Bioactive indanes: insight into the bioactivity of indane dimers related to the lead anti-inflammatory molecule PH46A.

OBJECTIVES: PH46A (1) demonstrates significant anti-inflammatory activity in phenotypic models but its mechanism and site of action have been elusive. Current study focused on the bioactivity of PH46 (2) and related novel indane dimers (6-10) to investigate the impact of changes in substitution and stereochemistry at the C-1 and C-2 positions of the PH46 (2) scaffold. METHODS: Cytotoxicity profiles of compounds were established using THP-1 macrophages and SW480 cells. Effects of the compounds were then evaluated at 10 µm using 5-lipoxygenase (LOX) and 15-LOX enzymes, and 5-LOX binding was evaluated in silico against NDGA, nitric oxide (NO) released from LPS-induced SW480 cells and cytokines in THP-1 macrophages (IL-6, IL-1ß, TNF-α and IFN-γ) and in SW480 cells (IL-8). KEY FINDINGS: PH46 (2) and 7 cause reduction in NO, inhibition of 5-LOX with high binding energy and no cytotoxicity effects in THP-1 macrophages and SW480 cell lines (up to 50 µm). The cytokine profiling of the series demonstrated inhibition of IL-6 and TNF-α in THP-1 macrophages together with IL-8 in SW480 cells. CONCLUSIONS: The observed profile of cytokine modulation (IL-6/ TNF-α, IL-8) and inhibition of release of NO and 5-LOX may contribute to the in vivo effects demonstrated by indane dimers and PH46A (1) in murine models of colitis.

The nature of antibody heavy chain residue H6 strongly influences the stability of a VH domain lacking the disulfide bridge.

Monoclonal antibody mAb 03/01/01, directed against the musk odorant traseolide, carries a serine residue instead of the conserved Cys H92 in the heavy chain variable domain, and is thus lacking the highly conserved disulfide bridge. We investigated the energetic consequence of restoring the disulfide bond and the nature of residue H6 (Glu or Gln), which is poised to interact with Ser H92 in the recombinant scFv fragment obtained from this antibody. In the scFv fragment derived from this antibody, the stabilizing effect of Gln H6 over Glu was found to be as large as the effect of reintroducing the disulfide bond. We have analyzed the conformation and hydrogen bond pattern of Gln H6 and Glu H6 in antibodies carrying these residues and suggest mechanisms by which this residue could contribute to VH domain stability. We also show that the unpaired cysteine H22 is buried, and conforms to the expected VH structure. The antibody appears to have acquired two somatic mutations (Ser H52 and Arg H66), which had been previously characterized as having a positive effect on VH stability. The overall domain stability is the decisive factor for generating functional, disulfide-free antibody domains, and several key residues play dominant roles.

Insight into odorant perception: the crystal structure and binding characteristics of antibody fragments directed against the musk odorant traseolide.

Monoclonal antibodies were elicited against the small hydrophobic hapten traseolide, a commercially available musk fragrance. Antibody variable region sequences were found to belong to different sequence groups, and the binding characteristics of the corresponding antibody fragments were investigated. The antibodies M02/01/01 and M02/05/01 are highly homologous and differ in the binding pocket only at position H93. M02/05/01 (H93 Val) binds the hapten traseolide about 75-fold better than M02/01/01 (H93 Ala). A traseolide analog, missing only one methyl group, does not have the characteristic musk odorant fragrance. The antibody M02/05/01 binds this hapten analog about tenfold less tightly than the original traseolide hapten, and mimics the odorant receptor in this respect, while the antibody M02/01/01 does not distinguish between the analog and traseolide. To elucidate the structural basis for the fine specificity of binding, we determined the crystal structure of the Fab fragment of M02/05/01 complexed with the hapten at 2.6 A resolution. The crystal structure showed that only van der Waals interactions are involved in binding. The somatic Ala H93 Val mutation in M02/05/01 fills up an empty cavity in the binding pocket. This leads to an increase in binding energy and to the ability to discriminate between the hapten traseolide and its derivatives. The structural understanding of odorant specificity in an antibody gives insight in the physical principles on how specificity for such hydrophobic molecules may be achieved.

Antigen-antibody interactions: binding studies with fluorescence and surface plasmon resonance exemplified by acid-traseolide as antigen.

The interaction between the musk fragrance acid-traseolide and monoclonal antibodies (mAB) generated against this odorant has been investigated with two different techniques. Fluorescence spectroscopy was used to study the quenching of tryptophan fluorescence of the antibody upon binding acid-traseolide. This spectroscopic approach is based on measurements under equilibrium conditions. The second technique exploited the surface plasmon resonance (SPR) phenomenon. The acid-traseolide was immobilized in the surface matrix and upon presenting mAB changes in SPR were recorded in real time during the association reaction. The SPR approach can be considered as a kinetic method. Although having a different origin, both methods lead to comparable equilibrium dissociation constants (Kd). However, the results obtained with fluorescence spectroscopy were more accurate and reproducible. Not only the association of acid-traseolide with antibody was evaluated, also Fab fragment and peptide (H3-peptide) mimicking the heavy chain CDR3 of this antibody were included in this study. The Kd-values, determined by both methods, increase in the order mAB < Fab < H3-peptide because of diminishing recognition.

Radioimmunoassays for the enantiomeric components of indacrinone and their phenolic metabolites.

MK-286 is a 90:10 mixture of the (+) and (-)-enantiomers of indacrinone, a combination which induces diuresis while maintaining isouricemia. The principal (phenolic) metabolites also possess pharmacological activity and assays for the four entities were needed for clinical studies. Antisera were produced with the stereospecificity required to measure one enantiomer in the presence of the others, but significant cross-reactivity between drug and metabolite necessitated the separation of these two species by means of Sep-Pak cartridges. Recovery was assessed by concurrently fractionated reference samples. The radioligand in each case was a [I-125]-L-iodotyrosine conjugate of the analyte. Absolute assay sensitivities ranged from 2-20 pg/assay tube, corresponding to 0.2-2.0 ng/ml in serum samples. The assay was also applied to urine with analytes at concentrations greater than 16 ng/ml.