Biomimetic analogs 1 of the microbial siderophore (iron carrier) ferrichrome were labeled with a fluorescent marker at a site which does not interfere with iron binding or receptor recognition to provide iron(III) carriers 5 and 10 (Figure ). These carriers were built from a tetrahedral carbon as an anchor which was symmetrically extended by three converging iron-binding chains and a single, exogenous anthracenyl residue. Carriers 5 varied in the nature of the amino acids (G = glycyl, A = alanyl and L = leucyl) linking the anchor with the iron-binding hydroxamate groups, while the alanyl derivative 10A differed from 5A in the spacer between the anthracenyl label and the anchor. Examination of these binders by 1H NMR confirmed that their conformations were analogous to those of the nonlabeled parent compounds. Titration experiments using UV/vis and fluorescence spectroscopy demonstrated the quenching of these compounds' fluorescence upon iron(III) loading and its recovery upon iron(III)'s release to a competing chelator. The quenching process fits the Perrin model for static quenching and was more efficient in derivatives 5, where the label could approach the iron-binding domain, than in derivative 10A, where the label's approach was prohibited. These data are in compliance with an intramolecular quenching process. In vivo examination of the labeled derivatives 5 with Pseudomonas putida as the indicator organism established that their behavior parallels that of the nonlabeled analogs 1, with the added benefit of signaling microbial activity by fluorescence emission. Thus incubation of P. putida with iron(III)-loaded (and therefore nonfluorescent) 5G caused buildup of the label's fluorescence in the culture medium. These observations provide direct evidence for a shuttle-mechanism of iron delivery where the fluorescent, iron-unloaded carrier is released to the medium. Inhibition of both phenomena by natural ferrichrome or NaN3 demonstrates the involvement of the microbial ferrichrome receptor and transport systems, respectively. On the other hand, 5A induced only modest iron(III) uptake by P. putida and failed to generate fluorescence in the culture medium, concurring with its action as an inhibitor. The fact that two strains of different Pseudomonas species did not respond to the ferrichrome analog 5G illustrates the specificity of these compounds. The performance of these carriers as structural and functional probes, paired with their high species specificity, encourage their consideration as diagnostic tools for the detection and identification of pathogenic bacteria and fungi.

Human monoclonal antibody (mAb) 447-52D neutralizes a broad spectrum of HIV-1 isolates, whereas murine mAb 0.5beta, raised against gp120 of the X4 isolate HIV-1(IIIB), neutralizes this strain specifically. Two distinct gp120 V3 peptides, V3(MN) and V3(IIIB), adopt alternative beta-hairpin conformations when bound to 447-52D and 0.5beta, respectively, suggesting that the alternative conformations of this loop play a key role in determining the coreceptor specificity of HIV-1. To test this hypothesis and to better understand the molecular basis underlying an antibody's breadth of neutralization, the solution structure of the V3(IIIB) peptide bound to 447-52D was determined by NMR. V3(IIIB) and V3(MN) peptides bound to 447-52D exhibited the same N-terminal strand conformation, while the V3(IIIB) peptide revealed alternative N-terminal conformations when bound to 447-52D and 0.5beta. Comparison of the three known V3 structures leads to a model in which a 180 degrees change in the orientation of the side chains and the resulting one-residue shift in hydrogen bonding patterns in the N-terminal strand of the beta-hairpins markedly alter the topology of the surface that interacts with antibodies and that can potentially interact with the HIV-1 coreceptors. Predominant interactions of 447-52D with three conserved residues of the N-terminal side of the V3 loop, K312, I314, and I316, can account for its broad cross reactivity, whereas the predominant interactions of 0.5beta with variable residues underlie its strain specificity.

We designed the N-methylanthranilic-desferrioxamine (MA-DFO) as a fluorescent iron(III) chelator with improved membrane permeation properties. Upon binding of iron(III), MA-DFO fluorescence is quenched, thus allowing traceability of drug-iron(III) interactions. MA-DFO is well tolerated by mammalian cells in culture. Its antimalarial activity is pronounced: IC50 values on in vitro (24-h) growth of Plasmodium fakciparum were 3±1 MM for MA-DFO compared with 30±8 for DFO. The onset of growth inhibition of rings or trophozoites occurs 2-4 h after exposure to 13 gM MA-DFO. This effect is commensurate with MA-DFO permeation into infected cells. In a 24-h exposure to MA-DFO or DFO, trophozoites take up either compound to -10% of the external concentration, rings to 5%, and noninfected cells to < 1%. Red cells encapsulated with millimolar concentrations of DFO or MA-DFO fully support parasite invasion and growth. We conclude that extracellular MA-DFO and DFO gain selective access into parasites by bypassing the host. The rate-limiting step is permeation through the parasite membrane, which MA-DVO accomplishes faster than DFO, in accordance with its iigher hydrophobicity. These views are consistent with the proposed duct, which apparently provides parasitized cells with a window to the external medium. (J. Clin. Invest. 1993. 91:218-224.)