Presentation_1_Crystal Structures of R-Type Bacteriocin Sheath and Tube Proteins CD1363 and CD1364 From Clostridium difficile in the Pre-assembled State.pdf

<p>Diffocins are high-molecular-weight phage tail-like bacteriocins (PTLBs) that some Clostridium difficile strains produce in response to SOS induction. Similar to the related R-type pyocins from Pseudomonas aeruginosa, R-type diffocins act as molecular puncture devices that specifically penetrate the cell envelope of other C. difficile strains to dissipate the membrane potential and kill the attacked bacterium. Thus, R-type diffocins constitute potential therapeutic agents to counter C. difficile-associated infections. PTLBs consist of rigid and contractile protein complexes. They are composed of a baseplate, receptor-binding tail fibers and an inner needle-like tube surrounded by a contractile sheath. In the mature particle, the sheath and tube structure form a complex network comprising up to 200 copies of a sheath and a tube protein each. Here, we report the crystal structures together with small angle X-ray scattering data of the sheath and tube proteins CD1363 (39 kDa) and CD1364 (16 kDa) from C. difficile strain CD630 in a monomeric pre-assembly form at 1.9 and 1.5 Å resolution, respectively. The tube protein CD1364 displays a compact fold and shares highest structural similarity with a tube protein from Bacillus subtilis but is remarkably different from that of the R-type pyocin from P. aeruginosa. The structure of the R-type diffocin sheath protein, on the other hand, is highly conserved. It contains two domains, whereas related members such as bacteriophage tail sheath proteins comprise up to four, indicating that R-type PTLBs may represent the minimal protein required for formation of a complete sheath structure. Comparison of CD1363 and CD1364 with structures of PTLBs and related assemblies suggests that several conformational changes are required to form complete assemblies. In the sheath, rearrangement of the flexible N- and C-terminus enables extensive interactions between the other subunits, whereas for the tube, such contacts are primarily established by mobile α-helices. Together, our results combined with information from structures of homologous assemblies allow constructing a preliminary model of the sheath and tube assembly from R-type diffocin.</p

α,β-Methylene-ADP (AOPCP) Derivatives and Analogues: Development of Potent and Selective <i>ecto</i>-5′-Nucleotidase (CD73) Inhibitors

<i>ecto</i>-5′-Nucleotidase (<i>e</i>N, CD73) catalyzes the hydrolysis of extracellular AMP to adenosine. <i>e</i>N inhibitors have potential for use as cancer therapeutics. The <i>e</i>N inhibitor α,β-methylene-ADP (AOPCP, adenosine-5′-<i>O</i>-[(phosphonomethyl)­phosphonic acid]) was used as a lead structure, and derivatives modified in various positions were prepared. Products were tested at rat recombinant <i>e</i>N. 6-(Ar)­alkylamino substitution led to the largest improvement in potency. <i>N</i>⁶-Monosubstitution was superior to symmetrical <i>N</i>⁶,<i>N</i>⁶-disubstitution. The most potent inhibitors were <i>N</i>⁶-(4-chlorobenzyl)- (<b>10l</b>, PSB-12441, <i>K</i>_i 7.23 nM), <i>N</i>⁶-phenylethyl- (<b>10h</b>, PSB-12425, <i>K</i>_i 8.04 nM), and <i>N</i>⁶-benzyl-adenosine-5′-<i>O</i>-[(phosphonomethyl)­phosphonic acid] (<b>10g</b>, PSB-12379, <i>K</i>_i 9.03 nM). Replacement of the 6-NH group in <b>10g</b> by O (<b>10q</b>, PSB-12431) or S (<b>10r</b>, PSB-12553) yielded equally potent inhibitors (<b>10q</b>, 9.20 nM; <b>10r</b>, 9.50 nM). Selected compounds investigated at the human enzyme did not show species differences; they displayed high selectivity versus other <i>ecto</i>-nucleotidases and ADP-activated P2Y receptors. Moreover, high metabolic stability was observed. These compounds represent the most potent <i>e</i>N inhibitors described to date