Infectious diseases represent a global public health concern due to increasing microbial resistance to currently available therapies. Identification of suitable targets and development of novel antimicrobial agents with alternative mechanisms of action are among the strategies to prevent and to lower the outburst of resistant strains. DNA gyrase is a clinically validated target for development of novel antibacterial drugs, while Hsp90 represents a promising target for development of novel antiviral drugs. Numerous scientific publications and patent applications and the large number of such compounds synthesized bear witness to the active research for novel inhibitors of these two targets. The especially large challenge is the resistance mechanisms of Gram-negative bacteria, among which the most important are low outer membrane permeability and complex efflux systems that expel structurally diverse compounds from the bacterial cell, thus lowering the effectiveness of many of the available antibacterial drugs. The possible strategies to circumvent these two resistance barriers are the use of efflux pump inhibitors and increased drug penetration across the bacterial cell wall through what is termed a Trojan horse strategy, in which bioactive compounds are conjugated with siderophores or siderophore mimics to yield antibacterial molecules known as sideromycins. Validation of this Trojan horse concept in the clinical setting was recently confirmed with the registration of cefiderocol as a first-in-class agent for the treatment of serious infections caused by resistant Gram-negative pathogens, such as Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In the present doctoral dissertation, we have successfully designed and synthesized novel pyrrolamide-based inhibitors of DNA gyrase B (GyrB) with an (S)-4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamine scaffold, based on their previously reported first generation. The design of these inhibitors and their synthetic strategy was guided by the known crystal structures of structurally similar pyrrolamides in complex with the ATP-binding site of GyrB. First, we prepared a series of compounds with differently substituted pyrroles attached at position 6 of the core scaffold, to explore the available chemical space in the binding site of GyrB. Secondly, we attached various substituents at position 2 of the core scaffold, to increase the strength of the interactions at this site of the molecule also. To further improve the antibacterial activities of these GyrB inhibitors, we successfully designed and prepared their conjugates with different siderophore mimics. Based on the above-mentioned crystal structures, the siderophore mimics were attached at the inhibitor region, for which we predicted that this would not decrease the binding affinity of the inhibitors, or will even contribute to their binding. Using the Trojan Andraž Lamut: Načrtovanje in sinteza novih protimikrobno delujočih zaviralcev DNA-giraze in Hsp90 4 horse concept, the aim here was to increase the concentrations of these GyrB inhibitors inside the bacterial cells, to potentially improve their in vitro antibacterial activities and diminish the effects of the bacterial efflux pumps, as the main drawbacks of the first generation 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamines. Enzyme inhibition assays showed that these second-generation compounds were indeed more potent than those of the first generation, with IC50 values of the most potent analogs from 0.016 [micro]M to 0.044 [micro]M against E. coli DNA gyrase and from 0.43 [micro]M to 0.71 [micro]M against Staphylococcus aureus DNA gyrase. These second-generation compounds were also more efficient in in vitro antibacterial assays against Gram-positive bacteria, with MICs of the most efficient compounds from 8.2 [micro]M to 30 [micro]M against vancomycin-resistant and methicillin-resistant S. aureus strains. The most potent GyrB inhibitor-siderophore mimic conjugates had IC50 values from 0.058 [micro]M to 0.11 [micro]M against E. coli DNA gyrase and thus the siderophore component of the conjugates did not disturb the binding of the inhibitors to the target site of GyrB. Antibacterial testing against E. coli [delta]tolC, which has a defective efflux pump, showed that both the second-generation compounds as well as prepared conjugates are good substrates for the efflux pumps, which is one of the limiting factors for sufficient efficiency against Gram-negative bacteria. Nevertheless, most of the compounds, which were tested against E. coli [delta]tolC, showed improved activity over the first generation, with the most efficient ones with MICs from 3.13 [micro]M to 6.25 [micro]M. However, the conjugates showed only minor improvements to their MICs against wild-type E. coli in a test system with low iron concentrations, which mimics the conditions present in the host during infection. Therefore, further studies are needed to evaluate the success of the Trojan horse concept for these 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamine-based sideromycins. Selected GyrB inhibitors with this 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2,6-diamine core were also tested for antiviral activities, as some of the other well-known GyrB inhibitors have antiviral properties as well as Hsp90 inhibitory activities. Some of these compounds were active against influenza A (H1N1, H3N2) and influenza B viruses, with EC50 values in the low micromolar range. They also showed binding to Hsp90, which suggests that Hsp90 inhibition might be the mechanism that underlies their antiviral activities. DNA gyrase and Hsp90 inhibitors that were synthesized in the scope of the presented doctoral dissertation represent an original contribution to science in this field of research, as these are innovative compounds with an alternative mechanism of action when compared to the currently available therapies. Thus, they offer the opportunity to cope with the challenge of bacterial and viral resistance in the future.
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