Cryptidases are Zinc dependent proteases that encapsulate their substrate in a large inner cavity called “crypt”, before hydrolysis. The members of the family differ one from the other by the shape, size and substrate recognition ability of their crypt. Members of this small family of enzymes are found in many living organisms including bacteria, plants and animals. Insulin-degrading enzyme (IDE) is the prototype of this family of proteases. It hydrolyses many different polypeptides involved in important signaling pathways: amyloid beta, insulin, glucagon, IGF-II,ubiquitin. Although the enzyme is ubiquitous and known for long, and its 3D structure recently solved with several substrates, its role remains poorly understood. The objective of this chemical biology project held at the Lille Pasteur Institute is to design and synthesize chemical probes that will modulate the substrate specificity of IDE. The probes will be designed using structural data from X-ray, molecular modeling and medicinal chemistry reasoning. The project will build on preliminary structure-activity relationships already obtained by the principal investigator. In particular, her team will exploit series that bind to the catalytic zinc but also to other places in the crypt including a genuine substrate binding exosite. Structural modifications will be made to optimize surface binding (classical) and volume filling (specific of crypt containing enzyme). The goal is to obtain several nanomolar compounds with various inhibition/activation profiles and physico-chemical properties suitable for a use in complex biological systems (whole cells assays and in vivo experiments). The probes will be useful to biologists for untangling the many roles of IDE in several biological systems. More generally, the concept and the methodology could be used for other cryptidases such as the recently discovered Presequence pepdidase.

In 2019, tuberculosis (TB) remains a global public health emergency as it is the deadliest infective disease above HIV. Moreover, despite increasing attention from the World Health Organization in the last decades, the global incidence only drops by 2% each year. On the opposite, the proportion of multi-drug resistant (MDR) and extensively resistant (XDR) cases of TB is constantly increasing, and the therapeutic alternatives for the patients with MDR and XDR TB are increasingly limited. In order to meet these therapeutic needs, new antibiotics with novel mechanisms of action are urgently needed. Indeed, apart from bedaquiline and delamanid that have been granted a temporary accelerated approval by the FDA or EMA for the treatment of MDR-TB, all the other drugs in clinical use have been discovered more than 40 years ago. One fundamental difficulty in the treatment of TB is the long therapy duration required for the complete eradication of the bacilli, that can enter a non-replicating state, insensitive to most of the known antitubercular agents. In this context, the NL4TB project aims at accelerating the development of novel, safe and highly effective new compounds with new modes of action in order to address the threatening issue of MDR- and XDR-TB. To this purpose, two phenotypic screenings have been performed with our unique library of compounds on the wild type strain of Mycobacterium tuberculosis H37Rv, but also on the streptomycin-starved 18b strain, one of the model that mimics the non-replicating state, in order to identify compounds also active on the dormant bacilli. We have identified several original chemical series active on H37Rv which have never been described so far for their anti-infective properties. Two families (NLP and Bztz families) were selected based on their structural novelty and chemical tractability for early structure-activity relationship studies (eSAR). Regarding the screening on ss18b, we identified a family of compounds (ATU family) that is also active on H37Rv, and was also advanced for eSAR. Preliminary medicinal chemistry work led to structure-activity and structure-property relationships in the three chemical series, highlighting more promising activities and physicochemical properties for the NLP family. In parallel, isolation of resistant mutants was successfully achieved for the NLP family. Whole Genome Sequencing led to the identification of the essential pathway likely targeted by these compounds. The main goals of the NL4TB project are first to optimize the NLP family to produce a lead compound for in vivo validation, and to validate target engagement. NL4TB also aim at identifying the biological targets of the two remaining families (ATU and Bztz). To achieve this, the structure-activity and structure-property relationships exploration of the NLP family will be deepen during the project. The most potent compounds will be selected for further multi-parametric lead optimization. Mice exposure of the best analogs will be evaluated to select a candidate for in vivo proof of concept. In parallel, target identification will be pursued for the two other chemical series through spontaneous resistant clones selection with more potent analogs and will also be supported by chemical biology and the use of affinity-based probes that will be designed thanks to the structure-activity relationships previously generated. The validation and engagement of identified biological targets will be performed by genetic modifications (knockout or overexpression). At completion of this project we will have a new drug-like chemical entities validated in vivo and new biological targets relevant for the treatment of TB. The introduction of such molecules in a new regimen containing new drugs with no pre-existing resistance would be a game-changer in the fight against both drug-susceptible and drug-resistant TB.

Multidrug resistant tuberculosis and gram +ve and -ve nosocomial bacteria are posing an ever increasing burden to global healthcare systems with high levels of associated mortality and morbidity. A major cause is that the antibiotics pipeline over the last 40 years has produced very few novel classes of antibiotics. To prevent us from entering a “post-antibiotic” era, it essential that concerted efforts are made, to both discovery novel antibiotics compounds, but also novel antibiotic drug targets. NAT-4-MDR will strive to address this pertinent problem and establish research to discover new small molecule and natural product antibacterials, and to elucidate novel vulnerable and drugable antibiotic targets in bacteria. In addition NAT-4-MDR will develop novel lines of research to address the rational modifications of natural products to exploit their potential as drugs. NAT-4-MDR is divided into three linked work-packages that are designed to tackle the antibiotic problem at different stages and from different angles. Work-package I, is setup to identify novel compounds with antibiotic activity (M. tuberculosis, S. aureus, and E. coli), either through the screening of locally available 73000 small molecules compound library, and through the targeted awakening of novel natural product antibiotics from cryptic biosynthetic gene clusters in the rare actinomycetes. The objective of work package 2 is to identify novel antibiotic targets using published potent antibiotic natural products. The elucidation of the mechanism of action of these natural products will uncover new vulnerable and drug-able targets in bacteria. In the final work-package, a rational approach to generate analogues of the natural product pyridomycin (by de novo chemistry and mutasynthesis) will seek to improve its drug-like qualities, and to investigate its potential in targeting various other targets in bacteria (here called target-switching). These work packages of NAT-4-MDR will establish a number of innovative and technical approaches in the field of antibiotics discovery and natural products that are currently not widely studied in France. This multidisciplinary approach tackles the issue of novel antibiotic development from a variety of angles, an approach considered essential to maximize the potential impact of the research. This work is clearly in line with the ambitions of the Institut Pasteur de Lille, which has the desire to investigate and act on the increasing burden of antimicrobial drug resistance. My personal experience in drug discovery for M. tuberculosis and target deconvolution within this bacterium give me the necessary expertise to drive this project. Personal preliminary data also strongly supports the feasibility of the proposed work and minimize the risk associated. Uniquely to France, and important for the proposed research is that Institut Pateur de Lille has a 73,000 small compound library, a state-of-the-art screening platform that allows for the automated nanolitre dispensing and biosafety 3 facilities that allows for the efficient screening and manipulation of M. tuberculosis. Close association of the Institut Pasteur de Lille and the University of Lille (Drugs and Molecules for Living Systems) also allows for efficient collaboration for the needed chemistry, while a strong collaboration with Prof. K-H Altmann (ETH Zurich in Switzerland) will allow for de novo chemistry of natural products (particularly pyridomycin). Overall therefore, NAT-4-MDR represents a unique package for France, with an ideally matched research project and local research infrastructure and knowledge that will maximize the potential and impact of this work.