Francis Bitsch

X-Ray Structure of the hRORα LBD at 1.63 Å: Structural and Functional Data that Cholesterol or a Cholesterol Derivative Is the Natural Ligand of RORα

Abstract The retinoic acid-related orphan receptor α (RORα) is an orphan member of the subfamily 1 of nuclear hormone receptors. No X-ray structure of RORα has been described so far, and no ligand has been identified. We describe the first crystal structure of the ligand binding domain (LBD) of RORα, at 1.63 A resolution. This structure revealed a ligand present in the ligand binding pocket (LBP), which was identified by X-ray crystallography as cholest-5-en-3β-ol (cholesterol). Moreover, RORα transcriptional activity could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. These findings suggest that RORα could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases.

Structural Basis for the Exceptional in vivo Efficacy of Bisphosphonate Drugs

To understand the structural basis for bisphosphonate therapy of bone diseases, we solved the crystal structures of human farnesyl pyrophosphate synthase (FPPS) in its unliganded state, in complex with the nitrogen‐containing bisphosphonate (N‐BP) drugs zoledronate, pamidronate, alendronate, and ibandronate, and in the ternary complex with zoledronate and the substrate isopentenyl pyrophosphate (IPP). By revealing three structural snapshots of the enzyme catalytic cycle, each associated with a distinct conformational state, and details about the interactions with N‐BPs, these structures provide a novel understanding of the mechanism of FPPS catalysis and inhibition. In particular, the accumulating substrate, IPP, was found to bind to and stabilize the FPPS–N‐BP complexes rather than to compete with and displace the N‐BP inhibitor. Stabilization of the FPPS–N‐BP complex through IPP binding is supported by differential scanning calorimetry analyses of a set of representative N‐BPs. Among other factors such as high binding affinity for bone mineral, this particular mode of FPPS inhibition contributes to the exceptional in vivo efficacy of N‐BP drugs. Moreover, our data form the basis for structure‐guided design of optimized N‐BPs with improved pharmacological properties.

Absolute Quantification of Monoclonal Antibodies in Biofluids by Liquid Chromatography−Tandem Mass Spectrometry

The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intra-assay coefficient of variation (CV) was lower than 4% at 4 microg/mL and 200 microg/mL HmAb (n = 3). The CV at 400 microg/mL corresponded to 9% (n = 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 microg/mL HmAb was 7% (n = 3). The CV obtained from the female pool was 8% (n = 3), at 4 microg/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 microL of serum, a lowest limit of quantification of 2 microg/mL HmAb was reached in both human and cynomolgus monkey samples.

Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Pagets disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.

Amino–acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies

Culture conditions for successful amino–acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells are described. The method was applied to the selective labeling of the catalytic domain of c-Abl kinase with 15N-phenylalanine, 15N-glycine, 15N-tyrosine or 15N-valine. For the essential amino acids phenylalanine, tyrosine and valine high 15N-label incorporation rates of ≥90% and approximately the expected number of resonances in the HSQC spectra were observed, which was not the case for the non-essential amino acid glycine. The method should be applicable to amino-acid-type selective isotope labeling of other recombinant proteins which have not been amenable to NMR analysis.

Microbial conversion of rapamycin

Abstract In order to obtain derivatives of rapamycin, a total of 28 bacterial and 72 fungal strains were screened for their ability to transform rapamycin. In the course of this screening, the already known derivatives 39-o-demethylrapamycin, 27-o-demethylrapamycin, 16-o-demethylrapamycin, and the 40-o-phosphoric ester of rapamycin were detected and isolated out of fermentations with Streptomyces rimosus ATCC 28893 or Thamnidium elegans ATCC 8997. Biotransformation of rapamycin using Syncephalastrum racemosus ATCC 1332B, Gliocladium deliquescens ATCC 10097, or Bacillus subtilis ATCC 55060 yielded the conversion products 24-hydroxyrapamycin, secorapamycin A, B, and C, and 16-o-demethylsecorapamycin B. None of these derivatives exhibited a stronger immunosuppressive effect than the parent compound; however, in the case of 24-hydroxyrapamycin and 40-o-phosphoric ester of rapamycin, a FKBP-binding affinity comparable to rapamycin was observed.

Disease activated drugs: a new concept for the treatment of asthma

Disease activated drugs (DAD) are pro-drugs of one active principle or combinations of two drugs, which have a proven efficacy for the treatment of the target disease. In opposition to pro-drugs, DAD are activated in inflamed but not normal tissues. Due to the disease specific activation, the amount of locally released drug(s) should be related directly to the severity of the inflammation. To test this concept in asthma a PDE4 inhibitor, an isoquinoline derivative, was chemically derivatized into pro-drugs or combined with corticosteroids. These new compounds were more readily cleaved into active PDE4 inhibitor, in bronchoalveolar lavage fluid (BALF) from Brown-Norway rats with lung inflammation than in BALF from rats without airway inflammation. The DAD concept (local selective release and improved therapeutic window) was validated in vivo using the inhibition of methacholine induced bronchoconstriction in guinea pigs with or without ozone induced lung inflammation. An example of DAD hydrolysis (isoquinoline-dexamethasone) was also examined in BALF from asthmatics and healthy volunteers.

Detection of dihydroxycholesterols in human plasma using HPLC–ESI-MS/MS

We report a straightforward sample preparation procedure and a direct liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the analysis of 7alpha,25-dihydroxycholesterol (7α25-OHC) and 7alpha,27-dihydroxycholesterol (7α27-OHC). By applying a slow protein precipitation approach using cold ethanol, we were able to detect and quantify 7α25-OHC and 7α27-OHC in a fast and reliable manner. The average concentrations from 20 healthy individuals were determined to be 0.21±0.05nM for 7α25-OHC and 3.4±0.1nM for 7α27-OHC. In addition, we are the first to report the average degrees of esterification (n=8) to be 73.8% and 82% for 7α25-OHC and 7α27-OHC, respectively. Using the established method, we achieved the sensitivity sufficient for detecting low abundant dihydroxylated oxysterols in healthy individuals. This result should enable extension of these studies towards a comprehensive analysis of oxysterol levels under disease conditions.

A natural ligand for the orphan receptor GPR15 modulates lymphocyte recruitment to epithelia

The identification of a natural ligand of the orphan chemoattractant receptor GPR15 provides mechanistic insight into the migration of lymphocytes in the skin. Deorphanizing a chemoattractant receptor The orphan G protein–coupled receptor GPR15 mediates the trafficking of lymphocytes to the colon and the skin and the recruitment of effector T cells to inflamed intestinal tissue. Suply et al. purified a natural ligand of GPR15 (GPR15L) from porcine colonic extracts. In vitro assays showed that GPR15L specifically activated GPR15, but not other chemoattractant receptors. Although migration assays suggested that GPR15L inhibited chemokine-induced T cell migration, mouse skin allotransplantations showed that GPR15L recruited CD8+ T cells to the graft and that loss of the ligand was associated with increased graft protection. Given that GPR15L mRNA is abundant in psoriatic lesions, these data suggest that targeting the GPR15-GPR15L axis may help in the treatment of inflammatory skin conditions. GPR15 is an orphan G protein–coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene C10ORF99 and has some features similar to the CC family of chemokines. GPR15L was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, GPR15L was also abundant in the cervix. In skin, GPR15L was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from Gpr15l-deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis.

Optimization of the anti-(human CD3) immunotoxin DT389-scFv(UCHT1) N-terminal sequence to yield a homogeneous protein

The production and regulatory approval processes for biopharmaceuticals require detailed characterization of potential products. Therapeutic proteins should preferably be homogeneous, although limited, reproducible, heterogeneity may be tolerated. A diphtheria toxin‐based anti‐(human CD3) immunotoxin, DT389–scFv(UCHT1), was expressed in Escherichia coli and purified following refolding [DT389 corresponds to amino acids 1–389 of diphtheria toxin, scFv is single‐chain variable‐region antibody fragment and UCHT1is an anti‐(human CD3) monoclonal antibody]. Biochemical characterization of this molecule by MS and N‐terminal sequencing by Edman degradation revealed that the protein was heterogeneous at the N‐terminus, containing species both with (60%) and without (40%) the initiator methionine residue. In an attempt to generate an N‐terminally homogeneous molecule, a panel of seven N‐terminal variants was designed, based on the published specificity of bacterial methionine aminopeptidase. Following bacterial expression, partial purification and separation on SDS/PAGE, these proteins were subjected to N‐terminal sequencing by Edman degradation. Three of the mutants yielded a 100% homogeneous amino acid sequence. By contrast, the original DT389–scFv(UCHT1) protein and four variant proteins yielded two sequences with varying ratios corresponding to species with and without methionine. The N‐terminal sequences of the three homogeneous clones were MLADD and MLDD, where the methionine was completely retained, and SADD, where the methionine was completely removed. One of the homogeneous mutants (SADD) was expressed, refolded and purified and found to be equipotent with the parent immunotoxin. Thus, using a rational mutagenesis approach, three N‐terminally homogeneous variants of DT389–scFv(UCHT1) have been identified, at least one of which is functionally indistinguishable from the parent immunotoxin. This approach is generally applicable to biopharmaceutical production and immunotoxin development in particular.