Trisubstituted-imidazoles induce apoptosis in human breast cancer cells by targeting the oncogenic PI3K/Akt/mTOR signaling pathway

Overactivation of PI3K/Akt/mTOR is linked with carcinogenesis and serves a potential molecular therapeutic target in treatment of various cancers. Herein, we report the synthesis of trisubstituted-imidazoles and identified 2-chloro-3-(4, 5-diphenyl-1H-imidazol-2-yl) pyridine (CIP) as lead cytotoxic agent. Naïve Base classifier model of in silico target prediction revealed that CIP targets RAC-beta serine/threonine-protein kinase which comprises the Akt. Furthermore, CIP downregulated the phosphorylation of Akt, PDK and mTOR proteins and decreased expression of cyclin D1, Bcl-2, survivin, VEGF, procaspase-3 and increased cleavage of PARP. In addition, CIP significantly downregulated the CXCL12 induced motility of breast cancer cells and molecular docking calculations revealed that all compounds bind to Akt2 kinase with high docking scores compared to the library of previously reported Akt2 inhibitors. In summary, we report the synthesis and biological evaluation of imidazoles that induce apoptosis in breast cancer cells by negatively regulating PI3K/Akt/mTOR signaling pathway

Supramolecular peptide nanofiber morphology affects mechanotransduction of stem cells

Chirality and morphology are essential factors for protein function and interactions with other biomacromolecules. Extracellular matrix (ECM) proteins are also similar to other proteins in this sense; however, the complexity of the natural ECM makes it difficult to study these factors at the cellular level. The synthetic peptide nanomaterials harbor great promise in mimicking specific ECM molecules as model systems. In this work, we demonstrate that mechanosensory responses of stem cells are directly regulated by the chirality and morphology of ECM-mimetic peptide nanofibers with strictly controlled characteristics. Structural signals presented on l-amino acid containing cylindrical nanofibers (l-VV) favored the formation of integrin β1-based focal adhesion complexes, which increased the osteogenic potential of stem cells through the activation of nuclear YAP. On the other hand, twisted ribbon-like nanofibers (l-FF and d-FF) guided the cells into round shapes and decreased the formation of focal adhesion complexes, which resulted in the confinement of YAP proteins in the cytosol and a corresponding decrease in osteogenic potential. Interestingly, the d-form of twisted-ribbon like nanofibers (d-FF) increased the chondrogenic potential of stem cells more than their l-form (l-FF). Our results provide new insights into the importance and relevance of morphology and chirality of nanomaterials in their interactions with cells and reveal that precise control over the chemical and physical properties of nanostructures can affect stem cell fate even without the incorporation of specific epitopes

Computational investigation of function of membrane proteins : Amt/Rh Ammonium transporters and SecY translocon

In this thesis, we studied the function of the Amt/Rh family of proteins and of the SecY/Sec61 translocons using computational methods. The Amt/Rh proteins mediate transport of ammonium across the lipid bilayer. SecY and Sec61 translocons facilitate the insertion of membrane proteins or translocation of secreted proteins in prokaryotes and eukaryotes, respectively. We investigated on the molecular details of ammonium transport in E.Coli AmtB and human RhCG proteins, and the effect of the hydrophobicity of the SecY translocon pore in membrane protein insertion. Functional studies have revealed that Amt proteins transport the charged form of ammonium (NH4+) while Rh proteins transport neutral ammonia (NH3). However, permeation mechanisms at a molecular level have not been understood clearly. Here, we present molecular details of ammonium transport in AmtB and RhCG proteins. Our calculations show that ammonium ion binds and deprotonates at the hydrophobic pore of AmtB. Then, ammonia diffuses down the hydrophobic pore while the excess proton is transported with the help of a highly conserved histidine dyad (H168 and H318). Ammonia gets re-protonated when it reaches the bottom of the pore and leaves the channel as ammonium. To recruit a new ammonium substrate the protonation states of the histidine dyad has to be reset. This is achieved through water molecules forming a single-file chain in the pore. Thus, hydration of the pore plays an important role in the transport mechanism in AmtB protein. Our simulations of RhCG protein have revealed that the pore of RhCG protein is not hydrated. Lack of hydration in the pore suggests that the excess proton cannot be transported across the hydrophobic pore as it is proposed for AmtB. We show that ammonium binds and deprotonates at a histidine residue (H185) lining the hydrophobic pore of RhCG. After deprotonation, ammonia diffuses down the pore. Then, the excess proton is circulated back to the extracellular site through a network of hydrogen bonds connecting H185 to D177. In conclusion, our calculations suggest that RhCG protein transports neutral ammonia while AmtB transports charged ammonium. Experimental findings showed that mutation of the pore-ring residues of Sec61 translocon changed the hydrophobicity threshold for membrane integration. Our free energy calculations suggested that mutation of the pore-ring residues influences the stability of peptides in the pore, thus affecting the probability of membrane integration. In addition, insertion experiments of oligo alanine peptides, which contain a cluster of three leucines at various positions, revealed an asymmetry in the membrane integration profile. In particular, a significant drop in membrane integration was observed when the three-leucine cluster aligns with the pore-ring residues. We simulated the wild-type SecY and its pore-ring mutants with the oligo-alanine peptides initially placed into the pores. Analysis of these simulations suggested that hydration of the leucine side-chains drops dramatically when the three-leucine cluster is aligned with the pore-ring residues. The reduced hydration of the leucine residues stabilizes the peptide in the translocon pore and favors its translocation

Different Hydration Patterns in the Pores of AmtB and RhCG Could Determine Their Transport Mechanisms

The ammonium transporters of the Amt/Rh family facilitate the diffusion of ammonium across cellular membranes. Functional data suggest that Amt proteins, notably found in plants, transport the ammonium ion (NH4(+)), whereas human Rhesus (Rh) proteins transport ammonia (NH3). Comparison between the X-ray structures of the prokaryotic AmtB, assumed to be representative of Amt proteins, and the human RhCG reveals important differences at the level of their pore. Despite these important functional and structural differences between Amt and Rh proteins, studies of the AmtB transporter have led to the suggestion that proteins of both subfamilies work according to the same mechanism and transport ammonia. We performed molecular dynamics simulations of the AmtB and RhCG proteins under different water and ammonia occupancy states of their pore. Free energy calculations suggest that the probability of finding NH3 molecules in the pore of AmtB is negligible in comparison to finding water. The presence of water in the pore of AmtB could support the transport of proton. The pore lumen of RhCG is found to be more hydrophobic due to the presence of a phenylalanine conserved among Rh proteins. Simulations of RhCG also reveal that the signature histidine dyad is occasionally exposed to the extracellular bulk, which is never observed in AmtB. These different hydration patterns are consistent with the idea that Amt and Rh proteins are not functionally equivalent and that permeation takes place according to two distinct mechanisms

Catch-bond mechanism of the bacterial adhesin FimH

Ligand-receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell-cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force

Ammonium Transporters Achieve Charge Transfer by Fragmenting Their Substrate

Proteins of the Amt/MEP family facilitate ammonium transport across the membranes of plants, fungi, and bacteria, and are essential for growth in nitrogen-poor environments. Some are known to facilitate the diffusion of the neutral NH3 while others, notably in plants, transport the positively charged NH4+. Based on the structural data for AmtB from Escherichia coli, we illustrate the mechanism by which proteins from the Amt family can sustain electrogenic transport. Free energy calculations show that NH4+ is stable in the AmtB pore, reaching a binding site from which it can spontaneously transfer a proton to a pore-lining histidine residue (His168). The substrate diffuses down the pore in the form of NH3 while the excess proton is co-transported through a highly conserved hydrogen-bonded His168-His318 pair. This constitutes a novel permeation mechanism that confers to the histidine dyad an essential mechanistic role that was so far unknown