Understanding the Differences of Danusertib's Residence Time in Aurora Kinases A/B: Dissociation Paths and Key Residues Identified using Conventional and Enhanced Molecular Dynamics Simulations.

The accurate experimental estimation of protein-ligand systems' residence time (τ) has become very relevant in drug design projects due to its importance in the last stages of refinement of the drug's pharmacodynamics and pharmacokinetics. It is now well-known that it is not sufficient to estimate the affinity of a protein-drug complex in the thermodynamic equilibrium process in in vitro experiments (closed systems), where the concentrations of the drug and protein remain constant. On the contrary, it is mandatory to consider the conformational dynamics of the system in terms of the binding and unbinding processes between protein and drugs in in vivo experiments (open systems), where their concentrations are in constant flux. This last model has been proven to dictate much of several drugs' pharmacological activities in vivo. At the atomistic level, molecular dynamics simulations can explain why some drugs are more effective than others or unveil the molecular aspects that make some drugs work better in one molecular target. Here, the protein kinases Aurora A/B, complexed with its inhibitor Danusertib, were studied using conventional and enhanced molecular dynamics (MD) simulations to estimate the dissociation paths and, therefore, the computational τ values and their comparison with experimental ones. Using classical molecular dynamics (cMD), three differential residues within the Aurora A/B active site, which seems to play an essential role in the observed experimental Danusertib's residence time against these kinases, were characterized. Then, using WT-MetaD, the relative Danusertib's residence times against Aurora A/B kinases were measured in a nanosecond time scale and were compared to those τ values observed experimentally. In addition, the potential dissociation paths of Danusertib in Aurora A and B were characterized, and differences that might be explained by the differential residues in the enzyme's active sites were found. In perspective, it is expected that this computational protocol can be applied to other protein-ligand complexes to understand, at the molecular level, the differences in residence times and amino acids that may contribute to it.

Crystal structure and molecular dynamics simulations of a promiscuous ancestor reveal residues and an epistatic interaction involved in substrate binding and catalysis in the ATP-dependent vitamin kinase family members.

Enzymes with hydroxymethylpyrimidine/phosphomethylpyrimidine kinase activity (HMPPK) are essential in the vitamin B1 (thiamine pyrophosphate) biosynthesis and recycling pathways. In contrast, enzymes with pyridoxal kinase activity (PLK) produce pyridoxal phosphate (vitamin B6), an essential cofactor for various biochemical reactions. In the ATP-dependent vitamin kinases family, the members of PLK/HMPPK-like subfamily have both enzymatic activities. It has been proposed that the promiscuous PLK activity of ancestral HMPPK enzymes could have been the starting point for this activity. In earlier work, we reconstructed the ancestral sequences of this family and characterized the substrate specificity of the common ancestor between PLK/HMPPK-like and HMPPK enzymes (AncC). From these studies, the Gln45Met mutation was proposed as a critical event for the PLK activity emergence. Here, we crystallize and determine the AncC structure by X-ray crystallography and assess the role of the Gln45Met mutation by site-directed mutagenesis. Kinetic characterization of this mutant shows a significant increase in the PL affinity. Through molecular dynamics simulation and MM/PBSA calculations some residues, important for substrate interactions and catalysis, were identified in the wild type and in the mutated ancestor. Interestingly, a strong epistatic interaction responsible for the evolutionary pathway of the PLK activity in PLK/HMPPK-like enzymes was revealed. Also, other putative mutations relevant to PLK activity in modern PLK/HMPPK-like enzymes were identified.

Cationic Carbosilane Dendritic Systems as Promising Anti-Amyloid Agents in Type†2 Diabetes.

The most common denominator of many of the neurodegenerative diseases is badly folded protein accumulation, which results in the formation of insoluble protein deposits located in different parts of the organism, causing cell death and tissue degeneration. Dendritic systems have turned out to be a promising new therapeutic approach for the treatment of these diseases due to their ability to modulate the folding of these proteins. With this perspective, and focused on type 2 diabetes (T2D), characterized by the presence of deposits containing the amyloidogenic islet amyloid polypeptide (IAPP), we demonstrate how different topologies of cationic carbosilane dendrimers inhibit the formation of insoluble protein deposits in pancreatic islets isolated from transgenic Tg-hIAPP mice. Also, the results obtained by the modification of dendritic carbosilane wedges with the chemical chaperone 4-phenylbutyric acid (4-PBA) at the focal point confirmed their potential as anti-amyloid agents with a concentration efficiency in their therapeutic action five orders of magnitude lower than that observed for free 4-PBA. Computational studies, which determined the main interaction between IAPP and dendrimers at the atomic level, support the experimental work.

The molecular nature of the 17ß-Estradiol binding site in the voltage- and Ca2+-activated K+ (BK) channel ß1 subunit.

The accessory ß1 subunit modulates the Ca2+- and voltage-activated K+ (BK) channel gating properties mainly by increasing its apparent Ca2+ sensitivity. ß1 plays an important role in the modulation of arterial tone and blood pressure by vascular smooth muscle cells (SMCs). 17ß-estradiol (E2) increases the BK channel open probability (Po) in SMCs, through a ß1 subunit-dependent modulatory effect. Here, using molecular modeling, bioinformatics, mutagenesis, and electrophysiology, we identify a cluster of hydrophobic residues in the second transmembrane domain of the ß1 subunit, including the residues W163 and F166, as the binding site for E2. We further show that the increase in Po induced by E2 is associated with a stabilization of the voltage sensor in its active configuration and an increase in the coupling between the voltage sensor activation and pore opening. Since ß1 is a key molecular player in vasoregulation, the findings reported here are of importance in the design of novel drugs able to modulate BK channels.

Engineering Atrazine Loaded Poly (lactic- co-glycolic Acid) Nanoparticles to Ameliorate Environmental Challenges.

The use of herbicides plays a vital role in controlling weeds and conserving crops; however, its usage generates both environmental and economic problems. For example, herbicides pose a financial issue as farmers must apply large quantities to protect crops due to absorption rates of less than 0.1%. Therefore, there is a great need for the development of new methods to mitigate these issues. Here, we report for the first time the synthesis of poly(lactic- co-glycolic-acid) (PLGA) nanoherbicides loaded with atrazine as an active ingredient. We used potato plants as a biological model to assess the herbicidal activity of the engineered PLGA nanoherbicides. Our method produced nanoherbicides with an average size of 110 ± 10 nm prior to lyophilization. Fifty percent of the loaded atrazine in the PLGA matrix is released in 72 h. Furthermore, we performed Monte Carlo simulations to determine the chemical interaction among atrazine, PLGA, and the solvent system. One of the most significant outcomes of these simulations was to find the formation of a hydrogen bond of 1.9 Å between PLGA and atrazine, which makes this interaction very stable. Our in vitro findings showed that as atrazine concentration is increased in PLGA nanoparticles, potato plants undergo a significant decrease in stem length, root length, fresh weight, dry weight, and the number of leaves, with root length being the most affected. These experimental results suggest the herbicidal effectiveness of atrazine-loaded PLGA nanoherbicides in inhibiting the growth of the potato plant. Hence, we present the proof-of-concept for using PLGA nanoherbicides as an alternative method for inhibiting weed growth. Future studies will involve a deep understanding of the mechanism of plant-nanoherbicide interaction as well as the role of PLGA as a growth potentiator.

Penicillium purpurogenum produces a novel endo-1,5-arabinanase, active on debranched arabinan, short arabinooligosaccharides and on the artificial substrate p-nitrophenyl arabinofuranoside.

Penicillium purpurogenum secretes numerous lignocellulose-degrading enzymes, including four arabinofuranosidases and an exo-arabinanase. In this work, the biochemical properties of an endo-arabinanase (ABN1) are presented. A gene, coding for a potential ABN was mined from the genome. It includes three introns. The cDNA is 975 bp long and codes for a mature protein of 324 residues. The cDNA was expressed in Pichia pastoris. The enzyme is active on debranched arabinan and arabinooligosaccharides. In contrast to other characterized ABNs, inactive on p-nitrophenyl-α-L-arabinofuranoside (pNPAra), ABN1 is active on this substrate. The enzyme has an optimal pH of 4.5 and an optimal temperature of 30-35 °C. Calcium does not activate ABN1. ABN1 belongs to GH family 43 sub-family 6, and a Clustal alignment with sequences of characterized fungal ABNs shows highest identity (54.6%) with an ABN from Aspergillus aculeatus. A three-dimensional model of ABN1 was constructed and the docking with pNPAra was compared with similar models of an enzyme very active on this substrate and another lacking activity, both from GH family 43. Differences in the number of hydrogen bonds between enzyme and substrate, and distance between the substrate and the catalytic residues may explain the differences in activity shown by these enzymes.

Location of TEMPO derivatives in micelles: subtle effect of the probe orientation.

Partition coefficients for six 4-substituted derivatives of the 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) derivatives in aqueous solutions of reduced Triton X-100 (RTX-100) were determined by measurements of the probe EPR g-factor and of the fluorescence quenching of pyrene by the radical in the micelle. The partition constant attained a maximum value and then decreased with increasing probe hydrophobicity. Simulation of the probes inside the micelle showed that this trend could be rationalized by a change in the orientation of the 4-substituted TEMPO derivatives with the increasing substituent chain-length. The use of the EPR g-factor for the determination of partition constants of radicals in micellar systems was thus validated as a reliable and sensitive method, capable of describing the probe orientation in its microenvironment.

A transient receptor potential ion channel in Chlamydomonas shares key features with sensory transduction-associated TRP channels in mammals.

Sensory modalities are essential for navigating through an ever-changing environment. From insects to mammals, transient receptor potential (TRP) channels are known mediators for cellular sensing. Chlamydomonas reinhardtii is a motile single-celled freshwater green alga that is guided by photosensory, mechanosensory, and chemosensory cues. In this type of alga, sensory input is first detected by membrane receptors located in the cell body and then transduced to the beating cilia by membrane depolarization. Although TRP channels seem to be absent in plants, C. reinhardtii possesses genomic sequences encoding TRP proteins. Here, we describe the cloning and characterization of a C. reinhardtii version of a TRP channel sharing key features present in mammalian TRP channels associated with sensory transduction. In silico sequence-structure analysis unveiled the modular design of TRP channels, and electrophysiological experiments conducted on Human Embryonic Kidney-293T cells expressing the Cr-TRP1 clone showed that many of the core functional features of metazoan TRP channels are present in Cr-TRP1, suggesting that basic TRP channel gating characteristics evolved early in the history of eukaryotes.

Emergence of pyridoxal phosphorylation through a promiscuous ancestor during the evolution of hydroxymethyl pyrimidine kinases.

In the family of ATP-dependent vitamin kinases, several bifunctional enzymes that phosphorylate hydroxymethyl pyrimidine (HMP) and pyridoxal (PL) have been described besides enzymes specific towards HMP. To determine how bifunctionality emerged, we reconstructed the sequence of three ancestors of HMP kinases, experimentally resurrected, and assayed the enzymatic activity of their last common ancestor. The latter has ∼ 8-fold higher specificity for HMP due to a glutamine residue (Gln44) that is a key determinant of the specificity towards HMP, although it is capable of phosphorylating both substrates. These results show how a specific enzyme with catalytic promiscuity gave rise to current bifunctional enzymes.

Bifunctional ADP-dependent phosphofructokinase/glucokinase activity in the order Methanococcales--biochemical characterization of the mesophilic enzyme from Methanococcus maripaludis.

In some archaea, the phosphorylation of glucose and fructose 6-phosphate (fructose 6P) is carried out by enzymes that are specific for either substrate and that use ADP as phosphoryl donor. In the hyperthermophilic archaeon Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphorylate glucose and fructose 6P has been described. To determine whether the ability to phosphorylate both glucose and fructose 6P is a common feature for all enzymes of the order Methanococcales, we expressed, purified and characterized the unique homologous protein of the mesophilic archaea Methanococcus maripaludis. Assay of the enzyme activity with different sugars, metals and nucleotides allows us to conclude that the enzyme is able to phosphorylate both fructose 6P and glucose in the presence of ADP and a divalent metal cation. Kinetic characterization of the enzyme revealed complex regulation by the free Mg(2+) concentration and AMP, with the latter appearing to be a key metabolite. To determine whether this enzyme could have a role in gluconeogenesis, we evaluated the reversibility of both reactions and found that glucokinase activity is reversible, whereas phosphofructokinase activity is not. To determine the important residues for glucose and fructose 6P binding, we modeled the bifunctional phosphofructokinase/glucokinase enzyme from M. maripaludis and its interactions with both sugar substrates using protein­ligand docking. Comparison of the active site of the phosphofructokinase/glucokinase enzyme from M. maripaludis with the structural models constructed for all the homology sequences present in the order Methanococcales shows that all of the ADP-dependent kinases from this order would be able to phosphorylate glucose and fructose 6P, which rules out the current annotation of these enzymes as specific phosphofructokinases.