Computational and biophysical approaches to protein-protein interaction inhibition of Plasmodium falciparum AMA1/RON2 complex.

Invasion of the red blood cell by Plasmodium falciparum parasites requires formation of an electron dense circumferential ring called the Moving Junction (MJ). The MJ is anchored by a high affinity complex of two parasite proteins: Apical Membrane Antigen 1 (PfAMA1) displayed on the surface of the parasite and Rhoptry Neck Protein 2 that is discharged from the parasite and imbedded in the membrane of the host cell. Structural studies of PfAMA1 revealed a conserved hydrophobic groove localized to the apical surface that coordinates RON2 and invasion inhibitory peptides. In the present work, we employed computational and biophysical methods to identify competitive P. falciparum AMA1-RON2 inhibitors with the goal of exploring the 'druggability' of this attractive antimalarial target. A virtual screen followed by molecular docking with the PfAMA1 crystal structure was performed using an eight million compound collection that included commercial molecules, the ChEMBL malaria library and approved drugs. The consensus approach resulted in the selection of inhibitor candidates. We also developed a fluorescence anisotropy assay using a modified inhibitory peptide to experimentally validate the ability of the selected compounds to inhibit the AMA1-RON2 interaction. Among those, we identified one compound that displayed significant inhibition. This study offers interesting clues to improve the throughput and reliability of screening for new drug leads.

Oligomeric-induced activity by thienyl pyrimidine compounds traps prion infectivity.

Accumulation of PrP(Sc), an abnormal form of cellular prion protein (PrP), in the brain of animals and humans leads to fatal neurodegenerative disorders known as prion diseases. Limited protease digestion of PrP(Sc) produces a truncated form called PrP(27-30) that retains prion infectivity and is the main marker of disease targeted in most diagnostic tests. In the search for new anti-prion molecules, drug-screening assays on prion-infected murine cells have been oriented toward decreasing levels of PrP(27-30). In contrast, we screened for drugs promoting multimers of PrP(27-30), illustrating a possible stabilization of mouse PrP(Sc) species, because recent studies aiming to characterize the conformational stability of various prion strains showed that stable recombinant amyloids produced more stable prion strain, leading to longest incubation time. We identified a family of thienyl pyrimidine derivatives that induce SDS-resistant dimers and trimers of PrP(27-30). Bioassays performed on mice brain homogenates treated with these compounds showed that these thienyl pyrimidine derivatives diminished prion infectivity in vivo. Oligomeric-induced activity by thienyl pyrimidine compounds is a promising approach not only to understanding the pathogenesis of prions but also for prion diagnostics. This approach could be extended to other neurodegenerative "prionopathies," such as Alzheimer's, Huntington, or Parkinson's diseases.

Llama single-domain antibodies directed against nonconventional epitopes of tumor-associated carcinoembryonic antigen absent from nonspecific cross-reacting antigen.

Single-domain antibodies (sdAbs), which occur naturally in camelids, are endowed with many characteristics that make them attractive candidates as building blocks to create new antibody-related therapeutic molecules. In this study, we isolated from an immunized llama several high-affinity sdAbs directed against human carcinoembryonic antigen (CEA), a heavily glycosylated tumor-associated molecule expressed in a variety of cancers. These llama sdAbs bind a different epitope from those defined by current murine mAbs, as shown by binding competition experiments using immunofluorescence and surface plasmon resonance. Flow cytometry analysis shows that they bind strongly to CEA-positive tumor cells but show no cross-reaction toward nonspecific cross-reacting antigen, a highly CEA-related molecule expressed on human granulocytes. When injected into mice xenografted with a human CEA-positive tumor, up to 2% of the injected dose of one of these sdAbs was found in the tumor, despite rapid clearance of this 15 kDa protein, demonstrating its high potential as a targeting moiety. The single-domain nature of these new anti-CEA IgG fragments should facilitate the design of new molecules for immunotherapy or diagnosis of CEA-positive tumors.

Directed mutagenesis in region 713-720 of human thyroperoxidase assigns 713KFPED717 residues as being involved in the B domain of the discontinuous immunodominant region recognized by human autoantibodies.

Autoantibodies (aAbs) to thyroid peroxidase (TPO), the hallmark of autoimmune thyroid disease (AITD), recognize conformational epitopes restricted to an immunodominant region (IDR), divided into two overlapping domains A and B. Despite numerous efforts aimed at localizing the IDR and identifying aAb-interacting residues on TPO, only two critical amino acids, Lys(713) and Tyr(772), have been characterized. Precise and complete delineation of the other residues involved in the IDR remains to be defined. By using a recombinant anti-TPO aAb T13, we demonstrated that four regions on TPO are part of the IDR/B; one of them, located between amino acids 713 and 720, is particularly important for the binding of sera from patients suffering from AITD. To precisely define critical residues implicated in the binding of aAb to human TPO, we used directed mutagenesis and expressed the mutants in stably transfected CHO cells. Then we assessed the kinetic parameters involved in the interactions between anti-TPO aAbs and mutants by real-time analysis. We identified (i) the minimal epitope 713-717 recognized by mAb 47 (a reference antibody) and (ii) the amino acids used as contact points for two IDR-specific human monoclonal aAbs TR1.9 (Pro(715) and Asp(717)) and T13 (Lys(713), Phe(714), Pro(715), and Glu(716)). Using a rational strategy to identify complex epitopes on proteins showing a highly convoluted architecture, this study definitively identifies the amino acids Lys(713)-Asp(717) as being the key residues recognized by IDR/B-specific anti-TPO aAbs in AITD.