Computational optimization of the SARS-CoV-2 receptor-binding-motif affinity for human ACE2

The coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the coronavirus disease 2019 pandemic, and the closely related SARS-CoV coronavirus enter cells by binding at the human angiotensin converting enzyme 2 (hACE2). The stronger hACE2 affinity of SARS-CoV-2 has been connected with its higher infectivity. In this work, we study hACE2 complexes with the receptor-binding domains (RBDs) of the human SARS-CoV-2 and human SARS-CoV viruses, using all-atom molecular dynamics simulations and computational protein design with a physics-based energy function. The molecular dynamics simulations identify charge-modifying substitutions between the CoV-2 and CoV RBDs, which either increase or decrease the hACE2 affinity of the SARS-CoV-2 RBD. The combined effect of these mutations is small, and the relative affinity is mainly determined by substitutions at residues in contact with hACE2. Many of these findings are in line and interpret recent experiments. Our computational protein design calculations redesign positions 455, 493, 494, and 501 of the SARS-CoV-2 receptor binding motif, which contact hACE2 in the complex and are important for ACE2 recognition. Sampling is enhanced by an adaptive importance sampling Monte Carlo method. Sequences with increased affinity replace CoV-2 glutamine by a negative residue at position 493; serine by a nonpolar or aromatic residue or an asparagine at position 494; and asparagine by valine or threonine at position 501. Substitutions at positions 455 and 501 have a smaller effect on affinity. Substitutions suggested by our design are seen in viral sequences encountered in other species, including bat and pangolin. Our results might be used to identify potential virus strains with higher human infectivity and assist in the design of peptide-based or peptidomimetic compounds with the potential to inhibit SARS-CoV-2 binding at hACE2.     

The 1.76 A resolution crystal structure of glycogen phosphorylase B complexed with glucose, and CP320626, a potential antidiabetic drug

CP320626, a potential antidiabetic drug, inhibits glycogen phosphorylase in synergism with glucose. To elucidate the structural basis of synergistic inhibition, we determined the structure of muscle glycogen phosphorylase b (MGPb) complexed with both glucose and CP320626 at 1.76 A resolution, and refined to a crystallographic R value of 0.211 (R(free)=0.235). CP320626 binds at a novel allosteric site, which is some 33 A from the catalytic site, where glucose binds. The high resolution structure allows unambiguous definition of the conformation of the 1-acetyl-4-hydroxy-piperidine ring supported by theoretical energy calculations. Both CP320626 and glucose promote the less active T-state, thereby explaining their synergistic inhibition. Structural comparison of MGPb--glucose--CP320626 complex with liver glycogen phosphorylase a (LGPa) complexed with a related compound (CP403700) show that the ligand binding site is conserved in LGPa.     

Self-Assembly of Phenylalanine Oligopeptides: Insights from Experiments and Simulations

Studies of peptide-based nanostructures provide general insights into biomolecular self-assembly and can lead material engineering toward technological applications. The diphenylalanine peptide (FF) self-assembles into discrete, hollow, well ordered nanotubes, and its derivatives form nanoassemblies of various morphologies. Here we demonstrate for the first time, to our knowledge, the formation of planar nanostructures with β-sheet content by the triphenylalanine peptide (FFF). We characterize these structures using various microscopy and spectroscopy techniques. We also obtain insights into the interactions and structural properties of the FF and FFF nanostructures by 0.4-μs, implicit-solvent, replica-exchange, molecular-dynamics simulations of aqueous FF and FFF solutions. In the simulations the peptides form aggregates, which often contain open or ring-like peptide networks, as well as elementary and network-containing structures with β-sheet characteristics. The networks are stabilized by polar and nonpolar interactions, and by the surrounding aggregate. In particular, the charged termini of neighbor peptides are involved in hydrogen-bonding interactions and their aromatic side chains form “T-shaped” contacts, as in three-dimensional FF crystals. These interactions may assist the FF and FFF self-assembly at the early stage, and may also stabilize the mature nanostructures. The FFF peptides have higher network propensities and increased aggregate stabilities with respect to FF, which can be interpreted energetically.     

Glucose-based spiro-isoxazolines: A new family of potent glycogen phosphorylase inhibitors

A series of glucopyranosylidene-spiro-isoxazolines was prepared through regio- and stereoselective [3+2]-cycloaddition between the methylene acetylated exo-glucal and aromatic nitrile oxides. The deprotected cycloadducts were evaluated as inhibitors of muscle glycogen phosphorylase b. The carbohydrate-based family of five inhibitors displays K_i values ranging from 0.63 to 92.5 μM. The X-ray structures of the enzyme–ligand complexes show that the inhibitors bind preferentially at the catalytic site of the enzyme retaining the less active T-state conformation. Docking calculations with GLIDE in extra-precision (XP) mode yielded excellent agreement with experiment, as judged by comparison of the predicted binding modes of the five ligands with the crystallographic conformations and the good correlation between the docking scores and the experimental free binding energies. Use of docking constraints on the well-defined positions of the glucopyranose moiety in the catalytic site and redocking of GLIDE-XP poses using electrostatic potential fit-determined ligand partial charges in quantum polarized ligand docking (QPLD) produced the best results in this regard.     

Glycogen phosphorylase inhibitors: a free energy perturbation analysis of glucopyranose spirohydantoin analogues

GP catalyzes the phosphorylation of glycogen to Glc-1-P. Because of its fundamental role in the metabolism of glycogen, GP has been the target for a systematic structure-assisted design of inhibitory compounds, which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan). In this work, we employ MD free energy simulations to calculate the relative binding affinities for GP of hydan and two spirohydantoin analogues, methyl-hydan and n-hydan, in which a hydrogen atom is replaced by a methyl- or amino group, respectively. The results are compared with the experimental relative affinities of these ligands, estimated by kinetic measurements of the ligand inhibition constants. The calculated binding affinity for methyl-hydan (relative to hydan) is 3.75 +/- 1.4 kcal/mol, in excellent agreement with the experimental value (3.6 +/- 0.2 kcal/mol). For n-hydan, the calculated value is 1.0 +/- 1.1 kcal/mol, somewhat smaller than the experimental result (2.3 +/- 0.1 kcal/mol). A free energy decomposition analysis shows that hydan makes optimum interactions with protein residues and specific water molecules in the catalytic site. In the other two ligands, structural perturbations of the active site by the additional methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water molecule for two well-defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water molecules in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in X-ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to estimate the order of relative binding strengths, in contrast to the rigorous free energy treatment.     

Low IL-23 levels in peripheral blood and bone marrow at diagnosis of acute leukemia in children increased with the elimination of leukemic burden

IL-23 is an IL-12 cytokine family member with pleiotropic functions that regulates tumour growth in various cancer types, exhibiting both anti-tumorigenic and pro-tumorigenic properties. Preclinical studies have shown a potential anti-leukemic action on childhood B-ALL cells. The study involved 65 children with acute leukemia [59 patients with acute lymphoblastic leukemia (ALL) and 6 patients with acute myeloid leukemia (AML)] and 27 healthy controls. Using an enzyme-linked immunosorbent assay, we aimed to determine the IL-23 levels in the peripheral blood (PB) and bone marrow (BM) of patients at diagnosis and at the end of the induction therapy (EIT). PB IL-23 levels were lower in leukemia patients compared to the healthy controls. In all acute leukemia patients, IL-23 levels were significantly lower at diagnosis both in PB (P = .015) and in BM (P = .037) compared to the PB and BM concentrations at the EIT. The same pattern was present in both subgroups of ALL and AML patients. The high leukemic burden at diagnosis was related with lower IL-23 levels, which were increased with the disease remission. Considering the anti-leukemic potential of this cytokine, the elevation of the IL-23 concentration at the disease remission indicates a beneficial role of IL-23 in paediatric acute leukemia.     

Kinetics, in silico docking, molecular dynamics, and MM-GBSA binding studies on prototype indirubins, KT5720, and staurosporine as phosphorylase kinase ATP-binding site inhibitors: the role of water molecules examined

With an aim toward glycogenolysis control in Type 2 diabetes, we have investigated via kinetic experiments and computation the potential of indirubin (IC?? > 50 μM), indirubin-3'-oxime (IC?? = 144 nM), KT5720 (K(i) = 18.4 nM) and staurosporine (K(i) = 0.37 nM) as phosphorylase kinase (PhKγtrnc) ATP-binding site inhibitors, with the latter two revealed as potent inhibitors in the low nM range. Because of lack of structural information, we have exploited information from homologous kinase complexes to direct in silico calculations (docking, molecular dynamics, and MMGBSA) to predict the binding characteristics of the four ligands. All inhibitors are predicted to bind in the same active site area as the ATP adenine ring, with binding dominated by hinge region hydrogen bonds to Asp104:O and Met106:O (all four ligands) and also Met106:NH (for the indirubins). The PhKγtrnc-staurosporine complex has the greatest number of receptor-ligand hydrogen bonds, while for the indirubin-3'-oxime and KT5720 complexes there is an important network of interchanging water molecules bridging inhibitor-enzyme contacts. The MM-GBSA results revealed the source of staurosporine's low nM potency to be favorable electrostatic interactions, while KT5720 has strong van der Waals contributions. KT5720 interacts with the greatest number of protein residues either by direct or 1-water bridged hydrogen bond interactions, and the potential for more selective PhK inhibition based on a KT5720 analogue has been established. Including receptor flexibility in Schr?dinger induced-fit docking calculations in most cases correctly predicted the binding modes as compared with the molecular dynamics structures; the algorithm was less effective when there were key structural waters bridging receptor-ligand contacts.     

Design of a modified mouse protein with ligand binding properties of its human analog by molecular dynamics simulations: the case of C3 inhibition by compstatin

The peptide compstatin and its derivatives inhibit the complement-component protein C3 in primate mammals and are potential therapeutic agents against the unregulated activation of complement in humans, but are inactive against C3 from lower mammals. Recent molecular dynamics (MD) simulations showed that the most potent compstatin analog comprised entirely of natural amino acids (W4A9) had a smaller affinity for rat C3, due to reproducible changes in the rat protein structure with respect to the human protein, which eliminated or weakened specific protein-ligand interactions seen in the human C3:W4A9 complex. Here, we study by MD simulations three W4A9 complexes with the mouse C3 protein, and two "transgenic" mouse derivatives, containing a small number (6-9) of human C3 substitutions. The mouse complex experiences the conformational changes and affinity reduction of the rat complex. In the "transgenic" complexes, the conformation remains closer to that of the human complex, the protein-ligand interactions are improved, and the affinity for compstatin becomes "human-like." The present work creates new avenues for a compstatin-sensitive animal model. A similar strategy, involving the comparison of a series of complexes by MD simulations, could be used to design "transgenic" sequences in other systems.     

Binding free energies and free energy components from molecular dynamics and Poisson-Boltzmann calculations. Application to amino acid recognition by aspartyl-tRNA synthetase

Specific amino acid binding by aminoacyl-tRNA synthetases (aaRS) is necessary for correct translation of the genetic code. Engineering a modified specificity into aminoacyl-tRNA synthetases has been proposed as a means to incorporate artificial amino acid residues into proteins in vivo. In a previous paper, the binding to aspartyl-tRNA synthetase of the substrate Asp and the analogue Asn were compared by molecular dynamics free energy simulations. Molecular dynamics combined with Poisson-Boltzmann free energy calculations represent a less expensive approach, suitable for examining multiple active site mutations in an engineering effort. Here, Poisson-Boltzmann free energy calculations for aspartyl-tRNA synthetase are first validated by their ability to reproduce selected molecular dynamics binding free energy differences, then used to examine the possibility of Asn binding to native and mutant aspartyl-tRNA synthetase. A component analysis of the Poisson-Boltzmann free energies is employed to identify specific interactions that determine the binding affinities. The combined use of molecular dynamics free energy simulations to study one binding process thoroughly, followed by molecular dynamics and Poisson-Boltzmann free energy calculations to study a series of related ligands or mutations is proposed as a paradigm for protein or ligand design.The binding of Asn in an alternate, "head-to-tail" orientation observed in the homologous asparagine synthetase is analyzed, and found to be more stable than the "Asp-like" orientation studied earlier. The new orientation is probably unsuitable for catalysis. A conserved active site lysine (Lys198 in Escherichia coli) that recognizes the Asp side-chain is changed to a leucine residue, found at the corresponding position in asparaginyl-tRNA synthetase. It is interesting that the binding of Asp is calculated to increase slightly (rather than to decrease), while that of Asn is calculated, as expected, to increase strongly, to the same level as Asp binding. Insight into the origin of these changes is provided by the component analyses. The double mutation (K198L,D233E) has a similar effect, while the triple mutation (K198L,Q199E,D233E) reduces Asp binding strongly. No binding measurements are available, but the three mutants are known to have no ability to adenylate Asn, despite the "Asp-like" binding affinities calculated here. In molecular dynamics simulations of all three mutants, the Asn ligand backbone shifts by 1-2 A compared to the experimental Asp:AspRS complex, and significant side-chain rearrangements occur around the pocket. These could reduce the ATP binding constant and/or the adenylation reaction rate, explaining the lack of catalytic activity in these complexes. Finally, Asn binding to AspRS with neutral K198 or charged H449 is considered, and shown to be less favorable than with the charged K198 and neutral H449 used in the analysis.     

Computational protein design with a generalized Born solvent model: application to Asparaginyl-tRNA synthetase

Computational Protein Design (CPD) is a promising method for high throughput protein and ligand mutagenesis. Recently, we developed a CPD method that used a polar-hydrogen energy function for protein interactions and a Coulomb/Accessible Surface Area (CASA) model for solvent effects. We applied this method to engineer aspartyl-adenylate (AspAMP) specificity into Asparaginyl-tRNA synthetase (AsnRS), whose substrate is asparaginyl-adenylate (AsnAMP). Here, we implement a more accurate function, with an all-atom energy for protein interactions and a residue-pairwise generalized Born model for solvent effects. As a first test, we compute aminoacid affinities for several point mutants of Aspartyl-tRNA synthetase (AspRS) and Tyrosyl-tRNA synthetase and stability changes for three helical peptides and compare with experiment. As a second test, we readdress the problem of AsnRS aminoacid engineering. We compare three design criteria, which optimize the folding free-energy, the absolute AspAMP affinity, and the relative (AspAMP-AsnAMP) affinity. The sequences and conformations are improved with respect to our previous, polar-hydrogen/CASA study: For several designed complexes, the AspAMP carboxylate forms three interactions with a conserved arginine and a designed lysine, as in the active site of the AspRS:AspAMP complex. The conformations and interactions are well maintained in molecular dynamics simulations and the sequences have an inverted specificity, favoring AspAMP over AsnAMP. The method is not fully successful, since experimental measurements with the seven most promising sequences show that they do not catalyze at a detectable level the adenylation of Asp (or Asn) with ATP. This may be due to weak AspAMP binding and/or disruption of transition-state stabilization.