Effect of polyethylene glycol modification on the circulatory stability and immunogenicity of recombinant human butyrylcholinesterase

The therapeutic value of human serum butyrylcholinesterase (Hu BChE) as a bioscavenger of chemical warfare agents is due to its high reactivity with organophosphorus compounds and prolonged circulatory stability. Native Hu BChE is mostly tetrameric in form while the enzyme produced using molecular cloning technology is a mixture of tetramers, dimers, and monomers. Previous studies revealed that monomers and dimers of recombinant human (rHu) BChE cleared rapidly from the circulation of mice compared to tetrameric rHu BChE and native Hu BChE, which have mean residence times (MRTs) of 18h and 45h, respectively. It was also shown that polyethylene glycol-20K (PEG) modification of tetrameric rHu BChE prolonged its circulatory stability and bioavailability in vivo. The goal of this study was to determine if modification with PEG could prolong the circulatory stability and eliminate the immunogenicity of monomeric rHu BChE. Monomeric rHu BChE was expressed in human 293A cells using a cDNA lacking the 45 amino acid tetramerization domain from the carboxyl terminus and the adenovirus expression system. The catalytic and inhibitory properties of purified monomeric rHu BChE were similar to those for native Hu BChE and were not affected by PEG modification. As expected, monomeric rHu BChE rapidly cleared from the circulation of mice (MRT=3.2+/-0.3h) while monomeric PEG-rHu BChE demonstrated significant improvement in its bioavailability and circulatory stability in blood (MRT=31.4+/-5.4h). However, a second injection of monomeric PEG-rHu BChE, 28 days after the first, displayed a much shorter MRT=11.6+/-0.4h, and circulating anti-monomeric PEG-rHu BChE antibodies were detected in the blood of mice. These results suggest that PEG modification increased the circulatory stability of monomeric rHu BChE but failed to reduce or eliminate its immunogenicity.     

Comparison of high pressure-induced dissociation of single-stranded DNA-binding protein (SSB) from high pressure-sensitive and high pressure-adapted marine Shewanella species

The effects of hydrostatic pressure on protein quaternary structure were compared for recombinant single-stranded DNA-binding protein (SSB) derived from piezosensitive, piezotolerant, and obligately piezophilic ("pressure-loving") marine Shewanella strains. The pressure-induced dissociation of the oligomeric SSB proteins was investigated using fluorescence anisotropy. The SSBs all exhibited striking similarity in the pressure-dependent behavior of the fluorescence intensity and emission spectrum as well as in their dissociation constants at atmospheric pressure. The free energies of subunit association into tetramers for all SSBs were between -27 and -30 kcal mol(-1). However, SSB from the piezosensitive Shewanella strain S. hanedai was more sensitive to pressure than that of the SSB proteins from the piezotolerant or piezophilic bacteria. The volume change of association obtained from the pressure dependence of dissociation at a fixed protein concentration (Delta V(p)) for SSB from S. hanedai was 394-402 ml mol(-1). The Delta V(p) values for SSB from the deeper-living Shewanellas were smaller and ranged from 253 to 307 ml mol(-1). Differences between the primary structures of the SSB proteins that could correlate with differences in sensitivity to pressure-induced dissociation were examined.     

Carotenoids of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum

The major carotenoid pigments of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum, were identified as zeaxanthin, beta-cryptoxanthin, and beta-carotene. Analysis was based on ultraviolet-visible spectroscopy, mass spectroscopy, and reversed-phase HPLC. Photoacoustic spectroscopy of intact bacterial cells revealed that the bulk of the pigments in S. antarcticus and S. multivorum was associated with the cell membrane. In vitro studies with synthetic membranes of phosphatidylcholine demonstrated that the major pigment was bound to the membranes and decreased their fluidity. The relative amounts of polar pigments were higher in cells grown at 5 degrees C than in cells grown at 25 degrees C. In the mesophilic strain, the synthesis of polar carotenoids was quantitatively less than that of the psychrotolerant strain.     

High-resolution solution structure of a designed peptide bound to lipopolysaccharide: transferred nuclear Overhauser effects, micelle selectivity, and anti-endotoxic activity

Designing peptides that would interact with lipopolysaccharides (LPS) and acquire a specific folded conformation can generate useful structural insights toward the development of anti-sepsis agents. In this work, we have constructed a 12-residue linear peptide, YW12, rich in aromatic and aliphatic amino acid residues with a centrally located stretch of four consecutive positively charged (KRKR) residues. In absence of LPS, YW12 is predominantly unstructured in aqueous solution. Using transferred nuclear Overhauser effect (Tr-NOE) spectroscopy, we demonstrate that YW12 adopts a well-folded structure as a complex with LPS. Structure calculations reveal that YW12 assumes an extended conformation at the N-terminus followed by two consecutive beta-turns at its C-terminus. A hydrophobic core is formed by extensive packing between number of aromatic and nonpolar residues, whereas the positively charged residues are segregated out to a separate region essentially stabilizing an amphipathic structure. In an in vitro LPS neutralization assay using NF-kappaB induction as the readout, YW12 shows moderate activity with an IC50 value of approximately 10 microM. As would be expected, tryptophan fluorescence studies demonstrate that YW12 shows selective interactions only with the negatively charged lipid micelles including sodium dodecyl sulfate (SDS), 1-palmitoyl-2-oleoylphosphatidyl-dl-glycerol (POPG), and LPS, and no significant interactions are detected with zwitterionic lipid micelles such as dodecyl-phosphocholine (DPC). Far-UV CD studies indicate the presence of beta-turns or beta-sheet-like conformations of the peptide in negatively charged micelles, whereas no structural transitions are apparent in DPC micelles. These results suggest that structural features of YW12 could be utilized to develop nontoxic antisepsis compounds.     

Preliminary evaluation of a 3H imidazoquinoline library as dual TLR7/TLR8 antagonists

Toll-like receptors (TLR) -7 and -8 are thought to play an important role in immune activation processes underlying the pathophysiology of HIV and several clinically important autoimmune diseases. Based on our earlier findings of TLR7-antagonistic activity in a 3H imidazoquinoline, we sought to examine a pilot library of 3H imidazoquinolines for dual TLR7/8 antagonists, since they remain a poorly explored chemotype. 2D-NOE experiments were employed to unequivocally characterize the compounds. A quinolinium compound 12, bearing p-methoxybenzyl substituents on N3 and N5 positions was identified as a lead. Compound 12 was found to inhibit both TLR7 and TLR8 at low micromolar concentrations. Our preliminary results suggest that alkylation with electron-rich substituents on the quinoline N5, or conversely, elimination of the fixed charge of the resultant quaternary amine on the quinolinium may yield more active compounds.     

Structure–activity relationships of lipopolysaccharide sequestration in N-alkylpolyamines

We have previously shown that simple N-acyl or N-alkyl polyamines bind to and sequester Gram-negative bacterial lipopolysaccharide, affording protection against lethality in animal models of endotoxicosis. Several iterative design-and-test cycles of SAR studies, including high-throughput screens, had converged on compounds with polyamine scaffolds which have been investigated extensively with reference to the number, position, and length of acyl or alkyl appendages. However, the polyamine backbone itself had not been explored sufficiently, and it was not known if incremental variations on the polymethylene spacing would affect LPS-binding and neutralization properties. We have now systematically explored the relationship between variously elongated spermidine [NH₂–(CH₂)₃–NH–(CH₂)₄–NH₂] and norspermidine [NH₂–(CH₂)₃–NH–(CH₂)₃–NH₂] backbones, with the N-alkyl group being held constant at C₁₆ in order to examine if changing the spacing between the inner secondary amines may yield additional SAR information. We find that the norspermine-type compounds consistently showed higher activity compared to corresponding spermine homologues.     

Structure–activity relationships of lipopolysaccharide sequestration in guanylhydrazone-bearing lipopolyamines

The toxicity of Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) resides in its structurally highly conserved glycolipid component called lipid A. Our major goal has been to develop small-molecules that would sequester LPS by binding to the lipid A moiety, so that it could be useful for the prophylaxis or adjunctive therapy of Gram-negative sepsis. We had previously identified in rapid-throughput screens several guanylhydrazones as potent LPS binders. We were desirous of examining if the presence of the guanylhydrazone (rather than an amine) functionality would afford greater LPS sequestration potency. In evaluating a congeneric set of guanylhydrazone analogues, we find that C₁₆ alkyl substitution is optimal in the N-alkylguanylhydrazone series; a homospermine analogue with the terminal amine N-alkylated with a C₁₆ chain with the other terminus of the molecule bearing an unsubstituted guanylhydrazone moiety is marginally more active, suggesting very slight, if any, steric effects. Neither C₁₆ analogue is significantly more active than the N-C₁₆-alkyl or N-C₁₆-acyl compounds that we had characterized earlier, indicating that basicity of the phosphate-recognizing cationic group, is not a determinant of LPS sequestration activity.     

Identification of Adjuvantic Activity of Amphotericin B in a Novel,Multiplexed, Poly-TLR/NLR High-Throughput Screen

Small-molecule agonists have been identified for TLR7, TLR8, TLR4 and TLR2 thus far, and chemotypes other than those of canonical ligands are yet to be explored for a number of innate immune receptors. The discovery of novel immunostimulatory molecules would enhance the repertoire of tools available for interrogating innate immune effector mechanisms, and provide additional venues for vaccine adjuvant development. A multiplexed, reporter gene-based high-throughput assay capable of detecting agonists of TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1 and NOD2 was utilized in screening 123,943 compounds, in which amphotericin B (AmpB) and nystatin were identified as prominent hits. The polyene antifungal agents act as TLR2- and TLR4-agonists. The TLR4-stimulatory activity of AmpB was similar to that of monophosphoryl lipid A, suggestive of TRIF-biased signaling. The adjuvantic activity of AmpB, at a dose of 100 micrograms, was comparable to several other candidate adjuvants in rabbit models of immunization. These results point to its potential applicability as a safe and effective adjuvant for human vaccines.     

Suppression of the enzymes of glutamate metabolism in cortical synaptosomes in methionine sulfoximine toxicity

Enzymes of glutamate metabolism were studied in synaptosomes prepared from normal rats and those treated with acute (300 mg/kg) and subacute (150 mg/kg) doses of the convulsant methionine sulfoximine (MSO). The activities of glutamine synthetase, glutamate dehydrogenase and aspartate aminotransferase were inhibited in the synaptosomes of drug treated animals. It is suggested that MSO would suppress the formation of glutamine and glutamate and consequently the releasable pool of glutamate, aspartate and GABA. These neurotransmitters would be depleted irom the nerve endings. It is also indicated that the ammonia accumulated would affect the cerebral functioning by interfering with the maintenance of ionic gradients.     

Potent Adjuvanticity of a Pure TLR7-Agonistic Imidazoquinoline Dendrimer

Engagement of toll-like receptors (TLRs) serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. We synthesized a dendrimeric molecule bearing six units of a potent TLR7/TLR8 dual-agonistic imidazoquinoline to explore if multimerization of TLR7/8 would result in altered activity profiles. A complete loss of TLR8-stimulatory activity with selective retention of the TLR7-agonistic activity was observed in the dendrimer. This was reflected by a complete absence of TLR8-driven proinflammatory cytokine and interferon (IFN)-γ induction in human PBMCs, with preservation of TLR7-driven IFN-α induction. The dendrimer was found to be superior to the imidazoquinoline monomer in inducing high titers of high-affinity antibodies to bovine α-lactalbumin. Additionally, epitope mapping experiments showed that the dendrimer induced immunoreactivity to more contiguous peptide epitopes along the amino acid sequence of the model antigen.