Periodic Reporting for period 1 - AnLeadOp (Antibiotic Lead Optimization)
Reporting period: 2023-06-01 to 2024-11-30
The prevalence of resistant bacteria is increasing rapidly and in contrast, antibiotic research and development (R & D) in big Pharma has become mostly abandoned. If the resistance problem is not addressed, it is estimated that as many as 10 million people will die each year due to infections caused by resistant bacteria in 2050 (the so called, silent Tsunami). The 2022 Global Antimicrobial Resistance report by the World Health Organization (WHO) underlines the worldwide alarming rise of drug-resistant bacteria, for which new antibiotics are urgently needed.
For instances, patients with infections due to the vancomycin-resistant Enterococci (VRE) or methicillin-resistant Staphylococcus aureus (MRSA) were found to be at high risk of mortality and prolonged hospitalization, burdening the healthcare systems and economy. Moreover, infections with the Gram-negative bacteria Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii are classified among the most serious and the pathogens are becoming increasingly multidrug-resistant (MDR). Furthermore, Escherichia coli causing urinary tract infections has become less susceptible to standard antibiotic treatment including ampicillin, co-trimoxazole, and fluoroquinolones in 20% of the patients in 2020.
Alarmingly, the antibiotic development pipeline only contains 32 antibacterials against the WHO priority pathogens and only two of these are active against MDR Gram-negative bacteria. Addressing Gram-negative bacteria is particularly difficult as they have an additional outer cell membrane that prevents the penetration of large, hydrophobic compounds while hydrophilic compounds are hindered by the inner membrane. In addition, bacteria have efflux pumps to rapidly remove toxic compounds that have found their way inside the cell. This makes antibiotic development extremely challenging. Additionally, most compounds in the pipeline are derivatives of antibiotics with a known mode-of-action (MoA), to which resistance mechanisms already exist. Thus, the development on innovative compound classes acting through an unprecedented MoA is urgently required to tackle the rising antibiotic resistance crisis.
In order to tackle this problem of antibiotic-innovation gap, we are concerned with developing novel small molecule antibiotics inhibiting new essential targets in bacteria. Our strategy can be beneficial for the treatment of bacterial infections in general and those caused by MDR pathogens in particular. In this context, we are interested in targeting the bacterial sliding clamp (DnaN), a replisome component pivotal for DNA synthesis and repair. DnaN is an emerging and very promising target in terms of efficacy, broad-spectrum activity, selectivity, and low frequency of resistance development. In this Proof-of-Concept project, we pursued to build on our promising findings from the ERC start grant (NovAnI) in order to advance our discovered novel class of DnaN inhibitors towards a lead candidate suitable for the preclinical development, under mentorship of a pharmaceutical industry partner.
For instances, patients with infections due to the vancomycin-resistant Enterococci (VRE) or methicillin-resistant Staphylococcus aureus (MRSA) were found to be at high risk of mortality and prolonged hospitalization, burdening the healthcare systems and economy. Moreover, infections with the Gram-negative bacteria Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii are classified among the most serious and the pathogens are becoming increasingly multidrug-resistant (MDR). Furthermore, Escherichia coli causing urinary tract infections has become less susceptible to standard antibiotic treatment including ampicillin, co-trimoxazole, and fluoroquinolones in 20% of the patients in 2020.
Alarmingly, the antibiotic development pipeline only contains 32 antibacterials against the WHO priority pathogens and only two of these are active against MDR Gram-negative bacteria. Addressing Gram-negative bacteria is particularly difficult as they have an additional outer cell membrane that prevents the penetration of large, hydrophobic compounds while hydrophilic compounds are hindered by the inner membrane. In addition, bacteria have efflux pumps to rapidly remove toxic compounds that have found their way inside the cell. This makes antibiotic development extremely challenging. Additionally, most compounds in the pipeline are derivatives of antibiotics with a known mode-of-action (MoA), to which resistance mechanisms already exist. Thus, the development on innovative compound classes acting through an unprecedented MoA is urgently required to tackle the rising antibiotic resistance crisis.
In order to tackle this problem of antibiotic-innovation gap, we are concerned with developing novel small molecule antibiotics inhibiting new essential targets in bacteria. Our strategy can be beneficial for the treatment of bacterial infections in general and those caused by MDR pathogens in particular. In this context, we are interested in targeting the bacterial sliding clamp (DnaN), a replisome component pivotal for DNA synthesis and repair. DnaN is an emerging and very promising target in terms of efficacy, broad-spectrum activity, selectivity, and low frequency of resistance development. In this Proof-of-Concept project, we pursued to build on our promising findings from the ERC start grant (NovAnI) in order to advance our discovered novel class of DnaN inhibitors towards a lead candidate suitable for the preclinical development, under mentorship of a pharmaceutical industry partner.
We pursued a structure-based drug design (SBDD) approach to design a new series of DnaN inhibitors with enhanced ligand–target interactions. Synthesis of the compounds was accomplished with the help of a contract research organization (CRO) for a productive and efficient cost–time management. We assessed the new compounds for DnaN binding using surface plasmon resonance (SPR) and X-ray crystallography. The antibacterial activities were tested against a panel of bacterial strains, including the Gram-positive (S. aureus, penicillin resistant S. pneumoniae DSM11865, and Enterococcus faecium) pathogens, the Gram-negative E. coli wild type and efflux mutant (ΔacrB) strains, as well as Mycobacterium tuberculosis H37Rv. Moreover, we measured the physicochemical properties such as solubility and logD7.4 of the active compounds, and profiled their in vitro ADME-T (absorption, distribution, metabolism, excretion, and toxicity) properties. Furthermore, we determined the in vivo pharmacokinetic (PK) parameters for the most promising 10 compounds in two cassette studies, and then we selected the best two compounds for a focused PK study to identify the optimum route of administration and dose for the pharmacodynamic (PD) studies. Based on these results, we carried out in vivo efficacy studies using mouse infection model.
We have synthesized 60 new small molecules for multiparameter optimization of our class of DnaN inhibitors. Our SBDD strategy turned to be efficient as indicated by the improved binding and, importantly, antibiotic activity achieving minimum inhibitory concentration (MIC) values of 0.25–4 µg/mL against Gram-positive pathogens. Interestingly, the optimized compounds display improved activity against Gram-negative bacteria such as E. coli, Moraxella catarrhalis, and Acinetobacter baumannii (MIC values of 2–8 µg/mL). Moreover, we successfully determined the crystal structures of two new derivatives in complex with the bacterial DnaN, revealing their binding mode and opening the stage for further structure-based optimization of this class. In addition, in vitro ADME-T characterization indicated outstanding metabolic stability and plasma stability for the compounds. However, high plasma protein binding (PPB) was observed, which might need further optimization. In agreement with the in vitro ADME-T data, the new compounds showed good in vivo PK parameters with half-life (t1/2) values up to 4.4 hours and oral bioavailability (F) of 23.5%. The in vivo proof-of-concept study in mice showed indeed some effect of our compound on the bacterial burden in the lung. These results enabled us to get deeper insight into the structure–activity relationships (SAR) for this class, generate valuable information about the in vivo PK and PD properties of the compounds, and to expand their chemical space for better intellectual property protection. With these promising findings in hand, we have successfully applied for the CARB-X grant for further optimization of our small molecule DnaN inhibitors towards an orally active antibiotic for the treatment of Gram-positive bacterial infections.