A new dawn for monoclonal antibodies against antimicrobial resistant bacteria

Antimicrobial resistance (AMR) is a quickly advancing threat for human health worldwide and almost 5 million deaths are already attributable to this phenomenon every year. Since antibiotics are failing to treat AMR-bacteria, new tools are needed, and human monoclonal antibodies (mAbs) can fill this role. In almost 50 years since the introduction of the first technology that led to mAb discovery, enormous leaps forward have been made to identify and develop extremely potent human mAbs. While their usefulness has been extensively proved against viral pathogens, human mAbs have yet to find their space in treating and preventing infections from AMR-bacteria and fully conquer the field of infectious diseases. The novel and most innovative technologies herein reviewed can support this goal and add powerful tools in the arsenal of weapons against AMR

A Click-Chemistry Linked 2’3’-cGAMP Analog

2’3’-cGAMP is an uncanonical cyclic dinucleotide where one A and one G base are connected via a 3’-5’ and a unique 2’-5’ linkage. The molecule is produced by the cyclase cGAS in response to cytosolic DNA binding. cGAMP activates STING and hence one of the most powerful pathways of innate immunity. cGAMP analogs with uncharged linkages that feature better cellular penetrability are currently highly desired. Here, we report the synthesis of a cGAMP analog with one amide and one triazole linkage. The molecule is best prepared via a first Cu(I) catalysed click reaction which establishes the triazole, while the cyclization is achieved by macrolactamization

Supersensitive Multifluorophore RNA‐FISH for Early Virus Detection and Flow‐FISH by Using Click Chemistry

The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid‐19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single‐fluorophore‐containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off‐target binding effects that create background noise. Here, we used click chemistry and alkyne‐modified DNA oligonucleotides to prepare multiple‐fluorophore‐containing probes. We found that these multiple‐dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5–10) of probe strands. The new method enabled the in situ detection of viral transcripts as early as 4 hours after infection

Design and synthesis of clickable nucleic acid analogues for cancer therapy and diagnosis

The use of nucleic acid molecules in cancer therapy and diagnosis represents a field in continuous growth. During this thesis, bioconjugation and click chemistry techniques were applied to biological system in order to provide new tools for cancer therapy (chapters 1-4) or diagnosis (chapter 5). In the first part the development of a series of STING agonists is described. STING is a key protein in the regulation of the innate immune system. The activation of the STING pathway begins when DNA is released in the cytosol. This DNA is seen by cells as a clear danger sign upon which the DNA sensor cGAS specifically recognizes and binds cytosolic DNA. Using ATP and GTP, cGAS can synthesise the second messenger 2’,3’-cGAMP (Figure 1). 2’,3’-cGAMP is then recognized by STING and this leads to a conformational change of the protein structure which ultimately triggers interferon expression. Because of their ability to activate the immune system, the use of 2’,3’-cGAMP analogues and STING agonists in medicine is gaining interest, with a constantly growing number of molecules currently in preclinical and clinical trials in the field of immunotherapy or vaccines. Because of the negative charge of 2’,3’-cGAMP, which impairs its uptake by cells and because of its metabolic instability, there is high need for new 2’,3’-cGAMP analogues that can cross the cell membrane or that are more stable towards the action of human or viral enzymes that are known to specifically degrade this cyclic dinucleotide. Therefore, we developed a series of cGAMP analogues that are based on a 2’,3’-cyclic dinucleotide scaffold and that contain adenosine or guanosine nucleosides. In addition to the natural cGAMP, we synthesised the 2’,3’-cyclic adenosine monophosphate-adenosine monophosphate (cAAMP), the dehydroxylated analogues 1, 2 and 3 and the methylated analogues 4 and 5 (Figure 2). In collaboration with Dr. D. Drexler (Hopfner group), the synthesised compounds were tested in thermal shift assays with the soluble portion of STING and then in isothermal titration calorimetry experiments in order to calculate the binding affinities of the synthesised analogues as well as the thermodynamic parameters of their interaction with the protein. Compounds 1, 2, 3, 4 and 5 were then further tested in cellular assays using THP-1 dual reporter cells, which allowed to measure the interferon expression triggered by these analogues and determine their EC50 value, reflecting the potency of the synthesised STING agonists. With these assays, the most potent STING agonist resulted to be compound 1 (EC50= 8.5 μM) while we calculated an EC50 value of 10.6 μM for cGAMP and a value of 60.5 and 106.5 μM for compounds 2 and 3. The methylated analogues 4 and 5 did not induce STING activation in our assays, likely because of the conformation of their ribose moieties. In the second part of this project we developed prodrug derivatives of compounds 2 and 5 described before in order to improve the cell permeability of these molecules and to achieve their efficient internalization. To do this, we modified the S-acylthioethyl (SATE) moiety, which is frequently used in prodrugs, to include a terminal alkyne which allows further late - stage functionalization by click chemistry to improve drug uptake (Figure 3). The synthesised analogue 31 was tested in THP-1 cells to determine its activity. With the introduction of the phosphate caging groups, we calculated an EC50 value of 47.6 nM indicating that 31 is approximately 200-fold more potent than cGAMP itself. In collaboration with W. Greulich (Hornung group) we studied the phosphorylation of the key proteins involved in the STING and interferon pathways (STING, TBK1 and STAT1) by western blot and we detected a much higher phosphorylation level of these proteins using compound 31 compared to cGAMP. Furthermore, the masked derivatives containing an alkyne were further functionalized by click chemistry with an anandamide azide leading to compound 44 and 45. Compound 44 was also tested in THP-1 cells, but, even if it proved to be more active than cGAMP, we measured a lower activity of derivative 44 compared to 31. In chapter 5 published work is presented, in which clickable dendrimers have been used in order to enhance the signal of a cell proliferation assay based on EdU incorporation. After cell feeding with EdU, a tetraazide dendrimer was employed to increase the number of reactive sites per each incorporated alkyne (Figure 5). In a second step, we reacted the multiple azides with a dye alkyne (double click procedure) or with another dendrimer with four alkynes, esponentially increasing the number of reactive alkynes for each incorporated EdU (triple click procedure). In this last procedure, we finally performed a click reaction with a dye azide to allow detection of proliferating cells by fluorescence microscopy. By employing these clickable dendrimers it was finally possible to achieve a 6-fold enhancement in the fluorescent signal

Synthesis of dicarba-cyclooctapeptide Somatostatin analogs by conventional and MW-assisted RCM: A study about the impact of the configuration at C α of selected amino acids

This work describes the synthesis of thirteen cyclooctapeptides dicarba-analogues of Somatostatin, containing l- or d-allylglycine (Agl) residues at the termini of the peptide chain, through on resin Ring Closing Metathesis (RCM) of the linear octapeptides. We investigated the influence of the stereochemistry of some strategic amino acids on the propensity to give the cyclic compounds in mild conditions (refluxing DCM). Systematic individual replacement of Phe6,7,11 residues with the corresponding enantiomers, strongly favoured the ring closure by conventional heating. The yield of the cyclic products was strictly correlated to the position of this amino acid on the peptide chain. In particular substitution of Phe⁶ by Tyr in peptides which did not give the cyclic compounds, allowed the ring formation. The effect of the phenolic −OH function of Tyr side chain on the proximity of the terminal Agl residue was studied by NMR techniques. All the linear precursors gave cyclic somatostatin dicarba-analogues, in good to high yields and in short reaction times, by microwave-assisted RCM, performed with the 2nd generation Grubbs catalyst. The unsaturated dicarba-tether resulted in a mixture of E and Z stereoisomers in a variable ratio, depending on the sequence and the cyclization method. The E isomer was largely the most abundant in all but one the described product

Novel Poxin Stable cGAMP-Derivatives Are Remarkable STING Agonists

2′,3′-cGAMP is a cyclic A- and G-containing dinucleotide second messenger, which is formed upon cellular recognition of foreign cytosolic DNA as part of the innate immune response. The molecule binds to the adaptor protein STING, which induces an immune response characterized by the production of type I interferons and cytokines. The development of STING-binding molecules with both agonistic as well as antagonistic properties is currently of tremendous interest to induce or enhance antitumor or antiviral immunity on the one hand, or to treat autoimmune diseases on the other hand. To escape the host innate immune recognition, some viruses encode poxin endonucleases that cleave 2′,3′-cGAMP. Here we report that dideoxy-2′,3′-cGAMP (1) and analogs thereof, which lack the secondary ribose-OH groups, form a group of poxin-stable STING agonists. Despite their reduced affinity to STING, particularly the compound constructed from two A nucleosides, dideoxy-2′,3′-cAAMP (2), features an unusually high antitumor response in mice