Libraries of DNA-tagged compounds are a validated screening technology for drug discovery. They are synthesized through combinatorial iterations of alternated coding and preparative synthesis steps. Thus, large chemical space can be accessed for target-based screening. However, the need to preserve the functionality of the DNA tag severely restricts the choice of chemical methods for library synthesis. Acidic organocatalysts, transition metals, and oxidants furnish diverse drug-like structures from simple starting materials, but cause loss of genetic information by depurination. A hexathymidine oligonucleotide, called “hexT” allows the chemist utilizing these classes of catalysts to access a potentially broad variety of structures in the initial step of library synthesis. We exploited its catalyst tolerance to efficiently synthesize diverse substituted β-carbolines, pyrazolines, and pyrazoles from readily available starting materials as hexT conjugates by acid- and Au(I)-catalysis, respectively. The hexT conjugates were ligated to coding DNA sequences yielding encoded screening libraries inspired by drug structures.

Background: Nusinersen is an intrathecally administered antisense oligonucleotide (ASO) that improves motor function in patients with spinal muscular atrophy (SMA). In addition to efficacy, the safety of a therapy is the decisive factor for the success of the treatment. For some ASOs, various organ toxicities have been described, such as thrombocytopenia, renal and liver impairment, or coagulation abnormalities. However, systematic data on laboratory parameters under treatment with nusinersen are mainly available from studies in infants and children. Therefore, our aim was to assess the safety of nusinersen therapy in adult SMA patients. Methods: Laboratory data from 404 nusinersen injections performed in 50 adult patients with SMA type 2 and type 3 were retrospectively analyzed. Results: The total observation period was 76.9 patient-years, and patients received up to 12 injections. Our data provides no new safety concerns. In cerebrospinal fluid (CSF), the mean white blood cell count and lactate remained stable over time. Total CSF protein increased by 2.9 mg/dL. No change in mean platelet count was observed under therapy. Only one patient showed sporadic mild thrombocytopenia. Coagulation parameters and inflammatory markers were stable. The mean creatinine level decreased by 0.09 mg/dL. Analysis of mean liver enzyme levels revealed no relevant changes during treatment. Conclusion: Our data demonstrate a favorable safety profile of nusinersen therapy in adult SMA patients under longer-term “real-world” conditions. In particular, we found no evidence of clinically relevant platelet declines, coagulopathies, or renal or hepatic organ toxicities, which are common concerns with the use of ASOs.

Background: Malignant ascites commonly occurs in advanced or recurrent stages of epithelial ovarian cancer during peritoneal carcinomatosis and is correlated with poor prognosis. Due to its complex composition of cellular and acellular components malignant ascites creates a unique tumor microenvironment, which mediates immunosuppression and promotes progression of disease. However, the immunosuppressive mechanisms remain poorly understood. Methods: In the present study, we explored the antitumor activity of healthy donor NK and T cells directed against ovarian cancer cells in presence of malignant ascites derived from patients with advanced or recurrent peritoneal carcinomatosis. A wide range of methods was used to study the effect of ascites on NK and T cells (FACS, ELISA, EliSpot, qPCR, Live-cell and confocal microscopy, Western blot and electrolyte flux assays). The ascites components were assessed using quantitative analysis (nephelometry, potentiometry and clinical chemistry) and separation methods (dialysis, ultracentrifugal filtration and lipid depletion). Results: Ascites rapidly inhibited NK cell degranulation, tumor lysis, cytokine secretion and calcium signaling. Similarly, target independent NK and T cell activation was impaired in ascites environment. We identified imbalanced electrolytes in ascites as crucial factors causing extensive immunosuppression of NK and T cells. Specifically, high sodium, low chloride and low potassium content significantly suppressed NK-mediated cytotoxicity. Electrolyte imbalance led to changes in transcription and protein expression of electrolyte channels and impaired NK and T cell activation. Selected inhibitors of sodium electrolyte channels restored intracellular calcium flux, conjugation, degranulation and transcript expression of signaling molecules. The levels of ascites-mediated immunosuppression and sodium/chloride/potassium imbalance correlated with poor patient outcome and selected molecular alterations were confirmed in immune cells from ovarian cancer patients. Conclusion: Our data suggest a novel electrolyte-based mechanism of immunosuppression in malignant ascites of patients with peritoneal carcinomatosis. We show for the first time that the immunosuppression of NK cytotoxicity in coculture assays is correlated to patient poor survival. Therapeutic application of sodium channel inhibitors may provide new means for restoring immune cell activity in ascites or similar electrolyte imbalanced environments.

A gold( i )-mediated reaction to a DNA-tagged spirocycle, and the tolerance of different nucleic acids to the reaction conditions are demonstrated.

An efficient, highly atom economic synthesis of hitherto unknown spirocyclic pyrazolidines in a one-pot process was developed. The gold-catalyzed three-component coupling of alkynols, hydrazines and aldehydes or ketones likely proceeds via cycloisomerization of the alkynol to an exocyclic enol ether and subsequent [3 + 2]-cycloaddition of an azomethine ylide. A library of 29 derivatives with a wide range of functional groups was synthesized in up to 97% yield. With this new method, every position in the final product can be substituted which renders the method ideal for applications in combinatorial or medicinal chemistry.

Various [O,O]- and [N,O]-spiroacetals were synthesized by gold-catalyzed spirocyclization of suitable functionalized alkynes. Whereas simple spiroacetals with two heteroatoms were readily obtained by regioselective cyclization of acetylenic diols or aminoalcohols, hitherto unknown spirocyclic isoxazolidines and pyrazolidines bearing three heteroatoms were formed by three-component coupling of alkynols, aldehydes, and protected hydroxylamine or hydrazine derivatives. The sustainability of these spirocyclizations was improved by using recyclable gold catalysts in water or nanomicelles as reaction medium.