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JPIAMR-JPI Antimikrobiell resistens

Development of novel Mycobacterial Tolerance Inhibitors (MTIs) against MDR/XDR tuberculosis

Alternative title: Utvikling av nye Mycobacterial Tolerance Inhibitors (MTI) mot multi-resistent tuberkulose

Awarded: NOK 5.8 mill.

Mycobacterium tuberculosis (Mtb) is the cause of the serious disease tuberculosis (TB). Multi-resistant Mtb is on the WHO's list of global pathogenic microbes. The list has been prepared to promote research and development of new treatments against multi-resistant microbes. In many countries, up to 48% of new TB cases are caused by multi-resistant Mtb. Today's treatment for TB includes a combination of antibiotics that were initially very effective against TB. However, they have a large degree of antibiotic resistance in the form of mono-, multi- and extensively-drug resistance developed in the previously sensitive Mtb bacterium. This failure in current TB therapy provides a great need for new treatment for Mtb infection, in order to achieve the WHO's sustainability goal of stopping the TB epidemic by 2030. Increased understanding of the mechanism that provides Mtb drug tolerance and antibiotic resistance is, therefore, essential to improve the current TB treatment. In this context, partners in the JPIAMR consortium MTI4MDR-TB has discovered and further developed a new component family of ring-formed 2-pyridones, so-called Mycobacterial Tolerance Inhibitors (MTIs). MTI has a unique mechanism of action that causes Mtb to become sensitized during stress, which occurs in the host cell during TB infection. They thereby restore the effect of the front-line drug isoniazid (INH) in INH-resistant Mtb reference isolates. Our goal was to test the activity and efficiency in sensitive and INH-resistant Mtb clinical isolates. We also aimed to define the mechanisms of action of these novel compounds (proof of concept). Thereby, new and even better MTIs can be developed. To do this, a combination of culture, molecular biology and –omics approaches in both Mtb reference strains and clinical isolates was employed. These combined approaches makes it possible to discover the molecular mechanisms involved in Mtb MTI drug tolerance and resistance. We aim to optimize the current version of MTI for new therapy that can be taken orally, and uncover mechanisms for the effect and development of tolerance and resistance. MTI drug activity were tested by resazurin microtiter assay (REMA) to determine the half maximal inhibitory concentration (IC50), followed by assessment of drug cytotoxicity of MTIs in human cell lines. The IC50 values of tested MTIs were low and exhibited no cytotoxicity in human cell lines. We have thus discovered that MTI works well against the clinical Mtb isolates with INH-resistance. We have also identified new active "lead" MTI candidates for further studies. In addition, we investigated the synergy of lead MTIs with various antibiotics used in TB treatment, such as INH and Bedaquiline using checkerboard analysis, in addition to other methods. We have performed proteome analyzes using mass spectrometry on Mtb laboratory strains and clinical isolates treated with: 1) MTI alone, 2) MTI and INH, 3) INH alone and 4) only DMSO as a control. The proteomics data sets were analyzed by statistical and bioinformatics tools for molecular annotations, were we also investigated the differences and similarities in the proteome between these four treatments. Thereby we have identified proteins that are involved in metabolic and cellular biological processes, as well as membrane and intracellular components. Our results show that MTIs sensitize Mtb to the frontline antibiotic isoniazid and re-sensitize isoniazid-resistant Mtb. The use of combined approaches has enabled the discovery of pathways involved in Mtb MTI drug tolerance and resistance, representing a novel basis for discovering how the MTIs work, in order to optimize TB treatment. Through collateral sensitivity action, MTIs thus hold great potential in the treatment against TB.

There is a substantial medical need and market potential for new TB therapy. The MTI4MDR-TB project has clearly demonstrated innovative potential towards unmet medical and patient needs and health impact. The MTI product will not only improve quality of life for users; it will save the lives of numerous patients infected with bacteria that otherwise would be resistant to the drugs available. MTIs also offer a low toxicity treatment alternative that will reduce the frequency of serious morbidity. The product will have a positive environmental impact because it is more difficult for the bacteria to develop resistance to such combination drugs. Saving lives, reducing morbidity due to side-effects and returning patients to work in shorter time, represents considerable socio-economic savings. The MTI4MDR-TB project scheme and team who has worked together in an interdisciplinary setting will promote health and innovation and secure contact between translational medicine, basic science and industry, amalgamating our ideas and potential products in an already interactive setting.

In 2017, the WHO published the Global priority pathogen lists with the aim to promote research and development of new treatments that are effective against microbes resistant to multiple antibiotics. Among them, multi- and extensively drug-resistant Mycobacterium tuberculosis (Mtb) caused 48% of new tuberculosis (TB) cases in some countries in 2016. Current regimens for the treatment of TB include a combination of antibiotics developed for their strong efficacy against drug sensitive bacterium. The inadequacies of present TB therapies demand discovery of new agents with unique mechanisms of action to treat Mtb infection. Towards this end, we have discovered and developed a new family of ringfused 2-pyridones (termed Mycobacterial Tolerance Inhibitors, MTIs) that invoke collateral sensitivity in Mtb by potently sensitizing Mtb to stresses encountered during infection and restoring activity to the frontline antibiotic isoniazid (INH) in otherwise INH-resistant Mtb isolates. Our short-term objectives are to demonstrate preclinical proof-of-concept for MTIs to combat Mtb infection, optimize the current lead MTIs for translation to a therapeutic, and reveal new insights into pathways of drug tolerance and resistance. Our long-term objective is to develop a new orally available antibiotic that improves the current regimens for patients with drug-resistant TB. We will also generate a deeper understanding of the MTI mode of action and their potential in synergistic interactions with other drugs. Importantly, we will also study how likely it will be for Mtb to develop resistance to combinations of MTIs and INH and other antibiotics.

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JPIAMR-JPI Antimikrobiell resistens