Axis 3: Chemical solutions to impermeability and efflux

Chemical synthesis

Jean-Michel Brunel

Antimicrobial resistance (AMR) represents a global health threat to humanity. Since their introduction, antibiotics have been the cornerstone of modern medicine but over time there has been a surge in the incidence of antibiotic-resistant bacteria. The increase has been driven in part by inappropriate prescribing by medical experts as well as by the ability of bacteria to adapt to and overcome, the action of antibiotics. Of concern, is the clinical prevalence of difficult-to-treat infections with Gram-negative bacteria including Pseudomonas aeruginosa, a common hospital-acquired pathogen that causes pneumonia. A strategy for overcoming Gram-negative bacterial resistance is to identify compounds that can circumvent drug resistance by enhancing the activity of antibiotics that are currently ineffective. Our mission is to design and develop targeted antibiotic adjuvants and antimicrobial agents effective against a variety of Gram-positive and Gram-negative bacteria. Initially, these compounds are tested in vitro, followed by trials in animal models. These compounds will first serve as pharmacological probes to validate therapeutic approaches and subsequently as drug prototypes. Our expertise encompasses microbiology and medicinal chemistry, enabling us to achieve our goals in the organoselective synthesis of biologically active molecules.

In this ambitious project, New Zealand-based experts in natural products and biologically active small molecules (Copp, Auckland) and French leaders in the investigation of marine natural products (Bourguet-Kondracki, Paris) and antibiotic enhancers (Brunel, Aix-Marseille), are collaborating to demonstrate that marine natural products and synthetic analogs can be a source of novel molecules that can restore the action of antibiotics towards difficult to treat Gram-negative bacteria. Such drug ‘rehabilitation’ could help halt the spread of antibiotic resistance. Thus, the marine environment has emerged as a promising source for the discovery of new classes of antimicrobials. Natural products from marine animals, such as squalamine a polyamine-containing aminosterol isolated from the dogfish Squalus acanthias (a common shark species) have shown broad-spectrum activity against both Gram-positive and Gram-negative bacteria. The marine sponge antimicrobial natural product ianthelliformisamine C, has been shown to enhance the activity of conventional antibiotics against drug-resistant Gram-negative bacteria. Samples of purified natural products previously isolated from marine organisms, as well as a library of pre-fractionated extracts from marine organisms, were screened as part of this project for antimicrobial activities. These biological assays led to the selection of marine polyamines for further investigations because they were found to act as antibiotic enhancers (Figure 1). A diverse set of changes were made to the original ‘hit’ compounds, leading to the synthesis of over 100 analogs, all of which were evaluated for their abilities to inhibit the growth of bacteria, enhance the action of old legacy antibiotics, and for detrimental cytotoxicity/hemolytic properties. One series, the indole-carboxamide-polyamines, is now the focus of ongoing development. Derivatives can restore the activity of unused antibiotics against resistant bacteria at levels of activity to those observed against non-resistant bacteria. We have been able to investigate their mechanism of action involved against both Gram-negative and positive bacteria. Serendipitous discoveries suggest that exploring commercial bio-waste streams as sources of raw materials for new generations of naturally derived biocides would be worthwhile.

Cholesterol is an essential and pleiotropic biological component in cells. We gathered a bundle of evidence pointing to a functional link between cholesterol and cancer. First, we documented that a genetic alteration found in several common cancers leads to the overexpression of a cholesterol transfer protein called STARD3. Second, overexpression of this protein participates in an oncogenic program, in which the intracellular repartition of cholesterol is specifically regulated. Thus, STARD3 represents a genetic vulnerability that can be exploited for targeted cancer therapy. The sterol transport function of STARD3 is essential for its oncogenic action therefore inhibiting this function will stop the growth of cancer cells. The objective of this project is to identify small molecules to prevent STARD3 sterol transfer function, that will be used as targeted therapeutic molecules against specific cancers. This proposal is based on a proof-of-concept project that allowed the identification and characterization of primary hits. The objectives of this full project proposal are to use the methods and technologies that we have established in the proof-of-concept phase to extend hit discovery to additional chemical families, and to evaluate the mode of interaction and the biological activity of already identified hits and new ones. To apply iterative drug design, synthesis, and testing cycles to optimize the activity of the most promising compounds to identify suitable therapeutic molecules inhibiting STARD3 activity.

Intravascular clotting is a major issue that threatens quality of life and survival and represents a significant economic burden. It is a key event in deep venous thrombosis. Still, it contributes to the severity of many chronic infectious and inflammatory diseases by generating microthrombi and participating in the immunothrombosis phenomenon. The mechanisms responsible for excessive clotting include changes in the vascular endothelium, whose key function is to prevent unwanted intraluminal blood clotting and the formation of occlusive thrombi. Because they act by blocking critical coagulation steps, currently used antithrombotic drugs expose patients to a substantially increased risk of bleeding (1). It is therefore essential to identify new antithrombotic strategies that preserve physiologic hemostasis. In this context, improving the physiological antithrombotic defense could be a way to prevent venous thrombosis without reducing coagulation in the first instance, which could avoid triggering bleeding. We will focus on thrombomodulin (TM) a key antithrombotic molecule. We have been working for several years to better understand the unusual properties of selected aminosterols. We have recently discovered that Claramine (Fig. 1A) and some of its derivatives increase the FIIa cofactor activity of TM (PC activation on the surface of endothelial cells) (Fig. 1B). Our unpublished results indicate that this effect involves the uncommon property of Claramine to increase the amount of TM (and that of EPCR and PAR2; data not shown) in lipid raft-like microdomains of the membranes (Fig. 1C). Isolation of lipid rafts by traditional isopycnic centrifugation of detergent-resistant membranes is time-consuming and not suitable for screening. We have demonstrated that using the “Ultra Ripa” kit (sold by Funakoshi) we can rapidly isolate “lipid rafts” that are comparable, at least in terms of TM content, to those obtained with traditional long and cumbersome techniques (Fig. 1D; Claramine, but not Trodusquemine increases the amount of TM in lipid rafts).

Our preliminary data indicate that the nature of the polyamine group linked at position 6 (Fig. 1A) of the sterol platform determines the efficiency of the Claramine derivative in increasing the amount of TM in lipid rafts. This property is poorly affected by protecting the hydroxyl group at position 3 of the cholesterol moiety. However, this does not mean that this position has no function; indeed, it could be targeted to create compounds with modified secondary properties (e.g. stability, toxicity, pharmacokinetics, bioavailability).

In a previous study, we identified novel polyamine-isoprene compounds that act synergistically with doxycycline (DOX) against P. aeruginosa. The thesis work of Margot Draveny further described the impact of NV716 on antibiotic accumulation and its mode of action on bacterial envelopes. To quantify accumulation, we used spectrofluorimetry and DUV microscopy, utilizing the intrinsic fluorescence properties of DOX. First, we observed that the presence of NV716 significantly increases the accumulation of DOX. Moreover, this effect was observed in both the wild-type strain and also in a derivative mutant deleted of four major efflux pumps, suggesting that NV716 does not target efflux. Subsequently, the initial accumulation rates were measured in the presence of various external concentrations of DOX, and it was concluded that NV716 increased the permeation of DOX by a factor of five. NV716 exhibits no cellular lytic activity per se, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the outer but not the inner membrane of P. aeruginosa. The outer membrane damage in single cells treated with NV716 was further confirmed by using high-resolution cryo soft-X-ray tomography available at the MISTRAL beamline of ALBA Synchrotron. The selective targeting of the outer membrane most probably occurs through interactions of NV716 with the lipid A portion of the lipopolysaccharide (LPS), as shown by bodipy-cadaverin fluorescence displacement experiments and antibiotic susceptibility assays in the presence of LPS. Finally, NV716 demonstrated efficacy in enhancing the activity of DOX against P. aeruginosa in a Galleria mellonella infection model, resulting in a significantly higher survival rate compared to the control group. These findings collectively suggest a promising avenue for the use of compounds to renew sensitivity for non-permeable antibiotics by targeting the primary outer membrane barrier to accumulation.

Project: Studying the mode of action of an outer membrane disruptor

Jean Michel Brunel, Muriel Masi

In a previous study, we identified novel polyamine-isoprene compounds that act synergistically with doxycycline (DOX) against P. aeruginosa. The thesis work of Margot Draveny further described the impact of NV716 on antibiotic accumulation and its mode of action on bacterial envelopes. To quantify accumulation, we used spectrofluorimetry and DUV microscopy, utilizing the intrinsic fluorescence properties of DOX. First, we observed that the presence of NV716 significantly increases the accumulation of DOX. Moreover, this effect was observed in both the wild-type strain and also in a derivative mutant deleted of four major efflux pumps, suggesting that NV716 does not target efflux. Subsequently, the initial accumulation rates were measured in the presence of various external concentrations of DOX, and it was concluded that NV716 increased the permeation of DOX by a factor of five. NV716 exhibits no cellular lytic activity per se, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the outer but not the inner membrane of P. aeruginosa. The outer membrane damage in single cells treated with NV716 was further confirmed by using high-resolution cryo soft-X-ray tomography available at the MISTRAL beamline of ALBA Synchrotron. The selective targeting of the outer membrane most probably occurs through interactions of NV716 with the lipid A portion of the lipopolysaccharide (LPS), as shown by bodipy-cadaverin fluorescence displacement experiments and antibiotic susceptibility assays in the presence of LPS. Finally, NV716 demonstrated efficacy in enhancing the activity of DOX against P. aeruginosa in a Galleria mellonella infection model, resulting in a significantly higher survival rate compared to the control group. These findings collectively suggest a promising avenue for the use of compounds to renew sensitivity for non-permeable antibiotics by targeting the primary outer membrane barrier to accumulation.