of the photocatalyst demonstrated. The mechanism of the reaction is also investigated
Accelerating Covalent Binding Studies: Direct mass shift measurement with acoustic ejection and TOF-MS.
No full textTracking chemical reactions by measuring incurred mass shifts upon successful binding is a direct and attractive alternative to existing assays based on chemical tags. Traditional methods use liquid chromatography-mass spectrometry (LC-MS), and because the required buffers are not amenable to direct MS injection, sample pre-treatment is needed to desalt. This leads to analysis times from ten seconds to minutes per sample, limiting throughput and preventing widespread application. Combining an acoustic ejection (AE) interface with a time-of-flight mass spectrometer (MS) removes this bottleneck, as samples can be directly introduced at rates of up to one second per sample. This article describes a complete workflow for measuring the covalent binding of compounds to proteins in real-time, from assay to data evaluation. It is noteworthy that this is the first instance of using SCIEX Echo® MS+ system with ZenoTOF 7600 system to study the kinetic regimes of covalent binding
Discovery and Optimization of First-in-Class Molecular Glue Degraders of the WIZ Transcription Factor for Fetal Hemoglobin In-duction to Treat Sickle Cell Disease
No full textSickle cell disease (SCD) is a prevalent, life-threatening condition with few treatment options, attributed to a heritable mutation in β-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) with small molecules has been pursued as a treatment to ameliorate many disease complications, but with limited success. Herein we report the discovery of 10, a novel, potent and selective molecular glue degrader of the transcription factor WIZ that robustly induces HbF expression as a potential treatment for SCD. 10 was optimized from a phenotypic screening hit utilizing insights from x-ray crystallography and computational modeling to improve potency, selectivity and in vivo exposure. In an hNBSGW mouse xenograft model, 10 demonstrated robust WIZ degradation and HbF induction, superior to the standard-of-care
Discovery of Darovasertib (NVP-LXS196), a Pan-PKC Inhibitor for the Treatment of Metastatic Uveal Melanoma.
No full textUveal melanoma (UM) is the most common primary intraocular malignancy in the adult eye. Despite the aggressive local management of primary UM, the development of metastases is common with no effective treatment options for metastatic disease. Genetic analysis of UM samples reveals the presence of mutually exclusive activating mutations in the Gq alpha subunits GNAQ and GNA11. One of the key downstream targets of the constitutively active Gq alpha subunits is the protein kinase C (PKC) signaling pathway. Herein, we describe the discovery of darovasertib (NVP-LXS196), a potent pan-PKC inhibitor with high whole kinome selectivity. The lead series was optimized for kinase and off target selectivity to afford a compound that is rapidly absorbed and well tolerated in preclinical species. LXS196 is being investigated in the clinic as a monotherapy and in combination with other agents for the treatment of uveal melanoma (UM), including primary UM and metastatic uveal melanoma (MUM)
Use of Pharmacokinetic and Pharmacodynamic Data to Develop the CDK4/6 Inhibitor Ribociclib for Patients with Advanced Breast Cancer.
No full textRibociclib is an orally bioavailable, selective cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor. CDK4/6 inhibition by ribociclib leads to retinoblastoma tumor suppressor protein (Rb) reactivation, thereby restoring Rb-mediated cell cycle arrest. Ribociclib is approved for the treatment of patients with hormone receptor-positive/human epidermal growth factor receptor-2-negative (HR+/HER2-) advanced breast cancer (ABC), at the dose of 600 mg once daily (QD) during cycles of 21 days on/7 days off, with optional dose reduction to 400 mg and 200 mg. Ribociclib is rapidly absorbed with a median time to reach maximum plasma concentration of 2.4 h, mean half-life of 32.0 h and oral bioavailability of 65.8% at 600 mg. It is eliminated mainly by hepatic metabolism (~ 84% of total elimination), mostly by cytochrome P450 (CYP) 3A4. Age, body weight, race, baseline Eastern Cooperative Oncology Group status, food, mild hepatic impairment, mild-to-moderate renal impairment, proton pump inhibitors, and combination partners (non-steroidal aromatase inhibitors or fulvestrant) have no clinically relevant impact on ribociclib exposure. Ribociclib inhibits CYP3A at 600 mg leading to increased exposure of CYP3A substrates. Strong CYP3A inhibitors or inducers increase or decrease, respectively, ribociclib exposure. Exposure-safety and exposure-efficacy analyses support the clinical benefit of the 600 mg QD starting dose, with potential individualized dose reductions to 400 mg and 200 mg for effective management of the adverse events neutropenia and QTcF interval prolongation, while maintaining efficacy, in patients with HR+/HER2- ABC. Overall, these clinical pharmacology data informed ribociclib dose justification and clinical development, as well as its prescribing information for clinical use in advanced breast cancer patients