IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD

Approaches in Enhancing Antioxidant Defense in Plants

This Special Issue, “Approaches in Enhancing Antioxidant Defense in Plants” published 13 original research works and a couple of review articles that discuss the various aspects of plant oxidative stress biology and ROS metabolism, as well as the physiological mechanisms and approaches to enhancing antioxidant defense and mitigating oxidative stress. These papers will serve as a foundation for plant oxidative stress tolerance and, in the long term, provide further research directions in the development of crop plants’ tolerance to abiotic stress in the era of climate change

The effects of photosymbiosis on gene expression in the facultatively symbiotic coral Astrangia poculata, with a focus on NF-kappaB signaling and antioxidant enzymes

Corals are critical to marine biodiversity and human welfare. Coral reefs cover <1% of the seafloor but support ~1/3 of all marine species. Approximately 1.5 billion people live within 100 km of coral reefs, relying upon them for food, income from tourism, and protection from storms. Their economic value has been estimated at $375 billion annually. The foundation of coral reefs is the intracellular symbiosis between corals and photosynthetic dinoflagellates of the family Symbiodiniaceae. Tropical corals satisfy up to 95% of their nutritional requirements through photosynthesis, and their ability to construct reefs is biochemically coupled to photosynthesis. While permitting corals to thrive, photosymbiosis also increases their exposure to environmental stressors and vulnerability to climate change. Reliance on photosynthesis restricts reef-building corals to shallow, clear, tropical waters, where they experience higher temperatures and UV exposure. The generation of reactive oxygen species by the symbiont also exposes corals to greater oxidative stress. The symbiosis is particularly sensitive to climate change: all of the mass coral bleaching events have occurred since 1982, driven by elevated ocean temperatures. Molecular cross-talk between host and symbiont impacts resilience of the coral holobiont and resistance to bleaching. Unfortunately, we know little about how photosymbiosis impacts expression or activity of coral genes. Tropical corals engage in an obligate symbiosis with Symbiodiniaceae, so we cannot study their gene expression in a stable aposymbiotic state. However, the northern star coral, Astrangia poculata, engages in a facultative symbiosis with Symbiodiniaceae. I used RNA sequencing to investigate how symbiosis impacts gene expression in A. poculata, focusing on genes implicated in photosymbiosis: antioxidant enzymes (specifically superoxide dismutases) and the NF-κB signaling pathway. From an improved transcriptome assembly, I recovered core elements of a primitively simple NF-κB signaling pathway and a rich complement of SOD proteins. 273 coral transcripts—many associated with protein metabolism and vesicle-mediated transport— were differentially expressed in symbiotic versus aposymbiotic corals. Unlike in the facultatively symbiotic sea anemone Exaiptasia, symbiosis was not associated with depressed NF-κB transcript levels. IKKε, a potential positive regulator of NF-κB activity, was strongly up-regulated, as was one particular superoxide dismutase

In vitro and in vivo protein folding under stress

Proteins are subject to a variety of stresses in biological organisms, including pressure and temperature, which are the easiest stresses to simulate by molecular dynamics simulations. The thesis will focus on discussing the effect of pressure and thermal stress on proteins including some of the fast-folding model proteins, who’s in vitro folding can be fully simulated on computers and compared directly with experiments. Pressure and temperature are prototypical perturbations that illustrate how close many proteins are to instability, a property that cells can exploit to control protein function. I will conclude with some recent in-cell experiments, and progress being made in measuring protein stability and function inside live cells under high pressure conditions. In chapters 2 and 3, fast-folding WW domains were studied (best-characterized systems for comparing experiments with simulations) by T-jump relaxation in conjunction with protein engineering. Chapter 1 is a comprehensive data set of mutational Φ-values (ΦM) as indicators for folding transition-state structure of 65 side chain, 7 backbone hydrogen bond, and 6 deletion and /or insertion mutants within loop 1 of the 34-residue hPin1 WW domain. We probed the robustness of the two hydrophobic clusters in the folding transition state, and discussed how local backbone disorder in the native-state can lead to non-classical ΦM‐values (ΦM > 1) in the rate-determining loop 1 substructure, and conclusively identify mutations and positions along the sequence that perturb the folding mechanism from loop 1-limited toward loop 2-limited folding. In chapter 2 we mutated the FBP 28 WW domain (formin-binding protein; Leu26 by Asp26 or Trp26) to alter the folding scenario from three-state folding toward two-state or downhill folding at temperatures below the melting point of the protein. The investigation was conducted using a combination of simulations over a broad temperature range with experimental temperature-jump data. Chapter 4 is focused on how attaching fluorescent protein tags to a host protein in vitro has a large non-additive effect on its folding free energy. We compared an unlabeled, three singly-labeled, and a doubly-labeled enzyme PGK (phosphoglycerate kinase). Two mechanisms for non-additivity were proposed. In the “quinary interaction” mechanism, two tags interact transiently with one another, relieving the host protein from unfavorable tag–protein interactions. In the “crowding” mechanism, adding two tags provides the minimal crowding necessary to overcome destabilizing interactions of individual tags with the host protein. Both of these mechanisms affect protein stability in cells; they must also be considered for tagged proteins used for reference in vitro. In Chapter 5 we showed that the protein unfolding/refolding reaction can be driven by a periodic thermal excitation above the reaction threshold. We were also able to speed up the reaction from an undetectable to a detectable rate by the addition of artificial thermal noise. A maximum in the recovered signal as a function of thermal noise was seen, a stochastic resonance. The study alluded that correlated noise is a physically and chemically plausible mechanism by which cells could modulate biomolecular dynamics during threshold processes such as signaling. Chapter 6 explores folding competing with misfolding or aggregation on the μs time scale using tethered WW domains. Tethered protein construct was engineered by linking two or more copies of the fast folding Fip35 WW domain with a flexible linker. We observed that adding more monomer units led to thermodynamic destabilization and slower folding rates, along with an abrupt onset of protein-protein interaction. Kinetics were determined by performing ultrafast laser temperature jump experiments at different temperatures and denaturant concentration. A simple multimeric network model is also proposed for globally fitting the thermodynamics and kinetics data. In the final chapter 7 of this thesis folding of an enzyme phosphoglycerate kinase (PGK) was studied under high pressure stress in different bacterial cytoplasm. The motivation was to understand how cell is capable of modulating the stability of its proteome when subjected to external stress especially high hydrostatic pressure. The thermodynamic stability of PGK was measured in two different strains Wildtype MG1655 and known pressure resistant J1 strain. These results were compared to in vitro experiments to reveal that cellular environment has an overall stabilizing effect on the protein thermodynamic stability but different cellular cytoplasm doesn’t affect the stability of PGK significantly

Natural Medicine in Therapy

Enter For a long time, natural medicine has been used as a therapeutic therapy based on generations of indigenous practices. Today the rise in natural remedies has been largely driven by public demand and billions of dollars are spent annually on herbal medicines. It is therefore important to document the effectiveness of natural medicine, its potential side effects, and potential interactions