Srinivasaraghavan Kannan

Enhanced sampling of peptide and protein conformations using replica exchange simulations with a peptide backbone biasing-potential.

During replica exchange molecular dynamics (RexMD) simulations, several replicas of a system are simulated at different temperatures in parallel allowing for exchange between replicas at frequent intervals. This technique allows significantly improved sampling of conformational space and is increasingly being used for structure prediction of peptides and proteins. A drawback of the standard temperature RexMD is the rapid increase of the replica number with increasing system size to cover a desired temperature range. In an effort to limit the number of replicas, a new Hamiltonian‐RexMD method has been developed that is specifically designed to enhance the sampling of peptide and protein conformations by applying various levels of a backbone biasing potential for each replica run. The biasing potential lowers the barrier for backbone dihedral transitions and promotes enhanced peptide backbone transitions along the replica coordinate. The application on several peptide cases including in all cases explicit solvent indicates significantly improved conformational sampling when compared with standard MD simulations. This was achieved with a very modest number of 5–7 replicas for each simulation system making it ideally suited for peptide and protein folding simulations as well as refinement of protein model structures in the presence of explicit solvent. Proteins 2007.

Structural basis for the inhibition of histone deacetylase 8 (HDAC8), a key epigenetic player in the blood fluke Schistosoma mansoni.

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/β HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.

Small Molecule Inhibitors of Histone Acetyltransferases as Epigenetic Tools and Drug Candidates

Alteration of the acetylation state of histone proteins contributes to transcriptional regulation and epigenetic inheritance. Dysregulation of these processes may lead to human diseases, especially cancer. One of the major chromatin modifications is histone acetylation and this review gives an overview of the role of histone acetyltransferases, their structural aspects, as well as of chemical modulators targeting their enzymatical activities. Inhibitors and activators of histone acetyltransferases are presented and their capability to influence histone and non‐histone protein acetylation levels is discussed. Development of small molecules as epigenetic tools that alter histone acetyltransferase activity will be helpful to better understand the consequences of histone and generally protein acetylation and potentially offer novel therapeutic approaches for the treatment of cancer and other diseases.

Folding simulations of Trp-cage mini protein in explicit solvent using biasing potential replica-exchange molecular dynamics simulations.

Replica exchange molecular dynamics (RexMD) simulations are frequently used for studying structure formation and dynamics of peptides and proteins. A significant drawback of standard temperature RexMD is, however, the rapid increase of the replica number with increasing system size to cover a desired temperature range. A recently developed Hamiltonian RexMD method has been used to study folding of the Trp‐cage protein. It employs a biasing potential that lowers the backbone dihedral barriers and promotes peptide backbone transitions along the replica coordinate. In two independent applications of the biasing potential RexMD method including explicit solvent and starting from a completely unfolded structure the formation of near‐native conformations was observed after 30–40 ns simulation time. The conformation representing the most populated cluster at the final simulation stage had a backbone root mean square deviation of ∼1.3 Å from the experimental structure. This was achieved with a very modest number of five replicas making it well suited for peptide and protein folding and refinement studies including explicit solvent. In contrast, during five independent continuous 70 ns molecular dynamics simulations formation of collapsed states but no near native structure formation was observed. The simulations predict a largely collapsed state with a significant helical propensity for the helical domain of the Trp‐cage protein already in the unfolded state. Hydrogen bonded bridging water molecules were identified that could play an active role by stabilizing the arrangement of the helical domain with respect to the rest of the chain already in intermediate states of the protein. Proteins 2009.

PDK1-SGK1 Signaling Sustains AKT-Independent mTORC1 Activation and Confers Resistance to PI3Kα Inhibition

Summary PIK3CA, which encodes the p110α subunit of PI3K, is frequently mutated and oncogenic in breast cancer. PI3Kα inhibitors are in clinical development and despite promising early clinical activity, intrinsic resistance is frequent among patients. We have previously reported that residual downstream mTORC1 activity upon treatment with PI3Kα inhibitors drives resistance to these agents. However, the mechanism underlying this phenotype is not fully understood. Here we show that in cancer cells resistant to PI3Kα inhibition, PDK1 blockade restores sensitivity to these therapies. SGK1, which is activated by PDK1, contributes to the maintenance of residual mTORC1 activity through direct phosphorylation and inhibition of TSC2. Targeting either PDK1 or SGK1 prevents mTORC1 activation, restoring the antitumoral effects of PI3Kα inhibition in resistant cells.

Discovery of inhibitors of Schistosoma mansoni HDAC8 by combining homology modeling, virtual screening, and in vitro validation.

Schistosomiasis, caused by S. mansoni, is a tropical disease that affects over 200 million people worldwide. A novel approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during their life cycle. We recently identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here we present results from a virtual screening campaign on smHDAC8. Besides hydroxamates, several sulfonamide-thiazole derivatives were identified by a target-based virtual screening using a homology model of smHDAC8. In vitro testing of 75 compounds identified 8 hydroxamates as potent and lead-like inhibitors of the parasitic HDAC8. Solving of the crystal structure of smHDAC8 with two of the virtual screening hits confirmed the predicted binding mode.

Simulated annealing coupled replica exchange molecular dynamics—An efficient conformational sampling method

Molecular dynamics simulated annealing (SA-MD) simulations are frequently used for refinement and optimization of peptide and protein structures. Depending on the simulation conditions and simulation length SA-MD simulations can be trapped in locally stable conformations far from the global optimum. As an alternative replica exchange molecular dynamics (RexMD) simulations can be used which allow exchanges between high and low simulation temperatures at all stages of the simulation. A significant drawback of RexMD simulations is, however, the rapid increase of the replica number with increasing system size to cover a desired temperature range. A combined SA-MD and RexMD approach termed SA-RexMD is suggested that employs a small number of replicas (4) and starts initially with a set of high simulation temperatures followed by gradual cooling of the set of temperatures until a target temperature has been reached. The protocol has been applied for the folding of several peptide systems and for the refinement of protein model structures. In all the cases, the SA-RexMD method turned out to be significantly more efficient in reaching low energy structures and also structures close to experiment compared to continuous MD simulations at the target temperature and to SA-MD simulations at the same computational demand. The approach is well suited for applications in structure refinement and for systematic force field improvement.

Application of biasing‐potential replica‐exchange simulations for loop modeling and refinement of proteins in explicit solvent

Comparative or homology modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. Depending on the target‐template similarity these model structures may contain regions of limited structural accuracy. In principle, molecular dynamics (MD) simulations can be used to refine protein model structures and also to model loop regions that connect structurally conserved regions but it is limited by the currently accessible simulation time scales. A recently developed biasing potential replica exchange (BP‐REMD) method was used to refine loops and complete decoy protein structures at atomic resolution including explicit solvent. In standard REMD simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. In a BP‐REMD simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. The method requires much fewer replicas for efficient sampling compared with T‐REMD. Application of the approach to several protein loops indicated improved conformational sampling of backbone dihedral angle of loop residues compared to conventional MD simulations. BP‐REMD refinement simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional MD simulations. Proteins 2010.

Carbamate prodrug concept for hydroxamate HDAC inhibitors.

Reversible acetylation of histones and other proteins has emerged over the last 10 years as an important mechanism for cell proliferation and has been identified as a valuable target for anticancer drug design. Acetylation is executed and maintained by the histone acetyltransferases and reversed by their counterparts, histone deacetylases (HDACs). The first HDAC inhibitors have already been approved for therapeutic use, and many additional clinical studies are currently under way. Herein, we describe virtual screening efforts that identified several novel HDAC6 inhibitors with cellular isoform selectivity. In particular, a carbamate-protected hydroxamic acid exhibited improved effects with respect to protein hyperacetylation compared with the parent hydroxamate, possibly because of improved cell permeability. The carbamate structure therefore represents a potential prodrug concept for hydroxamic acidcontaining HDAC inhibitors. HDACs are zinc-dependent amidohydrolases, and 11 human subtypes are known. Among the dozens of nonhistone substrates of HDACs, tubulin has attracted a lot of attention because it is a validated target for established anticancer drugs, such as taxanes, the vinca-alkaloids and their derivatives. Tubulin is deacetylated by a single zinc-dependent HDAC subtype, HDAC6, 7] and by the NAD-dependent histone deacetylase Sirt2. HDAC6-specific inhibitors and nonselective HDAC inhibitors synergize in cytotoxicity with the proteasome inhibitor bortezomib (Velcade), which makes HDAC6 an interesting target for inhibitor development. So far, reports of HDAC6selective inhibitors are limited 11, 12, 13] compared to nonselective or class I-selective inhibitors. Aiming at novel selective HDAC6 inhibitors, we were particularly interested in whether structure-based virtual screening using a HDAC6 homology model could deliver such compounds. We recently reported a homology model for HDAC6 used for docking studies of hydroxamates. The model was generated by exploiting multiple solved crystal structures of related HDACs as templates. Based on a substructure search for hydroxamates and related zinc binding groups, we identified 252 potential compounds by virtually screening the Maybridge compound collection comprising 55 000 molecules. The docking and subsequent visual inspection of the docking poses showed that, among the compounds with the highest docking scores, several hydroxamate and hydrazide derivatives could be identified. Finally, the five top-ranked compounds (1–5) were selected for in vitro testing.

Binding of the Bacteriophage P22 N-Peptide to the boxB RNA Motif Studied by Molecular Dynamics Simulations

Protein-RNA interactions are important for many cellular processes. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics simulations were used to investigate the dynamics of the P22 N-peptide-boxB complex and to elucidate the energetic contributions to binding. In addition, the free-energy changes of RNA and peptide conformational adaptation to the bound forms, as well as the role of strongly bound water molecules at the peptide-RNA interface, were studied. The influence of peptide amino acid substitutions and the salt dependence of interaction were investigated and showed good agreement with available experimental results. Several tightly bound water molecules were found at the RNA-binding interface in both the presence and absence of N-peptide. Explicit consideration of the waters resulted in shifts of calculated contributions during the energetic analysis, but overall similar binding energy contributions were found. Of interest, it was found that the electrostatic field of the RNA has a favorable influence on the coil-to-alpha-helix transition of the N-peptide already outside of the peptide-binding site. This result may have important implications for understanding peptide-RNA complex formation, which often involves coupled folding and association processes. It indicates that electrostatic interactions near RNA molecules can lead to a shift in the equilibrium toward the bound form of an interacting partner before it enters the binding pocket.