Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation.

We report the structure-based design and synthesis of a unique NOS inhibitor, called nanoshutter NS1, with two-photon absorption properties. NS1 targets the NADPH site of NOS by a nucleotide moiety mimicking NADPH linked to a conjugated push-pull chromophore with nonlinear absorption properties. Because NS1 could not provide reducing equivalents to the protein and competed with NADPH binding, it efficiently inhibited NOS catalysis. NS1 became fluorescent once bound to NOS with an excellent signal-to-noise ratio because of two-photon excitation avoiding interference from the flavin-autofluorescence and because free NS1 was not fluorescent in aqueous solutions. NS1 fluorescence enhancement was selective for constitutive NOS in vitro, in particular for endothelial NOS (eNOS). Molecular dynamics simulations suggested that two variable residues among NOS isoforms induced differences in binding of NS1 and in local solvation around NS1 nitro group, consistent with changes of NS1 fluorescence yield. NS1 colocalized with eNOS in living human umbilical vein endothelial cells. Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800-950-nm range, with great perspectives for eNOS imaging in living tissues.

Synthesis of new N-(arylcyclopropyl)acetamides and N-(arylvinyl)acetamides as conformationally-restricted ligands for melatonin receptors.

N-(Arylcyclopropyl)acetamides and N-(arylvinyl)acetamides or methyl ureas have been prepared as constrained analogues of melatonin. The affinity of these new compounds for chicken brain melatonin receptors and recombinant human MT(1) and MT(2) receptors was evaluated using 2-[(125)I]-iodomelatonin as radioligand. Strict ethylenic or cyclopropyl analogues of the commercialized agonist agomelatine (Valdoxan®) were equipotent to agomelatine in binding bioassays. However, the ethylenic analogue was more effective than the cyclopropyl one in the melanophore aggregation bioassay, but was still less potent than the disubstituted 2,7-dimethoxy-naphtalenic compounds.

CD4 mimetic miniproteins: potent anti-HIV compounds with promising activity as microbicides.

OBJECTIVES: The antiviral activity of CD4 miniproteins was evaluated as potential HIV microbicides, using relevant in vitro models. METHODS: Compounds were tested in a single-cycle HIV-1 pseudovirus assay and against replication competent HIV-1 in co-cultures of monocyte-derived dendritic cells (MO-DC) and CD4+ T cells. Cytotoxic activity was evaluated in an MTT assay. RESULTS: Monomeric miniproteins (M47 and M48) showed 50% effective concentration (EC(50)) values of 79-105 nM against a subtype B, CCR5 co-receptor-using Ba-L pseudovirus. Higher activity was found for the dimeric miniproteins M48D30, M48D50 and M48D100 (EC(50) between 15 and 30 nM), in contrast to the tetrameric miniproteins M48T30, M48T50 and M48T100 (EC(50) between 107 and 377 nM). The hetero-bivalent miniprotein M48-Hep and miniproteins that targeted the Phe-43 cavity on gp120 (M48-U1, M48-U2 and M48-U3) were highly active, with EC(50) values as low as 2 nM for M48-U1. All miniproteins showed high activity against CCR5 or CXCR4 co-receptor-using subtype B and CRF-01_A/E pseudoviruses. Many early M48-based compounds were much less active against subtype C pseudoviruses, whereas M48-U compounds that targeted the Phe-43 cavity were very active against all pseudoviruses, including subtype C. In MO-DC/CD4+ T cell co-cultures with replication-competent HIV-1 Ba-L, EC(50) values ranged between 13 and 1719 nM depending on the miniprotein, with M48-U1, M48-U2 and M48-U3 again being the most potent. Importantly, the latter compounds completely prevented viral replication by treating the cultures from 2 h before until 24 h after infection, at non-toxic concentrations of 66-6564 nM. CONCLUSIONS: These novel CD4 miniproteins might constitute a promising class of HIV microbicides.

Screening and in situ synthesis using crystals of a NAD kinase lead to a potent antistaphylococcal compound.

Making new ligands for a given protein by in situ ligation of building blocks (or fragments) is an attractive method. However, it suffers from inherent limitations, such as the limited number of available chemical reactions and the low information content of usual chemical library deconvolution. Here, we describe a focused screening of adenosine derivatives using X-ray crystallography. We discovered an unexpected and biocompatible chemical reactivity and have simultaneously identified the mode of binding of the resulting products. We observed that the NAD kinase from Listeria monocytogenes (LmNADK1) can promote amide formation between 5'-amino-5'-deoxyadenosine and carboxylic acid groups. This unexpected reactivity allowed us to bridge in situ two adenosine derivatives to fully occupy the active NAD site. This guided the design of a close analog showing micromolar inhibition of two human pathogenic NAD kinases and potent bactericidal activity against Staphylococcus aureus in vitro.

Synthesis of new arylalkoxy amido derivatives as melatoninergic ligands.

Amido derivatives 10-18 of the corresponding oxyamines were synthesised as melatoninergic ligands by the reaction of hydroxyphtalimide with the halogeno derivatives or the corresponding alcohols using Mitsunobu reaction conditions. The affinity of the compounds for chicken brain melatonin receptors and recombinant human MT(1) and MT(2) receptors was evaluated using 2-[125I]-iodomelatonin as the radioligand. Overall, the introduction of an oxygen atom in the amido chain was not a favourable parameter as the compounds were less potent than the corresponding deoxy derivatives. However, nanomolar compounds were obtained with the arylethyloxy derivatives (13c (R'=nPr), chicken brain, hMT(1), hMT(2), K(i) values: 4.8, 3.86, 2.4 nM, respectively) and the 2,7-dimethoxynaphthalene derivatives (17c (R'=nPr), chicken brain, hMT(1), hMT(2), K(i) values: 0.04, 0.13, 0.1 nM, respectively). The functional activity of these compounds was evaluated by the aggregation of melanophores in Xenopus laevis tadpoles and the potency was related to the affinity of the molecules for melatonin receptors. The compounds were found to be full agonists and compound 17a was 20-fold more potent than melatonin in this bioassay.