Periodic Reporting for period 5 - resistance evolution (Bacterial evolution of hypersensitivity and resistance against antimicrobial peptides) Reporting period: 2021-04-01 to 2022-03-31 Summary of the context and overall objectives of the project Multi-drug resistant bacterial infections have been recognized as a major public health concern, and are responsible for a significant proportion of deaths worldwide. Alarmingly, the World Health Organization estimates that the mortality of drug-resistant bacterial infections might exceed that of malignant tumours by 2050, unless the current trends are reversed. Paradoxically, however, many pharmaceutical companies have discontinued their antibiotic research programs. This is principally due to the rapid spread of multi-drug resistant bacteria, which makes the commercial success of new antimicrobial drugs unpredictableAntimicrobial peptides are promising alternatives of traditional antibiotics currently used in clinics, however, the potential of bacterial resistance is a major concern. Our work (Spohn et al. Nature Communications 2019) indicates that the evolution of resistance against certain antimicrobial peptides is limited. Additionally, we showed (Kintses et al. Nature Microbiology 2018), that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts in the human gut microbiome. Our results suggest that AMPs induce highly specific changes in the composition of the human microbiota, with implications for disease risks. Finally, we have rationally designed two novel antimicrobial peptide analogs (Bhaumik et al. MSDE 2022). When co-administered as an adjuvant, the resulting compounds have substantially reduced the level of antibiotic resistance of multi-drug resistant clinical isolates. These findings indicate that manipulating bacterial membrane electrophysiology by antimicrobial peptides could be a valuable tool to overcome antimicrobial resistance.In sum, our work serves as a promising source for the development of new antimicrobial peptide-based therapeutics less prone to resistance, a feature necessary to avoid any possible interference with our innate immune system. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Antimicrobial peptides - promising antibiotic candidates with limited resistance We applied an integrated systems biology approach to study the susceptibilities of antibiotic resistant Escherichia coli strains towards antimicrobial peptides (AMPs). When a bacterial population faces a single antibiotic and evolves resistance against it, they frequently become multidrug resistant. By contrast, as we show in our work, antibiotic resistance often renders bacteria more susceptible to AMPs. We also identified the underlying molecular mechanisms leading to an increased sensitivity to AMPs. We show that a canonical multidrug resistance conferring mutation in marR elevates the susceptibility to AMPs up to 40%. This finding is of extreme importance since mutations in marR are clinically relevant and usually responsible for multi-drug resistance. In a follow-up study we also have found that AMPs with different physicochemical properties and cellular targets vary considerably in their resistance determinants. As a consequence, cross-resistance is prevalent only between AMPs with similar modes of action. We anticipate that chemical-genetic approaches could inform future efforts to minimize cross-resistance between therapeutic and human host antimicrobial peptides.Our work serves as a promising source for the development of new antimicrobial peptide-based therapeutics less prone to resistance, a feature necessary to avoid any possible interference with our innate immune system.DiVERGE Multi-drug resistant bacterial infections have been recognized as a major public health concern, however, many pharmaceutical companies have discontinued their antibiotic research programs. This is principally due to the rapid spread of multi-drug resistant bacteria, which makes the commercial success of new antimicrobial drugs unpredictable. Antibiotic developers do not have the appropriate toolsets to appropriately test antibiotic candidates for resistance development. At an early phase of drug discovery, researchers typically identify numerous molecules with antibacterial activities. However, identification of resistance-free antibacterial compounds is a complex problem. Standard microbial protocols used by the Pharmaceutical Companies or offered by Contract Research Organizations (CROs) are slow, have low coverage, and fail to predict the frequency and molecular mechanisms of antibiotic resistance by genomic mutations. Therefore, it is common that companies waste considerable resources on less promising drug candidates that are prone to resistance formation during clinical trials. Our team has recently developed a unique proprietary targeted mutagenesis technology, termed DIvERGE. DIvERGE is under strict IP protection (PCT/EP2017/082574). It allows generating mutant bacteria at unprecedented speed and accuracy, and is potentially applicable to a wide range of bacterial species with immediate biotechnological or biomedical interest. The market has shown immediate interest in the technology. Our team has recently managed to out-license the technology to a biotech company in a form of a non-exclusive contract with the aim to jointly develop bacteriophage mutant libraries.Development of new antibiotic candidatesIn a collaborative effort, the labs of Csaba Pal and Lucija Peterlin Mašič demonstrated that rational design of balanced multitargeting antibiotics is feasible by combining DIvERGE-based resistance screens and a medicinal chemistry workflow (Nyerges et al. Plos Biology 2019). Specifically, the teams aimed to develop antibiotic leads that are efficient against the methicillin-resistant Staphylococcus aureus (MRSA) bacterium. MRSA is one of the most dangerous bacterial pathogens in Europe. According to certain estimates, the related global healthcare costs reach USD 5 billion per annum. The antibiotics currently commercially available or in the development phase, do not offer a satisfactory solution as MRSA is extremely fast in adapting to new antibiotics. The resultant lead compounds, ULD1 and ULD2, belonging to a novel chemical class, and almost equipotently inhibit bacterial DNA gyrase and DNA topoisomerase IV (PCT/EP2019/073412). Antibiotics ULD1 and ULD2 are excellently potent against a broad panel of multidrug-resistant Staphylococcus aureus clinical strains. As a consequence, resistance mutations against these compounds were exceptionally rare and substantially reduced bacterial growth. Based on their efficacy and lack of toxicity demonstrated in murine infection models, these compounds could translate into new therapies against multidrug-resistant S. aureus bacterial infections. Of note, these compounds show also in vitro antibacterial potency against the most dangerous Gram-negative ESKAPE pathogens with critical clinical importance, such as A. baumannii. E. coli and P. aureoginosa. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Identification of antimicrobial peptide-antibiotic combinations which are effective against multidrug resistant pathogens (Lazar et al. Nature Microbiology 2018). Identification of antimicrobial peptides with limited resistance (Spohn et al. Nature Communications 2019)Development of a new bacterial genome engineering tool (PortMAGE, Nyerges et al. PNAS 2016)Development of a unique proprietary targeted mutagenesis technology(DIvERGE, Nyerges et al. PNAS 2018, PCT/EP2017/082574). Development of new antibiotic candidates (Nyerges et al. Plos Biology 2019, PCT/EP2019/073412). szbk5000-1959-k.jpg szbk5000-1956-k.jpg szbk5000-1976-k.jpg
