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Die COVID-19 Pandemie ist die bisher verheerendste Pandemie des 21. Jahrhunderts. Durch die Einführung neuer mRNA-basierter Impfstoffe sowie der hohen Rate natürlicher Infektionen konnte die weltweite SARS-CoV-2-Immunität gesteigert werden. Trotz aller Erfolge zur Eindämmung der Pandemie kann eine Infektion auch heute noch zu schweren Verläufen und Tod führen. Eine adäquate COVID-19-Therapie ist folglich auf potente Virostatika angewiesen. Eine durch Umgehung zeitaufwändiger klinischer Studien schnell verfügbare Alternative zu neu entwickelten Arzneimitteln ist die Anwendung etablierter Medikamente. Wir isolierten und charakterisierten ein von einem Patienten stammendes SARS-CoV-2-Virus. Dieses Virusisolat wurde bisher in elf Publikationen verwendet. Mittels quantitativer Echtzeit-Polymerasekettenreaktion untersuchten wir eine Substanzbibliothek mit mehr als 300 neuen und bereits zugelassenen Wirkstoffen auf ihre Wirksamkeit gegen SARS-CoV-2. Dabei konnten wir zeigen, dass der selektive Serotonin-Wiederaufnahmehemmer Fluoxetin die SARS-CoV-2-Replikation ab einer Dosis von 0,8 μg/ml signifikant inhibiert, einer bei der Behandlung von Depressionen häufig angewandten Dosierung. Der EC50-Wert lag bei 387 ng/ml. Die Behandlung mit Fluoxetin resultierte in einer reduzierten Zahl an Virusprotein-produzierenden Zellen, was darauf hindeutet, dass es die virale Reinfektion und/oder Proteinexpression inhibiert. Fluoxetin ist ein racemisches Gemisch, wobei das (S)-Enantiomer der potentere Serotonin-Wiederaufnahmehemmer ist. Wir konnten zeigen, dass beide Enantiomere einen vergleichbaren antiviralen Effekt gegen SARS-CoV-2 aufweisen, wodurch das (R)-Enantiomer bei virologischer Indikation gegebenenfalls präferiert werden sollte. Fluoxetin hat keinen Einfluss auf die Replikation des Tollwut-Virus und des Humanen Respiratorischen Synzytial-Virus, was auf eine Virusspezifität hindeutet. Weitere aus der Bibliothek stammende signifikante Inhibitoren der SARS-CoV-2-Replikation sind die am Institut für Organische Chemie Würzburg entwickelten Substanzen AKS 232 und AKS 128. Neben der medikamentösen Therapie ist die akkurate Bestimmung neutralisierender Antikörper gegen SARS-CoV-2 zur Quantifizierung des bestehenden (Re-) Infektionsschutzes sowie zur Planung zukünftiger Impfstrategien von großer Bedeutung. Im Rahmen dieser Arbeit entwickelten wir unter Verwendung der quantitativen Echtzeit-Polymerasekettenreaktion erfolgreich ein zuverlässiges Testverfahren zur Detektion neutralisierender anti-SARS-CoV-2 Antikörper.
To circumvent time-consuming clinical trials, testing whether existing drugs are effective inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this path and screened approved medications "off-label" against SARS-CoV-2. Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8 mu g/ml significantly in these screenings, and the EC50 was determined with 387 ng/ml. Furthermore, Fluoxetine reduced viral infectivity in precision-cut human lung slices showing its activity in relevant human tissue targeted in severe infections. Fluoxetine treatment resulted in a decrease in viral protein expression. Fluoxetine is a racemate consisting of both stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that both isomers show similar activity on the virus, indicating that the R-form might specifically be used for SARS-CoV-2 treatment. Fluoxetine inhibited neither Rabies virus, human respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1 gene expression, indicating that it acts virus-specific. Moreover, since it is known that Fluoxetine inhibits cytokine release, we see the role of Fluoxetine in the treatment of SARS-CoV-2 infected patients of risk groups.
Marine organisms and their associated microbes are rich in diverse chemical leads. With the development of marine biotechnology, a considerable number of research activities are focused on marine bacteria and fungi-derived bioactive compounds. Marine bacteria and fungi are ranked on the top of the hierarchy of all organisms, as they are responsible for producing a wide range of bioactive secondary metabolites with possible pharmaceutical applications. Thus, they have the potential to provide future drugs against challenging diseases, such as cancer, a range of viral diseases, malaria, and inflammation. This review aims at describing the literature on secondary metabolites that have been obtained from Scleractinian-associated organisms including bacteria, fungi, and zooxanthellae, with full coverage of the period from 1982 to 2020, as well as illustrating their biological activities and structure activity relationship (SAR). Moreover, all these compounds were filtered based on ADME analysis to determine their physicochemical properties, and 15 compounds were selected. The selected compounds were virtually investigated for potential inhibition for SARS-CoV-2 targets using molecular docking studies. Promising potential results against SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and methyltransferase (nsp16) are presented.
Mass critical care caused by the severe acute respiratory syndrome corona virus 2 pandemic poses an extreme challenge to hospitals. The primary goal of hospital disaster preparedness and response is to maintain conventional or contingency care for as long as possible. Crisis care must be delayed as long as possible by appropriate measures. Increasing the intensive care unit (ICU) capacities is essential. In order to adjust surge capacity, the reduction of planned, elective patient care is an adequate response. However, this involves numerous problems that must be solved with a sense of proportion. This paper summarises preparedness and response measures recommended to acute care hospitals.
Due to the lack of data on asymptomatic SARS-CoV-2-positive persons in healthcare institutions, they represent an inestimable risk. Therefore, the aim of the current study was to evaluate the first 1,000,000 reported screening tests of asymptomatic staff, patients, residents, and visitors in hospitals and long-term care (LTC) facilities in the State of Bavaria over a period of seven months. Data were used from the online database BayCoRei (Bavarian Corona Screening Tests), established in July 2020. Descriptive analyses were performed, describing the temporal pattern of persons that tested positive for SARS-CoV-2 by real-time polymerase chain reaction (RT-PCR) or antigen tests, stratified by facility. Until 15 March 2021, this database had collected 1,038,146 test results of asymptomatic subjects in healthcare facilities (382,240 by RT-PCR, and 655,906 by antigen tests). Of the RT-PCR tests, 2.2% (n = 8380) were positive: 3.0% in LTC facilities, 2.2% in hospitals, and 1.2% in rehabilitation institutions. Of the antigen tests, 0.4% (n = 2327) were positive: 0.5% in LTC facilities, and 0.3% in both hospitals and rehabilitation institutions, respectively. In LTC facilities and hospitals, infection surveillance using RT-PCR tests, or the less expensive but less sensitive, faster antigen tests, could facilitate the long-term management of the healthcare workforce, patients, and residents.
As central components of life, DNA and RNA encode the genetic information. However, RNA performs several functions that exceed the competences stated in the ‘central dogma of life‘. RNAs undergo extensive post-transcriptional processing like chemical modifications. Among all classes of RNA, tRNAs are the most extensively modified. Their modifications are chemically diverse and vary from simple methylations (e.g. m3C, m6A) to more complex residues, like isopentenyl group (e.g. i6A, hypermodifications: e.g. ms2i6A) or even amino acids (e.g. t6A). Depending on their location within the overall structure, modifications can have an impact on tRNA stability and structure, as well as affinity for the ribosome and translation efficiency and fidelity. Given the importance of tRNA modifications new tools are needed for their detection and to study their recognition by proteins and enzymatic transformations.
The chemical synthesis of these naturally occurring tRNA modifications as phosphoramidite building blocks is a prerequisite to incorporate the desired modification via solid-phase synthesis into oligonucleotides. With the help of the m3C, (ms2)i6A, and t6A oligonucleotides, the importance and impact of tRNA modifications was investigated in this thesis. To this end, the role of METTL8 as the methyltransferase responsible for the installation of the methyl group at C32 for mt-tRNAThr and mt-tRNASer(UCN) was resolved. Thereby, the respective adenosine modification on position 37 is essential for the effectiveness of the enzyme. Besides, by means of NMR analysis, CD spectroscopy, thermal denaturation experiments, and native page separation, the impact of m3C32 on the structure of the tRNA ASLs was shown. The modification appeared to fine-tune the tRNA structure to optimize mitochondrial translation. To investigate the regulation of the dynamic modification pathway of m3C, demethylation assays were performed with the modified tRNA-ASLs and the (α-KG)- and Fe(II)-dependent dioxygenase ALKBH1 and ALKHB3. A demethylation activity of ALKBH3 on the mt-tRNAs was observed, even though it has so far only been described as a cytoplasmic enzyme. Whether this is physiologically relevant and ALKBH3 present a mitochondrial localization needs further validation. In addition, ALKBH1 was confirmed to not be able to demethylate m3C on mt-tRNAs, but indications for a deprenylation and exonuclease activity were found. Furthermore, the aforementioned naturally occurring modifications were utilized to find analytical tools that can determine the modification levels by DNAzymes, which cleave RNA in the presence of a specific modification. Selective DNA enzymes for i6A, as well as the three cytidine isomers m3C, m4C, and m5C have been identified and characterized.
Besides the naturally occurring tRNA modifications, the investigation on artificially modified nucleosides is also part of this thesis. Nucleosides with specific properties for desired applications can be created by modifying the scaffold of native nucleosides.
During the pandemic, the potential of antiviral nucleoside analogues was highlighted for the treatment of the SARS-CoV-2 infection. For examinations of the potential drug-candidate Molnupiravir, the N4-hydroxycytidine phosphoramidite building block was synthesized and incorporated into several RNA oligonucleotides. A two-step model for the NHC-induced mutagenesis of SARS-CoV-2 was proposed based on RNA elongation, thermal denaturation, and cryo-EM experiments using the modified RNA strands with the recombinant SARS-CoV-2 RNA-dependent RNA polymerase. Two tautomeric forms of NHC enable base pairing with guanosine in the amino and with adenosine in the imino form, leading to error catastrophe after the incorporation into viral RNA. These findings were further corroborated by thermal melting curve analysis and NMR spectroscopy of the NHC-containing Dickerson Drew sequence. In conclusion, the anti-amino form in the NHC-G base pair was assigned by NMR analysis using a 15N-labeld NHC building block incorporated into the Dickerson Drew sequence.
This thesis also addressed the synthesis of a 7-deazaguanosine crosslinker with a masked aldehyde as a diol linker for investigations of DNA-protein interactions. The diol functional group can be unmasked to release the reactive aldehyde, which can specifically form a covalent bond with amino acids Lys or Arg within the protein complex condensin. The incorporation of the synthesized phosphoramidite and triphosphate building blocks were shown and the functionality of the PCR product containing the crosslinker was demonstrated by oxidation and the formation of a covalent bond with a fluorescein label.
The development of assays that detect changes in this methylation pattern of m6A could provide new insights into important biological processes. In the last project of this thesis, the influence of RNA methylation states on the structural properties of RNA was analyzed and a fluorescent nucleoside analog (8-vinyladenosine) as molecular tools for such assays was developed. Initial experiments with the fluorescent nucleoside analog N6-methyl-8-vinyladenosine (m6v8A) were performed and revealed a strong fluorescence enhancement of the free m6v8A nucleoside by the installation of the vinyl moiety at position 8.
Overall, this thesis contributes to various research topics regarding the application of naturally occurring and artificial nucleoside analogues. Starting with the chemical synthesis of RNA and DNA modifications, this thesis has unveiled several open questions regarding the dynamic (de-)methylation pathway of m3C and the mechanism of action of molnupiravir through in-depth analysis and provided the basis for further investigations of the protein complex condensin, and a new fluorescent nucleoside analog m6v8A.
At the beginning of the COVID-19 pandemic, patients with primary and secondary immune disorders — including patients suffering from cancer — were generally regarded as a high-risk population in terms of COVID-19 disease severity and mortality. By now, scientific evidence indicates that there is substantial heterogeneity regarding the vulnerability towards COVID-19 in patients with immune disorders. In this review, we aimed to summarize the current knowledge about the effect of coexistent immune disorders on COVID-19 disease severity and vaccination response. In this context, we also regarded cancer as a secondary immune disorder. While patients with hematological malignancies displayed lower seroconversion rates after vaccination in some studies, a majority of cancer patients’ risk factors for severe COVID-19 disease were either inherent (such as metastatic or progressive disease) or comparable to the general population (age, male gender and comorbidities such as kidney or liver disease). A deeper understanding is needed to better define patient subgroups at a higher risk for severe COVID-19 disease courses. At the same time, immune disorders as functional disease models offer further insights into the role of specific immune cells and cytokines when orchestrating the immune response towards SARS-CoV-2 infection. Longitudinal serological studies are urgently needed to determine the extent and the duration of SARS-CoV-2 immunity in the general population, as well as immune-compromised and oncological patients.
(1) Background: Data on coronavirus 2 infection during pregnancy vary. We aimed to describe maternal characteristics and clinical presentation of SARS-CoV-2 positive women requiring intensive care treatment for COVID-19 during pregnancy and postpartum period based on data of a comprehensive German surveillance system in obstetric patients. (2) Methods: Data from COVID-19 Related Obstetric and Neonatal Outcome Study (CRONOS), a prospective multicenter registry for SARS-CoV-2 positive pregnant women, was analyzed with respect to ICU treatment. All women requiring intensive care treatment for COVID-19 were included and compared regarding maternal characteristics, course of disease, as well as maternal and neonatal outcomes. (3) Results: Of 2650 cases in CRONOS, 101 women (4%) had a documented ICU stay. Median maternal age was 33 (IQR, 30–36) years. COVID-19 was diagnosed at a median gestational age of 33 (IQR, 28–35) weeks. As the most invasive form of COVID-19 treatment interventions, patients received either continuous monitoring of vital signs without further treatment requirement (n = 6), insufflation of oxygen (n = 30), non-invasive ventilation (n = 22), invasive ventilation (n = 28), or escalation to extracorporeal membrane oxygenation (n = 15). No significant clinical differences were identified between patients receiving different forms of ventilatory support for COVID-19. Prevalence of preterm delivery was significantly higher in women receiving invasive respiratory treatments. Four women died of COVID-19 and six fetuses were stillborn. (4) Conclusions: Our cohort shows that progression of COVID-19 is rare in pregnant and postpartum women treated in the ICU. Preterm birth rate is high and COVID-19 requiring respiratory support increases the risk of poor maternal and neonatal outcome.
Mit dem Auftreten des SARS-CoV-2 Virus im Jahr 2020 war der Informationsgewinn für vulnerable Patientengruppen essentiell. Ziel dieser Arbeit war es maternale Charakteristika und das klinische Bild SARS-CoV-2 positiver Frauen mit Notwendigkeit einer intensivmedizinischen Behandlung während der Schwangerschaft und postpartal darzustellen, und diese Kohorte mit den SARS-CoV-2 positiven Schwangeren ohne intensivmedizinischen Handlungsbedarf zu vergleichen. Die Daten stammten aus dem deutschen CRONOS-Register, einem prospektiven, multizentrischen Register für SARS-CoV-2 positive schwangere Frauen. Eingeschlossen wurden alle schwangeren und postpartalen Frauen, die während ihrer SARS-CoV-2 Infektion auf eine ITS aufgenommen wurden. Diese wurden hinsichtlich maternaler Charakteristika, Krankheitsverlauf, sowie Outcomes verglichen.
In 101 von 2650 Fällen (4%) der Patientinnen des CRONOS-Registers, kam es zu einer Aufnahme auf die ITS. Als invasivste Form der COVID-19 Behandlung war bei 6 Patientinnen nur eine Überwachung notwendig, 30 Patientinnen benötigten eine Sauerstoffinsufflation, 22 wurden nicht-invasiv beatmet, 28 erhielten eine invasive Beatmung und bei 15 Frauen wurde die Behandlung zur ECMO-Therapie eskaliert. Es wurden keine klinisch signifikanten Unterschiede zwischen Patientinnen gefunden, die unterschiedliche Behandlungsformen benötigten. Die Gruppe der ITS und Non-ITS Patientinnen unterschied sich statistisch signifikant beim Einfluss von Alter, BMI bei Einschluss und der Herkunft. Die Prävalenz der Frühgeburtlichkeit war unter den invasiv behandelten Patientinnen signifikant höher und auch im Vergleich der ITS mit den Non-ITS Patientinnen zeigte sich ein signifikanter Unterschied. Vier Frauen verstarben an COVID-19 und sechs Feten der ITS-Gruppe waren Totgeburten.
Diese Kohorte zeigt, dass schwere COVID-19 Erkrankung bei schwangeren Frauen und Wöchnerinnen selten sind. Die Frühgeburtenrate ist hoch und COVID-19 mit Notwendigkeit einer Atemunterstützung erhöht das Risiko für ein schlechtes maternales und neonatales Outcome. Unter anderem ein höheres Alter und BMI sind mit einem höheren Risiko für eine ITS-Aufnahme verbunden.
Beyond the four canonical nucleosides as primary building blocks of RNA, posttranscriptional modifications give rise to the epitranscriptome as a second layer of genetic information. In eukaryotic mRNA, the most abundant posttranscriptional modification is N6-methyladenosine (m6A), which is involved in the regulation of cellular processes. Throughout this thesis, the concept of atomic mutagenesis was employed to gain novel mechanistic insights into the substrate recognition by human m6A reader proteins as well as in the oxidative m6A demethylation by human demethylase enzymes. Non-natural m6A atomic mutants featuring distinct steric and electronic properties were synthesized and incorporated into RNA oligonucleotides. Fluorescence anisotropy measurements using these modified oligonucleotides revealed the impact of the atomic mutagenesis on the molecular recognition by the human m6A readers YTHDF2, YTHDC1 and YTHDC2 and allowed to draw conclusions about structural prerequisites for substrate recognition. Furthermore, substrate recognition and demethylation mechanism of the human m6A demethylase enzymes FTO and ALKBH5 were analyzed by HPLC-MS and PAGE-based assays using the modified oligonucleotides synthesized in this work.
Modified nucleosides not only expand the genetic alphabet, but are also extensively researched as drug candidates. In this thesis, the antiviral mechanism of the anti-SARS-CoV-2 drug remdesivir was investigated, which causes delayed stalling of the viral RNA-dependent RNA polymerase (RdRp). Novel remdesivir phosphoramidite building blocks were synthesized and used to construct defined RNA-RdRp complexes for subsequent studies by cryogenic electron microscopy (cryo-EM). It was found that the 1'-cyano substituent causes Rem to act as a steric barrier of RdRp translocation. Since this translocation barrier can eventually be overcome by the polymerase, novel derivatives of Rem with potentially improved antiviral properties were designed.