Ramakrishna Vadrevu

Multiple e-Pharmacophore Modeling Combined with High-Throughput Virtual Screening and Docking to Identify Potential Inhibitors of β-Secretase(BACE1)

β‐Secretase (BACE1) is an aspartate protease involved in the production of amyloid‐β a major peptide responsible for the pathogenesis of Alzheimer’s disease. Given its role in the formation of amyloids leading to Alzheimer’s disease, it has been a major therapeutic target for intervention and has been a challenge in the past and the progress has been very slow. More than hundred crystal structures with inhibitors are available in the protein data bank. Many strategies for drug design have been employed in the design of numerous diverse ligands for this target and many have failed due to undesirable drug properties primarily the inability to cross the blood‐brain barrier. In the present work we attempted to consider multiple crystal structures with bound inhibitors showing affinity in the range of 2–210 nM efficacy and optimize the pharmacophoric requirement based on the energy involved in binding termed as e‐pharmacophore mapping. A high throughput screening combined with molecular docking, ADMET predictions, logP values and in vitro assay led to the identification of 7 potential compounds showing inhibition at 10µM which could be further developed as novel inhibitors for β‐secretase.

NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein.

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (alpha TS) from Escherichia coli, a (beta alpha)(8) TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several (15)N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of alpha TS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several beta-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact beta-barrel and a frayed alpha-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of alpha TS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.

Partial NMR assignments and secondary structure mapping of the isolated α subunit of Escherichia coli tryptophan synthase, a 29-kD TIM barrel protein

The α subunit of tryptophan synthase (αTS) from S. typhimurium belongs to the triosephosphate isomerase (TIM) or the (β/α)8 barrel fold, one of the most common structures in biology. To test the conservation of the global fold in the isolated Escherichia coli homolog, we have obtained a majority of the backbone assignments for the 29‐kD αTS by using standard heteronuclear multidimensional NMR methods on uniformly 15N‐ and 15N/13C‐labeled protein and on protein selectively 15N‐labeled at key hydrophobic residues. The secondary structure mapped by chemical shift index, nuclear Overhauser enhancements (NOEs), and hydrogen‐deuterium (H‐D) exchange, and several abnormal chemical shifts are consistent with the conservation of the global TIM barrel fold of the isolated E. coli αTS. Because most of the amide protons that are slow to exchange with solvent correspond to the β‐sheet residues, the β‐barrel is likely to play an important role in stabilizing the previously detected folding intermediates for E. coli αTS. A similar combination of uniform and selective labeling can be extended to other TIM barrel proteins to obtain insight into the role of the motif in stabilizing what appear to be common partially folded forms.

Rapid NMR Assignments of Proteins by Using Optimized Combinatorial Selective Unlabeling

A new approach for rapid resonance assignments in proteins based on amino acid selective unlabeling is presented. The method involves choosing a set of multiple amino acid types for selective unlabeling and identifying specific tripeptides surrounding the labeled residues from specific 2D NMR spectra in a combinatorial manner. The methodology directly yields sequence specific assignments, without requiring a contiguously stretch of amino acid residues to be linked, and is applicable to deuterated proteins. We show that a 2D [15N,1H] HSQC spectrum with two 2D spectra can result in ∼50 % assignments. The methodology was applied to two proteins: an intrinsically disordered protein (12 kDa) and the 29 kDa (268 residue) α‐subunit of Escherichia coli tryptophan synthase, which presents a challenging case with spectral overlaps and missing peaks. The method can augment existing approaches and will be useful for applications such as identifying active‐site residues involved in ligand binding, phosphorylation, or protein–protein interactions, even prior to complete resonance assignments.

βα-Hairpin Clamps Brace βαβ Modules and Can Make Substantive Contributions to the Stability of TIM Barrel Proteins

Non-local hydrogen bonding interactions between main chain amide hydrogen atoms and polar side chain acceptors that bracket consecutive βα or αβ elements of secondary structure in αTS from E. coli, a TIM barrel protein, have previously been found to contribute 4–6 kcal mol−1 to the stability of the native conformation. Experimental analysis of similar βα-hairpin clamps in a homologous pair of TIM barrel proteins of low sequence identity, IGPS from S. solfataricus and E. coli, reveals that this dramatic enhancement of stability is not unique to αTS. A survey of 71 TIM barrel proteins demonstrates a 4-fold symmetry for the placement of βα-hairpin clamps, bracing the fundamental βαβ building block and defining its register in the (βα)8 motif. The preferred sequences and locations of βα-hairpin clamps will enhance structure prediction algorithms and provide a strategy for engineering stability in TIM barrel proteins.

Long-range side-chain–main-chain interactions play crucial roles in stabilizing the (βα)8 barrel motif of the alpha subunit of tryptophan synthase

The role of hither‐to‐fore unrecognized long‐range hydrogen bonds between main‐chain amide hydrogens and polar side chains on the stability of a well‐studied (βα)8, TIM barrel protein, the alpha subunit of tryptophan synthase (αTS), was probed by mutational analysis. The F19–D46 and I97–D124 hydrogen bonds link the N terminus of a β‐strand with the C terminus of the succeeding antiparallel α‐helix, and the A103–D130 hydrogen bond links the N terminus of an α‐helix with the C terminus of the succeeding antiparallel β‐strand, forming clamps for the respective βα or αβ hairpins. The individual replacement of these aspartic acid side chains with alanine leads to what appear to be closely related partially folded structures with significantly reduced far‐UV CD ellipticity and thermodynamic stability. Comparisons with the effects of eliminating another main‐chain–side‐chain hydrogen bond, G26–S33, and two electrostatic side‐chain–side‐chain hydrogen bonds, D38–H92 and D112–H146, all in the same N‐terminal folding unit of αTS, demonstrated a unique role for the clamp interactions in stabilizing the native barrel conformation. Because neither the asparagine nor glutamic acid variant at position 46 can completely reproduce the spectroscopic, thermodynamic, or kinetic folding properties of aspartic acid, both size and charge are crucial to its unique role in the clamp hydrogen bond. Kinetic studies suggest that the three clamp hydrogen bonds act in concert to stabilize the transition state leading to the fully folded TIM barrel motif.

Identification of abelson tyrosine kinase inhibitors as potential therapeutics for Alzheimer’s disease using multiple e-pharmacophore modeling and molecular dynamics

Efforts to combat Alzheimer’s disease are focused predominantly on inhibiting the activity of the enzyme(s) that have been identified to be responsible for the production of the amyloid-forming peptide. However, the inherent complexity associated with the network of pathways leading to the disease may involve additional targets for designing effective therapies. Recent experimental findings have identified abelson tyrosine kinase, a non-receptor kinase as a new target for Alzheimer’s. In this work, we employed energy optimized multiple pharmacophore modeling strategy from multiple c-Abl structures bound with ligands in the inactive ATP binding conformation (DFG-out). Virtual screening followed by docking of molecules from ChemBridge resulted in the identification of 10 best scoring molecules. MD simulations of the top three complexes revealed that Compound A, C are the most stable complexes with the most persistent protein–ligand interactions consistent with the calculated binding affinities for the top three compounds. Given the implied role of c-Abl not only in AD but in Parkinson’s disease, the identified compounds may serve as leads for effective neurotherapeutics.

Pharmacophore based 3D-QSAR modeling, virtual screening and docking for identification of potential inhibitors of -secretase

The enzyme β-secretase-1 is responsible for the cleavage of the amyloid precursor protein, a vital step in the process of the formation of amyloid-β peptides which are known to lead to neurodegeneration causing Alzheimers disease. Challenges associated with toxicity and blood brain permeation inability of potential inhibitors, continue to evade a successful therapy, thus demanding the search and development of highly active and effective inhibitors. Towards these efforts, we used a ligand based pharmacophore model generation from a dataset of known inhibitors whose activities against β-secretase hovered in the nano molar range. The identified 5 feature pharmacophore model, AHHPR, was validated via three dimensional quantitative structure activity relationship as indicated by r2, q2 and Pearson R values of 0.9013, 0.7726 and 0.9041 respectively. For a dataset of compounds with nano molar activity, the important pharmacophore features present in the current model appear to be similar with those observed in the models resulting from much wider activity range of inhibitors. Virtual screening of the ChemBridge CNS-Set™, a database having compounds with a better suitability for central nervous system based disorders followed by docking and analysis of the ligand protein interactions resulted in the identification of eight prospective compounds with considerable diversity. The current pharmacophore model can thus be useful for the identification, design and development of potent β-secretase inhibitors which by optimization can be potential therapeutics for Alzheimers disease.

Crowding interactions perturb structure and stability by destabilizing the stable core of the α‐subunit of tryptophan synthase

The consequences of crowding derived from relatively small and intrinsically disordered proteins are not clear yet. We report the effect of ficoll‐70 on the structure and stability of native and partially folded states of the 29 kDa alpha subunit of tryptophan synthase (αTS). Overall, combining the changes in the circular dichroism and fluorescence spectra, in conjunction with the gradual loss of cooperativity under urea denaturation in the presence of increasing amounts of ficoll, it may be concluded that the crowding agent perturbs not only the native state but also the partially folded state of αTS. Importantly, NMR data indicate that ficoll interacts with the residues that constitute the stable core of the protein thus shedding light on the origin of the observed perturbation.

Diversity in αβ and βα Loop Connections in TIM Barrel Proteins: Implications for Stability and Design of the Fold

The (βα)8/TIM barrel is one of the most common folds of known protein structures facilitating diverse catalytic functions. The fold is formed by the repetition of the basic βαβ building block in which the β-strands are followed by α-helices eight times alternating in sequence and structure. αβ and βα loops connecting α-helices to the β-strands and the β-strands to the α-helices contribute to stability and function, respectively, an inherent imposition by the TIM barrel architecture itself. In this study, αβ and βα loops from a data set of 430 non-redundant, high-resolution triosephosphate isomerase (TIM) barrels bearing sequence homology of <30% were analyzed for their amino acid propensities, sequence profiles, and positional preferences of amino acids. While the distribution of short connections is significantly higher in αβ loops, there appears to be no such preference in βα loops. Glycine, proline, lysine, and arginine tend to show greater preference to occur in αβ loops, whereas serine, threonine, cysteine, tryptophan, and histidine occur more frequently in βα loops. In addition, striking dissimilarities in sequence and positional preferences of amino acids, especially, in short, αβ and βα loops are observed. Together, the analysis suggests the role for short loops and charged residues in promoting both non-polar and polar interactions and in β strand registry. The observed diversity, perhaps, dictates the distinct role of αβ and βα loops in stability and function, respectively. In summary, the overall observations and reasoning, in addition to steering protein engineering efforts on TIM barrel design and stabilization can provide the basis for incorporating consensus loop sequences for designing independently folding βαβ modules.