The early phase of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was exacerbated by a diagnostic challenge of unprecedented magnitude. In the absence of effective therapeutics or vaccines, breaking the chain of transmission through early disease detection and patient isolation was the only means to control the growing pandemic. While polymerase chain reaction (PCR)-based methods and rapid-antigen tests rose to the occasion, the analytical challenge of rapid and sequence-specific nucleic acid-sensing at a point-of-care or home setting stimulated intense developments. Herein we report a method that combines recombinase polymerase amplification and a DNA-templated reaction to achieve a dual readout with either fluorescence (microtiter plate) or naked eye (lateral flow assay: LFA) detection. The nucleic acid templated reaction is based on an SN Ar that simultaneously transfers biotin from one Peptide Nucleic Acid (PNA) strand to another PNA strand, enabling LFA detection while uncaging a coumarin for fluorescence readout. This methodology has been applied to the detection of a DNA or RNA sequence uniquely attributed to the SARS-CoV-2.

Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms - a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition - apart from its radiosensitizing potential - obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC.

Lipids are essential components of eukaryotic cell membranes and play crucial roles in cellular signaling and metabolism. While increasing evidence shows that the activities of lipids are dependent upon subcellular localization, tools to study local lipid metabolism and signaling are limited. Herein, we report an approach that enabled us to selectively deliver photo-caged lipids into lysosomes and thereafter to quickly release the lipid molecules by illumination. On combining this method with genetic techniques and lipidomics, we were able to investigate the localization-dependent metabolism of an important intermediate of sphingolipid metabolism, sphingosine. Our data reveal a distinct metabolic pattern of lysosomal sphingosine. In general, this method has the potential to serve as a platform to study lysosomal metabolism and signaling of various lipids and metabolites in living cells.

The chaperone heat shock protein 90 (HSP90) crucially supports the maturation, folding, and stability of a variety of client proteins which are of pivotal importance for the survival and proliferation of cancer cells. Consequently, targeting of HSP90 has emerged as an attractive strategy of anti-cancer therapy, and it appears to be particularly effective in the context of molecular sensitization towards radiotherapy as has been proven in preclinical models of different cancer entities. However, so far the clinical translation has largely been hampered by suboptimal pharmacological properties and serious hepatotoxicity of first- and second-generation HSP90 inhibitors. Here, we report on NW457, a novel radicicol-derived member of the pochoxime family with reduced hepatotoxicity, how it inhibits the DNA damage response and how it synergizes with ionizing irradiation to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer in vitro and in vivo.

Withaferin A, a natural steroidal lactone found in the extracts of Withania somnifera, is used extensively in traditional medicine and part of an ancient remedy in ayurvedic medicine. Prior investigations into its mode of action have shown withaferin to be a polyfunctional pharmacophore with the covalent engagement of a multitude of therapeutic targets. Herein, we report that withaferin A is also a covalent inhibitor of IPO5, an importin that translocates cargos from the cytosol to the nucleus. We show that withaferin inhibits influenza A replication in epithelial cells (A549). Using a panel of inhibitors that selectively recapitulate part of withaferin A's pharmacological profile (goyazensolide, withaferin A derivatives, FiVe1, and bardoxolone methyl), we show that IPO5 inhibition contributes to the influenza replication inhibition but is not essential for the observed activity of withaferin A. We show that bardoxolone methyl, a semisynthetic triterpenoid in clinical development to treat chronic kidney disease and that shares some of the pharmacological profile of withaferin, also inhibits influenza A replication effectively. The inhibitory activity against influenza A replication should stimulate further studies to repurpose this therapeutic.

Unravelling autoimmune targets triggered by SARS-CoV-2 infection may provide crucial insights into the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining (a) the association between anti-SARS-CoV-2 and anti-apoA-1 humoral response and (b) the degree of linear homology between SARS-CoV-2, apoA-1 and Toll-like receptor 2 (TLR2) epitopes.

A Ru(bpy)3 Cl2 photocatalyst is applied to the rapid trans to cis isomerization of a range of alkene-containing pharmacological agents, including combretastatin A-4 (CA-4), a clinical candidate in oncology, and resveratrol derivatives, switching their configuration from inactive substances to potent cytotoxic agents. Selective in cellulo activation of the CA-4 analog Res-3M is demonstrated, along with its potent cytotoxicity and inhibition of microtubule dynamics.

Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in S. cerevisiae To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signaling through Ypk1 is therefore more closely related to PKA- and Sch9-signaling than previously appreciated and may perform functions previously only attributed to the latter kinases.

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. The ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate (QPD-OTf) for kinesin-1 that yields a precipitating dye along its walking path on microtubules (MTs). QPD-OTf enables to monitor native kinesin-1 transport activity in cellulo without external modifications. In vitro assays show that kinesin-1 and MTs are sufficient to yield fluorescent crystals; in cells, kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. These findings are further supported by docking studies, which suggest the binding of the activity-based substrate in the nucleotide binding site of kinesin-1.

Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damage response factors on the protein level, distinct transcriptomic alterations, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in glioblastoma cells in vitro. In vivo, HSP90 inhibition by NW457 improved the therapeutic outcome of fractionated CBCT-based irradiation in an orthotopic, syngeneic GBM mouse model, both in terms of tumor progression and survival. Nevertheless, in view of the promising in vitro results the in vivo efficacy was not as strong as expected, although apart from the radiosensitizing effects HSP90 inhibition also reduced irradiation-induced GBM cell migration and tumor invasiveness. Hence, our findings identify the combination of HSP90 inhibition and radiotherapy in principle as a promising strategy for GBM treatment whose performance needs to be further optimized by improved inhibitor substances, better formulations and/or administration routes, and fine-tuned treatment sequences.

Radiotherapy is an essential part of multi-modal treatment for soft tissue sarcomas. Treatment failure is commonly attributed to radioresistance, but comprehensive analyses of radiosensitivity are not available, and suitable biomarkers or candidates for targeted radiosensitization are scarce. Here, we systematically analyzed the intrinsic radioresistance of a panel of soft tissue sarcoma cell lines, and extracted scores of radioresistance by principal component analysis (PCA). To identify molecular markers of radioresistance, transcriptomic profiling of DNA damage response regulators was performed. The expression levels of HSP90 and its clients ATR, ATM, and NBS1 revealed strong, positive correlations with the PCA-derived radioresistance scores. Their functional involvement was addressed by HSP90 inhibition, which preferentially sensitized radioresistant sarcoma cells and was accompanied by delayed γ-H2AX foci clearance and HSP90 client protein degradation. The induction of apoptosis and necrosis was not significantly enhanced, but increased levels of basal and irradiation-induced senescence upon HSP90 inhibition were detected. Finally, evaluation of our findings in the TCGA soft tissue sarcoma cohort revealed elevated expression levels of HSP90, ATR, ATM, and NBS1 in a relevant subset of cases with particularly poor prognosis, which might preferentially benefit from HSP90 inhibition in combination with radiotherapy in the future.

The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90α and Hsp90β. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90β die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90α gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90α are apparently normal, with one major exception. Mutant male mice, whose Hsp90β levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90α may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects.

Photoactivation ('uncaging') is a powerful approach for releasing bioactive small-molecules in living cells. Current uncaging methods are limited by the random distribution of caged molecules within cells. We have developed a mitochondria-specific photoactivation method, which permitted us to release free sphingosine inside mitochondria and thereafter monitor local sphingosine metabolism by lipidomics. Our results indicate that sphingosine was quickly phosphorylated into sphingosine 1-phosphate (S1P) driven by sphingosine kinases. In time-course studies, the mitochondria-specific uncaged sphingosine demonstrated distinct metabolic patterns compared to globally-released sphingosine, and did not induce calcium spikes. Our data provide direct evidence that sphingolipid metabolism and signaling are highly dependent on the subcellular location and opens up new possibilities to study the effects of lipid localization on signaling and metabolic fate.

A dual Bcl-XL / Bcl-2 inhibitor was discovered from DNA-encoded libraries using a two steps process. In the first step, DNA was used to pair PNA-encoded fragments exploring > 250 000 combinations. In the second step, a focused library combining the selected fragments with linkers of different lengths and geometries led to the identification of tight binding adducts that were further investigated for their selective target engagement in pull-down assays, for their affinity by SPR, and their selectivity in a cytotoxicity assay. The best compound showed comparable cellular activity to venetoclax, the first-in-class therapeutic targeting Bcl-2.

Bioluminescence resonance energy transfer (BRET) is extensively used to study dynamic systems and has been utilized in sensors for studying protein proximity, metabolites, and drug concentrations. Herein, we demonstrate that BRET can activate a ruthenium-based photocatalyst which performs bioorthogonal reactions. BRET from luciferase to the ruthenium photocatalyst is used to uncage effector molecules with up to 64 turnovers of the catalyst, achieving concentrations >0.6 μM effector with 10 nM luciferase construct. Using a BRET sensor, we further demonstrate that the catalysis can be modulated in response to an analyte, analogous to allosterically controlled enzymes. The BRET-induced reaction is used to uncage small-molecule drugs (ibrutinib and duocarmycin) at biologically effective concentrations in cellulo.

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.