Summary Miniproteins provide a bridge between proteins and small molecules. Here we adapt methods from combinatorial chemistry to optimize CD4M33, a synthetic miniprotein into which we had previously transplanted the HIV-1 gp120 binding surface of the CD4 receptor. Iterative deconvolution of generated libraries produced CD4M47, a derivative of CD4M33 that had been optimized at four positions. Surface-plasmon resonance demonstrated 4-to-6-fold improvement in CD4M47 affinity for gp120 to a level about 3-fold tighter than that of CD4 itself. Assessment of the neutralization properties of CD4M47 against a diverse range of isolates spanning from HIV-1 to SIVcpz showed that CD4M47 retained the extraordinary breadth of the parent CD4M33, but yielded only limited improvements in neutralization potencies. Crystal structures of CD4M47 and a phenylalanine variant ([Phe23]M47) were determined at 2.4 and 2.6 Å resolution, in ternary complexes with HIV-1 gp120 and the 17b antibody. Analysis of these structures revealed a correlation between mimetic affinity for gp120 and overall mimetic-gp120 interactive surface. A correlation was also observed between CD4- and mimetic-induced gp120-structural similarity and CD4- and mimetic-induced gp120 affinity for the CCR5 co-receptor. Despite mimetic substitutions, including a glycine to (D)-proline change, the gp120 conformation induced by CD4M47 was as close or closer to the conformation induced by CD4 as the one induced by the parent CD4M33. Our results demonstrate the ability of combinatorial chemistry to optimize a disulfide-containing miniprotein and of structural biology to decipher the resultant interplay between binding affinity, neutralization breadth, molecular mimicry, and induced affinity for CCR5.
P947 (DOTA-Gd-peptide) was recently identified as an MRI contrast agent for the detection and characterization of the matrix metalloproteinases (MMP)-rich atherosclerotic plaques. Because this product displays a broad spectrum affinity for the MMP family, we hypothesized that it may also recognize other metalloproteinases overactivated in vulnerable atherosclerotic plaques. Therefore, this study aimed at describing, at the molecular and cellular level, the interactions between P947 and proteases of atherosclerotic plaques. Fluorimetric assays were used to measure the in vitro affinity of P947 toward recombinant and purified MMPs, angiotensin-converting enzyme (ACE), endothelin-converting enzyme (ECE-1), neutral endopeptidase (NEP), and both aminopeptidases A and N (APA and APN). Using similar fluorimetric assays associated with specific substrates, enzymatic activities were measured in vulnerable and stable plaques collected from human atherosclerotic carotid arteries. Ex vivo affinity of P947 for metalloproteinases in vulnerable lesions was subsequently determined. Interaction between P947 and major cell types present in atherosclerotic plaques was also investigated in different cell lines: PMA-1-differentiated THP-1 (macrophage), Ox-LDL-treated THP-1 (foam cell), Jurkat cell line (lymphocyte), and human umbilical vein endothelial cell (HUVEC, endothelial cell). Molecular targeting of P947 was confirmed by fluorimetry, ICP-MS, and in vitro MRI approaches. Potential application of P947 for detecting atherosclerotic plaques by in vivo MRI was tested in a rabbit model of atherosclerosis. In vitro, P947 displayed affinities for purified MMPs, ACE, ECE-1, NEP, APA, and APN in the micromolar range. Interestingly, MMPs, ACE, and APN exhibited higher activities in vulnerable plaques from human atherosclerotic carotid samples, as compared to stable plaques. ECE-1, NEP, and APA had either no activity or the same low activity in both vulnerable and stable plaques. P947 showed micromolar affinities for MMPs, ACE, and APN secreted by plaque samples. Moreover, P947 bound to THP-1 macrophages and THP-1 foam cells in a concentration-dependent manner and with a higher intensity than the control contrast agents DOTA-Gd or P1135 (DOTA-Gd coupled to a scrambled peptide). In THP-1 macrophages, P947 inhibited largely (70%) and almost completely (95%) MMP and APN activities, respectively, which strongly suggested an MMP- and APN-dependent binding of P947 to these cells. This enzyme-specific binding was confirmed with in vitro MRI. Indeed, the T1 value of THP-1 cells decreased from 2.094 s (macrophages w/o P947) to 2.004 s (macrophages with 1 mM of P947). In addition, the Gd content measured by ICP-MS was 11.01 ± 1.05 fg Gd/macrophage when cells were incubated in the presence of P947 and only 5.18 ± 0.43 fg Gd/macrophage with the control product P1135. The difference of Gd concentration between both contrast agents corresponded to a specific accumulation of 5.83 fg Gd/cell, which may be detected by MRI. MR imaging in the atherosclerosi...
Background: Legionella pneumophila secretes a protease without known specific substrate and three-dimensional structure.Results: Analysis of a quenched peptide library identified a lead substrate, ameliorated by rational design, using an Msp structural model obtained by the x-ray structure of pseudolysin. Conclusion:The study identifies the first selective substrate for Msp. Significance: This substrate could be useful for Legionella detection.
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