Identification and Characterization of PDE8 Inhibitors Using a Fission Yeast Based High-throughput Screening Platform

Thesis advisor: Charles S. HoffmanIn this thesis, I describe the development of a screening platform for detecting PDE8A inhibitors using the cAMP-dependent glucose sensing pathway of the fission yeast Schizosaccharomyces pombe, which led us to discover several PDE8A selective inhibitors. In this system, the only PDE of the fission yeast is replaced with mammalian PDE8A1 in strains that have been engineered such that PDE inhibition is required to allow cell growth. Using this system, I screened 56 compounds obtained from PDE4 and PDE7 high throughput screens (HTSs) and identified a PDE4-PDE8 dual specificity inhibitor. Using this as a positive control, I developed a robust high-throughput screen (HTS) for PDE8A inhibitors and screened 240,267 compounds at the Harvard Medical School ICCB Screening Facility. Approximately 0.2 % of the screened compounds were potential PDE8A inhibitors with 0.03% displaying significant potency. Secondary assays of 367 of the most effective compounds against strains expressing PDE8A (both full length and catalytic domain), PDE4A and PDE7A or PDE7B led to the selection of structurally diverse compounds for further testing. To profile the selectivity of twenty-eight of these compounds, dose response assays were conducted using 16 yeast strains that express different PDE isoforms (representing all PDE families with the exception of the PDE6 family). These assays identified compounds with different patterns of inhibition, including structurally-distinct PDE8A-specific inhibitors. By evaluating the effects of these compounds for steroid production in mouse Leydig cells, biologically active compounds that can elevate steroid production were identified.Thesis (PhD) — Boston College, 2011.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology

Galactose epimerase deficiency: lessons from the GalNet registry.

BACKGROUND Galactose epimerase (GALE) deficiency is a rare hereditary disorder of galactose metabolism with only a few cases described in the literature. This study aims to present the data of patients with GALE deficiency from different countries included through the Galactosemia Network to further expand the existing knowledge and review the current diagnostic strategy, treatment and follow-up of this not well characterized entity. METHODS Observational study collecting medical data from December 2014 to April 2022 of 22 not previously reported patients from 14 centers in 9 countries. Patients were classified as generalized or non-generalized based on their genotype, enzyme activities in different tissues and/or clinical picture and professional judgment of the treating physician. RESULTS In total 6 patients were classified as generalized and 16 as non-generalized. In the generalized group, acute neonatal illness was reported in 3, cognitive and developmental delays were present in 5 and hearing problems were reported in 3. Four generalized patients were homozygous for the genetic variant NM_001008216.2:c.280G > A (p.Val94Met). In the non-generalized group, no clearly related symptoms were found. Ten novel genetic variants were reported in this study population. CONCLUSION The phenotypic spectrum of GALE deficiency ranges from asymptomatic to severe. The generalized patients have a phenotype that is in line with the 9 described cases in the literature and prescribing dietary interventions is the cornerstone for treatment. In the non-generalized group, treatment advice is more difficult. To be able to offer proper counseling, in addition to red blood cell enzyme activity, genetic studies, transferrin glycoform analysis and enzymatic measurements in fibroblasts are recommended. Due to lack of facilities, additional enzymatic testing is not common practice in many centers nor a tailored long-term follow-up is performed

Galactokinase deficiency:lessons from the GalNet registry

PURPOSE Galactokinase (GALK1) deficiency is a rare hereditary galactose metabolism disorder. Beyond cataract, the phenotypic spectrum is questionable. Data from affected patients included in the Galactosemias Network registry were collected to better characterize the phenotype. METHODS Observational study collecting medical data of 53 not previously reported GALK1 deficient patients from 17 centers in 11 countries from December 2014 to April 2020. RESULTS Neonatal or childhood cataract was reported in 15 and 4 patients respectively. The occurrence of neonatal hypoglycemia and infection were comparable with the general population, whereas bleeding diathesis (8.1% versus 2.17-5.9%) and encephalopathy (3.9% versus 0.3%) were reported more often. Elevated transaminases were seen in 25.5%. Cognitive delay was reported in 5 patients. Urinary galactitol was elevated in all patients at diagnosis; five showed unexpected Gal-1-P increase. Most patients showed enzyme activities ≤1%. Eleven different genotypes were described, including six unpublished variants. The majority was homozygous for NM_000154.1:c.82C>A (p.Pro28Thr). Thirty-five patients were diagnosed following newborn screening, which was clearly beneficial. CONCLUSION The phenotype of GALK1 deficiency may include neonatal elevation of transaminases, bleeding diathesis, and encephalopathy in addition to cataract. Potential complications beyond the neonatal period are not systematically surveyed and a better delineation is needed

Effect of UV aging on the thermo-mechanical properties of C-B-a and G-B-a hybrid composites: A study using TMA

In this study, C-B-A and G-B-A new hybrid composites are produced by compression molding using Carbon (C), Basalt (B), Aramid (A), and Glass (G) fibers, and the produced samples are scanned using micro-computed tomography and Micro-CT. The coefficient of thermal expansion (CTE), glass transition temperature (Tg), and dimensional changes are determined by Thermomechanical Analysis (TMA) technique and thus the limits of thermal expansion and contraction dimensions, softening, and thermomechanical properties of newly produced hybrid composites are determined. In addition, the produced composites are exposed to UV radiation in different cycles and the results are compared with UV radiation non-exposed samples.</p

Tensile behavior of functionally graded sandwich PLA-ABS produced via fused filament fabrication process

The study investigated the tensile behavior of Sandwich Functionally Graded Material (SFGM) fabricated using Additive Manufacturing (AM) technology experimentally and numerically. SFGMs are characterized by a gradual variation in composition and structure with respect to the forming volume from the lower and upper surfaces of the structure toward the center, resulting in a corresponding change in material properties. Fused Filament Fabrication (FFF), a widely used AM process, was used in the present work to fabricate the thermoplastic polymer-based SFGM specimens. SFGM were produced by the FFF method using ABS and PLA materials and subjected to tensile tests according to ASTMD638

Numerical Modeling of Mechanical Behavior of Functionally Graded Polylactic Acid–Acrylonitrile Benzidine Styrene Produced via Fused Deposition Modeling: Experimental Observations

Functionally graded materials (FGM) have attracted considerable attention in the field of composite materials and rekindled interest in research on composite materials due to their unique mechanical response achieved through material design and optimization. Compared to conventional composites, FGMs offer several advantages and exceptional properties, including improved deformation resistance, improved toughness, lightness properties, and excellent recoverability. This study focused on the production of functionally graded (FG) polymer materials by the additive manufacturing (AM) method. FG structures were produced by the fused deposition modeling (FDM) method using acrylonitrile benzidine styrene (ABS) and polylactic acid (PLA) materials, and tensile tests were performed according to ASTM D638. The effects of different layer thicknesses, volume ratios, and total thicknesses on mechanical behavior were investigated. The tensile standard of materials produced by additive manufacturing introduces geometric differences. Another motivation in this study is to reveal the differences between the results according to the ASTM standard. In addition, tensile tests were carried out by producing single-layer samples at certain volume ratios to create a numerical model with the finite element method to verify the experimental data. As a result of this study, it is presented that the FG structure produced with FDM improves mechanical behavior

A yeast-based chemical screen identifies a PDE inhibitor that elevates steroidogenesis in mouse Leydig cells via PDE8 and PDE4 inhibition.

A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC₅₀ value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems