Periodic Reporting for period 2 - INTEGRATE (Interdisciplinary Training Network for Validation of Gram-Negative Antibacterial Targets) Reporting period: 2017-01-01 to 2018-12-31 Summary of the context and overall objectives of the project Antimicrobial resistance is posing a continuously-rising threat to global health. One key recommendation from the recent “Action plan against the rising threats from Antimicrobial Resistance” report (the Commission to the European Parliament and Council (15.11.2011)) is the development of effective antimicrobials or alternatives for treatment of human and animal infections. The INTEGRATE project is a direct response to this.We have assembled a team of ten beneficiaries from eight EU member states, encompassing both academic and nonacademic sectors and different disciplines, to form a consortium committed to train Early Stage Researchers (ESRs) in the discovery and preclinical validation of novel Gram-negative antibacterial targets and agents. The principal objective of the consortium was to provide a training platform where students are exposed to every aspect of the antimicrobial discovery process, ranging from target identification and validation, through organic synthesis, in silico design and compound screening, to mode-of-action and possible resistance mechanisms. This exposure was supported by a concrete secondment plan, coupled with a series of high-level consortium-wide training events and networking programs.The INTEGRATE project foresaw four main scientific objectives to tackle down multidrug resistant bacteria: 1) identifying and optimizing novel chemotypes; 2) establishing new tools and resources for antibacterial drug discovery; 3) validating non-canonical targets for combating Gram-negative bacterial infections; 4) strengthening inter-sectorial and multidisciplinary collaborations towards discovery of new antibacterial agents.Through this, INTEGRATE contributed to overcoming the fragmentation of approaches that often characterize drug discovery and trained 11 ESRs in a multi-disciplinary environment. The training-through-research part of INTEGRATE focused on several gram-negative targets, not yet exploited clinically, for which we intended to clarify their potential relevance in the bacterial fitness, and for which we intended to progress a series of small-molecules lead candidates for further development. The training-through-research plan is flanked by an active program of soft-skill acquisition and by a dissemination plan to increase the general awareness on the importance of basic research to fight bacterial infections Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The scientific efforts of INTEGRATE were carried out in three work-packages involving concerted action of several beneficiaries. ‘Computer-aided molecular design’ work package developed, and distributed among partners molecular models for LsrK, glyoxylate shunt, GyrB, OASS-B, SurA and ClpP. The molecular models served as the guide for the rational design of new chemotypes and for the interpretation of the molecular basis of the ligand’s activity. Virtual screening campaigns to identify and optimizenew LsrK, glyoxylate shunt and gyrase B inhibitors were taken.‘Medicinal and synthetic organic chemistry’ work-package exploited medicinal and synthetic organic chemistry activities and synthesize low molecular-weight compounds to target gyrase B, OASS and ClpP. The design of the compounds was based on the modeling results of work package 1. The activity within this work package included heavy medicinal chemistry optimization campaign for improving the identified target inhibitor activities to development of novel synthetic approaches for the synthesis of novel chemotypes. The synthesized compounds were tested for target inhibition in a biochemical assay. Proof-of-concept was obtained that inhibitory compounds acting at (low) micromolar concentration could be developed for each of these targets.The activities within the ‘Molecular microbiology and bioactivity screening’ work package characterized and validated the targets for antimicrobial intervention and characterized the impact of the novel low-molecular weight compounds on a previously validated target. To this end, target-based, automation-compatible and scalable assays for high-throughput screening (HTS) of chemical libraries were set-up, validated, and used to perform several screening campaigns for OASS, LsrK, malate synthase and ClpP. Potent low molecular weight compounds inhibitors were discovered for several targets and the antimicrobial activity of the most promising compounds was validated. Notably, advanced structural and functional characterization of the purified target proteins was performed to understand the targets biochemical features and potential as antibacterial drug targets. This included solved X-ray crystals structures for malate synthase. Additionally, the activities provided enabling technologies that facilitate HTS, mechanism of action, resistance mechanism(s) and in vivo validation of low-molecular weight compound hits.The scientific highlights of the consortium wide activities resulted in numerous conference presentations and, so far, 16 scientific publications, with several manuscripts being currently in a drawing up phase. 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) INTEGRATE scientific efforts discovered and characterized a novel discrete set of gram-negative targets that have not been previously clinically exploited. These include targets for the multidrug resistant pathogens Pseudomonas aeruginosa and Staphylococcus aureus, which remain among the most important therapeutic challenge of antimicrobial resistance. INTEGRATE provided proof-of-principle for the relevance of the novel targets in the bacterial fitness, and progressed a series of small molecules lead inhibitory candidates for the targets for further development. As such the project extends beyond state-of-the-art.INTEGRATE trained 11 early stage researchers in a multidisciplinary environment. The training-through-research plan flanked by an active program of soft-skill acquisition. During the reporting period two workshops and a summer school were organised. The INTEGRATE Outreach Conference promoted the findings of the project with approximately 70 registered conference participants.The contribution of the INTEGRATE the expected impact continues to be very relevant. The need for new approaches to the discovery of gram-negative antibacterials remains highly pressing. Newer case of superbugs are continuously reporting and there is a strong consensus that only multidisciplinary approaches will help tackling the threat. Furthermore, over these 24 months, synergies with other EU funded projects (such as TRANSLOCATE or the IMI ENABLE projects) have been identified. Their exploitation will maximize the impact of the EU funded research in the field of antibacterials.Through this, INTEGRATE contributed to overcoming the fragmentation of approaches that often characterize drug discovery and trained 11 ESRs in multi-disciplinary environment. work packages INTEGRATE logo in computer class during summer school 2016 beneficiaries logos INTEGRATE Outreach Conference homepage modeling image
