Remdesivir: Mechanism of Metabolic Conversion from Prodrug to Drug

Background: Remdesivir (GS-5734) has emerged as a promising drug during the challenging times of COVID-19 pandemic. Being a prodrug, it undergoes several metabolic reactions before converting to its active triphosphate metabolite. It is important to establish the atomic level details and explore the energy profile of the prodrug to drug conversion process. Methods: In this work, Density Functional Theory (DFT) calculations were performed to explore the entire metabolic path. Further, the potential energy surface (PES) diagram for the conversion of prodrug remdesivir to its active metabolite was established. The role of catalytic triad of Hint1 phosphoramidase enzyme in P-N bond hydrolysis was also studied on a model system using combined molecular docking and quantum mechanics approach. Results: The overall energy of reaction is 11.47 kcal/mol exergonic and the reaction proceeds through many steps requiring high activation energies. In the absence of a catalyst, the P-N bond breaking step requires 41.78 kcal/mol, which is reduced to 14.26 kcal/mol in a catalytic environment. Conclusion: The metabolic pathways of model system of remdesivir (MSR) were completely explored completely and potential energy surface diagrams at two levels of theory, B3LYP/6-311++G(d, p) and B3LYP/6-31+G(d), were established and compared. The results highlight the importance of an additional water molecule in the metabolic reaction. The P-N bond cleavage step of the metabolic process requires the presence of an enzymatic environment.

TM2D genes regulate Notch signaling and neuronal function in Drosophila

TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each model organism species that has been sequenced. Rare variants in TM2D3 are associated with Alzheimer’s disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. We knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the γ-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive motor and electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD.

Study on Microstructure and Fatigue Properties of FGH96 Nickel-Based Superalloy

In this study, using synchrotron radiation X-ray imaging, the microstructure, tensile properties, and fatigue properties of FGH96 nickel-based superalloy were tested, and the fatigue damage mechanism was analyzed. An analysis of the experimental results shows that the alloy structure is dense without voids or other defects. It was observed that the primary γ′ phase is distributed on the grain boundary in a chain shape, and the secondary γ′ phase is found inside the crystal grains. The X-ray diffraction (XRD) pattern indicates that no other phases were seen except for the γ and γ′ phases. The tensile strength of the alloy is 1570 MPa and the elongation is 12.1%. Using data fitting and calculation, it was found that the fatigue strength of the alloy under the condition of 5 × 106 cycles is 620.33 MPa. A fatigue fracture has the characteristics of secondary crack, cleavage step, fatigue stripe, tire indentation, and dimple. The fracture is a mix of cleavage fracture and ductile fracture. Through a three-dimensional reconstruction of the alloy synchrotron radiation imaging area, it was found that the internal defects are small and mostly distributed at the edge of the sample. The dimple morphology is formed by cavity aggregation and cavity germination resulting from defects in the material itself, fracture of the second-phase particles, and separation of the second-phase particles from the matrix interface. By analyzing the damage mechanism of fatigue fractures, it is concluded that the cleavage step is formed by the intersection of cleavage planes formed by branch cracks, with the main crack of the confluence extending forward to form a cleavage fracture. The crack propagation path was also analyzed, and under the action of cyclic load and tip passivation, the crack shows Z-shaped propagation.

In Vitro and In Silico Characterization of an Antimalarial Compound with Antitumor Activity Targeting Human DNA Topoisomerase IB

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.

Alzheimer's disease-associated TM2D genes regulate Notch signaling and neuronal function in Drosophila

TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each species. Rare variants in TM2D3 are associated with Alzheimer's disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. We knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the γ-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD.

An improved ChEC-seq method accurately maps the genome-wide binding of transcription coactivators and sequence-specific transcription factors

Mittal and colleagues have raised questions about mapping transcription factor locations on DNA using the MNase-based ChEC-seq method (Mittal et al., 2021). Partly due to this concern, we modified the experimental conditions of the MNase cleavage step and subsequent computational analyses, resulting in more stringent conditions for mapping protein-DNA interactions (Donczew et al., 2020). The revised method (dx.doi.org/10.17504/protocols.io.bizgkf3w) answers questions raised by Mittal et al. and, without changing earlier conclusions, identified widespread promoter binding of the transcription coactivators TFIID and SAGA at active genes. The revised method is also suitable for accurately mapping the genome-wide locations of DNA sequence-specific transcription factors.

Mechanistic Insight into the Ring-Opening Polymerization of ɛ-Caprolactone and L-Lactide Using Ketiminate-Ligated Aluminum Catalysts

The reactivity and the reaction conditions of the ring-opening polymerization of ɛ-caprolactone (ɛ-CL) and L-lactide (LA) initiated by aluminum ketiminate complexes have been shown differently. Herein, we account for the observation by studying the mechanisms on the basis of density functional theory (DFT) calculations. The calculations show that the ring-opening polymerization of ɛ-CL and LA are rate-determined by the benzoxide insertion and the C–O bond cleavage step, respectively. Theoretical computations suggest that the reaction temperature of L–LA polymerization should be higher than that of ɛ-CL one, in agreement with the experimental data. To provide a reasonable interpretation of the experimental results and to give an insight into the catalyst design, the influence of the electronic, steric, and thermal effects on the polymerization behaviors will be also discussed in this study.

Asymmetrical cleavages ofSleeping Beautytransposons generate multiple excised transposon fragments during transposition

ABSTRACTAlthough theSleeping Beauty(SB) transposon is the most validated DNA transposon used as a gene delivery vehicle in vertebrates, many details of the excision and integration steps in the transposition process are unclear. We have probed in detail the products of the excision step and apparent selective integration of a subset of those products during transposition. The standard model of SB transposase-mediated transposition includes symmetrical cleavages at both ends of the transposon for excision and re-integration in another DNA sequence. In our analysis of excised transposon fragments (ETFs), we found evidence for the requirement of certain flanking sequences for efficient cleavage and a significant rate of asymmetrical cleavage during the excision process that generates multiple ETFs. Our results suggest that the cleavage step by SB transposase is not as precise as indicated in most models. Repair of the donor ends can produce eight footprint sequences (TACTGTA, TACAGTA, TACATA, TACGTA, TATGTA, TACTA, TAGTA and TATA). Our data also suggest that mismatch repair (MMR) is not an essential requirement for footprint formation. Among the twenty liberated ETFs, only eight appear to effectively re-integrate into TA sites distributed across the genome, supporting earlier findings of unequal rates of excision and reintegration during SB transposition. These findings may be important in considerations of efficiency of SB transposon remobilization, selection of TA integration sites and detection of SB excision and integration loci, all of which may be important in human gene therapy.