Antonio Morreale

Cooperativity Between T Cell Receptor Complexes Revealed by Conformational Mutants of CD3ɛ

A mutant CD3ɛ subunit that cannot change conformation in response to ligand binding exerts dominant-negative inhibition of T cell responses. Must Change Shape The T cell antigen receptor (TCR) complex consists of the TCR αβ subunits noncovalently associated with the δ, ɛ, γ, and ζ subunits of CD3. Engagement of the TCR by peptide bound to the major histocompatibility complex (MHC) on the surface of an antigen-presenting cell triggers the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 subunits, and these motifs recruit the proteins that mediate TCR signaling. With a molecular dynamics model, Martínez-Martín et al. investigated how extracellular binding of antigen to the TCR is transduced to the inside of the cell. They identified two residues in CD3ɛ that were critical for the transmission of ligand-induced conformational changes to the intracellular portions of this subunit. CD3ɛ subunits with mutations in either of these residues blocked transmission of the conformational change, and one of these variants functioned as a dominant-negative inhibitor of TCR signaling and T cell activation even when present at very low abundance. The authors propose that the initial interaction of an antigen with a TCR may influence the conformation of oligomeric TCR complexes so that TCRs act cooperatively to transmit signals from peptide-MHC. The CD3ɛ subunit of the T cell receptor (TCR) complex undergoes a conformational change upon ligand binding that is thought to be important for the activation of T cells. To study this process, we built a molecular dynamics model of the transmission of the conformational change within the ectodomains of CD3. The model showed that the CD3 dimers underwent a stiffening effect that was funneled to the base of the CD3ɛ subunit. Mutation of two relevant amino acid residues blocked transmission of the conformational change and the differentiation and activation of T cells. Furthermore, this inhibition occurred even in the presence of excess endogenous CD3ɛ subunits. These results emphasize the importance of the conformational change in CD3ɛ for the activation of T cells and suggest the existence of unforeseen cooperativity between TCR complexes.

The amino terminal domain from Mrt4 protein can functionally replace the RNA binding domain of the ribosomal P0 protein

In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein.

Computational Approaches to Model Ligand Selectivity in Drug Design

To be effective, a designed drug must discriminate successfully the macromolecular target from alternative structures present in the organism. The last few years have witnessed the emergence of different computational tools aimed to the understanding and modeling of this process at molecular level. Although still rudimentary, these methods are shaping a coherent approach to help in the design of molecules with high affinity and specificity, both in lead discovery and in lead optimization. It is the purpose of this review to illustrate the array of computational tools available to consider selectivity in the design process, to summarize the most relevant applications, and to sketch the challenges ahead.

MM-ISMSA: An Ultrafast and Accurate Scoring Function for Protein-Protein Docking.

An ultrafast and accurate scoring function for protein-protein docking is presented. It includes (1) a molecular mechanics (MM) part based on a 12-6 Lennard-Jones potential; (2) an electrostatic component based on an implicit solvent model (ISM) with individual desolvation penalties for each partner in the protein-protein complex plus a hydrogen bonding term; and (3) a surface area (SA) contribution to account for the loss of water contacts upon protein-protein complex formation. The accuracy and performance of the scoring function, termed MM-ISMSA, have been assessed by (1) comparing the total binding energies, the electrostatic term, and its components (charge-charge and individual desolvation energies), as well as the per residue contributions, to results obtained with well-established methods such as APBSA or MM-PB(GB)SA for a set of 1242 decoy protein-protein complexes and (2) testing its ability to recognize the docking solution closest to the experimental structure as that providing the most favorable total binding energy. For this purpose, a test set consisting of 15 protein-protein complexes with known 3D structure mixed with 10 decoys for each complex was used. The correlation between the values afforded by MM-ISMSA and those from the other methods is quite remarkable (r(2) ∼ 0.9), and only 0.2-5.0 s (depending on the number of residues) are spent on a single calculation including an all vs all pairwise energy decomposition. On the other hand, MM-ISMSA correctly identifies the best docking solution as that closest to the experimental structure in 80% of the cases. Finally, MM-ISMSA can process molecular dynamics trajectories and reports the results as averaged values with their standard deviations. MM-ISMSA has been implemented as a plugin to the widely used molecular graphics program PyMOL, although it can also be executed in command-line mode. MM-ISMSA is distributed free of charge to nonprofit organizations.

Protein-protein interaction antagonists as novel inhibitors of non-canonical polyubiquitylation.

Background Several pathways that control cell survival under stress, namely RNF8-dependent DNA damage recognition and repair, PCNA-dependent DNA damage tolerance and activation of NF-κB by extrinsic signals, are regulated by the tagging of key proteins with lysine 63-based polyubiquitylated chains, catalyzed by the conserved ubiquitin conjugating heterodimeric enzyme Ubc13-Uev. Methodology/Principal Findings By applying a selection based on in vivo protein-protein interaction assays of compounds from a combinatorial chemical library followed by virtual screening, we have developed small molecules that efficiently antagonize the Ubc13-Uev1 protein-protein interaction, inhibiting the enzymatic activity of the heterodimer. In mammalian cells, they inhibit lysine 63-type polyubiquitylation of PCNA, inhibit activation of NF-κB by TNF-α and sensitize tumor cells to chemotherapeutic agents. One of these compounds significantly inhibited invasiveness, clonogenicity and tumor growth of prostate cancer cells. Conclusions/Significance This is the first development of pharmacological inhibitors of non-canonical polyubiquitylation that show that these compounds produce selective biological effects with potential therapeutic applications.

Structure-Based Discovery of Novel Non-nucleosidic DNA Alkyltransferase Inhibitors: Virtual Screening and in Vitro and in Vivo Activities

The human DNA-repair O (6)-alkylguanine DNA alkyltransferase (MGMT or hAGT) protein protects DNA from environmental alkylating agents and also plays an important role in tumor resistance to chemotherapy treatment. Available inhibitors, based on pseudosubstrate analogs, have been shown to induce substantial bone marrow toxicity in vivo. These deficiencies and the important role of MGMT as a resistance mechanism in the treatment of some tumors with dismal prognosis like glioblastoma multiforme, the most common and lethal primary malignant brain tumor, are increasing the attention toward the development of improved MGMT inhibitors. Here, we report the identification for the first time of novel non-nucleosidic MGMT inhibitors by using docking and virtual screening techniques. The discovered compounds are shown to be active in both in vitro and in vivo cellular assays, with activities in the low to medium micromolar range. The chemical structures of these new compounds can be classified into two families according to their chemical architecture. The first family corresponds to quinolinone derivatives, while the second is formed by alkylphenyl-triazolo-pyrimidine derivatives. The predicted inhibitor protein interactions suggest that the inhibitor binding mode mimics the complex between the excised, flipped out damaged base and MGMT. This study opens the door to the development of a new generation of MGMT inhibitors.

Tubulin-based Structure-affinity Relationships for Antimitotic Vinca Alkaloids

The Vinca alkaloids are a group of widely used anticancer drugs, originally extracted from the Madagascar periwinkle, that disrupt microtubule dynamics in mammalian cells by interfering with proper assembly of α,β-tubulin heterodimers. They favor curved tubulin assemblies that destabilize microtubules and induce formation of spiral aggregates. Their binding energy profiles have been characterized by means of sedimentation velocity assays and the binding site of vinblastine at the interface between two tubulin dimers (α1β1 � α2β2) has been ascertained by X-ray crystallographic studies on a complex of tubulin with the stathmin-like domain of protein RB3, albeit at relatively low resolution. Here we use molecular modeling and simulation techniques to build, refine and perform a comparative analysis of the three-dimensional complexes of vinblastine, vincristine, vinorelbine and vinflunine with a β1α2-tubulin interface in explicit water to rationalize the binding affinity differences in structural and energetic terms. Our results shed some more light into the binding determinants and the structure-activity relationships of these clinically useful agents.

Synthesis, biological assessment and molecular modeling of 14-aryl-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]quinolin-13-amines

The synthesis and pharmacological evaluation of racemic 14-aryl-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]quinolin-13-amines (19-28), prepared by Friedländer reaction of 3-amino-1-aryl-1H-benzo[f]chromene-2-carbonitriles (10-18) with suitable cycloalkanones is described. These molecules are potent, in the nanomolar range [IC(50) (EeAChE)=7-101 nM], and selective inhibitors of acetylcholinesterase (AChE). The most potent inhibitor, 4-(13-amino-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]quinolin-14-yl)phenol (20) [IC(50) (EeAChE)=7±2 nM] is four-fold more active than tacrine. Kinetic studies on compound 20 showed that this is a mixed-type inhibitor of EeAChE with a K(i) of 5.00 nM. However, racemic 20 was unable to displace propidium iodide, suggesting that the inhibitor does not strongly bind to the peripheral anionic site (PAS) of AChE. Docking, molecular dynamics stimulations, and MM-GBSA calculations agree well with this behavior.

A new implicit solvent model for protein–ligand docking

A new implicit solvent model for computing the electrostatics binding free energy in protein–ligand docking is proposed. The new method is based on an adaptation of the screening coulombic potentials proposed originally by Hassan et al. (J Phys Chem B 2000;104:6490–6498). In essence, it relies on two basic assumptions; (i) solvent screening can be accounted for by means of radially dependent sigmoidal dielectric functions and; (ii) the effective atom Born radii can be expressed only as a function of the exposed atom surface. Parameters of the model other than radii and charges are generic. These were optimized for a dataset of 826 protein–ligand complexes, comprising both X‐ray complexes for 23 receptors as well as decoys generated by docking computations. We show that the new model provides satisfactory results when benchmarked against reference values based on the numerical solution of the Poisson equation, with a root mean square error of 4.2 kcal/mol over a range of ∼40 kcal/mol in electrostatics binding free energies, a cross‐validated r2 of 0.81, a slope of 0.97, and an intercept of 1.06 kcal/mol. We show that the model is appropriate for ligands of different sizes, polarities, overall charge, and chemical composition. Furthermore, not only the total value of the electrostatic contribution to the binding free energy, but also its components (coulombic term, receptor desolvation, and ligand desolvation) are reasonably well reproduced. Computation times of ∼0.030 s per pose are obtained on a single processor desktop workstation. Proteins 2007.

Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis

Background: Reactive arthritis is an HLA-B27-associated disease triggered by Chlamydia trachomatis. Results: Three chlamydial peptides endogenously presented by HLA-B27 were identified. All were homologous to human-derived sequences, and one showed conformational similarity to a self-derived HLA-B27 ligand. Conclusion: Molecular mimicry between chlamydial and self-derived HLA-B27 ligands is not uncommon. Significance: Molecular mimicry may contribute to the pathology of reactive arthritis. Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na+-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27+ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.