Cis-isomerism and other chemical requirements of steroidal agonists and partial agonists acting at TRPM3 channels

BACKGROUND AND PURPOSE

The transient receptor potential melastatin-3 (TRPM3) channel forms calcium-permeable, non-selective, cationic channels that are stimulated by pregnenolone sulphate (PregS). Here, we aimed to define chemical requirements of this acute steroid action and potentially reveal novel stimulators with physiological relevance.

EXPERIMENTAL APPROACH

We used TRPM3 channels over-expressed in HEK 293 cells, with intracellular calcium measurement and whole-cell patch-clamp recording techniques.

KEY RESULTS

The stimulation of TRPM3 channels was confined to PregS and closely related steroids and not mimicked by other major classes of steroids, including progesterone. Relatively potent stimulation of TRPM3-dependent calcium entry was observed. A sulphate group positioned at ring A was important for strong stimulation but more striking was the requirement for a cis (β) configuration of the side group, revealing previously unrecognized stereo-selectivity and supporting existence of a specific binding site. A cis-oriented side group on ring A was not the only feature necessary for high activity because loss of the double bond in ring B reduced potency and loss of the acetyl group at ring D reduced efficacy and potency. Weak steroid stimulators of TRPM3 channels inhibited effects of PregS, suggesting partial agonism. In silico screening of chemical libraries for non-steroid modulators of TRPM3 channels revealed the importance of the steroid backbone for stimulatory effects.

CONCLUSIONS AND IMPLICATIONS

Our data defined some of the chemical requirements for acute stimulation of TRPM3 channels by steroids, supporting the existence of a specific and unique steroid binding site. Epipregnanolone sulphate was identified as a novel TRPM3 channel stimulator.     

CD20 monoclonal antibody (rituximab) for therapy of Epstein-Barr virus lymphoma after hemopoietic stem-cell transplantation

After bone marrow transplantation (BMT) using T-cell-depleted marrow from an unrelated donor or HLA-mismatched related donor, the risk of developing lymphoproliferative disease associated with the Epstein-Barr virus (EBV) ranges from 1% to 25%. We have shown that administration of donor-derived EBV-specific cytotoxic T lymphocytes (CTL) is effective prophylaxis and treatment for this complication, and we routinely generate CTL for high-risk patients. However, EBV lymphoma can occur in recipients of matched-sibling transplants for whom CTL are unavailable or in patients for whom CTL administration is contraindicated. We report on 3 such patients, who were successfully and safely treated with rituximab, a CD20 monoclonal antibody. The patients remain disease free 7, 8, and 9 months, respectively, after therapy. We conclude that CD20 antibody may be a useful alternative treatment strategy in patients with EBV lymphoma after BMT. (Blood. 2000;95:1502-1505)     

Generation of autologous Epstein-Barr virus-specific cytotoxic T cells for adoptive immunotherapy in solid organ transplant recipients

BACKGROUND: Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disorders (PTLD) affect 2%-27% of solid organ transplant (SOT) recipients. Adoptive immunotherapy may have therapeutic potential in this setting, but there is little experience in generating autologous EBV-specific cytotoxic T-cell lymphocytes (EBV-CTLs) from SOT recipients, and their efficacy and persistence in an immunosuppressed environment is unknown. METHODS: EBV-CTLs were generated from eight SOT recipients, using weekly stimulations with autologous lymphoblastoid cell lines (LCLs) and interleukin-2. CTL phenotype and function were evaluated in the presence of therapeutic concentration of cyclosporin A or FK506. RESULTS: In all cases, CTLs expanded with normal kinetics. The majority was CD3+CD8+ (mean, 76%), with less than 3% of natural killer cells. All ex vivo-generated CTLs produced significantly higher killing of autologous LCLs than of HLA-mismatched LCLs (mean, 56% vs. 14% at 20:1 ratio). No lysis of autologous or allogeneic PHA blasts was observed. The CTL expansion rate was reduced in a concentration-dependent manner in the presence of immunosuppressive drugs; however, neither lytic activity nor phenotype was affected. CONCLUSIONS: Using methods that are approved for clinical application, EBV-CTLs can be generated from SOT recipients, even those with frank lymphoma, or who are receiving immunosuppressive drugs. These CTLs retain their function in the presence of immunosuppressive agents. Although in vivo efficacy, safety, and persistence can be assessed only in clinical trials, our results suggest that CTLs can be effective for the treatment of PTLD, even when immunosuppression cannot be reduced because of the high risk of graft rejection.