DEVELOPMENT OF CARBON MONOXIDE-RELEASING MOLECULES CONJUGATED TO POLYSACCHARIDE CARRIERS TARGETING ADIPOSE TISSUE FOR THE TREATMENT OF OBESITY – SWEET-CO

Chemistry. The synthesis of Glyco-CORMs results from the grafting of metal carbonyl complexes onto defined molecules A and A1. Three synthetic strategies were investigated: (i) synthesis of a functionalized molecule A capable of coordinating directly onto carbonyl complexes, (ii) modification of molecule A and installation on the backbone of a spacer allowing the grafting of carbonyl complexes, (iii) synthesis of a piperazine-derived CO-RM capable of conjugating with an activated molecule A. While the first two strategies proved unsuccessful or limited, the third one gave with good yields molecules A- and A1-derived conjugates, presenting various CO-RMs loading. In addition, the third strategy was successfully applied either to a sulphated compoun that is the proprietary drug provided by the industry partner OTR3. Biology. The biological testing and characterization of new CORMs derivatives synthesized in collaboration with the chemist is achieved through a series of methods that allow us to: 1) assess the release of CO from the new compounds in vitro in cuvette, using HemoCD1 and hemoglobin which bind CO with high affinity and change their absorption spectra once bound to CO; 2) determine the ability of Glyco-CORMs to cause accumulation of CO in preadipocytes and adipocytes. In this assay we use a probe which avidly and specifically binds CO and that changes its spectral absorbance once bound to CO, which can be measured spectrophotometrically. This probe, HemoCD1, is available to the biology group thanks to a collaboration with the Japanese Prof Hiroaki Kitagishi from Doshisha University in Kyoto, after having developed a method to detect CO in cells and tissues; 3) determine the time-dependent accumulation of CO in the most important organs/tissues after oral administration of CORM-401; 4) to assess whether Glyco-CORMs are effective against obesity, using a model of high fat diet already established in our laboratory. To study the mechanisms of action of CO in obesity, we focused on the gut microbiota, where we observed a big accumulation of CO when animals were given CORM-401. DNA and RNA sequencing of the microbiota as well as metabolomics analysis of plasma and fecal samples were carried out in collaboration with platforms at our institute and in Paris.

Using the methods described above, we quantified the CO released by our compounds and determined that the Glyco-CORMs obtained with the 3rd strategy were those releasing CO with predicted kinetics. Glyco-CORMs with 25, 50, 75 and 100% metal carbonyls grafted on dextran released CO in proportional amounts, e.g. GlycoCORM 100% released approximately double the amount of CO than GlycoCORM 50%. We found that the CO released was lower than expected, a result likely due to steric hindrance when dextran is bound to a very large number of metal carbonyls. This first part was important to determine the best solvent to obtain good solubilization of the new compounds. Indeed, dextran is soluble in water and CORM-401 is soluble in DPBS. However, the GlycoCORM complex could only be solubilized in DMSO (but not completely). We worked to find formulations that allow a complete solubilization of the compounds, considering the in vivo work in mouse models of high-fat diet. In the characterization in preadipocytes and adipocytes, we also found that intracellular CO increased strongly after incubation of the cells with Glyco-CORMs. Adipocytes had a strong CO retention and, if Glyco-CORMS was used at very low concentrations (=0.5 µM), they did not affect cell viability. In a first series of in vivo experiments where CORM-401 was administered to mice, we showed that CO levels increased significantly in the liver, kidneys, intestine and adipose tissue with a peak at 6h and decreasing to near-basal levels at 24 or 48h.

We also tested the glyco-CORMs and found that in vivo they did not release large amounts of CO, neither in the blood nor in the tissues. This is likely due to the large size of the glyco-CORMs. They were still relatively effective in improving high-fat diet-induced obesity and metabolic dysfunction, but they were not superior to CORM-401 and were more difficult to administer. The compounds also did not deliver CO specifically to adipose tissue. We then decided to focus on CORM-401, which was ultimately the most promising compound. By focusing on its effect on the gut microbiota, we demonstrated that CO improved the diversity of the gut microbiota of animals on the high-fat diet, increasing the abundance of bacterial species, such as Akkermanisa muciniphila, which are beneficial to the organism but are strongly decreased by the high-fat diet. We also showed that many metabolites in the plasma and feces of animals that were altered during the high-fat diet were improved by CORM-401.

Glyco-CORMS were synthesized and tested with good but not superior results compared to the original CORM-401 in the mouse obese model. As a result, our focus is to continue using CORM-401 and characterize as much as possible its positive, anti-obesity properties in the organism. This work will involve analyzing its effects in major metabolic organs and try to dissect how CO is preventing fat accumulation and metabolic dysfunction during a high fat diet. This is indeed the most interesting aspect for us: how can animals that are eating a diet rich in fat not gain weight when they are also treated with CORM-401? How is CO affecting affecting metabolism in the organism, from an organ and tissue-specific focus to the whole body? In addition, becuase we found that CORM-401 is significnalty affecting the gut microbiota, we would like to further investigate how CO interacts with this system to promote a healthy outcome during high fat diet.

Article: Highly sensitive quantification of carbon monoxide (CO) in vivo reveals a protective role of circulating hemoglobin in CO intoxication. Mao Q, Kawaguchi AT, Mizobata S, Motterlini R, Foresti R, Kitagishi H.. Communications Biology, 4(1):425, 2021.
Book chapter: Metal-based carbon monoxide-releasing molecules (CO-RMs) as pharmacologically active therapeutics. Roberta Foresti, Djamal Edine Benrahla, Shruti Mohan, Roberto Motterlini. In: Carbon Monoxide in Drug Discovery: Basics, Pharmacology, and Therapeutic Potential (B. Wang, and L. Otterbein Editors), Wiley Series in Drug Discovery and Development, by John Wiley and Sons, 2022.

Carbon monoxide (CO), which is produced in tissues by the inducible defensive protein heme oxygenase-1 (HO-1), is a known anti-inflammatory and cytoprotective gas mediator. Our group has pioneered the discovery of CO-releasing molecules (CO-RMs) to deliver controlled amounts of CO to biological systems and confirmed their therapeutic applicability in vascular, ischemic and inflammatory diseases. We have recently published that CO-RMs act as mild uncouplers of mitochondrial respiration in different cell types. In this context, our data just accepted for publication show that oral administration of a manganese-based CO carrier (CORM-401) stimulates weight loss and counteracts the impairment of metabolism in obese mice via a mechanism that involves mitochondrial uncoupling in adipose tissues. This effect is reminiscent of that exerted by uncoupling agents known to induce weight loss. Because the delivery of CO in vivo is not specific, we aim to synthesize and evaluate novel molecules that direct CO specifically to adipocytes in order to maximize the efficacy of CO-RMs. To achieve this, we propose a strategy to develop CO-RMs conjugated to bioactive polysaccharides known to enhance the delivery of drugs to adipose tissue. We will refer to these new chemical entities as ‘glyco-CORMs’.

Building on our preliminary findings, the main OBJECTIVES of this proposal are:

1) to synthesize glyco-CORMs by conjugating CORM-401 homologs with bioactive sugars for targeted delivery of CO to adipose tissue;

2) to characterize in vitro the biological activities of glyco-CORMs by focusing on their effects in adipocytes metabolism and inflammation;

3) to assess in vitro and in vivo the ability of glyco-CORMs to preferentially target adipose cells and tissues;

4) to evaluate the efficacy of glyco-CORMs to reduce metabolic dysfunction in obese mice.

The successful accomplishment of this project will rely on the expertise and synergistic collaboration between biologists with a strong background on the pharmacological effects of CO/CO-RMs in metabolism (Dr Foresti-Coordinator-Partner 1), organic chemists having acquired experience in the synthesis of hybrid CO-RMs (Dr Rivard-Partner 2) and an industrial partner (CarboMimetics) with long-standing experience on sugar chemistry and drug discovery (Dr El Hadri-Partner 3). The synthesis of glyco-CORMs is an achievable goal because Dr Foresti and Dr Rivard have established a long-standing collaboration based on the successful synthesis of CO-RMs hybridized to bioactive molecules that are being investigated as anti-inflammatory agents. The consortium will apply state-of the art techniques developed to study the biological role of CO in cell metabolism (Seahorse Analyzer) and to evaluate its preferential accumulation in adipose tissue in vivo. Moreover, for future translational applications, we are partnering with CarboMimetics, a company dedicated to the discovery and development of fully synthetic active saccharide fragments that exert therapeutic action in diabetes and can be conjugated to CO-RMs. Although CO-RMs have undisputed pharmacological effects, we expect that glyco-CORMs will act specifically on adipose tissue, creating new chemical entities to be applied within the defined clinical indication of obesity and metabolic derangement.