Structural insights and binding of a natural ligand, succinic acid with serine and cysteine proteases.

In the age of growing infectious diseases, there is a great demand for new inhibitors which can exhibit minimum side effects. Owing to the importance of proteases in life cycle and invasion, they have been projected as attractive targets for structure based drug designing against microbes including viruses. Here we report the inhibitory activity of a well known natural compound succinic acid against both serine and cysteine proteases. The ligand is found co-crystallized with Bovine pancreatic trypsin in one of our crystallization trials and the diffraction data up to1.9 Å reveal its interactions with the catalytic triad residues Histidine 57 and Serine 195. Binding of the ligand with these proteases have been validated using caseinolysis inhibition. With trypsin, ITC analysis showed tight binding of the ligand, resulting in change in Gibb's free energy (ΔG) by -20.31 kJ/mol. To understand the existence of succinic acid at the active site, molecular docking was performed and it revealed binding of it with trypsin and papain at corresponding active sites. This dual inhibitory activity of natural ligand, succinic acid can be accounted for the recent reports on anti-viral property of plant extracts where dicarboxilic fatty acids are normally abundant.

Flavonoids modulate multidrug resistance through wnt signaling in P-glycoprotein overexpressing cell lines.

BACKGROUND: Wnt signaling has been linked with P-glycoprotein (P-gp) overexpression and which was mainly mediated by ß-catenin nuclear translocation. Flavonoids have already been reported as modulators of the Wnt/ß-catenin pathway and hence they may serve as promising agents in the reversal of P-gp mediated cancer multi drug resistance (MDR). METHODS: In this study, we screened selected flavonoids against Wnt/ß-catenin signaling molecules. The binding interaction of flavonoids (theaflavin, quercetin, rutin, epicatechin 3 gallate and tamarixetin) with GSK 3ß was determined by molecular docking. Flavonoids on P-gp expression and the components of Wnt signaling in drug-resistant KBCHR8-5 cells were analyzed by western blotting and qRT-PCR. The MDR reversal potential of these selected flavonoids against P-gp mediated drug resistance was analyzed by cytotoxicity assay in KBCHR8-5 and MCF7/ADR cell lines. The chemosensitizing potential of flavonoids was further analyzed by observing cell cycle arrest in KBCHR8-5 cells. RESULTS: In this study, we observed that the components of Wnt/ß-catenin pathway such as Wnt and GSK 3ß were activated in multidrug resistant KBCHR8-5 cell lines. All the flavonoids selected in this study significantly decreased the expression of Wnt and GSK 3ß in KBCHR8-5 cells and subsequently modulates P-gp overexpression in this drug-resistant cell line. Further, we observed that these flavonoids considerably decreased the doxorubicin resistance in KBCHR8-5 and MCF7/ADR cell lines. The MDR reversal potential of flavonoids were found to be in the order of theaflavin > quercetin > rutin > epicatechin 3 gallate > tamarixetin. Moreover, we observed that flavonoids pretreatment significantly induced the doxorubicin-mediated arrest at the phase of G2/M. Further, the combinations of doxorubicin with flavonoids significantly modulate the expression of drug response genes in KBCHR8-5 cells. CONCLUSION: The present findings illustrate that the studied flavonoids significantly enhances doxorubicin-mediated cell death through modulating P-gp expression pattern by targeting Wnt/ß-catenin signaling in drug-resistant KBCHR8-5 cells.

Structural basis for the hydrolysis of ATP by a nucleotide binding subunit of an amino acid ABC transporter from Thermus thermophilus.

ATP-binding cassette (ABC) transporters are a major family of small molecule transporter proteins, and their deregulation is associated with several diseases, including cancer. Here, we report the crystal structure of the nucleotide binding domain (NBD) of an amino acid ABC transporter from Thermus thermophilus (TTHA1159) in its apo form and as a complex with ADP along with functional studies. TTHA1159 is a putative arginine ABC transporter. The apo-TTHA1159 was crystallized in dimeric form, a hitherto unreported form of an apo NBD. Structural comparison of the apo and ADP-Mg(2+) complexes revealed that Phe14 of TTHA1159 undergoes a significant conformational change to accommodate ADP, and that the bound ADP interacts with the P-loop (Gly40-Thr45). Modeling of ATP-Mg(2+):TTHA1159 complex revealed that Gln86 and Glu164 are involved in water-mediated hydrogen bonding contacts and Asp163 in Mg(2+) ion-mediated hydrogen bonding contacts with the γ-phosphate of ATP, consistent with the findings of other ABC transporters. Mutational studies confirmed the necessity of each of these residues, and a comparison of the apo/ADP Mg(2+):TTHA1159 with its ATP-complex model suggests the likelihood of a key conformational change to the Gln86 side chain for ATP hydrolysis.

GAP: towards almost 100 percent prediction for ß-strand-mediated aggregating peptides with distinct morphologies.

MOTIVATION: Distinguishing between amyloid fibril-forming and amorphous ß-aggregating aggregation-prone regions (APRs) in proteins and peptides is crucial for designing novel biomaterials and improved aggregation inhibitors for biotechnological and therapeutic purposes. RESULTS: Adjacent and alternate position residue pairs in hexapeptides show distinct preferences for occurrence in amyloid fibrils and amorphous ß-aggregates. These observations were converted into energy potentials that were, in turn, machine learned. The resulting tool, called Generalized Aggregation Proneness (GAP), could successfully distinguish between amyloid fibril-forming and amorphous ß-aggregating hexapeptides with almost 100 percent accuracies in validation tests performed using non-redundant datasets. CONCLUSION: Accuracies of the predictions made by GAP are significantly improved compared with other methods capable of predicting either general ß-aggregation or amyloid fibril-forming APRs. This work demonstrates that amino acid side chains play important roles in determining the morphological fate of ß-mediated aggregates formed by short peptides. AVAILABILITY AND IMPLEMENTATION: http://www.iitm.ac.in/bioinfo/GAP/.

1-[2-Hy-droxy-4-(prop-2-yn-1-yl-oxy)phen-yl]ethanone.

In the title compound, C11H10O3, there is an intra-molecular O-H⋯O hydrogen bond generating an S(6) ring motif. The O atom of the hy-droxy group deviates by 0.0200 (1) Šfrom the benzene ring to which it is attached. The propyne group is almost linear, the C-C C angle being 177.83 (15)°, and is almost coplanar with the benzene ring; the C-C-O-C torsion angle being only -1.1 (2)°. In the crystal, mol-ecules are linked via C-H⋯O hydrogen bonds, forming infinite C(11) chains running parallel to [103]. These chains are linked by a pair of C-H⋯O hydrogen bonds, enclosing R 2 (2)(8) inversion dimers, forming a corrugated two-dimensional network lying parallel to (103).

(2E)-2-Benzyl-idene-9-phenyl-3,4-di-hydro-acridin-1(2H)-one.

In the title compound, C26H19NO, the plane of the aromatic heterocycle makes a dihedral angle of 75.22 (4)° with that of the attached phenyl ring. In the crystal, mol-ecules are connected by C-H⋯O inter-actions, generating R 2 (2)(12) dimers. These dimers are further connected by C-H⋯π inter-actions, linking the mol-ecules into chains running along the a-axis direction.

Crystal structure of bis[4-(di-methyl-amino)-pyridinium] bis(2-nitro-benzoate) trihydrate.

The title salt, 2C7H11N2 (+)·2C7H4NO4 (-)·3H2O, crystallized with two anions and two cations in the asymmetric unit, together with three water mol-ecules. Both 4-di-methyl-amino-pyridinium cations are protonated at their pyridine N atoms with the plane of the N(CH3)2 hetero atoms inclined to the pyridine ring by 4.5 (2) and 1.4 (2)°. In the 2-nitro-benzoate anions, the carboxyl and nitro groups are inclined to their respective benzene rings by 77.1 (3) and 20.0 (3)°, and 75.8 (2) and 20.9 (3)°. In the crystal, the anions are linked via O-H⋯O hydrogen bonds involving the water mol-ecules, forming chains along [100]. The cations are linked to these chains by N-H⋯O hydrogen bonds. The chains are linked via C-H⋯O hydrogen bonds and C-H⋯π and π-π inter-actions [inter-centroid distances range from 3.617 (1) to 3.851 (1) Å], forming a three-dimensional structure.

(E)-3-Isopropyl-1-methyl-2,6-di-phenyl-piperidin-4-one O-nicotinoyl oxime.

In the title compound, C27H29N3O2, the piperidine ring exists in a chair conformation with an equatorial orientation of the phenyl and methyl substituents. The C-C=N bond angles are significantly different [119.1 (2) and 127.2 (2)°]. The phenyl rings are inclined to one another by 44.90 (14)°, and by 80.85 (13) and 79.62 (12)° to the mean plane of the piperidine ring. The terminal pyridine ring is inclined to the piperidine ring mean plane by 74.79 (15)°. In the crystal, mol-ecules are linked by C-H⋯π inter-actions, forming a three-dimensional network.

Crystal structure analysis of L-fuculose-1-phosphate aldolase from Thermus thermophilus HB8 and its catalytic action: as explained through in silico.

Fuculose phosphate aldolase catalyzes the reversible cleavage of fuculose-1-phosphate to dihydroxyacetone phosphate and L-lactaldehyde. A tetramer by nature, this enzyme from Thermus thermophilus HB8 represents the group of Class II aldolases. The structure was solved in two different space groups using the crystals obtained from slow evaporation vapour-diffusion and microbatch techniques. The detailed crystallization description has been reported previously. In this study, the structural features of fuculose phosphate aldolase from T. thermophilus have been explored extensively through sequence and structure comparisons with fuculose phosphate aldolases of different species. Finally, an in silico analysis using induced fit docking was attempted to deduce the binding mode of fuculose phosphate aldolase with its natural substrate fuculose-1-phosphate along with a substrate analog dihydroxyacetone phosphate and phosphoglycolohydroxymate--a potential aldolase inhibitor. The results show the mechanism of action may be similar to that of Escherichia coli fuculose aldolase.

Distinct position-specific sequence features of hexa-peptides that form amyloid-fibrils: application to discriminate between amyloid fibril and amorphous ß-aggregate forming peptide sequences.

BACKGROUND: Comparison of short peptides which form amyloid-fibrils with their homologues that may form amorphous ß-aggregates but not fibrils, can aid development of novel amyloid-containing nanomaterials with well defined morphologies and characteristics. The knowledge gained from the comparative analysis could also be applied towards identifying potential aggregation prone regions in proteins, which are important for biotechnology applications or have been implicated in neurodegenerative diseases. In this work we have systematically analyzed a set of 139 amyloid-fibril hexa-peptides along with a highly homologous set of 168 hexa-peptides that do not form amyloid fibrils for their position-wise as well as overall amino acid compositions and averages of 49 selected amino acid properties. RESULTS: Amyloid-fibril forming peptides show distinct preferences and avoidances for amino acid residues to occur at each of the six positions. As expected, the amyloid fibril peptides are also more hydrophobic than non-amyloid peptides. We have used the results of this analysis to develop statistical potential energy values for the 20 amino acid residues to occur at each of the six different positions in the hexa-peptides. The distribution of the potential energy values in 139 amyloid and 168 non-amyloid fibrils are distinct and the amyloid-fibril peptides tend to be more stable (lower total potential energy values) than non-amyloid peptides. The average frequency of occurrence of these peptides with lower than specific cutoff energies at different positions is 72% and 50%, respectively. The potential energy values were used to devise a statistical discriminator to distinguish between amyloid-fibril and non-amyloid peptides. Our method could identify the amyloid-fibril forming hexa-peptides to an accuracy of 89%. On the other hand, the accuracy of identifying non-amyloid peptides was only 54%. Further attempts were made to improve the prediction accuracy via machine learning. This resulted in an overall accuracy of 82.7% with the sensitivity and specificity of 81.3% and 83.9%, respectively, in 10-fold cross-validation method. CONCLUSIONS: Amyloid-fibril forming hexa-peptides show position specific sequence features that are different from those which may form amorphous ß-aggregates. These positional preferences are found to be important features for discriminating amyloid-fibril forming peptides from their homologues that don't form amyloid-fibrils.

2-Hy-droxy-4-(prop-2-yn-yloxy)benz-alde-hyde.

The asymmetric unit of the title compound, C10H8O3, contains two independent mol-ecules, both of which are almost planar (r.m.s deviations for all non-H atoms of 0.044 and 0.053 Å). The dihedral angles between the benzene ring and the prop-1-yne group are 3.47 (1) and 3.07 (1)° in the two mol-ecules, and the prop-1-yne groups adopt extended conformations. In each mol-ecule, an intra-molecular O-H⋯O hydrogen bond involving the OH and aldehyde substituents forms an S(6) ring. In the crystal, mol-ecules are linked into cyclic centrosymmetric dimers via C-H⋯O hydrogen bonds, generating R2(2)(14) ring motifs. The crystal structure is further stabilized by aromatic π-π stacking inter-actions between the benzene rings [centroid-centroid distances = 3.813 (2) and 3.843 (2) Å].

6-Phenyl-benzo[d]naphtho-[2,3-b]thio-phene.

In the title compound, C22H14S, the r.m.s. deviation from the mean plane of the four-fused-ring naphtho-thio-phene unit is 0.056 Å. The dihedral angle between the naphtho-thio-phene plane and the pendant phenyl ring is 67.24 (6)°. In the crystal, weak C-H⋯π and π-π stacking [minimum centroid-centroid separation = 3.7466 (10) Å] inter-actions are observed, which together lead to (010) sheets.

[(4E)-1-Methyl-2,6-diphenyl-3-(propan-2-yl)piperidin-4-yl-idene]amino 3-methyl-benzoate.

In the title compound, C29H32N2O2, the piperidine ring exists in a chair conformation (the bond-angle sum at the sp (2)-hybridized C atom is 359.79°). The phenyl rings and the methyl group substituted on the heterocyclic ring are in equatorial orientations. In the crystal, pairs of C-H⋯π inter-actions result in the formation of inversion dimers.

[(4E)-3-Ethyl-1-methyl-2,6-di-phenyl-piperidin-4-yl-idene]amino 3-methyl-benzoate.

In the title compound, C28H30N2O2, the piperidine ring exists in a chair conformation with an equatorial orientation of the phenyl rings and methyl group substituted on the heterocycle. In the crystal, C-H⋯π inter-actions result in chains of mol-ecules running parallel to the a-axis direction.

3-Hy-droxy-1-[(morpholin-4-yl)meth-yl]pyridazin-6(1H)-one.

In the title compound, C9H13N3O3, the morpholine ring adopts a chair conformation and its mean plane makes a dihedral angle of 68.00 (11)° with the pyridazine ring. The carbonyl O atom deviates from the plane of the pyridazine ring by 0.0482 (12) Å. An intra-molecular C-H⋯O hydrogen bond occurs. In the crystal, mol-ecules are linked by O-H⋯O and C-H⋯O hydrogen bonds, forming chains along [1-10].

4-[2-(4-But-oxy-phen-yl)ethen-yl]-1-methyl-pyridinium tosylate.

In the title mol-ecular salt, C18H22NO(+)·C7H7O3S(-), the dihedral angle between the aromatic rings in the cation is 10.00 (9)°; its alkyl side chain adopts an extended conformation. In the crystal, weak C-H⋯O and π-π [centroid-centroid distance = 3.7658 (17) Å] inter-actions link the components, generating a three-dimensional network.

Ethyl (E)-3-[1'-ethyl-2-oxo-4'-(phenyl-sulfon-yl)-2H-spiro-[acenaphthyl-ene-1,2'-pyrrolidine]-3'-yl]acrylate.

In the title compound, C(28)H(27)NO(5)S, the five-membered pyrrolidine ring, which exhibits an envelope conformation (the C atom at the spiral junction being the flap atom), makes dihedral angles of 57.37 (10) and 86.84 (8)°, respectively, with the phenyl ring and the acenaphthyl-ene ring system. In the crystal, mol-ecules associate via two C-H⋯O hydrogen bonds, forming R(2) (2)(20) and R(2) (2)(10) graph-set motifs.

Methyl (2E)-2-{[(2-methyl-quinolin-8-yl)-oxy]meth-yl}-3-(thio-phen-2-yl)acrylate.

In the mol-ecule of the title compound, C(19)H(17)NO(3)S, the dihedral angle formed by the quinoline ring system and the thio-phene ring is 83.15 (8)°. In the crystal, C-H⋯O hydrogen bonds link the mol-ecules into a C(8) chain running along the b axis. The packing of the mol-ecules is further influenced by C-H⋯π inter-actions.