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Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump. Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, the authors identify pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through allosteric inhibition of its primary RND transporter.

Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-β. Knockout and genetic studies have linked IDE to Alzheimer’s disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisingly impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes. Inhibiting insulin-degrading enzyme (IDE) has been proposed as a potential therapeutic strategy for the treatment of patients with diabetes. Here, the authors develop a novel IDE inhibitor but find that, surprisingly, IDE inhibition has negative effects on glucose tolerance in mice.

Tuberculosis (TB) is a leading infectious cause of death worldwide. The use of ethionamide (ETH), a main second line anti-TB drug, is hampered by its severe side effects. Recently discovered “booster” molecules strongly increase the ETH efficacy, opening new perspectives to improve the current clinical outcome of drug-resistant TB. To investigate the simultaneous delivery of ETH and its booster BDM41906 in the lungs, we co-encapsulated these compounds in biodegradable polymeric nanoparticles (NPs), overcoming the bottlenecks inherent to the strong tendency of ETH to crystallize and the limited water solubility of this Booster. The efficacy of the designed formulations was evaluated in TB infected macrophages using an automated confocal high-content screening platform, showing that the drugs maintained their activity after incorporation in NPs. Among tested formulations, “green” β-cyclodextrin (pCD) based NPs displayed the best physico-chemical characteristics and were selected for in vivo studies. The NPs suspension, administered directly into mouse lungs using a Microsprayer®, was proved to be well-tolerated and led to a 3-log decrease of the pulmonary mycobacterial load after 6 administrations as compared to untreated mice. This study paves the way for a future use of pCD NPs for the pulmonary delivery of the [ETH:Booster] pair in TB chemotherapy.

Background Nonsense mutations are at the origin of many cancers and inherited genetic diseases. The consequence of nonsense mutations is often the absence of mutant gene expression due to the activation of an mRNA surveillance mechanism called nonsense-mediated mRNA decay (NMD). Strategies to rescue the expression of nonsense-containing mRNAs have been developed such as NMD inhibition or nonsense mutation readthrough. Methods Using a dedicated screening system, we sought molecules capable to block NMD. Additionally, 3 cell lines derived from patient cells and harboring a nonsense mutation were used to study the effect of the selected molecule on the level of nonsense-containing mRNAs and the synthesis of proteins from these mutant mRNAs. Results We demonstrate here that amlexanox, a drug used for decades, not only induces an increase in nonsense-containing mRNAs amount in treated cells, but also leads to the synthesis of the full-length protein in an efficient manner. We also demonstrated that these full length proteins are functional. Conclusions As a result of this dual activity, amlexanox may be useful as a therapeutic approach for diseases caused by nonsense mutations.

Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.

Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in M. tuberculosis, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.

Chemical biology and drug discovery are two scientific activities that pursue different goals but complement each other. The former is an interventional science that aims at understanding living systems through the modulation of its molecular components with compounds designed for this purpose. The latter is the art of designing drug candidates, i.e., molecules that act on selected molecular components of human beings and display, as a candidate treatment, the best reachable risk benefit ratio. In chemical biology, the compound is the means to understand biology, whereas in drug discovery, the compound is the goal. The toolbox they share includes biological and chemical analytic technologies, cell and whole-body imaging, and exploring the chemical space through state-of-the-art design and synthesis tools. In this article, we examine several tools shared by drug discovery and chemical biology through selected examples taken from research projects conducted in our institute in the last decade. These examples illustrate the design of chemical probes and tools to identify and validate new targets, to quantify target engagement in vitro and in vivo, to discover hits and to optimize pharmacokinetic properties with the control of compound concentration both spatially and temporally in the various biophases of a biological system.

Insulin-degrading enzyme (IDE) is a multifunctional protease due to the variety of its substrates, its various cellular locations, its conservation between species and its many non-proteolytic functions. Numerous studies have successfully demonstrated its implication in two main therapeutic areas: metabolic and neuronal diseases. In recent years, several reports have underlined the overexpression of this enzyme in different cancers. Still, the exact role of IDE in the physiopathology of cancer remains to be elucidated. Known as the main enzyme responsible for the degradation of insulin, an essential growth factor for healthy cells and cancer cells, IDE has also been shown to behave like a chaperone and interact with the proteasome. The pharmacological modulation of IDE (siRNA, chemical compounds, etc.) has demonstrated interesting results in cancer models. All these results point towards IDE as a potential target in cancer. In this review, we will discuss evidence of links between IDE and cancer development or resistance, IDE’s functions, catalytic or non-catalytic, in the context of cell proliferation, cancer development and the impact of the pharmacomodulation of IDE via cancer therapeutics.

Ethionamide (Eto) and prothionamide (Pto) are second-line antibiotics used for tuberculosis (TB) treatment. Both are prodrugs whose antibacterial activity depends on bioactivation by oxidases in Mycobacterium tuberculosis , including the Baeyer-Villiger monooxygenase MymA. Through biophysical, genetic, and cellular assays, we show that the clinical candidate alpibectir (Alp, BVL-GSK098) binds the transcriptional regulator VirS, increasing MymA expression and potentiating Eto and Pto activity. Alpibectir also boosts the activity of the corresponding host-derived sulfoxide metabolites. We additionally show that alpibectir exhibits intrinsic antibacterial activity via overexpression of the mymA operon. The alpibectir/Eto (AlpE) combination is rapidly bactericidal in vitro and in mice, lowers the frequency of spontaneous resistance of Eto, and remains active on Eto- and isoniazid-resistant strains, including isolates with inhA promoter mutations. Alpibectir was safe in a Phase 1 human clinical trial. Together with the potentiation data presented here, these findings highlight its potential to optimize TB chemotherapy by reducing Eto/Pto doses, which can minimize dose-related side effects, enhancing adherence. In this work, authors show that alpibectir boosts ethionamide efficacy against Mycobacterium tuberculosis by enhancing its bioactivation and also shows intrinsic activity. The ethionamide-alpibectir combination (AlpE) is bactericidal in mice and active against drug-resistant strains.