Kailas D. Sonawane

Amyloid cascade hypothesis: Pathogenesis and therapeutic strategies in Alzheimer's disease

Alzheimers disease is an irreversible, progressive neurodegenerative disorder. Various therapeutic approaches are being used to improve the cholinergic neurotransmission, but their role in AD pathogenesis is still unknown. Although, an increase in tau protein concentration in CSF has been described in AD, but several issues remains unclear. Extensive and accurate analysis of CSF could be helpful to define presence of tau proteins in physiological conditions, or released during the progression of neurodegenerative disease. The amyloid cascade hypothesis postulates that the neurodegeneration in AD caused by abnormal accumulation of amyloid beta (Aβ) plaques in various areas of the brain. The amyloid hypothesis has continued to gain support over the last two decades, particularly from genetic studies. Therefore, current research progress in several areas of therapies shall provide an effective treatment to cure this devastating disease. This review critically evaluates general biochemical and physiological functions of Aβ directed therapeutics and their relevance.

Molecular Dynamics Simulation and Molecular Docking Studies of Angiotensin Converting Enzyme with Inhibitor Lisinopril and Amyloid Beta Peptide

Angiotensin converting enzyme (ACE) cleaves amyloid beta peptide. So far this cleavage mechanism has not been studied in detail at atomic level. Keeping this view in mind, we performed molecular dynamics simulation of crystal structure complex of testis truncated version of ACE (tACE) and its inhibitor lisinopril along with Zn2+ to understand the dynamic behavior of active site residues of tACE. Root mean square deviation results revealed the stability of tACE throughout simulation. The residues Ala 354, Glu 376, Asp 377, Glu 384, His 513, Tyr 520 and Tyr 523 of tACE stabilized lisinopril by hydrogen bonding interactions. Using this information in subsequent part of study, molecular docking of tACE crystal structure with Aβ-peptide has been made to investigate the interactions of Aβ-peptide with enzyme tACE. The residues Asp 7 and Ser 8 of Aβ-peptide were found in close contact with Glu 384 of tACE along with Zn2+. This study has demonstrated that the residue Glu 384 of tACE might play key role in the degradation of Aβ-peptide by cleaving peptide bond between Asp 7 and Ser 8 residues. Molecular basis generated by this attempt could provide valuable information towards designing of new therapies to control Aβ concentration in Alzheimer’s patient.

Structural analysis of membrane-bound hECE-1 dimer using molecular modeling techniques: insights into conformational changes and Aβ1–42 peptide binding

The human endothelin converting enzyme-1 (hECE-1) is a homodimer linked by a single disulfide bridge and has been identified as an important target for Alzheimer’s disease. Structural analysis of hECE-1 dimer could lead to design specific and effective therapies against Alzheimer’s disease. Hence, in the present study homology model of transmembrane helix has been constructed and patched with available crystal structure of hECE-1 monomer. Then, membrane-bound whole model of hECE-1 dimer has been developed by considering biophysical properties of membrane proteins. The explicit molecular dynamics simulation revealed that the hECE-1 dimer exhibits conformational restrains and controls total central cavity by regulating the degree of fluctuations in some residues (238–226) for substrate/product entrance/exit sites. In turn, conformational rearrangements of interdomain linkers as well as helices close to the inner surface are responsible for increasing total central cavity of hECE-1 dimer. Further, the model of hECE-1 dimer was docked with Aβ1–42 followed by MD simulation to investigate possible orientation and interactions of Aβ1–42 in catalytic groove of hECE-1 dimer. The free energy calculations exposed the stability of complex and helped us to identify key residues of hECE-1 involved in interactions with Aβ1–42 peptide. Hence, the present study might be useful to understand structural significance of membrane-bound dimeric hECE-1 to design therapies against Alzheimer’s disease.

Homology modeling, molecular docking and MD simulation studies to investigate role of cysteine protease from Xanthomonas campestris in degradation of Aβ peptide

Cysteine protease is known to degrade amyloid beta peptide which is a causative agent of Alzheimers disease. This cleavage mechanism has not been studied in detail at the atomic level. Hence, a three-dimensional structure of cysteine protease from Xanthomonas campestris was constructed by homology modeling using Geno3D, SWISS-MODEL, and MODELLER 9v7. All the predicted models were analyzed by PROCHECK and PROSA. Three-dimensional model of cysteine protease built by MODELLER 9v7 shows similarity with human cathepsin B crystal structure. This model was then used further for docking and simulation studies. The molecular docking study revealed that Cys17, His87, and Gln88 residues of cysteine protease form an active site pocket similar to human cathepsin B. Then the docked complex was refined by molecular dynamic simulation to confirm its stable behavior over the entire simulation period. The molecular docking and MD simulation studies showed that the sulfhydryl hydrogen atom of Cys17 of cysteine protease interacts with carboxylic oxygen of Lys16 of Aβ peptide indicating the cleavage site. Thus, the cysteine protease model from X. campestris having similarity with human cathepsin B crystal structure may be used as an alternate approach to cleave Aβ peptide a causative agent of Alzheimers disease.

Insights into the molecular interactions between aminopeptidase and amyloid beta peptide using molecular modeling techniques

Amyloid beta (Aβ) peptides play a central role in the pathogenesis of Alzheimer’s disease. The accumulation of Aβ peptides in AD brain was caused due to overproduction or insufficient clearance and defects in the proteolytic degradation of Aβ peptides. Hence, Aβ peptide degradation could be a promising therapeutic approach in AD treatment. Recent experimental report suggests that aminopeptidase from Streptomyces griseus KK565 (SGAK) can degrade Aβ peptides but the interactive residues are yet to be known in detail at the atomic level. Hence, we developed the three-dimensional model of aminopeptidase (SGAK) using SWISS-MODEL, Geno3D and MODELLER. Model built by MODELLER was used for further studies. Molecular docking was performed between aminopeptidase (SGAK) with wild-type and mutated Aβ peptides. The docked complex of aminopeptidase (SGAK) and wild-type Aβ peptide (1IYT.pdb) shows more stability than the other complexes. Molecular docking and MD simulation results revealed that the residues His93, Asp105, Glu139, Glu140, Asp168 and His255 are involved in the hydrogen bonding with Aβ peptide and zinc ions. The interactions between carboxyl oxygen atoms of Glu139 of aminopeptidase (SGAK) with water molecule suggest that the Glu139 may be involved in the nucleophilic attack on Ala2–Glu3 peptide bond of Aβ peptide. Hence, amino acid Glu139 of aminopeptidase (SGAK) might play an important role to degrade Aβ peptides, a causative agent of Alzheimer’s disease.

Conformational Preferences of Anticodon 3′-Adjacent Hypermodified Nucleic Acid Base cis-or trans-Zeatin and its 2-methylthio Derivative, cis-or trans-ms2Zeatin

Abstract Conformational preferences of the hypermodified nucleic acid bases N6-(Δ2 -cis-hydroxyisopentenyl)adenine, cis-io6Ade also known as cis-zeatin, and N6-(Δ2 -trans-hydroxyisopentenyl)adenine, trans-io6ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms2io6Ade or cis-ms2zeatin, and trans-ms2io6Ade or trans-ms2zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AMI and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io6Ade, trans-io6Ade, cis-ms2io6Ade and trans-ms2io6Ade are such that in each of these molecules the isopentenyl substituent spreads away (has “dista” conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io6Ade as well as cis- ms2io6Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io6Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms2io6Ade, possible O-H…S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms2io6Ade as well as trans-ms2io6Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this nonobstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i6Ade and ms2i6Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms2-zeatin (ms2io6Ade) modification.

Purification and characterization of novel organic solvent tolerant 98kDa alkaline protease from isolated Stenotrophomonas maltophilia strain SK.

Ability of microorganisms to grow at alkaline pH makes them an attractive target for several industrial applications. Thus, search for new extremozyme producing microorganisms must be a continuous exercise. Hence, we isolated a potent alkaline protease producing bacteria from slaughter house soil. The morphological, biochemical and 16S rDNA gene sequencing studies revealed that the isolated bacteria is Stenotrophomonas maltophilia strain SK. Alkaline protease from S. maltophilia strain SK was purified by using ammonium sulphate precipitation and DEAE-cellulose ion exchange column chromatography. The purified enzyme was optimally active at pH 9.0 and temperature 40°C with broad substrate specificity. It was observed that the metal ions such as Ca(++), Mg(++) and Fe(+++) completely repressed the enzyme activity. The enzyme was stable in presence of various water miscible solvents like ethanol, methanol, isopropanol at 25% (v/v) concentration and less stable at 37.5% (v/v) concentration. These robust properties of enzyme might be applicable for various applications in detergent and pharmaceutical industries.

Conformational flipping of the N(6) substituent in diprotonated N6‐(N‐glycylcarbonyl)adenines: The role of N(6)H in purine‐ring‐protonated ureido adenines

Conformational transitions of the N(6) substituent, in hypermodified nucleic acid base N6-(N-glycylcarbonyl)adenine, gc6Ade, on diprotonation of the adenine ring at any two of N(1), N(3), and N(7) sites, are studied using the quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. The N(6) substituent retains the usual “distal” orientation (α=0°) in (N(1),N(3)) diprotonated gc6Ade, but the “proximal” orientation (α=180°) is preferred instead, for (N(3),N(7)) and (N(7),N(1)) diprotonated gc6Ade. The proximal orientation may alter the reading frame during translation. Intramolecular N(6)HO(13b) hydrogen bonding is the key common feature, present in the preferred structure, for each of these variously diprotonated gc6Ade.

Insight into molecular interactions of Aβ peptide and gelatinase from Enterococcus faecalis: a molecular modeling approach

Alzheimers disease is characterized by the presence of extracellular deposition of amyloid beta (Aβ) peptides. The Aβ levels are maintained by a suitable balance between its production and clearance via receptor-mediated cellular uptake and direct enzymatic degradation. However, decreased production of Aβ degrading enzymes and loss of Aβ degrading activity in the human brain initiates the process of accumulation of Aβ peptides. Mutations in amyloid beta peptides are also known to reduce clearance of Aβ peptides. So it becomes necessary to understand molecular interactions involved in the process of Aβ degradation in detail at the atomic level. Hence, in this study, we used molecular docking and molecular dynamics simulation techniques to explore molecular interactions between gelatinase from Enterococcus faecalis and Aβ peptide. The comparison between wild type and mutant Aβ peptides revealed that the docked complex of gelatinase–wild type Aβ peptide is more stable as compared to mutant complex. These results showed that the residue Glu 328 of gelatinase from E. faecalis might play a catalytic role by serving as a proton shuttle to cleave wild type Aβ peptide in between Leu 34–Met 35. Thus, the results obtained from this study might be useful to design new strategies against Aβ peptide degradation.

Exploring mode of phosphoramidon and Aβ peptide binding to hECE-1 by molecular dynamics and docking studies.

Human Endothelin converting enzyme (hECE-1) has been widely known for its involvement in hydrolyzing Aβ peptides at multiple sites. In the present study we have performed molecular dynamics (MD) simulation of crystal structure complex of hECE-1 and its inhibitor phosphoramidon with Zn ion to understand the dynamic behavior of active site residues. Root Mean Square Deviation (RMSD) results revealed that enzyme hECE-1 structure was highly stable throughout the simulation period. The L-leucyl-L-tryptophan moiety and N-phosphoryl moiety of phosphoramidon was found in the S1 and S2 pockets of hECE-1 respectively. The inhibitor was stabilized by hydrogen bonding interactions with residues Arg 145, Asn 566, Pro 731 and His 732 of hECE-1. Based on this information molecular docking of hECE- 1 crystal structure with three different structures of Aβ peptides has been performed. Zinc ion interacts with His 607(NE2), His 611(NE2), Glu 667 (OE1, OE2) and backbone oxygen atom of Phe 19 showing catalytic coordination between Aβ peptide and hECE-1. The unusual orientation of Aβ peptide residues affects hydrophobic interactions and hydrogen bonding network between hECE-1 and Aβ peptide. The molecular basis of amyloid beta peptide cleavage by hECE-1 could aid in designing enzyme based therapies to control Aβ peptide concentration in Alzheimers patient.