Discovery and development of next generation sGC stimulators with diverse multidimensional pharmacology and broad therapeutic potential.

Nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC), an enzyme that catalyzes the conversion of guanosine-5'-triphosphate (GTP) to cyclic guanosine-3',5'-monophophate (cGMP), transduces many of the physiological effects of the gasotransmitter NO. Upon binding of NO to the prosthetic heme group of sGC, a conformational change occurs, resulting in enzymatic activation and increased production of cGMP. cGMP modulates several downstream cellular and physiological responses, including but not limited to vasodilation. Impairment of this signaling system and altered NO-cGMP homeostasis have been implicated in cardiovascular, pulmonary, renal, gastrointestinal, central nervous system, and hepatic pathologies. sGC stimulators, small molecule drugs that synergistically increase sGC enzyme activity with NO, have shown great potential to treat a variety of diseases via modulation of NO-sGC-cGMP signaling. Here, we give an overview of novel, orally available sGC stimulators that Ironwood Pharmaceuticals is developing. We outline the non-clinical and clinical studies, highlighting pharmacological and pharmacokinetic (PK) profiles, including pharmacodynamic (PD) effects, and efficacy in a variety of disease models.

Crystal structure of the principal neutralization site of HIV-1.

The crystal structure of a complex between a 24-amino acid peptide from the third variable (V3) loop of human immunodeficiency virus-type 1 (HIV-1) gp 120 and the Fab fragment of a broadly neutralizing antibody (59.1) was determined to 3 angstrom resolution. The tip of the V3 loop containing the Gly-Pro-Gly-Arg-Ala-Phe sequence adopts a double-turn conformation, which may be the basis of its conservation in many HIV-1 isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of synthetic vaccines and drugs to inhibit HIV-1 entry and virus-related cellular fusion.

Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant.

The principal neutralizing determinant (PND) of human immunodeficiency virus HIV-1 is part of a disulfide bridged loop in the third variable region of the external envelope protein, gp120. Analysis of the amino acid sequences of this domain from 245 different HIV-1 isolates revealed that the PND is less variable than thought originally. Conservation to better than 80 percent of the amino acids in 9 out of 14 positions in the central portion of the PND and the occurrence of particular oligopeptide sequences in a majority of the isolates suggest that there are constraints on PND variability. One constraining influence may be the structural motif (beta strand--type II beta turn--beta strand--alpha helix) predicted for the consensus PND sequence by a neural network approach. Isolates with a PND similar to the commonly investigated human T cell lymphoma virus IIIB (HTLV-IIIB) and LAV-1 (BRU) strains were rare, and only 14 percent of sera from 86 randomly selected HIV-1 seropositive donors contained antibodies that recognized the PND of these virus isolates. In contrast, over 65 percent of these sera reacted with peptides containing more common PND sequences. These results suggest that HIV vaccine immunogens chosen because of their similarity to the consensus PND sequence and structure are likely to induce antibodies that neutralize a majority of HIV-1 isolates.

Broadly neutralizing antibodies elicited by the hypervariable neutralizing determinant of HIV-1.

The principal neutralizing determinant (PND) of human immunodeficiency virus (HIV)-1 resides within the V3 loop of the envelope protein. Antibodies elicited by peptides of this region were able to neutralize diverse isolates. Serum from one of three animals immunized with the human T cell lymphoma virus (HTLV)-IIIMN PND peptide, RP142, neutralized MN and the sequence-divergent HTLV-IIIB isolate. Serum from one of three animals immunized with a 13-amino acid IIIB PND peptide (RP337) also neutralized both of these isolates. Characterization of these sera revealed that the cross-neutralizing antibodies bound the amino acid sequence GlyProGlyArgAlaPhe (GPGRAF) that is present in both isolates. This sequence is frequently found in the PNDs analyzed in randomly selected HIV-1 isolates. Sera from two rabbits immunized with a peptide containing only the GPGRAF residues neutralized divergent isolates, including IIIB and MN.

Predictive power of an in vitro system to assess drug interactions of an antimuscarinic medication: a comparison of in vitro and in vivo drug-drug interaction studies of trospium chloride with digoxin.

The authors studied a potential drug-drug interaction via findings from in vitro and in vivo studies, to assess whether the in vitro system was predictive of in vivo clinical pharmacokinetic outcomes. An in vitro experiment and a clinical study were performed to assess the potential for interaction. The effect of trospium chloride on human P-glycoprotein-mediated transport of [3H]-digoxin was determined in vitro. A randomized, crossover clinical trial in 40 subjects was performed to evaluate the effect of trospium on the pharmacokinetics of digoxin in vivo. The findings from the studies were then compared. The in vitro findings in this study were corroborated by the clinical study via assessment of inhibition and impact on pharmacokinetic parameters. The in vitro system for assessment of a potential interaction of 2 drugs excreted primarily through the kidney was predictive of the pharmacokinetic outcomes obtained from a clinical setting.

Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing fabs.

BACKGROUND: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. RESULTS: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II beta turn. CONCLUSIONS: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.

Major antigen-induced domain rearrangements in an antibody.

BACKGROUND: Recent structural results have shown that antibodies use an induced fit mechanism to recognize and bind their antigens. Here we present the crystallographically determined structure of an Fab directed against an HIV-1 peptide (Fab 50.1) in the unliganded state and compare it with the peptide-bound structure. We perform a detailed analysis of the components that contribute to enhanced antigen binding and recognition. RESULTS: Induced fit of Fab 50.1 to its peptide antigen involves a substantial rearrangement of the third complementarity determining region loop of the heavy chain (H3), as well as a large rotation of the variable heavy (VH) chain relative to the variable light (VL) chain. Analysis of other Fab structures suggests that the extent of the surface area buried at the VL-VH interface correlates with the ability to alter antibody quaternary structure by reorientation of the VL-VH domains. CONCLUSION: Fab 50.1 exhibits the largest conformational changes yet observed in a single antibody. These can be attributed to the flexibility of the variable region. Comparisons of new data with previous examples lend to the general conclusion that a small VL-VH interface, due in part to a short H3 loop, permits substantial alterations to the antigen-binding pocket. This has major implications for the prediction, engineering and design of antibody-combining sites.

Affinity and kinetics of the interactions between an alphabeta T-cell receptor and its superantigen and class II-MHC/peptide ligands.

Immune activation is mediated by a specific interaction between the T-cell receptor (TCR) and an antigenic peptide bound to the major histocompatibility complex (MHC). T-cell activation can also be stimulated by superantigens which bind to germline-encoded variable domain sequences of certain TCR beta-chains. We have used a surface plasmon resonance biosensor to characterize the molecular interactions between a class II-restricted alphabeta TCR and its superantigen and MHC/peptide ligands. The extracellular domains of the murine D10 TCR (Valpha2, Vbeta8.2) were expressed in insect cells and secreted as a disulfide-linked heterodimer. In the absence of MHC class II, purified soluble D10 TCR bound to Staphylococcus aureus enterotoxin C2 with an association rate of 1.69+/-0.12 x 10(4)M(-1) sec(-1) and a dissociation rate of 1.9+/-0.47 x 10(-2) sec(-1), giving a dissociation constant of 1.1 microM. Binding of the TCR to S. aureus enterotoxin B was barely detectable and could not be measured accurately due to the rapid dissociation rate. Soluble D10 TCR also bound to a soluble murine MHC class II I-A(k) molecule containing a fused antigenic conalbumin peptide and complementary leucine zipper sequences to facilitate efficient chain pairing. The purified I A(k) chimera specifically stimulated proliferation of the D10 T-cell clone, and bound to immobilized soluble D10 TCR with an association rate of 1.07+/-0.19 x 10(4)M(-1)sec(-1) and a dissociation rate of 2.2+/-0.65 x 10(-2) sec(-1), giving a dissociation constant of 2.1 microM.

Immunoassay to measure staphylococcal protein A in the presence of murine immunoglobulins.

Immunoassays designed to measure low concentrations of staphylococcal protein A are subject to varying degrees of interference by excess IgG. We have developed an enzyme-linked immunosorbent assay (ELISA) that overcomes this problem by analyzing IgG-containing protein A samples in solutions buffered at pH 3.5. Under these carefully selected conditions, protein A is absorbed efficiently by solid-phase chicken anti-protein A antibodies, but protein A-IgG complexes are largely dissociated. The assay has a protein A detection limit of 0.1 ng/ml, and the response is unaffected by 0.25 mg/ml murine IgG. The method should be useful for determining protein A contamination levels in antibodies purified by affinity chromatography on immobilized protein A resins.

A sulfhydryl presumed essential is not required for catalysis by an aminoacyl-tRNA synthetase.

Several aminoacyl-tRNA synthetases are sensitive to reagents that modify sulfhydryl groups. We report here the significance of N-ethylmaleimide (NEM)-mediated inactivation of Escherichia coli glycyl-tRNA synthetase, and alpha 2 beta 2 enzyme. We confirmed earlier observations that NEM abolishes synthetase-catalyzed aminoacylation with pseudo-first order kinetics and provided a second method of proof that the site of inactivation is located in the beta-subunit. Using oligonucleotide-directed mutagenesis of the glyS gene, each beta-subunit cysteine codon (positions 98, 395, and 450) was replaced, individually, by an alanine codon. The three resulting mutant proteins are each active in vivo, and their in vitro aminoacylation activities are comparable to that of the native enzyme. A mutant incorporating all three amino acid substitutions is also active in vivo and in vitro. These results establish conclusively that a beta-subunit cysteine thiol is not required for the catalysis of aminoacylation. The Cys98----Ala and Cys450----Ala mutants are inactivated by NEM with the same kinetics as the wild-type protein. However, the Cys395----Ala mutant is refractory to NEM. This suggests that NEM inactivation of the native enzyme is due to alkylation of Cys395. Aware that inactivation may result from steric effects, we constructed a mutant with a bulkier amino acid residue at position 395 (Cys395----Gln). The aminoacylation activity of this protein is less than 10% of that of the wild-type enzyme. The glutamine substitution affects only the tRNA-dependent step of the reaction--the rate of glycyl adenylate synthesis is not lowered. In these features, the mutant resembles the NEM-inactivated protein. We propose that the NEM sensitivity of glycyl-tRNA synthetase, and possibly of other synthetases, arises from steric or conformational effects of the alkylated cysteine side chain.

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides of DL-alanine and N-(tert-butoxycarbonyl)-DL-alanine.

The aminoacylation of diinosine monophosphate (IpI) was studied. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-DL-alanine, a 40% enantiomeric excess of the L isomer was incorporated at the internal 2' site and the positions of equilibrium for the 2' in equilibrium with 3' migration reaction differed for the D and L enantiomers. The reactivity of the nucleoside hydroxyl groups decreased in the order 2'(3') greater than internal 2' greater than 5', and the extent of reaction was affected by the concentration of the imidazole buffer (pH 7.1). In contrast, reaction of IpI with the imidazolide of unprotected DL-alanine led to an excess of the D isomer at the internal 2' site, while reaction with the N-carboxy anhydride of DL-alanine proceeded without detectable stereoselection. The relevance of these results to the evolution of optical activity and the origin of genetically directed protein synthesis is discussed.

Solution conformational preferences of immunogenic peptides derived from the principal neutralizing determinant of the HIV-1 envelope glycoprotein gp120.

With standard one- and two-dimensional proton NMR techniques, a common structural motif has been identified in water solutions of short peptide sequences derived from the envelope glycoprotein gp120 of HIV-1. Three peptides of lengths 12, 24, and 40 residues (termed RP342, RP142, and RP70, respectively) were synthesized, each containing a central amino acid sequence common to many HIV-1 isolates. In addition, RP70 contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to constitute an important structural and immunological component of the intact glycoprotein. Peptides RP70 and RP142 showed evidence for the presence of a significant population of conformations containing a beta-turn in the conserved sequence Gly-Pro-Gly-Arg. Strong nuclear Overhauser effect (NOE) connectivities were observed between the amide protons of the arginine and the adjacent glycine. A weak NOE connectivity was observed between the C alpha H of the proline residue and the NH of the Arg [a d alpha N(i,i + 2) NOE connectivity], confirming the presence of a conformational preference for a turn conformation in this sequence. The remainder of the peptide showed evidence of conformational averaging: no NMR evidence for a uniquely folded structure was obtained for any of the peptides in water solution. Circular dichroism (CD) spectra indicated that no ordered helix was present in water solutions of RP70, although a CD spectrum that indicated the presence of approximately 30% helix could be induced by the addition of trifluoroethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Two cases of complicated intestinal lipoma. Review of small intestinal lipomas.

Two cases of solitary small intestinal lipoma are presented. One occured in the duodenum as a submucosal pedunculated polyp with a bleeding ulcerated surface. This was diagnosed by means of the fiberoptic duodenoscope (Olympus D1G2). The second involved a large massive submucosal lipoma which formed the leading edge of an intussusception resulting in small bowel obstruction. A brief review of the pathology, complications, symptoms, radiographic diagnosis and treatment of small intestinal lipomas is given.

Synthesis of 2'(3')-O-DL-alanyl hexainosinic acid using T4 RNA ligase: suppression of the enzymic reverse transfer reaction by alkaline phosphatase.

2'(3')-O-DL-Alanyl (Ip)5I was synthesized by a new method. An alanine ortho ester of inosine 5'-phosphate was added to (Ip)4I using the ATP-independent reaction of T4 RNA ligase, and the product was converted smoothly to the desired ester. The enzymic reverse transfer reaction was conveniently suppressed by the dephosphorylation of the adenosine 5'-phosphate coproduct, catalyzed in situ by alkaline phosphatase.

Crystal structure of a human immunodeficiency virus type 1 neutralizing antibody, 50.1, in complex with its V3 loop peptide antigen.

The crystal structure of the Fab fragment of a human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody Fab has been determined at 2.8 A resolution in complex with a linear 16-residue peptide from the third hypervariable region (V3) of gp120. The first 9 residues of the peptide are ordered in the electron density maps, and their conformation is in partial agreement with the beta-strand-type II beta-turn structure predicted for this portion of the V3 loop. Notably, several of the peptide residues that are well conserved among different HIV-1 isolates contact a nonpolar 25-A-long groove in the antibody-combining site. The largely extended structure of the peptide differs from the beta-turns seen as the primary determinants in other published anti-peptide Fab structures. Analysis of the specific Fab-peptide interactions only partially explains the MN isolate specificity shown by this antibody.

A chemically defined synthetic vaccine model for HIV-1.

Multiple Ag peptide (MAP) system without the use of a protein carrier was used as a vaccine model in three species of animals. Synthetic peptides from the V3 region of the gp120 of IIIB, RF and MN HIV-1 isolates were used as the Ag. MAP consisting of various chain lengths, from 11 to 24 residues, were prepared in a monoepitope configuration containing four repeats of each individual peptide. In parallel, they were synthesized in a diepitope configuration adding at the carboxyl-terminus of the V3 peptides a conserved sequence, known to be a Th cell epitope of gp120. The antibody response elicited by the monoepitope constructs was species-dependent. Rabbits produced immunity against all nine peptides, whereas mice were strongly reactive mainly to the longest sequence of the IIIB isolate. The immune response of guinea pigs was intermediate to those of rabbits and mice. Diepitope MAPs were immunogenic in all three species and elicited significantly higher titers than those raised by the immunization with the monoepitope MAPs. The response was type specific; the high-titered antibodies were reactive mostly against the isolate from which the peptides were derived, with a small cross-reactivity in ELISA between IIIB and RF strains. The dominant antigenic site of the B cell epitope, IIIB sequence, was located at the amino and central part of the MAP and a sequence overlapping the putative V3 reverse-turn was particularly reactive with the raised antibodies. Moreover, sera from the immunized animals inhibited virus-dependent cell fusion. These results show that MAP, with a chemically defined structure and without the use of a protein carrier, can be potentially useful for the design of synthetic HIV-1 vaccine candidates.

The topical microbicide PRO 2000 protects against genital herpes infection in a mouse model.

Vaginal gel formulations containing the naphthalene sulfonate polymer PRO 2000 are being developed as topical microbicides to protect against infection with sexually transmitted disease (STD) pathogens. A mouse model was used to determine whether PRO 2000 could protect against genital herpes in vivo. Animals received a single intravaginal application of 15 microL of a 10% PRO 2000 aqueous solution or a 4.0% or 0.5% PRO 2000 vaginal gel formulation 20 s prior to intravaginal challenge with 4.0 log10 pfu of herpes simplex virus type 2. Treatment with the 4.0% gel provided complete protection against infection; treatment with the 0.5% gel or 10% solution provided 81% and 80% protection, respectively. Furthermore, the 4% gel provided significant protection even when viral challenge was delayed until 60 min after treatment. This is the first report to show that PRO 2000 can protect against infection with an STD pathogen in vivo.