D3R Grand Challenge 4: prospective pose prediction of BACE1 ligands with AutoDock-GPU.

In this paper we describe our approaches to predict the binding mode of twenty BACE1 ligands as part of Grand Challenge 4 (GC4), organized by the Drug Design Data Resource. Calculations for all submissions (except for one, which used AutoDock4.2) were performed using AutoDock-GPU, the new GPU-accelerated version of AutoDock4 implemented in OpenCL, which features a gradient-based local search. The pose prediction challenge was organized in two stages. In Stage 1a, the protein conformations associated with each of the ligands were undisclosed, so we docked each ligand to a set of eleven receptor conformations, chosen to maximize the diversity of binding pocket topography. Protein conformations were made available in Stage 1b, making it a re-docking task. For all calculations, macrocyclic conformations were sampled on the fly during docking, taking the target structure into account. To leverage information from existing structures containing BACE1 bound to ligands available in the PDB, we tested biased docking and pose filter protocols to facilitate poses resembling those experimentally determined. Both pose filters and biased docking resulted in more accurate docked poses, enabling us to predict for both Stages 1a and 1b ligand poses within 2 Å RMSD from the crystallographic pose. Nevertheless, many of the ligands could be correctly docked without using existing structural information, demonstrating the usefulness of physics-based scoring functions, such as the one used in AutoDock4, for structure based drug design.

Conformational Dynamics and Binding Free Energies of Inhibitors of BACE-1: From the Perspective of Protonation Equilibria.

BACE-1 is the ß-secretase responsible for the initial amyloidogenesis in Alzheimer's disease, catalyzing hydrolytic cleavage of substrate in a pH-sensitive manner. The catalytic mechanism of BACE-1 requires water-mediated proton transfer from aspartyl dyad to the substrate, as well as structural flexibility in the flap region. Thus, the coupling of protonation and conformational equilibria is essential to a full in silico characterization of BACE-1. In this work, we perform constant pH replica exchange molecular dynamics simulations on both apo BACE-1 and five BACE-1-inhibitor complexes to examine the effect of pH on dynamics and inhibitor binding properties of BACE-1. In our simulations, we find that solution pH controls the conformational flexibility of apo BACE-1, whereas bound inhibitors largely limit the motions of the holo enzyme at all levels of pH. The microscopic pKa values of titratable residues in BACE-1 including its aspartyl dyad are computed and compared between apo and inhibitor-bound states. Changes in protonation between the apo and holo forms suggest a thermodynamic linkage between binding of inhibitors and protons localized at the dyad. Utilizing our recently developed computational protocol applying the binding polynomial formalism to the constant pH molecular dynamics (CpHMD) framework, we are able to obtain the pH-dependent binding free energy profiles for various BACE-1-inhibitor complexes. Our results highlight the importance of correctly addressing the binding-induced protonation changes in protein-ligand systems where binding accompanies a net proton transfer. This work comprises the first application of our CpHMD-based free energy computational method to protein-ligand complexes and illustrates the value of CpHMD as an all-purpose tool for obtaining pH-dependent dynamics and binding free energies of biological systems.

Molecular modeling studies and comparative analysis on structurally similar HTLV and HIV protease using HIV-PR inhibitors.

Retroviruses are most perilous viral family, which cause much damage to the Homo sapiens. HTLV-1 mechanism found to more similar with HIV-1 and both retroviruses are causative agents of severe and fatal diseases including adult T-cell leukemia (ATL) and the acquired immune deficiency syndrome (AIDS). Both viruses code for a protease (PR) that is essential for replication and therefore represents a key target for drugs interfering with viral infection. In this work, the comparative study of HIV-1 and HTLV-1 PR enzymes through sequence and structural analysis is reported along with approved drugs of HIV-PR. Conformation of each HIV PR drugs have been examined with different parameters of interactions and energy scorings parameters. MD simulations with respect to timescale event of 20 ns favors that, few HIV-PR inhibitors can be more active inside the HTLV-1 PR binding pocket. Overall results suggest that, some of HIV inhibitors like Tipranavir, Indinavir, Darunavir and Amprenavir are having good energy levels with HTLV-1. Due to absence of interactions with MET37, here we report that derivatives of these compounds can be much better inhibitors for targeting HTLV-1 proteolytic activity.

In Silico Inhibition of BACE-1 by Selective Phytochemicals as Novel Potential Inhibitors: Molecular Docking and DFT Studies.

BACKGROUND: Alzheimer's Disease (AD) has become the most common age-dependent disease of dementia. The trademark pathologies of AD are the presence of amyloid aggregates in neurofibrils. Recently phytochemicals being considered as potential inhibitors against various neurodegenerative, antifungal, antibacterial and antiviral diseases in human beings. OBJECTIVE: This study targets the inhibition of BACE-1 by phytochemicals using in silico drug discovery analysis. METHODS: A total of 3150 phytochemicals were collected from almost 25 different plants through literature assessment. The ADMET studies, molecular docking and density functional theory (DFT) based analysis were performed to analyze the potential inhibitory properties of these phytochemicals. RESULTS: The ADMET and docking results exposed seven compounds that have high potential as an inhibitory agent against BACE-1 and show binding affinity >8.0 kcal/mol against BACE-1. They show binding affinity greater than those of various previously reported inhibitors of BACE-1. Furthermore, DFT based analysis has shown high reactivity for these seven phytochemicals in the binding pocket of BACE- 1, based on ELUMO, EHOMO and Kohn-Sham energy gap. All seven phytochemicals were testified (as compared to experimental ones) as novel inhibitors against BACE-1. CONCLUSION: Out of seven phytochemicals, four were obtained from plant Glycyrrhiza glabra i.e. Shinflavanone, Glabrolide, Glabrol and PrenyllicoflavoneA, one from Huperzia serrate i.e. Macleanine, one from Uncaria rhynchophylla i.e. 3a-dihydro-cadambine and another one was from VolvalerelactoneB from plant Valeriana-officinalis. It is concluded that these phytochemicals are suitable candidates for drug/inhibitor against BACE-1, and can be administered to humans after experimental validation through in vitro and in vivo trials.

Flavonoids as BACE1 inhibitors: QSAR modelling, screening and in vitro evaluation.

Alzheimer's disease (AD) is marked by the presence of amyloid plaques, neurofibrillary tangles, oxidatively damaged neuronal macromolecules and redox sensitive ions. Reduction of amyloid plaques and oxidative stress emerge as a convincing treatment strategy. Plaque reduction is achieved by inhibition of BACE1, the rate limiting enzyme generating the prime constituent of plaques, Aß, through proteolysis of the amyloid precursor protein. Here, we report a QSAR model with five descriptors, developed to screen natural compounds as potent BACE1 inhibitors. Seven compounds out of which five flavonols namely isorhamnetin, syringetin, galangin, tamarixetin, rhamnetin and two flavanonols namely dihydromyricetin, taxifolin were screened. The ability of these compounds were validated using the BACE1 activity assay. The antioxidant property were estimated by the DPPH and ABTS assay. Although inhibition assay implied syringetin to be a promising BACE1 inhibitor, its poor antioxidant activity leaves it less effective as a multitarget ligand. Exhibiting moderate dual ability, isorhamnetin and taxifolin qualified as multi-target scaffolds for AD therapeutics. Our study reveals the importance of 4'-OH in the B ring of flavonols and the lack of any effect of 5'-OH in flavanonols for BACE1 inhibition. In case of antioxidant activity favourable association of 3'-O-methylation derivatives was observed in flavonols.

BACE2 is stored in secretory granules of mouse and rat pancreatic beta cells.

BACE2 is a protease homologous to BACE1 protein, an enzyme involved in the amyloid formation of Alzheimer disease (AD). However, despite the high homology between these two proteins, the biological role of BACE2 is still controversial, even though a few studies have suggested a pathogenetic role in sporadic inclusion-body myositis and hereditary inclusion-body myopathy, which are characterized by vacuolization of muscular fibers with intracellular deposits of proteins similar to those found in the brain of AD patients. Although BACE2 has also been identified in the pancreas, its function remains unknown and its specific localization in different pancreatic cell types has not been definitively ascertained. For these reasons, the authors have investigated the cellular and subcellular localization of BACE2 in normal rodent pancreases. BACE2 immunoreactivity was found in secretory granules of beta cells, co-stored with insulin and IAPP, while it was lacking in the other endocrine and exocrine cell types. The presence of BACE2 in secretory granules of beta cells suggests that it may play a role in diabetes-associated amyloidogenesis.

Crystal structure of the aspartic proteinase from Rhizomucor miehei at 2.15 A resolution.

The crystal structure of the aspartic proteinase from Rhizomucor miehei (RMP, EC 3. 4. 23. 23) has been refined to 2.15 A resolution to a crystallographic R-value of 0.215 and an Rfree of 0.281. The root-mean-square (r.m.s.) error for the atomic coordinates estimated from a Luzzati plot is 0.2 A. The r.m.s. deviations for the bond distances and bond angles from ideality are 0.01 A and 1.7 degrees, respectively. RMP contains two domains that consist predominantly of beta-sheets. A large substrate-binding cleft is clearly visible between the two domains, and the two catalytic residues Asp38 and Asp237 are located in the middle of the cleft with a water molecule bridging the carboxyl groups of Asp38 and Asp237. Due to crystal packing, the C-terminal domain is more mobile than the N-terminal domain. Most of the aspartic proteinases (except renin) reach their maximum activity at acidic pH. We propose that the optimum pH of each aspartic proteinase is determined by the electrostatic potential at the active site, which, in turn, is determined by the positions and orientations of all the residues near the active site. RMP is the most glycosylated among the aspartic proteinases. The carbohydrate moieties are linked to Asn79 and Asn188. Asn79 is in the middle of a beta-strand and Asn188 is on a surface loop in contrast to the previous hypothesis proposed by Brown and Yada that they are both on surface beta-turns. RMP has a very high thermal stability. The high thermal stability is probably due to the high level of glycosylation. We propose that the highly flexible carbohydrates act as heat reservoirs to stabilize the conformation of RMP and therefore give the enzyme a high level of thermal stability. Three-dimensional structural and sequence alignments of RMP with other aspartic proteinases show that RMP is most structurally homologous to that of Mucor pusillus (MPP), and differs from other fungal enzymes as much as it does from the mammalian enzymes. This suggests that RMP and MPP diverged from the main stream of aspartic proteinases at an early stage of evolution. The present study adds a second member to this subfamily of aspartic proteinases.

X-ray analyses of aspartic proteinases. V. Structure and refinement at 2.0 A resolution of the aspartic proteinase from Mucor pusillus.

The structure of mucor pusillus pepsin (EC 3.4.23.6), the aspartic proteinase from Mucor pusillus, has been refined to a crystallographic R-factor of 16.2% at 2.0 A resolution. The positions of 2638 protein atoms, 221 solvent atoms and a sulphate ion have been determined with an estimated root-mean-square (r.m.s.) error of 0.15 to 0.20 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0.022 A, and for angle distances it is 0.050 A. Comparison of the overall three-dimensional structure with other aspartic proteinases shows that mucor pusillus pepsin is as distant from the other fungal enzymes as it is from those of mammalian origin. Analysis of a rigid body shift of residues 190 to 302 shows that mucor pusillus pepsin displays one of the largest shifts relative to other aspartic proteinases (14.4 degrees relative to endothiapepsin) and that changes have occurred at the interface between the two rigid bodies to accommodate this large shift. A new sequence alignment has been obtained on the basis of the three-dimensional structure, enabling the positions of large insertions to be identified. Analysis of secondary structure shows the beta-sheet to be well conserved whereas alpha-helical elements are more variable. A new alpha-helix hN4 is formed by a six-residue insertion between positions 131 and 132. Most insertions occur in loop regions: -5 to 1 (five residues relative to porcine pepsin): 115 to 116 (six residues); 186 to 187 (four residues); 263 to 264 (seven residues); 278 to 279 (four residues); and 326 to 332 (six residues). The active site residues are highly conserved in mucor pusillus pepsin; r.m.s. difference with rhizopuspepsin is 0.37 A for 25 C alpha atom pairs. However, residue 303, which is generally conserved as an aspartate, is changed to an asparagine in mucor pusillus pepsin, possibly influencing pH optimum. Substantial changes have occurred in the substrate binding cleft in the region of S1 and S3 due to the insertion between 115 and 116 and the rearrangement of loop 9-13. Residue Asn219 necessitates a shift in position of substrate main-chain atoms to maintain hydrogen bonding pattern. Invariant residues Asp11 and Tyr14 have undergone a major change in conformation apparently due to localized changes in molecular structure. Both these residues have been implicated in zymogen stability and activation.

Crystallization of inhibited aspartic proteinase from Candida albicans.

A major secreted aspartic proteinase from Candida albicans has been crystallized in the presence of inhibitors to prevent autodegradation. With pepstatin a cleaved form of the enzyme was nevertheless found in the crystals whereas with the inhibitor A70450 the enzyme remained intact. The crystals containing pepstatin were not suitable for X-ray data collection while the crystals containing A70450 grew by vapour diffusion as tetragonal bipyramids, space group P4(3)2(1)2 (or P4(1)2(1)2), a = b = 76.2 A, c = 126.1 A, with one molecule in the asymmetric unit and they diffract to beyond 2.2 A.

X-ray analyses of aspartic proteinases. III Three-dimensional structure of endothiapepsin complexed with a transition-state isostere inhibitor of renin at 1.6 A resolution.

The aspartic proteinase, endothiapepsin (EC 3.4.23.6), was complexed with a highly potent renin inhibitor, H-261 (t-Boc-His-Pro-Phe-His-LeuOHVal-Ile-His), where OH denotes a hydroxyethylene (-(S) CHOH-CH2-) transition-state isostere in the scissile bond surrogate. Crystals were grown in a form that has the same space group P2(1) as the uncomplexed enzyme, but with a 10 A decrease in the length of the alpha-axis and a 13 degrees decrease in the beta-angle. X-ray data have been collected to a resolution of 1.6 A. The rotation and translation parameters defining the position of the enzyme in the unit cell were determined previously using another enzyme-inhibitor complex that crystallized isomorphously with that of H-261. The molecule was refined using restrained least-squares refinement and the positions of non-hydrogen atoms of the inhibitor and water molecules were defined by difference Fourier techniques. The enzyme-inhibitor complex and 322 water molecules were further refined to a crystallographic R-factor of 0.14. Apart from a small rigid group rotation of a domain comprising residues 190 to 302 and small movements in the flap, there is little difference in conformation between the complexed and uncomplexed forms of the enzyme. The inhibitor is bound in an extended conformation along the active site cleft, and the hydroxyl group of the hydroxyethylene moiety is hydrogen-bonded to both catalytic aspartate carboxylates. The complex is stabilized by hydrogen bonds between the main-chain of the inhibitor and the enzyme. All side-chains of the inhibitor are in van der Waals' contact with groups in the enzyme and define a series of specificity pockets along the active site cleft. The study provides useful clues as to how this potent renin inhibitor (IC50 value of 0.7 x 10(-9) M) may bind renin. In particular it defines the interactions of the hydroxyethylene transition-state isostere with the enzyme more precisely than has been previously possible and therefore provides a useful insight into interactions in the transition state complex.

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases.

Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied, mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development.

Structure of the protease from simian immunodeficiency virus: complex with an irreversible nonpeptide inhibitor.

A variant of the simian immunodeficiency virus protease (SIV PR), covalently bound to the inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), was crystallized. The structure of the inhibited complex was determined by X-ray crystallography to a resolution of 2.4 A and refined to an R factor of 19%. The variant, SIV PR S4H, was shown to diminish the rate of autolysis by at least 4-fold without affecting enzymatic parameters. The overall root mean square (rms) deviation of the alpha-carbons from the structure of HIV-1PR complexed with a peptidomimetic inhibitor (7HVP) was 1.16 A. The major differences are concentrated in three surface loops with rms differences between 1.2 and 2.1 A. For 60% of the molecule the rms deviation was only 0.6 A. The structure reveals one molecule of EPNP bound per protease dimer, a stoichiometry confirmed by mass spectral analysis. The epoxide moiety forms a covalent bond with either of the active site aspartic acids of the dimer, and the phenyl moiety occupies the P1 binding site. The EPNP nitro group interacts with Arg 8. This structure suggests a starting template for the design of nonpeptide-based irreversible inhibitors of the SIV and related HIV-1 and HIV-2 PRs.

Three-dimensional structure of a simian immunodeficiency virus protease/inhibitor complex. Implications for the design of human immunodeficiency virus type 1 and 2 protease inhibitors.

Simian immunodeficiency virus (SIV) proteins have considerable amino acid sequence homology to those from human immunodeficiency virus (HIV); thus monkeys are considered useful models for the preclinical evaluation of acquired immune deficiency syndrome (AIDS) therapeutics. We have crystallized and determined the three-dimensional structure of SIV protease bound to the hydroxyethylene isostere inhibitor SKF107457. Crystals of the complex were grown from 25-32% saturated sodium chloride, by the hanging drop method of vapor diffusion. They belong to the orthorhombic space group I222, with a = 46.3 A, b = 101.5 A, and c = 118.8 A. The structure has been determined at 2.5-A resolution by molecular replacement and refined to a crystallographic discrepancy factor, R (= sigma parallel Fo magnitude of - magnitude of Fc parallel/sigma magnitude of Fo magnitude of), of 0.189. The overall structure of the complex is very similar to previously reported structures of HIV-1 protease bound to inhibitors. The inhibitor is bound in a conformation that is almost identical to that found for the same inhibitor bound to HIV-1 protease, except for an overall translation of the inhibitor, varying along the backbone atoms from about 1.0 A at the termini to about 0.5 A around the scissile bond surrogate. The structures of the SIV and HIV-1 proteins vary significantly only in three surface loops composed of amino acids 15-20, 34-45, and 65-70. Superposition of the 1188 protein backbone atoms from the two structures gives an rms deviation of 1.0 A; this number is reduced to 0.6 A when atoms from the three surface loops are eliminated from the rms calculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of misfolded protein aggregates leads to the formation of russell body-like dilated endoplasmic reticulum in yeast.

RNAP-1, an aspartic proteinase from a filamentous fungus Rhizopus niveus, is secreted very efficiently in Saccharomyces cerevisiae. It is synthesized first as a precursor form with signal sequence and prosequence in its amino-terminus. Our previous study indicated that the prosequence of RNAP-I had important roles in its correct folding and secretion in yeast, and that a prosequence-deleted derivative of RNAP-I, delta pro, was not secreted but was retained and degraded in the yeast endoplasmic reticulum (ER). In the present study, we show that the accumulation of delta pro in the yeast ER caused elevated synthesis of ER resident chaperones, indicating that delta pro is recognized as an unfolded protein species in the ER. Our biochemical data demonstrated that delta pro formed aggregates which contained BiP, but not protein disulfide isomerase (PDI), in the ER. Immunoelectron microscopical analysis revealed that the delta pro aggregates were indeed visible as electron-dense regions in the ER and nuclear envelope. Such 'chaperone-associated misfolded protein bodies' were observed for the first time in yeast. Morphologies of the ER and nucleus were drastically altered by the accumulation of the delta pro aggregates. The ER lost its flat cisternal shape; the ER lumen extended aberrantly and the ER membrane irregularly proliferated. The misfolded delta pro proteins are probably sorted from the ordinary ER lumen to form the aggregates so that the ER function would not be grossly impaired, and the dilated ER may represent an ER subcompartment where the delta pro aggregates are degraded.