The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase--GW311616A a development candidate.

The discovery of a potent intracellular inhibitor of human neutrophil elastase which is orally active and has a long duration of action is described. The pharmacodynamic and pharmacokinetic properties of a trans-lactam development candidate, GW311616A, are described.

Intracellular inhibition of human neutrophil elastase by orally active pyrrolidine-trans-lactams.

Described are the acylation binding of trans-lactam 1 to porcine pancreatic elastase, the selection of the SO2Me activating group for the lactam N which also confers metabolic stability in hamster liver microsomes, the introduction of aqueous solubility through the piperidine salt 9, the in vivo oral activity of 9 and its bioavailability, and the introduction of 9 as an intracellular neutrophil elastase inhibitor.

Identification of novel transmembrane gene sequence and its use for cell-surface targeting of beta subunit of human chorionic gonadotropin.

We identified a 685-nucleotide gene fragment that codes for the transmembrane and cytoplasmic domains of glycoprotein of the LEP strain rabies virus and carried out experiments designed to express a novel fusion protein on the cell surface. The cDNA encoding the membrane anchor sequence was fused in the correct reading frame to the 3' end of the cDNA encoding the beta subunit of human chorionic gonadotropin (beta(h)CG), a secretory glycoprotein that is used as an antigen for a contraceptive vaccine being developed in our laboratory. The fusion gene cassette was placed under the control of a vaccinia virus early promoter and cloned in a host-restricted fowlpox viral vector. The recombinants, when used to infect mammalian cells that do not allow the replication of fowlpox virus, expressed the N-terminal 135 amino acid residues of beta(h)CG anchored in the cell membrane by the 75-amino acid C-terminal sequence derived from rabies virus glycoprotein. This hybrid protein is correctly processed post-translationally and transported efficiently to the plasma membrane of non-permissive cells such that the anchored beta(h)CG molecule retains the correctly folded native antigenic epitope(s).

The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors, clorobiocin.

Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24-kDa N-terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP-binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3' position of the noviose sugar is substituted by a 5-methyl-2-pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24-kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5'-adenylyl-beta, gamma-imidodiphosphate, and with information on the bound conformation of novobiocin in the p24-novobiocin complex obtained by heteronuclear isotope-filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented.

The HSD-hCG vaccine prevents pregnancy in women: feasibility study of a reversible safe contraceptive vaccine.

PROBLEM: To develop a vaccine for reversible control of fertility in women. MATERIALS AND PROTOCOLS: Purified beta subunit of hCG annealed to purified alpha subunit of ovine LH linked chemically to tetanus toxoid (TT) and diphtheria (DT); vaccine employed at 300 micrograms gonadotropin equivalent per injection adsorbed on alhydrogel with 1 mg SPLPS added in the first injection; Phase I safety trials in 47 women with elective tubal ligation; Phase II efficacy studies in 148 proven fertile women (2 children), sexually active, desirous of family planning using IUD; IUD removed when anti-hCG titres exceed 50 ng/ml hCG bioneutralization capacity; boosters given to maintain above threshold antibody levels; post coital tests conducted in 8 volunteers; sera of protected women analysed for immuno-determinants recognized by competitive enzyme immunoassays employing a panel of monoclonal antibodies and by direct binding to synthetic peptides; recombinant vaccines expressing beta hCG as a secreted product or as a fused protein anchored on membrane. RESULTS: Immunization was well tolerated with no significant changes in endocrine, metabolic and hematological indices. Normal ovulatory cycles were maintained as indicated by menstrual regulation. The vaccine was highly effective in preventing pregnancy (1 pregnancy in 1224 cycles ) at and above antibody titres of 50 ng/ml. Antibodies declined in course of time in absence of boosters, with conceptions occurring below 35 ng/ml titres indicating regain of fertility. Ability of antibodies to prevent pregnancy was confirmed by post coital tests. High avidity (10(10) M-1) and other characteristics of antibodies generated by the vaccine are described and compared with those induced by two other hCG vaccines having undergone Phase I trials. The antibody response of the HSD vaccine in humans is characterized predominantly to an epitope recognized by the monoclonals 206 and P3W80. The antibodies had low or no reactivity with the carboxy terminal peptide and 38-57 region peptide. Live recombinant vaccines expressing beta hCG as a membrane anchored peptide generated antibody response to hCG in all animals following a single injection. CONCLUSIONS: Reversible fertility control is feasible with the HSD-hCG vaccine without impairment of ovulation or disturbance of menstrual regularity. Suggestions have been made for further optimization of the vaccine, which include replacement of TT and DT by a panel of T non B determinants communicating with the entire MHC spectrum and development of recombinant vaccine expressing beta hCG along with membrane anchored carrier.

The nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray crystallography.

This study describes the first crystal structures of a complex between a DNA topoisomerase and a drug. We present the structures of a 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein in complexes with two different inhibitors of the ATPase activity of DNA gyrase, namely the coumarin antibiotic, novobiocin, and GR122222X, a member of the cyclothialidine family. These structures are compared with the crystal structure of the complex with an ATP analogue, adenylyl-beta-gamma-imidodiphosphate (ADPNP). The likely mechanism, by which mutant gyrase B proteins become resistant to inhibition by novobiocin are discussed in light of these comparisons. The three ligands are quite dissimilar in chemical structure and bind to the protein in very different ways, but their binding is competitive because of a small degree of overlap of their binding sites. These crystal structures consequently describe a chemically well characterized ligand binding surface and provide useful information to assist in the design of novel ligands.

Crystallization of inhibitor complexes of an N-terminal 24 kDa fragment of the DNA gyrase B protein.

A 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein has been crystallized in the presence of novobiocin. One crystal form has been obtained that is orthorhombic, P2(1)2(1)2(1), with unit cell dimensions a = 40.3 A, b = 47.7 A, c = 111.9 A. The asymmetric unit of this crystal form contains one molecule (Vm = 2.24 A3/Da). Complete native data have been collected to 2.5 A resolution. This same protein fragment has also been crystallized in the presence of GR122222X, an inhibitor that is structurally related to cyclothialidine. These crystals also exhibit P2(1)2(1)2(1) symmetry but have unit cell dimensions of a = 68.8 A, b = 68.6 A, c = 48.6 A. The Vm value of this crystal form is 2.39 A3/Da, assuming one molecule in the asymmetric unit, and native data have been collected to 2.0 A resolution. Molecular replacement studies of both complexes are underway.

X-ray crystallographic studies of a series of penicillin-derived asymmetric inhibitors of HIV-1 protease.

In the development of a treatment for AIDS, the HIV-1 protease has been identified as a good target enzyme for inhibitor design. We previously reported a series of dimeric penicillin-derived C2-symmetric HIV-1 protease inhibitors [Humber, D., et al. (1993) J. Med. Chem. 36, 3120-3128]. In an attempt to reduce the size and optimize the binding of these C2-symmetric inhibitors, molecular modeling studies led to a novel series of monomeric penicillin-derived inhibitors of HIV-1 protease. The binding modes of these monomeric inhibitors have been characterized by X-ray crystallographic and NMR studies. Crystal structures of HIV-1 protease complexed to three inhibitors (GR123976, GR126045, and GR137615) from this series identify the molecular details of the interactions. The binding of GR123976 (IC50 = 2.3 microM) exhibits good hydrophobic contacts but few electrostatic interactions. A strategy of structure-based design and chemical synthesis led to the elaboration of GR123976 to optimize interactions with the protein. Crystallographic analysis of HIV-1 protease complexed to GR126045 and GR137615 identified these interactions with the catalytic aspartates and the protein binding pockets. The crystal structures of the three complexes confirm the presence of the major interactions modeled in order to optimize potency and reveal details of the molecular recognition by HIV-1 protease of this novel series of nonpeptidic inhibitors.

A series of penicillin-derived C2-symmetric inhibitors of HIV-1 proteinase: structural and modeling studies.

The binding modes of a series of penicillin-derived C2 symmetric dimer inhibitors of HIV-1 proteinase were investigated by NMR, protein crystallography, and molecular modeling. The compounds were found to bind in a symmetrical fashion, tracing and S-shaped course through the active site, with good hydrophobic interactions in the S1/S1' and S2/S2' pockets and hydrogen bonding of inhibitor amide groups. Interactions with the catalytic aspartates appeared poor and the protein conformation was very similar to that seen in complexes with peptidomimetics, in spite of the major differences in ligand structure.

Active immunization of baboons (Papio anubis) with the bovine LH receptor.

Four baboons (Papio anubis) were actively immunized with bovine LH receptor for periods of 6-22 months. Serum antibody levels were measured by an enzyme immunoabsorbant assay (ELISA). Antibodies against the receptor were detected 2 weeks after the first injection. Antisera caused an inhibition in the binding of human chorionic gonadotropin (hCG) to its receptor as well as inhibited the production of hCG induced testosterone by rat Leydig cells in culture. Serum estradiol and progesterone levels were determined by radioimmunoassay (RIA). Progesterone levels were suppressed during the post immunization period. Two baboons experienced periods of anovulation. Serum estradiol levels were cyclic and appeared elevated. Baboons were mated with males of proven fertility; none of the immunized females conceived over eight cycles of observation. Fertility parameters returned to normal, when antibodies against LH-receptor were undetectable in the serum.

Large scale expression and purification of recombinant HIV-1 proteinase from Escherichia coli.

The availability of target proteins in sufficient quantity is a limiting factor in crystallographic studies and therefore in rational drug design. Even after optimisation, expression of recombinant proteins may be low and the only way to produce enough protein is by large scale cell growth/purification. HIV-1 proteinase in Escherichia coli, which due to its toxicity is expressed as a soluble protein only at around 0.1% of total protein, is a paradigm for this. In this paper a detailed process for large scale expression and purification of HIV-1 proteinase which delivers material of suitable quantity (30 mg from 500 g of wet weight of cells) and quality for crystallographic studies is described.

Expression of an autoprocessing CAT-HIV-1 proteinase fusion protein: purification to homogeneity of the release 99 residue proteinase.

The 99 residue human immunodeficiency virus type 1 proteinase has been expressed in Escherichia coli as part of an autocleaving fusion protein. Expression of the fusion protein is toxic to the host cells, however yields of the released proteinase have been improved by optimising induction nad harvest times to increase culture biomass, and decrease degradation of the proteinase. Soluble proteinase was extracted from these cells by a simple and highly efficient three step process. N-terminal sequence analysis confirms that the enzyme preparation is highly pure and correctly autoprocessed. The proteinase cleaves peptide substrate IGCTLNFPISPIETV between F and P at pH 6.0 with a Km of 310 microM and a Kcat of 14s-1. The enzyme is sensitive to its ionic environment, showing stimulation of activity at high salt concentrations, and shows a pH optimising 5.5.

The phospholipid-dependence of UDP glucuronosyltransferase. Purification, delipidation and reconstitution of microsomal enzyme from guinea-pig liver.

In order to study the interaction of liver microsomal UDPglucuronosyltransferase and microsomal phospholipids under closely defined conditions, guinea-pig enzyme was purified to homogeneity (as judged by sodium dodecyl sulphate gel electrophoresis) by detergent-solubilisation, salt precipitation, chromatography on DEAE-cellulose and DEAE-Sephadex, and affinity chromatography on UDPglucuronosyl-diaminohexanyl--Sepharose 4B. The purified transferase, which catalysed the glucuronidation of p-nitrophenol with high specific activity, was associated with microsomal phospholipids, and phosphatidylcholine was the major species present; the transferase protein had a subunit molecular weight of about 55 000. The enzyme was almost completely inactivated by delipidation of the protein by hydroxyapatite chromatography and efficient reconstitution of high activity was observed only with fluid (microsomal and egg-yolk) phosphatidylcholines. These results confirm that microsomal UDPglucuronosyltransferase is phospholipid-dependent with a specific requirement for phosphatidylcholine.