EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance.

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.

Mapping and characterization of the epitope(s) of Sch 55700, a humanized mAb, that inhibits human IL-5.

mAb against human IL-5 inhibit pulmonary eosinophilia, tissue damage and airway hyper-reactivity in allergic animal models. Sch 55700 is a humanized, neutralizing anti-IL-5 antibody. To better understand the molecular mechanism by which Sch 55700 blocks IL-5 bioactivity, we have mapped its epitope by scanning IL-5 with synthetic peptides. Those peptides containing a region, ERRRV, corresponding to amino acids 89-93 of IL-5 specifically interact with both Sch 55700 and its parental rat IgG, 39D10. Among the five residues of this region, all three arginine residues were particularly critical for interaction of these peptides with Sch 55700. We further characterized this region by alanine scanning using site-directed mutagenesis. Examination of COS-expressed IL-5 mutants by Western blot showed that single mutations of E(89), R(90), R(91) or R(92) to alanine caused a loss of IL-5 binding to both Sch 55700 and 39D10. We further demonstrated in surface plasmon resonance studies using a BIAcore biosenosor that E(89), R(90) or R(91) are involved in the interaction between IL-5 and its receptor alpha subunit. Based upon the findings here and previously reported structures of the IL-5 and 39D10 variable region, we propose a model suggesting that the molecular interactions between the IL-5 and Sch 55700 mainly involve several ion pair interactions. We conclude that Sch 55700 occupies a region, ERRR, on IL-5 that is essential for its interaction with the receptor and thereby blocks IL-5 bioactivity.

Effect of Sch 55700, a humanized monoclonal antibody to human interleukin-5, on eosinophilic responses and bronchial hyperreactivity.

This report describes the development and the biology of Sch 55700, a humanized monoclonal antibody to human IL-5 (hIL-5). Sch 55700 was synthesized using CDR (complementarity determining regions) grafting technology by incorporating the antigen recognition sites for hIL-5 onto consensus regions of a human IgG4 framework. In vitro, Sch 55700 displays high affinity (Kd = 20 pmol/l) binding to hIL-5, inhibits the binding of hIL-5 to Ba/F3 cells (IC50 = 0.5 nmol/l) and blocks IL-5 mediated proliferation of human erythroleukemic TF-1 cells. In allergic mice, Sch 55700 (0.1-10 mg/kg, i.p. or i.m.) inhibits the influx of eosinophils in the lungs, demonstrates long duration of activity and the anti-inflammatory activity of this compound is additive with oral prednisolone. In allergic guinea pigs, Sch 55700 (0.03-30 mg/kg i.p.) inhibits both the pulmonary eosinophilia and airway hyperresponsiveness and at 30 mg/kg, i.p. inhibited allergic, but not histamine-induced bronchoconstriction. In allergic rabbits, Sch 55700 blocks cutaneous eosinophilia. Sch 55700 (0.1-1 mg/kg i.p.) also blocks the pulmonary eosinophilia and neutrophilia caused by tracheal injection of hIL-5 in guinea pigs. In allergic cynomolgus monkeys, a single dose of Sch 55700 (0.3 mg/kg i.v.) blocks the pulmonary eosinophilia caused by antigen challenge for up to six months. Sch 55700 is, therefore, a potent antibody against IL-5 in vitro and in a variety of species in vivo that could be used to establish the role of IL-5 in human eosinophilic diseases such as asthma.

The neurokinin-1 and neurokinin-2 receptor binding sites of MDL103,392 differ.

Several small molecule non-peptide antagonists of the NK-1 and NK-2 receptors have been developed. Mutational analysis of the receptor protein sequence has led to the conclusion that the binding site for these non-peptide antagonists lies within the bundle created by transmembrane domains IV-VII of the receptor and differs from the binding sites of peptide agonists and antagonists. The current investigation uses site-directed mutagenesis of the NK-1 and NK-2 receptors to elucidate the amino acids that are important for binding and functional activity of the first potent dual NK-1/NK-2 antagonist MDL103,392. The amino acids found to be important for MDL103,392 binding to the NK-1 receptor are Gln-165, His-197, Leu-203, Ile-204, Phe-264, His-265 and Tyr-272. The amino acids found to be important for MDL103,392 binding to the NK-2 receptor are Gln-166, His-198, Tyr-266 and Tyr-289. While residues in transmembrane (TM) domains IV and V are important in both receptors (Gln-165/166 and His-197/198), residues in TM V and VI are more important for the NK-1 receptor and residues in TM VII play a more important role in the NK-2 receptor. These data are the first report of the analysis of the binding site of a dual tachykinin receptor antagonist and indicate that a single compound (MDL103,392) binds to each receptor in a different manner despite there being a high degree of homology in the transmembrane bundles. In addition, this is the first report in which a model for the binding of a non-peptide antagonist to the NK-2 receptor is proposed.

Structural and biological stability of the human interleukin 10 homodimer.

Human interleukin 10 (huIL-10) is a cytokine that regulates the synthesis of type 1 helper T cell derived cytokines such as gamma-interferon, interleukin 2, and tumor necrosis factor alpha. The potential immunosuppressive activities of huIL-10 suggest that this protein may be clinically useful for treating autoimmune diseases. Due to the potential clinical value of this cytokine, physicochemical studies have been performed regarding its association state and biological/structural stability. These studies include performing size-exclusion chromatography, chemical cross-linking, equilibrium ultracentrifugation, and circular dichroism spectroscopy. The results indicate huIL-10 is predominantly a noncovalent homodimer at neutral pH and 4 degreesC for concentrations greater than 0.003 mg/mL (0.08 microM dimer). An apparent pKa value of approximately 4.8 was calculated for both the pH-dependent subunit dissociation and pH-induced loss in MC/9 biological activity. A temperature analysis revealed a linear relationship between the percent dimer and relative MC/9 activity, thus, these results and the pH-dependent activity results suggest that the huIL-10 dimer is the active species. The GndHCl-induced unfolding of rhuIL-10, monitored by far-UV circular dichroism, revealed a unique biphasic unfolding process which contained both a subunit dissociation process (<1.6 M GndHCl) as well as the unfolding of a highly alpha-helical monomer intermediate ([GndHCl]1/2 = 3.5 M). The monomer intermediates generated with 1.6 M GndHCl or pH 2.5 retained approximately 80% and 89% of the alpha-helical content of the native protein, respectively. Although a soluble and highly helical monomer state can be generated, the observed correlation between unfolding studies and biological activity suggests the dimer is the active species. These results are consistent with both the recent observation that the three-dimensional structure of rhuIL-10 is a 2-fold symmetric homodimer and that a complex between the extracellular domain of the recombinant human IL-10 receptor and IL-10 is consistent with two IL-10 homodimers and four receptors.

Two related neurokinin-1 receptor antagonists have overlapping but different binding sites.

The neuropeptide substance P binds to the G protein-coupled neurokinin-1 (NK-1) receptor and elicits cellular responses thought to be involved in pain, neurogenic inflammation, vasodilatation, and plasma exudation. Several small molecule nonpeptide antagonists of the substance P/NK-1 receptor interaction have been developed. Mutational analysis of the receptor protein sequence has led to the conclusion that the binding site for these nonpeptide antagonists lies within the bundle created by transmembrane domains IV-VII of the receptor. This current investigation employs site directed mutagenesis of the NK-1 receptor to compare the binding site of CP-96,345 with that of a related compound CP-99,994. The data demonstrate that while both compounds appear to bind within the transmembrane domain bundle, the contribution of individual amino acid residues to the binding of each compound differs.

The three-dimensional solution structure of the SRC homology domain-2 of the growth factor receptor-bound protein-2.

A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 A for all backbone atoms and 0.40 A for the backbone atoms of the central beta-sheet. The core structure of the SH2 domain contains an antiparallel beta-sheet flanked by two parallel alpha-helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions.

Pulmonary biology of anti-interleukin 5 antibodies

Interleukin 5 (IL-5) is a critical cytokine for the maturation of eosinophil precursors to eosinophils in the bone marrow and those eosinophils then accumulate in the lungs during asthma. We have studied anti-bodies on allergic responses in mice, guinea pigs anf monkeys and are extending this experiment into humans with a humanized antibody. In a monkey model of pulmonary inflammation and airway hyperreactivity, we found that the TRFK-5 antibody blocked both responses for three months following a single dose of 0.3 mg/kg i.v. This antibody also blocked lung eosinophilia in mice by inhibiting release from the bone marrow. To facilitate multiple dosing and to reduce immunogenicity in humans, we prepared Sch 55700, humanized antibody against IL-5. Sch 55700 was also active against lung eosinophilia in allergic monkeys and mice and against pulmonary eosinophilia and airway hyperresponsiveness in guinea pigs. Furthermore, as opposed to steroids, Sch 55700 dis not cause immunosuppression in guinea pigs. Studies with antibody in humans will be critical to establishing the therapeutic potential of IL-5 inhibition.

Pulmonary biology of anti-interleukin 5 antibodies.

Interleukin-5 (IL-5) is a critical cytokine for the maturation of eosinophil precursors to eosinophils in the bone marrow and those eosinophils then accumulated in the lungs during asthma. We have studied anti IL-5 antibodies on allergic responses in mice, guinea pigs and monkeys and are extending this experiment into humans with a humanized antibody. In a monkey model of pulmonary inflammation and airway hyperreactivity, we found that the TRFK-5 antibody blocked both responses for three months following a single does of 0.3 mg/kg, i.v. This antibody also blocked lung eosinophilia in mice by inhibiting release from the bone marrow. To facilitate multiple dosing and to reduce immunogenicity in humans, we prepared Sch 55700, a humanized antibody against IL-5. Sch 55700 was also active against lung eosinophilia in allergic monkeys and mice and against pulmonary eosinophilia and airway hyperresponsiveness in guinea pigs. Furthermore, as opposed to steroids, Sch 55700 did not cause immunosuppression in guinea pigs. Studies with this antibody in humans will be critical to establishing the therapeutic potential of IL-5 inhibition.

Structure and humanization of a rat monoclonal Fab to human interleukin-5.

The X-ray crystal structure of a rat monoclonal Fab JES1-39D10, raised against recombinant human interleukin-5, has been determined with the use of molecular replacement techniques and refined at 2.7 A resolution by simulated annealing. The overall structure is similar to a murine Fab HyHEL-10 that is specific for hen egg white lysozyme. An interesting feature of the structure is the presence of leucine residues to support the H1 complementarity-determining region (CDR) loop. To our knowledge this is the first Fab crystal structure containing this unusual H1 loop support pattern. The activity of three humanized versions of 39D10 is explained by analysis of Fv interface residues and H1 support patterns of 39D10 and the human template HIL.

Crystallization and preliminary X-ray investigation of recombinant human interleukin 10.

Crystals of recombinant human interleukin 10 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 36.5 A and c = 221.9 A. There is the equivalent of one polypeptide chain in the asymmetric unit. The crystals are stable to X-rays and diffract to at least 2.5 A resolution.

Inhibition of pulmonary eosinophilia and hyperreactivity by antibodies to interleukin-5.

Eosinophils infiltrate into the lungs during asthma and may cause the damage associated with pulmonary inflammation. In allergic animal models, antibodies to interleukin (IL)-5 inhibit pulmonary eosinophilia, tissue damage and hyperreactivity. Sch 55700, a humanized antibody against human IL-5, inhibits eosinophilia in these models with an extended biological duration. On the basis of this dosing regimen and the humanized nature of Sch 55700, it is anticipated that the host response leading to tolerance would be minimized.

A homology model of human interferon alpha-2.

An atomic coordinate five alpha-helix three-dimensional model is presented for human interferon alpha-2 (HuIFN alpha 2). The HuIFN alpha 2 structure was constructed from murine interferon beta (MuIFN beta) by homology modeling using the STEREO and IMPACT programs. The HuIFN alpha 2 model is consistent with its known biochemical and biophysical properties including epitope mapping. Lysine residues predicted to be buried in the model were primarily unreactive with succinimidyl-7-amino-4-methylcoumarin-3-acetic acid (AMCA-NHS), a lysine modification agent, as shown by mass spectrometric analysis of tryptic digests. N-terminal sequence analysis of polypeptides generated by limited digestion of HuIFN alpha 2 with endoproteinase Lys-C demonstrated rapid cleavage at K31, which is consistent with the presence of this residue in a loop in the proposed HuIFN alpha 2 model. Based on this model structure potential receptor binding sites are identified.

Engineering the substrate specificity of glutathione reductase toward that of trypanothione reduction.

Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione reductase (CAS registry number 102210-35-5), which are related flavoprotein disulfide oxidoreductases, have marked specificities for glutathione and trypanothione, respectively. A combination of primary sequence alignments and molecular modeling, together with the high-resolution crystal structure of human glutathione reductase, identified certain residues as potentially being responsible for substrate discrimination. Site-directed mutagenesis of Escherichia coli glutathione reductase was used to test these predictions. The mutation of Asn-21 to Arg demonstrated that this single change was insufficient to generate the greater discrimination against trypanothione shown by human glutathione reductase compared with the E. coli enzyme. However, the mutation of Ala-18, Asn-21, and Arg-22 to the amino acid residues (Glu, Trp, and Asn, respectively) in corresponding positions in Trypanosoma congolense trypanothione reductase confirmed that this region of polypeptide chain is intimately involved in substrate recognition. It led to a mutant form of E. coli glutathione reductase that possessed essentially no activity with glutathione but that was able to catalyze trypanothione reduction with a kcat/Km value that was 10% of that measured for natural trypanothione reductases. These results should be of considerable importance in the design of trypanocidal drugs targeted at the differences between glutathione and trypanothione metabolism in trypanosomatids and their hosts.

X-ray structure of trypanothione reductase from Crithidia fasciculata at 2.4-A resolution.

Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidine conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. We report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from -2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4 degrees in the domains that form the site, with relative shifts of as much as 2-3 A in residue positions. These results provide a detailed view of the residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

Preliminary crystallographic analysis of trypanothione reductase from Crithidia fasciculata.

Trypanothione reductase, a flavoprotein disulfide reductase specific to trypanosomatid parasites, has been crystallized by vapor diffusion of a protein solution (10 mg/ml) against 22% polyethylene glycol (average Mr 8000) containing 100 mM-ammonium sulfate. Crystals of a size suitable for structure determination by X-ray diffraction have been obtained by seeding protein solutions with smaller crystals. The space-group is P21 (a = 60.9 A, b = 161.8 A, c = 58.4 A, beta = 99.1 degrees). The molecular mass and volume of the unit cell suggest that there is a dimer of the enzyme in the asymmetric unit, and this is confirmed by self-rotation functions calculated using data to 4.5 A resolution. The crystals diffract to beyond 3 A resolution. Crystals of another P21 form (a = 91.3 A, b = 114.4 A, c = 92.0 A, beta = 141.3 degrees) are observed to grow under similar conditions.

The conformation of dolichol.

An understanding of the natural conformation of dolichol is important for the elucidation of the mechanism of protein glycosylation and dolichol's other as yet undisclosed biological functions. Since the molecular mechanics method has been shown to be well suited for the prediction of alcohol and alkene conformations, we have employed it to study the conformations of apparent least energy of dolichol-19 and smaller polymers of isoprene, namely, squalene, trans,trans-farnesol, and cis,cis-farnesol. Additionally, the small-angle X-ray scattering (SAXS) method was employed to determine the validity of the apparent least energy conformer of dolichol-19 derived by the molecular mechanics method. The results indicate that the solution conformation of dolichol-19 is comprised of a central coiled region flanked by two arms. The central coiled region has two and a half turns of dimensions 9.84 x 16.55 x 51.66 A3. The arms of dimensions 3.99 x 5.89 x 17.47 A3 and 4.49 x 9.23 x 11.14 A3 are approximately diametrically opposed. Measurement of the intrinsic viscosity of dolichol in both isopentyl alcohol and oleyl alcohol showed that the natural conformation of dolichol is capable of increasing solution fluidity (i.e., lowering solution viscosity). Thus, while examination of the conformation of dolichol in a membrane-mimetic solvent by SAXS is not possible, the quantitative measure of the effect of dolichol on solution viscosity (and thus solution fluidity) is possible. The results are consistent with dolichol acting as a membrane-fluidizing agent and provide the first quantitative measure of the effect of dolichol on solution fluidity of a membrane-mimetic solvent.