Final Report Summary - NABARSI (New AntiBacterials with Inhibitory activity on Aminoacyl-tRNA Synthetases)
Executive Summary:
Widespread resistance to antimicrobials is now a global threat. However, the number of new antimicrobials that have been brought to market has declined considerably in recent decades. In this respect, the NABARSI project has attempted to identify inhibitors of aminoacyl-tRNA synthetases (aaRS), enzymes necessary for the production of proteins within bacterial cells (the production of proteins being necessary for survival of bacterial cells). The NABARSI consortium has succeeded in obtaining three new and potentially interesting aaRS antibiotics. We have been able to establish that they are working through the intended mechanism of action and that they have a spectrum of activity against Gram-negative bacteria, including clinical isolates. Funding is currently being sought in order to further evaluate, develop and better characterise these NCEs.
The newly identified compounds are structurally novel and two patent applications have been filed, with further applications expected to be made in the future.
In addition to the patent applications, the results of the NABARSI consortium have been presented at international and national meetings, and several papers have been published in the scientific literature.
Project Context and Objectives:
Widespread resistance to antimicrobials is now a global threat. However, the number of new antimicrobials that have been brought to market has declined considerably in recent decades. Manyof the antibiotics that are now in clinical development belong to existing families of compounds, where antibiotic resistance has already been reported. Therefore, there is an urgent need for chemically-novel antibacterial agents, preferably those which act upon previously unexploited bacterial targets. In this respect, the NABARSI project has attempted to identify inhibitors of aminoacyl-tRNA synthetases (aaRS), enzymes necessary for the production of proteins within bacterial cells (the production of proteins being necessary for survival of bacterial cells). Validation of this protein family as a target for antibacterials has been provided by the already marketed drug mupirocin and by other compounds in clinical development. The NABARSI consortium has undertaken a cutting-edge drug discovery programme of 36-month duration in order to try and identify one or more antimicrobial aaRS case inhibitors. Novel computational screening technologies, synthetic methodologies, advanced mechanistic studies and in vitro studies (using multi-drug resistant (MDR) pathogenic bacteria belonging to the ESKAPE group of bacteria - Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species and Escherichia coli) have been combined with a state-of-the-art drug discovery approach and several potential new antimicrobial candidates have been identified for further studies.
Project Results:
The primary experimental screening involved the establishment of 3 primary assays, including assays for the detection of interesting compounds using enzymatic and bacterial growth susceptibility assays. The enzymatic assays used were divided in two types; 1) the ‘In Omnia’ assay and 2) the ‘Biothema’ assay. The In Omnia assay is a powerful assay in which the aaRS enzyme activity of specific bacterial pathogens can be measured inside human cells. This assay provides several pieces of information in only a single assay, namely specific activity, permeability, toxicity and stability. The second assay is an in vitro enzymatic activity assay developed in Omnia using a commercial kit from the Biothema company (www.biothema.se). Bacterial growth susceptibility assays were being performed against MRSA (Methicillin resistant Staphylococcus. aureus), Escherichia coli and Acinetobacter baumannii. This assay provides valuable information regarding the bacterial ‘growth inhibitory/killing capacity’ of NCEs discovered in the project.
Compounds for test in these assays were identified using a variety of in silico database searching techniques and rational drug design techniques. In silico drug design and chemical synthesis has including the creation of the Scopius-CSpace database of commercially available chemical compounds, with a specific focus on creating targeted libraries of putative enzyme inhibitors. This database contains approximately 7,800,000 unique compounds that are available for purchase from chemical companies worldwide. Several complementary screening approaches have been adopted against this source of chemical diversity for the in silico discovery of new chemical entities (NCEs) that target the aminoacyl tRNA synthetases of LeuRS, ValRS and IleRS. These efforts have been supported by biophysical screening studies, including ITC, NMR and X-ray crystallography.
Over the lifetime of NABARSI, a total of 3301 were tested in the “In Omnia” and “Biothema” assays for LeuRS from S.aureus and E.coli and the “Biothema” assay for ValRS from S.aureus and E.coli.
In total, 102 of these compounds were inhibitors of one or more aaRS enzymes. The most potent compounds were inhibitors of the LeuRS from E. coli, but inhibitors of S. aureus LeuRS, inhibitors of IleRS, and weak inhibitors of TrpRS were also found. Three compounds inhibited both IleRS and LeuRS, with weak dual inhibition of ValRS and LeuRS being seen for one additional compound.
62 compounds were active in antibacterial assays. Most of these (59), however, were inactive in all of the enzyme assays. It is not uncommon for antibacterial drug discovery projects to identify compounds with non-specific antibacterial activity. Although the mechanism of action of these compounds was not investigated in detail, they were not taken forward because of concerns regarding toxicity and they were outside the objectives of the project. Thus, the major difficulty encountered during the project was the fact that the majority of NCEs that have activity against the relevant targets do not have an effect on bacterial growth, and conversely, NCEs that have an effect on bacterial growth tend to display non-specific (off-target) activity.
However, the NABARSI consortium has succeeded in obtaining three new and potentially interesting aaRS antibiotics. We have been able to establish that they are working through the intended mechanism of action and that they have a spectrum of activity against Gram-negative bacteria, including clinical solates. Funding is currently being sought in order to further evaluate, develop and better characterise these NCEs.
The newly identified compounds are structurally novel and two patent applications have been filed, with further applications expected to be made in the future.
In addition to the patent applications, the results of the NABARSI consortium have been presented at international and national meetings, and several papers have been published in the scientific literature.
Potential Impact:
The anti-infective market in general and antibiotics in particular is an international and globalized market, with global annual figures sales of around €16,000 million for oral antibiotics and €6,200 million for intravenous administration. However, over the last few decades the number of new antibiotics brought to market has declined, while the rate of resistance to existing antibiotics has steadily increased. The human and economic costs of these trends has caused policy-makers to apply increasing pressure on pharmaceutical companies to address this gap, and also to create new private-sector incentives in this area such as accelerated review and preferential pricing for new antibiotics. The human impact of these two trends has been to increase mortality and morbidity in hospital, especially among the growing population of elderly and immune-suppressed patients. According to data from the Centres for Disease Control, the six ESKAPE bacteria are responsible for two-thirds of all Healthcare Associated Infections. Most hospital-acquired infections are resistant to at least one drug, and the incidence of MDR is rising. The inability to rapidly get these infections under control results in nearly 10,000 deaths every year in the United States, and many more in Europe and in the developing world. The economic impact of MDR infections is equally dramatic. The cost of hospital prolonged by MDR infection has been estimated at €7,000 million a year in Europe (Strategic Council on Resistance in Europe, SCORE; Utrecht, The Netherlands, SCORE, 2004). In this context, the NABARSI project aimed to discover compounds that have both antimicrobial and on-target (specific) activity against bacterial amino-acyl tRNA synthetases (aaRS), an activity that could be effective against clinically relevant Gram-positive and Gram-negative bacterial pathogens. The result of the project is the discovery of several new potentially clinically relevant antibiotics, which can be taken forward all further in vitro and in vivo testing. The further development and testing of these new antibiotics couldl have a major impact on reducing the current global morbidity and mortality associated with multi-resistant bacterial pathogens, and ultimately provide wider economic benefits to EU healthcare systems.
List of Websites:
www.nabarsi.eu
Partner email addresses -
Erasmus MC - John Hays, Wil Goessens
InhibOx Ltd - Paul Finn
Latvian Institute of Organic Synthesis - Aigars Jirgensons ,aigars@osi.lv>
University of Leeds - Alex O'Neill
Omnia Molecular S.L. (via IRB Barcelona)
Widespread resistance to antimicrobials is now a global threat. However, the number of new antimicrobials that have been brought to market has declined considerably in recent decades. In this respect, the NABARSI project has attempted to identify inhibitors of aminoacyl-tRNA synthetases (aaRS), enzymes necessary for the production of proteins within bacterial cells (the production of proteins being necessary for survival of bacterial cells). The NABARSI consortium has succeeded in obtaining three new and potentially interesting aaRS antibiotics. We have been able to establish that they are working through the intended mechanism of action and that they have a spectrum of activity against Gram-negative bacteria, including clinical isolates. Funding is currently being sought in order to further evaluate, develop and better characterise these NCEs.
The newly identified compounds are structurally novel and two patent applications have been filed, with further applications expected to be made in the future.
In addition to the patent applications, the results of the NABARSI consortium have been presented at international and national meetings, and several papers have been published in the scientific literature.
Project Context and Objectives:
Widespread resistance to antimicrobials is now a global threat. However, the number of new antimicrobials that have been brought to market has declined considerably in recent decades. Manyof the antibiotics that are now in clinical development belong to existing families of compounds, where antibiotic resistance has already been reported. Therefore, there is an urgent need for chemically-novel antibacterial agents, preferably those which act upon previously unexploited bacterial targets. In this respect, the NABARSI project has attempted to identify inhibitors of aminoacyl-tRNA synthetases (aaRS), enzymes necessary for the production of proteins within bacterial cells (the production of proteins being necessary for survival of bacterial cells). Validation of this protein family as a target for antibacterials has been provided by the already marketed drug mupirocin and by other compounds in clinical development. The NABARSI consortium has undertaken a cutting-edge drug discovery programme of 36-month duration in order to try and identify one or more antimicrobial aaRS case inhibitors. Novel computational screening technologies, synthetic methodologies, advanced mechanistic studies and in vitro studies (using multi-drug resistant (MDR) pathogenic bacteria belonging to the ESKAPE group of bacteria - Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species and Escherichia coli) have been combined with a state-of-the-art drug discovery approach and several potential new antimicrobial candidates have been identified for further studies.
Project Results:
The primary experimental screening involved the establishment of 3 primary assays, including assays for the detection of interesting compounds using enzymatic and bacterial growth susceptibility assays. The enzymatic assays used were divided in two types; 1) the ‘In Omnia’ assay and 2) the ‘Biothema’ assay. The In Omnia assay is a powerful assay in which the aaRS enzyme activity of specific bacterial pathogens can be measured inside human cells. This assay provides several pieces of information in only a single assay, namely specific activity, permeability, toxicity and stability. The second assay is an in vitro enzymatic activity assay developed in Omnia using a commercial kit from the Biothema company (www.biothema.se). Bacterial growth susceptibility assays were being performed against MRSA (Methicillin resistant Staphylococcus. aureus), Escherichia coli and Acinetobacter baumannii. This assay provides valuable information regarding the bacterial ‘growth inhibitory/killing capacity’ of NCEs discovered in the project.
Compounds for test in these assays were identified using a variety of in silico database searching techniques and rational drug design techniques. In silico drug design and chemical synthesis has including the creation of the Scopius-CSpace database of commercially available chemical compounds, with a specific focus on creating targeted libraries of putative enzyme inhibitors. This database contains approximately 7,800,000 unique compounds that are available for purchase from chemical companies worldwide. Several complementary screening approaches have been adopted against this source of chemical diversity for the in silico discovery of new chemical entities (NCEs) that target the aminoacyl tRNA synthetases of LeuRS, ValRS and IleRS. These efforts have been supported by biophysical screening studies, including ITC, NMR and X-ray crystallography.
Over the lifetime of NABARSI, a total of 3301 were tested in the “In Omnia” and “Biothema” assays for LeuRS from S.aureus and E.coli and the “Biothema” assay for ValRS from S.aureus and E.coli.
In total, 102 of these compounds were inhibitors of one or more aaRS enzymes. The most potent compounds were inhibitors of the LeuRS from E. coli, but inhibitors of S. aureus LeuRS, inhibitors of IleRS, and weak inhibitors of TrpRS were also found. Three compounds inhibited both IleRS and LeuRS, with weak dual inhibition of ValRS and LeuRS being seen for one additional compound.
62 compounds were active in antibacterial assays. Most of these (59), however, were inactive in all of the enzyme assays. It is not uncommon for antibacterial drug discovery projects to identify compounds with non-specific antibacterial activity. Although the mechanism of action of these compounds was not investigated in detail, they were not taken forward because of concerns regarding toxicity and they were outside the objectives of the project. Thus, the major difficulty encountered during the project was the fact that the majority of NCEs that have activity against the relevant targets do not have an effect on bacterial growth, and conversely, NCEs that have an effect on bacterial growth tend to display non-specific (off-target) activity.
However, the NABARSI consortium has succeeded in obtaining three new and potentially interesting aaRS antibiotics. We have been able to establish that they are working through the intended mechanism of action and that they have a spectrum of activity against Gram-negative bacteria, including clinical solates. Funding is currently being sought in order to further evaluate, develop and better characterise these NCEs.
The newly identified compounds are structurally novel and two patent applications have been filed, with further applications expected to be made in the future.
In addition to the patent applications, the results of the NABARSI consortium have been presented at international and national meetings, and several papers have been published in the scientific literature.
Potential Impact:
The anti-infective market in general and antibiotics in particular is an international and globalized market, with global annual figures sales of around €16,000 million for oral antibiotics and €6,200 million for intravenous administration. However, over the last few decades the number of new antibiotics brought to market has declined, while the rate of resistance to existing antibiotics has steadily increased. The human and economic costs of these trends has caused policy-makers to apply increasing pressure on pharmaceutical companies to address this gap, and also to create new private-sector incentives in this area such as accelerated review and preferential pricing for new antibiotics. The human impact of these two trends has been to increase mortality and morbidity in hospital, especially among the growing population of elderly and immune-suppressed patients. According to data from the Centres for Disease Control, the six ESKAPE bacteria are responsible for two-thirds of all Healthcare Associated Infections. Most hospital-acquired infections are resistant to at least one drug, and the incidence of MDR is rising. The inability to rapidly get these infections under control results in nearly 10,000 deaths every year in the United States, and many more in Europe and in the developing world. The economic impact of MDR infections is equally dramatic. The cost of hospital prolonged by MDR infection has been estimated at €7,000 million a year in Europe (Strategic Council on Resistance in Europe, SCORE; Utrecht, The Netherlands, SCORE, 2004). In this context, the NABARSI project aimed to discover compounds that have both antimicrobial and on-target (specific) activity against bacterial amino-acyl tRNA synthetases (aaRS), an activity that could be effective against clinically relevant Gram-positive and Gram-negative bacterial pathogens. The result of the project is the discovery of several new potentially clinically relevant antibiotics, which can be taken forward all further in vitro and in vivo testing. The further development and testing of these new antibiotics couldl have a major impact on reducing the current global morbidity and mortality associated with multi-resistant bacterial pathogens, and ultimately provide wider economic benefits to EU healthcare systems.
List of Websites:
www.nabarsi.eu
Partner email addresses -
Erasmus MC - John Hays
InhibOx Ltd - Paul Finn
Latvian Institute of Organic Synthesis - Aigars Jirgensons ,aigars@osi.lv>
University of Leeds - Alex O'Neill
Omnia Molecular S.L. (via IRB Barcelona)