Administration of therapeutic proteins by methods other than injection is limited, in part, by inefficient penetration of epithelial barriers. Therefore, unique approaches to breaching these barriers are needed. The neonatal constant region fragment (Fc) receptor (FcRn), which is responsible for IgG transport across the intestinal epithelium in newborn rodents, is expressed in epithelial cells in adult humans and non-human primates. Here we show that FcRnmediated transport is functional in the lung of non-human primates and that this transport system can be used to deliver erythropoietin (Epo) when it is conjugated to the Fc domain of IgG1. FcRn-dependent absorption was more efficient when the EpoFc fusion protein was deposited predominantly in the upper and central airways of the lung, where epithelial expression of FcRn was most prominently detected. To optimize fusion protein absorption in the lung, we created a recombinant ''monomericEpo'' Fc fusion protein comprised of a single molecule of Epo conjugated to a dimeric Fc. This fusion protein exhibited enhanced pharmacokinetic and pharmacodynamic properties. The bioavailability of the EpoFc monomer when delivered through the lung was approximately equal to that reported for unconjugated Epo delivered s.c. in humans. These studies show that FcRn can be harnessed to noninvasively deliver bioactive proteins into the systemic circulation in therapeutic quantities.

The gene 2.5 protein (gp2.5) of bacteriophage T7 is a singlestranded DNA (ssDNA) binding protein that has essential roles in DNA replication and recombination. In addition to binding DNA, gp2.5 physically interacts with T7 DNA polymerase and T7 primasehelicase during replication to coordinate events at the replication fork. We have determined a 1.9-Å crystal structure of gp2.5 and show that it has a conserved OB-fold (oligosaccharide͞oligonucle-otide binding fold) that is well adapted for interactions with ssDNA. Superposition of the OB-folds of gp2.5 and other ssDNA binding proteins reveals a conserved patch of aromatic residues that stack against the bases of ssDNA in the other crystal structures, suggesting that gp2.5 binds to ssDNA in a similar manner. An acidic C-terminal extension of the gp2.5 protein, which is required for dimer formation and for interactions with the T7 DNA polymerase and the primase-helicase, appears to be flexible and may act as a switch that modulates the DNA binding affinity of gp2.5.

The neonatal Fc receptor FcRn provides IgG molecules with their characteristically long half-lives in vivo by protecting them from intracellular catabolism and then returning them to the extracellular space. Other investigators have demonstrated that mice lacking FcRn are protected from induction of various autoimmune diseases, presumably because of the accelerated catabolism of pathogenic IgGs in the animals. Therefore, targeting FcRn with a specific inhibitor may represent a unique approach for the treatment of autoimmune disease or other diseases where the reduction of pathogenic IgG will have a therapeutic benefit. Using phage display peptide libraries, we screened for ligands that bound to human FcRn (hFcRn) and discovered a consensus peptide sequence that binds to hFcRn and inhibits the binding of human IgG (hIgG) in vitro. Chemical optimization of the phage-identified sequences yielded the 26-amino acid peptide dimer SYN1436, which is capable of potent in vitro inhibition of the hIgG-hFcRn interaction. Administration of SYN1436 to mice transgenic for hFcRn induced an increase in the rate of catabolism of hIgG in a dose-dependent manner. Treatment of cynomolgus monkeys with SYN1436 led to a reduction of IgG by up to 80% without reducing serum albumin levels that also binds to FcRn. SYN1436 and related peptides thus represent a previously uncharacterized family of potential therapeutic agents for the treatment of humorally mediated autoimmune and other diseases.FcRn antagonist ͉ protein-protein interactions ͉ phage display ͉ autoimmune disease

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The H NMR‐determined structure and dynamics of a synthetic, amphiphilic α‐helical peptide, PH‐1.0 (LYQELQKLTQTLK), and several homologs were compared in 50% trifluoroethanol‐d2 (TFE‐d2)/H2O and in sodium dodecyW25 sulfate (SDS‐d25) micelles. The peptides were designed to test the influence on secondary structure of placement of favored and disfavored residues relative to a “longitudinal, hydrophobic strip‐of‐helix” defined by the repeating leucines. PH‐1.0 was highly ordered as an α‐helix in 50% TFE‐d2/H2O and in SDS‐d25 micelles. Homologs PH‐1.1, in which LI was replaced by T, and PH‐1.4, in which LI 2 was replaced by T, were found to be partially helical in both media. Calculated structures in SDS‐d25 revealed that the helix of PH‐1.1 was slightly disordered at the N‐terminus, but that of PH‐1.4 was completely disordered at the C‐terminus. Examination of distributions of hydrophobic residues in protein structures revealed that, when ♦= LIVFM and ⋄= nonLIVFM, the pattern ⋄♦♦⋄⋄ is favored and ⋄♦⋄♦⋄ is disfavored in α‐helices. Several analogs of PH‐1.0 incorporating these patterns were studied. Peptide PH‐1.12 (LYQELQKLLQTLK) retained a‐helical structure in both 50% TFE‐d2/H2O and in SDS‐d25 micelles. However, although PH‐1.13 (LYQELQKLLQTLK) was fully helical in 50% TFE, it was helical only through residue 6 in SDS micelles. Two homologs containing an additional loop of the helix and repeats of favored (PH‐5.0, NYLQTLLETLKTLLQK) or suppressed LL patterns (PH‐5.11, NYLQTLELTLKLTLQK) gave similar results, i.e. the latter peptide was helical only through residue 6 in SDS micelles. The degree of local order in these SDS micelle‐adsorbed peptides correlates to placement of hydrophobic residues in motifs which are favored or disfavored in proteins in general and in α‐helices specifically. © Munksgaard 1997.

The response of bacteriophage RB69 DNA polymerase to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP), related nucleotides and non-nucleoside inhibitors was measured and compared to values obtained for the closely related DNA polymerase from bacteriophage T4. Both enzymes showed similar responses to inhibitors in terms of Ki values and the ability to utilize BuPdGTP as a substrate. These results provide the relevance of using the recent crystal structure of RB69 DNA polymerase for analysis of BuPdGTP/B family DNA polymerase interactions.

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Nucleoside Carbonyl(di-and triphosphates).-The first examples of nucleoside di-and triphosphates containing the electrophilic and potentially reactive carbonyl group in place of a phosphoanhydride oxygen is reported. Both (I) and (II) are potent, competitive inhibitors of human DNA polymerase α. -(YANACHKOV, IVAN B.; STATTEL, JAMES M.; WRIGHT, GEORGE E.; Perkin 1 (2001) 22, 3080-3084; GLSynthesis, Inc., Worcester, MA 01605, USA; EN)