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.

Using the methods described above, we detected and quantified the CO liberated by our compounds and determined that Glyco-CORMs obtained with the 3rd strategy were the ones liberating CO with a predicted kinetic. Specifically, Glyco-CORMs with 25, 50, 75 and 100% metal carbonyls grafted onto molecule A released CO in proportional amounts, with GlycoCORM 100% releasing approximately double the amount of CO than GlycoCORM 50% and continuing with the same trend for Glyco-CORMs with a lower number of grafted CORMs. In many cases we found that the CO released was less than predicted, a result that is likely due to steric hindrance when molecule A is bound to a very high number of metal carbonyls. This initial part was also important to determine the best solvent to obtain a good solubilization of the new compounds. Indeed, molecule A is soluble in water while CORM-401 is soluble in DPBS. However, the GlycoCORM complex could only be solubilized in DMSO (although not fully) and we are working to find formulations that allow a full solubilization of the compounds, especially in view of the in vivo work that we are planning in mouse models of high fat diet. In our characterisation in preadipocytes and fully matured adipocytes we also found that intracellular CO strongly increased after incubation of cells for three hours with Glyco-CORMs at different concentrations, highlighting again a relationship between the number of metal carbonyls attached to molecule A and the quantity of CO detected in cells. Adipocytes had a high retention of CO and, if Glyco-CORMS were used at very low concentrations (=0.5 µM), they did not affect cell viability to a great extent. In a first set of experiments in vivo where CORM-401 was administred to mice, we found that CO levels increased significantly in liver, kidney, gut and adipose tissue with a peak at 6h for most organs and decreasing to near-basal levels at 24 or 48h. These data will be used as a reference to test the CO distribution using the most promising Glyco-CORMs.

Chemistry. Biological evaluations on in vivo models will require large amounts of Glyco-CORMS. As a result, compounds that revealed to be the most biologically promising among the dozen obtained according to the 3rd strategy will be re-synthesized on gram scale in the remaining time. Used as a reference-compound for these same experiments, CORM-401 will also have to be re-synthesized on multi-gram scale.
Biology. The following work is forecast for the remaining period: 1) We have to continue testing in cuvette the new complex composed of RGTA-CORM. This is in line with the objective set for our collaboration with the OTR3 company; 2) in vitro testing of selected Glyco-CORMs for their effect on metabolism in cells, focusing on glycolysis and mitochondrial respiration as the most important biochemical pathways for energy production that could be modulated by the compounds; 3) in vivo characterization of selected Glyco-CORMs, including accumulation of CO in different organs/tissues, effect on metabolism (insulin resistance-glucose tolerance) and weight gain in a mouse model of HFD-induced obesity. We hope this work will provide us with positive effects of Glyco-CORMs in counteracting the negative effects of a high fat diet on weight gain and metabolic dysfunction.

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.