The past decade has witnessed a paradigm shift in preclinical drug discovery with structure-based drug design (SBDD) making a comeback while high-throughput screening (HTS) methods have continued to ...generate disappointing results. There is a deficit of information between identified hits and the many criteria that must be fulfilled in parallel to convert them into preclinical candidates that have a real chance to become a drug. This gap can be bridged by investigating the interactions between the ligands and their receptors. Accurate calculations of the free energy of binding are still elusive; however progresses were made with respect to how one may deal with the versatile role of water. A corpus of knowledge combining X-ray structures, bioinformatics and molecular modeling techniques now allows drug designers to routinely produce receptor homology models of increasing quality. These models serve as a basis to establish and validate efficient rationales used to tailor and/or screen virtual libraries with enhanced chances of obtaining hits. Many case reports of successful SBDD show how synergy can be gained from the combined use of several techniques. The role of SBDD with respect to two different classes of widely investigated pharmaceutical targets: (a) protein kinases (PK) and (b) G-protein coupled receptors (GPCR) is discussed. Throughout these examples prototypical situations covering the current possibilities and limitations of SBDD are presented.
Understanding the mechanism and specificity of substrate binding in the cytochrome P450 (P450) superfamily is an important step toward explaining its key role in drug metabolism, toxicity, xenobiotic ...degradation, and several biosynthetic pathways. Here we investigate the ligand exit pathways and mechanisms of P450cam (CYP101), P450BM-3 (CYP102), and P450eryF (CYP107A1) by using random expulsion molecular dynamics and classical molecular dynamics simulations. Although several different pathways are found for each protein, one pathway is common to all three. The mechanism of ligand exit along this pathway is, however, quite different in the three different proteins. For P450cam, small backbone conformational changes, in combination with aromatic side chain rotation, allow for the passage of the rather rigid, compact, and hydrophobic substrate, camphor. In P450BM-3, larger transient backbone changes are observed on ligand exit. R47, situated at the entrance to the channel, appears important in guiding negatively charged fatty acid substrates in and out of the active site. In P450eryF, an isolated buried arginine, R185, stabilized by four hydrogen bonds to backbone carbonyl oxygen atoms, is located in the exit channel and is identified as having a particularly unusual functionality, dynamically gating channel opening. The results for these three P450s suggest that the channel opening mechanisms are adjusted to the physico-chemical properties of the substrate and can kinetically modulate protein-substrate specificity.
To bind at an enzyme's active site, a ligand must diffuse or be transported to the enzyme's surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. ...Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also influence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-influenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and β -lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as ``ionic tethering.'' We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme's surroundings even when the substrate is nonpolar.
Quantum chemical calculations on the isomerization of 24-methylenecycloartanol are described. An energetically viable mechanism, with a rate-determining protonation step, is proposed. This ...rearrangement may find applicability in tests for determining if an olive oil has been refined.
Graphical Abstract
The results of density functional calculations on the mechanism of a rearrangement of a sterol proposed to occur during olive oil heating are described. These results indicate that this rearrangement can indeed occur at a fast rate under refining conditions, but will not occur at an appreciable rate under conditions for producing extra virgin olive oil.
A long-standing puzzle in structure−function studies of cytochrome P450cam is how the substrate, camphor, reaches the buried active site. The crystal structure shows no channel from the surface to ...the active site large enough for substrate to pass through. Recent experiments indicate that access of the rather nonpolar substrate to the active site is controlled by electrostatic interactions and may involve rupture of the two salt links to Asp251 Deprez, E., Gerber, N. C., Di Primo, C., Douzou, P., Sligar, S. G., & Hui Bon Hoa, G. (1994) Biochemistry 33, 14464−14468. Consequently, we have computed the electrostatic strength of 53 ionic pairs, including 32 salt links, in cytochrome P450cam by numerical solution of the finite-difference linearized Poisson−Boltzmann equation. The calculated electrostatic free energies, ΔG tot, of the salt links range from −9 to +6 kcal/mol with approximately 60% of the salt links being energetically favorable and 40% being unfavorable with respect to mutation to their uncharged, nonpolar isosteres. Strikingly, of the four most stable salt links in the protein (ΔG tot < −6 kcal/mol), two involve the propionate groups of the heme and the other two involve Asp251. In the modeled D251N mutant, for which electrostatic effects on substrate binding are diminished, the latter two salt links lose their stability (ΔG tot > −2.4 kcal/mol). Thus it appears that cytochrome P450cam has evolved four unusually strong salt bridges, stabilized by surrounding charged and polar groups in the protein, to keep its heme cofactor in place and to regulate substrate binding.
We analyze here 7 very first strains of OMICRON the SARS-CoV2 new variant from South Africa, the USA (California and Minesota), Canada and Belgium. We applied, at the scale of the whole genome and ...the spike gene, the biomathematics method of Fibonacci meta-structure fractal analysis applied to the UA / CG proportions. We have evidenced the RUPTURE of OMICRON with respect to ALL the previous variants: D614G, ALPHA, BETA, GAMMA, DELTA. Remarkably, it is observed that the density of OMICRON mutations in the SPIKE PRION region is more than 8 times that of the rest of the Spike protein. In particular, we suggest that the mRNA stabilizing secondary structure ("hairpin" conformation) in the spike of all variants is degraded in OMICRON, probably making its mRNA more fragile. The loss of long-range fractal meta-structures in the OMICRON spike gene are in line with common knowledge on the mechanisms of epidemic ending, involving recombination of heavily mutated RNA fragments of the virus, with the possible inference of a distinct helper virus. This would indicate that the SARS-CoV2 is under very strong evolutionary pressure, possibly marking the end of the pandemic. We are studying more particularly the prion-like region of the spike, the mutations rate of which is 8 times higher in OMICRON than that of the whole spike protein.
An analysis of a molecular dynamics simulation of metmyoglobin in an explicit solvent environment of 3,128 water molecules has been performed. Both statics and dynamics of the protein-solvent ...interface are addressed in a comparison with experiment. Three-dimensional density distributions, temperature factors, and occupancy weights are computed for the solvent by using the trajectory coordinates. Analysis of the hydration leads to the localization of more than 500 hydration sites distributed into multiple layers of solvation located between 2.6 and 6.8 A from the atomic protein surface. After locating the local solvent density maxima or hydration sites we conclude that water molecules of hydration positions and hydration sites are distinct concepts. Both global and detailed properties of the hydration cluster around myoglobin are compared with recent neutron and X-ray data on myoglobin. Questions arising from differences between X-ray and neutron data concerning the locations of the protein-bound water are investigated. Analysis of water site differences found from X-ray and neutron experiments compared with our simulation shows that the simulation gives a way to unify the hydration picture given by the two experiments.
In molecular sciences, articles tend to revolve around 2D representations of 3D molecules, and sighted scientists often resort to 3D virtual reality software to study these molecules in detail. Blind ...and visually impaired (BVI) molecular scientists have access to a series of audio devices that can help them read the text in articles and work with computers. Reading articles published in this journal, though, is nearly impossible for them because they need to generate mental 3D images of molecules, but the article-reading software cannot do that for them. We have previously designed AsteriX, a web server that fully automatically decomposes articles, detects 2D plots of low molecular weight molecules, removes meta data and annotations from these plots, and converts them into 3D atomic coordinates. AsteriX–BVI goes one step further and converts the 3D representation into a 3D printable, haptic-enhanced format that includes Braille annotations. These Braille-annotated physical 3D models allow BVI scientists to generate a complete mental model of the molecule. AsteriX–BVI uses Molden to convert the meta data of quantum chemistry experiments into BVI friendly formats so that the entire line of scientific information that sighted people take for granted—from published articles, via printed results of computational chemistry experiments, to 3D models—is now available to BVI scientists too. The possibilities offered by AsteriX–BVI are illustrated by a project on the isomerization of a sterol, executed by the blind co-author of this article (HBW).
Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics. Cytochrome ...P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s. However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations. Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product. Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data. Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors. In contrast to large backbone motions (up to 4 A) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 A) in conjunction with side-chain rotations. Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed.
The dynamics of water at the protein-solvent interface is investigated through the analysis of a molecular dynamics simulation of metmyoglobin in explicit aqueous environment. Distribution implied ...dynamics, harmonic and quasi-harmonic, are compared with the simulated macroscopic dynamics. The distinction between distinguishable solvent molecules and hydration sites developed in the previous paper is used. The simulated hydration region within 7 A from the protein surface is analyzed using a set of 551 hydration sites characterized by occupancy weights and temperature B-factors determined from the simulation trajectory. The precision of the isotropic harmonic and anisotropic harmonic models for the description of proximal solvent fluctuations is examined. Residence times and dipole reorientation times of water around the protein surface are compared with NMR and ESR results. A correlation between diffraction experiment quantities such as the occupancy weights and temperature factors and the residence and correlation times resulting from magnetic resonance experiments is found via comparison with simulation.
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