Biomedical subjects

Amedeo Caflisch

Publications and source records attributed to Amedeo Caflisch.

At least 19 recordsLinked to original sources

PubMed2026

Targeting the METTL1/m7G axis as a therapeutic strategy in myeloid leukemia.

N7-methylguanosine (m7G), a prevalent modification in transfer RNAs (tRNAs), is primarily catalyzed by the methyltransferase METTL1. Although growing evidence supports a role for METTL1 in various tumors, its therapeutic potential and precise function in leukemia stem cell (LSC) homeostasis remain largely unexplored. Here, we identify METTL1 as a key regulator of LSC self-renewal and homing within bone marrow (BM) microenvironment through catalyzing m7G formation on a specific tRNA, tRNAPheGAA, thereby promoting leukemogenesis. Mechanistically, METTL1 loss significantly reduces m7G abundance and steady-state levels of tRNAPheGAA, leading to translation suppression and degradation of transcripts enriched with tRNAPheGAA-related codons, such as hematopoietic cell kinase (HCK). Decreased HCK expression disrupts CXCR4 signaling, impairing LSC self-renewal and BM homing. Therapeutically, we characterized a small-molecule METTL1 inhibitor (M1i; NSC137443), through high-throughput screening. Pharmacological inhibition of METTL1 demonstrated potent antitumor efficacy by reducing tRNA m7G levels and disrupting the tRNAPheGAA/HCK/CXCR4 cascade. Notably, targeting METTL1 significantly reduces LSC frequency, delays leukemogenesis, and prolongs survival in multiple acute myeloid leukemia models. Together, our findings establish a previously unrecognized role for METTL1 and its target tRNAPheGAA in LSC homeostasis and provide compelling proof-of-concept evidence that METTL1 is a druggable epitranscriptomic target for antileukemia therapy.

Humans↗

PubMed2007

Estimation of folding probabilities and phi values from molecular dynamics simulations of reversible Peptide folding.

Molecular dynamics simulations with an implicit model of the solvent have allowed to investigate the reversible folding of structured peptides. For a 20-residue antiparallel beta-sheet peptide, the simulation results have revealed multiple folding pathways. Moreover, the conformational heterogeneity of the denatured state has been shown to originate from high enthalpy, high entropy basins with fluctuating non-native secondary structure, as well as low enthalpy, low entropy traps. An efficient and simple approach to estimate folding probabilities from molecular dynamics simulations has allowed to isolate conformations in the transition state ensemble and to evaluate Phi values, i.e., the effects of mutations on the folding kinetics and thermodynamic stability. These molecular dynamics studies have provided evidence that, if interpreted by neglecting the non-native interactions, Phi values overestimate the amount of native-like structure in the transition state.

Computer Simulation↗

PubMed2006

Automatic and efficient decomposition of two-dimensional structures of small molecules for fragment-based high-throughput docking.

The computer program DAIM (Decomposition and Identification of Molecules) has been developed to automatically break up compounds in small-molecule libraries for fragment-based docking as well as database analysis. Here, DAIM is evaluated on 130 ligands derived from known crystal structures of ligand-protein complexes. The decomposition and a new fingerprint-based identification technique are used to select anchor fragments for docking. The docking results show that the DAIM selection is superior to size-based or random selection of fragments. To evaluate the usefulness for analyzing the fragment composition of a large library, DAIM is applied to a collection of about 1.85 million commercially available compounds. Interestingly, it is found that the set of most frequent cyclic and acyclic fragments originating from the decomposition of the 1.85 million molecules shows a large overlap with the most frequent fragments in a library of 5120 known drugs. DAIM has been successfully used in the in silico screening for inhibitors of beta-secretase and EphB4 kinase by fragment-based high-throughput docking. Possible future applications for de novo ligand design are briefly discussed.

Amyloid Precursor Protein Secretases↗

PubMed2006

Computational models for the prediction of polypeptide aggregation propensity.

In amyloid fibrils, beta-strand conformations of polypeptide chains, or segments thereof, are perpendicular to the fibril axis, but knowledge of their three dimensional structure at atomic level of detail is scarce. Two types of computational approaches have been developed recently for investigating the aggregation propensity of peptides and proteins and identifying the segments most prone to form fibrils (hot spots). The physicochemical properties of the natural amino acids (e.g. beta-propensity, hydrophobicity, aromatic content and charge) have been used to derive phenomenological models able to predict changes in aggregation rate upon mutation, as well as absolute rates and hot spots. Applications of these models to entire proteomes have provided evidence that intrinsically disordered proteins are less amyloidogenic than globular proteins. In the second type of approach, amyloidogenic polypeptides have been decomposed into overlapping segments, and atomistic simulations of three or more copies of each segment have been performed to obtain insights into aggregation propensity and structural details of the ordered aggregates (e.g. turn regions).

Chemical Phenomena↗

PubMed2006

Sequential unfolding of individual helices of bacterioopsin observed in molecular dynamics simulations of extraction from the purple membrane.

Multiple molecular dynamics simulations of bacterioopsin pulling from its C-terminus show that its alpha-helices unfold individually. In the first metastable state observed in the simulations, helix G is unfolded at its C-terminal segment while the rest of helix G (residues 200-216) is folded and opposes resistance because of a salt-bridge network consisting of Asp-212 and Lys-216 on helix G and Arg-82 and Asp-85 on helix C. Helix G unfolds inside the bundle because the external force is applied to its C-terminal end in a direction perpendicular to the surface of the membrane. Inversely, helix F has to flip by 180 degrees to exit from the membrane because the applied force and the helical N-C axis point in opposite directions. At the highest peak of the force, which cannot be interpreted in single-molecule force spectroscopy experiments, helix F has a pronounced kink at Pro-186. Mutation of Pro-186 and/or the charged side chains mentioned above, which are involved in very favorable electrostatic interactions in the low-dielectric region of the membrane, are expected to reduce the highest peak of the force. Helices E and D unfold in a similar way to helices G and F, respectively. Hence, the force-distance profile and sequence of events during forced unfolding of bacterioopsin are influenced by the up-and-down topology of the seven-helix bundle. The sequential extraction of individual helices from the membrane suggests that the spontaneous (un)folding of bacterioopsin proceeds through metastable bundles of fewer than seven helices. The metastable states observed in the simulations provide atomic level evidence that corroborates the interpretation of very recent force spectroscopy experiments of bacteriorhodopsin refolding.

Bacteriorhodopsins↗

PubMed2006

Unfolding transition state and intermediates of the tumor suppressor p16INK4a investigated by molecular dynamics simulations.

The ankyrin repeat is one of the most common protein motifs and is involved in protein-protein interactions. It consists of 33 residues that assume a beta-hairpin helix-loop-helix fold. Mutagenesis and kinetic experiments (Phi-value analysis of the folding transition state) have shown that the tumor suppressor p16(INK4a), a four-repeat protein, unfolds sequentially starting from the two N-terminal repeats. Here, the flexibility of p16(INK4a) at room temperature and its unfolding mechanism at high temperature have been investigated by multiple molecular dynamics runs in explicit water for a total simulation time of 0.65 micros. The transition state ensemble (TSE) of p16(INK4a) was identified by monitoring both the deviation from the experimental Phi values and sudden conformational changes along the unfolding trajectories. Conformations in the TSE have a mainly unstructured second repeat whereas the other repeats are almost completely folded. A rigid-body displacement of the first repeat involving both a rotation and translation is observed in all molecular dynamics simulations at high temperature. The Trp(15), Pro(75), and Ala(76) side-chains are more buried in the TSE than the native state. The sequential unfolding starting at the second repeat is in agreement with the mutagenesis studies whereas the displacement of the first repeat and the presence of nonnative interactions at the TSE are simulation results which supplement the experimental data. Furthermore, the unfolding trajectories reveal the presence of two on-pathway intermediates with partial alpha-helical structure. Finally, on the basis of the available experimental and simulation results we suggest that in modular proteins the shift of the folding TSE toward the native structure upon reduction of the number of tandem repeats is consistent with the Hammond effect.

Binding Sites↗

PubMed2006

Interpreting the aggregation kinetics of amyloid peptides.

Amyloid fibrils are insoluble mainly beta-sheet aggregates of proteins or peptides. The multi-step process of amyloid aggregation is one of the major research topics in structural biology and biophysics because of its relevance in protein misfolding diseases like Alzheimer's, Parkinson's, Creutzfeld-Jacob's, and type II diabetes. Yet, the detailed mechanism of oligomer formation and the influence of protein stability on the aggregation kinetics are still matters of debate. Here a coarse-grained model of an amphipathic polypeptide, characterized by a free energy profile with distinct amyloid-competent (i.e. beta-prone) and amyloid-protected states, is used to investigate the kinetics of aggregation and the pathways of fibril formation. The simulation results suggest that by simply increasing the relative stability of the beta-prone state of the polypeptide, disordered aggregation changes into fibrillogenesis with the presence of oligomeric on-pathway intermediates, and finally without intermediates in the case of a very stable beta-prone state. The minimal-size aggregate able to form a fibril is generated by collisions of oligomers or monomers for polypeptides with unstable or stable beta-prone state, respectively. The simulation results provide a basis for understanding the wide range of amyloid-aggregation mechanisms observed in peptides and proteins. Moreover, they allow us to interpret at a molecular level the much faster kinetics of assembly of a recently discovered functional amyloid with respect to the very slow pathological aggregation.

Amyloid↗

PubMed2006

In silico discovery of beta-secretase inhibitors.

Alzheimer's disease, the most common amyloid-associated disorder, accounts for the majority of the dementia diagnosed after the age of 60. The cleavage of the beta-amyloid precursor protein is initiated by beta-secretase (BACE-1), a membrane-bound aspartic protease, which has emerged as an important but difficult protein target. Here, an in silico screening approach consisting of fragment-based docking, ligand conformational search by a genetic algorithm, and evaluation of free energy of binding was used to identify low-molecular-weight inhibitors of BACE-1. More than 300,000 small molecules were docked and about 15,000 prioritized according to a linear interaction energy model with evaluation of solvation by continuum electrostatics. Eighty-eight compounds were tested in vitro, and 10 of them showed an IC(50) value lower than 100 microM in a BACE-1 enzymatic assay. Interestingly, the 10 active compounds shared a triazine scaffold. Moreover, four of them were active in an assay with mammalian cells (EC(50) < 20 microM), indicating that they are cell-permeable. Therefore, these triazine derivatives are very promising lead candidates for BACE-1 inhibition. The discoveries of this series and two other series of nonpeptidic BACE-1 inhibitors demonstrate the usefulness of our in silico high-throughput screening approach.

Amyloid Precursor Protein Secretases↗

PubMed2006

Network and graph analyses of folding free energy surfaces.

Protein folding is governed by a complex free energy surface whose entropic contributions are relevant because of the large number of degrees of freedom involved. Such complexity, in particular the conformational heterogeneity of the denatured state, is hidden in projections onto one or two order parameters (e.g. fraction of native contacts and/or radius of gyration), which usually results in relatively smooth surfaces. Recent approaches borrowed from network and graph theory have yielded quantitative unprojected representations of the free energy surfaces of a beta-hairpin and a three-stranded beta-sheet peptide using equilibrium folding-unfolding molecular dynamics simulations. Interestingly, the network and graph analyses of these structured peptides have revealed a very heterogeneous denatured state ensemble. It includes high-enthalpy, high-entropy conformations with fluctuating non-native secondary structure, as well as low-enthalpy, low-entropy traps.

Computational Biology↗

PubMed2005

Local modularity measure for network clusterizations.

Many complex networks have an underlying modular structure, i.e., structural subunits (communities or clusters) characterized by highly interconnected nodes. The modularity has been introduced as a measure to assess the quality of clusterizations. has a global view, while in many real-world networks clusters are linked mainly locally among each other (local cluster connectivity). Here we introduce a measure of localized modularity , which reflects local cluster structure. Optimization of and on the clusterization of two biological networks shows that the localized modularity identifies more cohesive clusters, yielding a complementary view of higher granularity.

Journal Article↗

PubMed2005

Organism complexity anti-correlates with proteomic beta-aggregation propensity.

We introduce a novel approach to estimate differences in the beta-aggregation potential of eukaryotic proteomes. The approach is based on a statistical analysis of the beta-aggregation propensity of polypeptide segments, which is calculated by an equation derived from first principles using the physicochemical properties of the natural amino acids. Our analysis reveals a significant decreasing trend of the overall beta-aggregation tendency with increasing organism complexity and longevity. A comparison with randomized proteomes shows that natural proteomes have a higher degree of polarization in both low and high beta-aggregation prone sequences. The former originates from the requirement of intrinsically disordered proteins, whereas the latter originates from the necessity of proteins with a stable folded structure.

Amino Acid Sequence↗

PubMed2005

Discovery of cell-permeable non-peptide inhibitors of beta-secretase by high-throughput docking and continuum electrostatics calculations.

A fragment-based docking procedure followed by substructure search were used to identify active-site beta-secretase inhibitors from a composite set of about 300 000 and a library of nearly 180 000 small molecules, respectively. EC(50) values less than 10 microM were measured in at least one of two different mammalian cell-based assays for 12 of the 72 purchased compounds. In particular, the phenylureathiadiazole 2 and the diphenylurea derivative 3 are promising lead compounds for beta-secretase inhibition.

Amyloid Precursor Protein Secretases↗

PubMed2005

Estimation of protein folding probability from equilibrium simulations.

The assumption that similar structures have similar folding probabilities (p(fold)) leads naturally to a procedure to evaluate p(fold) for every snapshot saved along an equilibrium folding-unfolding trajectory of a structured peptide or protein. The procedure utilizes a structurally homogeneous clustering and does not require any additional simulation. It can be used to detect multiple folding pathways as shown for a three-stranded antiparallel beta-sheet peptide investigated by implicit solvent molecular dynamics simulations.

Biophysics↗

PubMed2005

Spontaneous formation of detergent micelles around the outer membrane protein OmpX.

The structure and flexibility of the outer membrane protein X (OmpX) in a water-detergent solution and in pure water are investigated by molecular dynamics simulations on the 100-ns timescale and compared with NMR data. The simulations allow for an unbiased determination of the structure of detergent micelles and the protein-detergent mixed micelle. The short-chain lipid dihexanoylphosphatidylcholine, as a detergent, aggregates into pure micelles of approximately 18 molecules, or alternatively, it binds to the protein surface. The detergent binds in the form of a monolayer ring around the hydrophobic beta-barrel of OmpX rather than in a micellar-like oblate; approximately 40 dihexanoylphosphatidylcholine lipids are sufficient for an effective suppression of water from the surface of the beta-barrel region. The phospholipids bind also on the extracellular, protruding beta-sheet. Here, polar interactions between charged amino acids and phosphatidylcholine headgroups act as condensation seed for detergent micelle formation. The polar protein surface remains accessible to water molecules. In total, approximately 90-100 detergent molecules associate within the protein-detergent mixed micelle, in agreement with experimental estimates. The simulation results indicate that OmpX is not a water pore and support the proposed role of the protruding beta-sheet as a "fishing rod".

Bacterial Outer Membrane Proteins↗

PubMed2005

Phi-value analysis by molecular dynamics simulations of reversible folding.

In Phi-value analysis, the effects of mutations on the folding kinetics are compared with the corresponding effects on thermodynamic stability to investigate the structure of the protein-folding transition state (TS). Here, molecular dynamics (MD) simulations (totaling 0.65 ms) have been performed for a large set of single-point mutants of a 20-residue three-stranded antiparallel beta-sheet peptide. Between 57 and 120 folding events were sampled at near equilibrium for each mutant, allowing for accurate estimates of folding/unfolding rates and stability changes. The Phi values calculated from folding and unfolding rates extracted from the MD trajectories are reliable if the stability loss upon mutation is larger than approximately 0.6 kcal/mol, which is observed for 8 of the 32 single-point mutants. The same heterogeneity of the TS of the wild type was found in the mutated peptides, showing two possible pathways for folding. Single-point mutations can induce significant TS shifts not always detected by Phi-value analysis. Specific nonnative interactions at the TS were observed in most of the peptides studied here. The interpretation of Phi values based on the ratio of atomic contacts at the TS over the native state, which has been used in the past in MD and Monte Carlo simulations, is in agreement with the TS structures of wild-type peptide. However, Phi values tend to overestimate the nativeness of the TS ensemble, when interpreted neglecting the nonnative interactions.

Computer Simulation↗

PubMed2005

Functional plasticity in the substrate binding site of beta-secretase.

The aspartic protease beta-secretase (BACE) cleaves the amyloid precursor protein into a 42 residue beta-peptide, which is the principal biochemical marker of Alzheimer's disease. Multiple explicit-water molecular dynamics simulations of the apo and inhibitor bound structures of BACE indicate that both open- and closed-flap conformations are accessible at room temperature and should be taken into account for inhibitor design. Correlated motion is observed within each of the two lobes of BACE, as well as for the interfacial region. A self-inhibited conformation with the side chain of Tyr71 occupying the S(1) pocket is present in some of the unbound simulations. The reversible loss of the side chain hydrogen bond between the catalytic Asp32 and Ser35, due to the concomitant reorientation of the Ser35 hydroxyl group and a water molecule conserved in pepsin-like enzymes, provides further evidence for the suggestion that Ser35 assists in proton acceptance and release by Asp32 during catalysis.

Alzheimer Disease↗

PubMed2005

Change of the unbinding mechanism upon a mutation: a molecular dynamics study of an antibody-hapten complex.

We study forced unbinding of fluorescein from the wild type (WT) and a mutant [H(H58)A] of the single-chain variable-fragment (scFv) anti-fluorescein antibody FITC-E2 by molecular dynamics simulations using various pulling techniques. A large number of long simulations were needed to obtain statistically meaningful results as both the wild type and the H(H58)A mutant unbinding occurs through multiple pathways, often with metastable intermediates. For the wild type, the rate-limiting step in the unbinding process corresponds to the breaking of the non-native interactions characteristic of a specific intermediate. The H(H58)A mutation disfavors the occurrence of this intermediate. Two events where the hapten partially unbinds in the absence of pulling force are observed in extensive equilibrium simulations of the wild type, and their analysis indicates that forced unbinding and spontaneous unbinding proceed along similar pathways. The different unbinding mechanisms observed in the simulations suggest a possible reason for the difference in the experimental off-rate between the two antibodies. We predict mutations that are expected to modulate the occurrence of the unbinding intermediate. For two such new mutants [H(H58)A and S(H52)A], our predictions are validated in silico by additional simulations. The accompanying paper in this issue by Honegger et al. reports the X-ray structure of FITC-E2 with a derivative of fluorescein, which was used as the starting conformation for the work presented here.

Amino Acid Substitution↗

PubMed2005

Prediction of aggregation rate and aggregation-prone segments in polypeptide sequences.

The reliable identification of beta-aggregating stretches in protein sequences is essential for the development of therapeutic agents for Alzheimer's and Parkinson's diseases, as well as other pathological conditions associated with protein deposition. Here, a model based on physicochemical properties and computational design of beta-aggregating peptide sequences is shown to be able to predict the aggregation rate over a large set of natural polypeptide sequences. Furthermore, the model identifies aggregation-prone fragments within proteins and predicts the parallel or anti-parallel beta-sheet organization in fibrils. The model recognizes different beta-aggregating segments in mammalian and nonmammalian prion proteins, providing insights into the species barrier for the transmission of the prion disease.

Alzheimer Disease↗