The efficacy of coumarin derivatives against persistent mycobacterial biofilms

Abstract

Numerous strides have been made in the treatment and diagnosis of Tuberculosis (TB), but it still remains one of the deadliest infectious diseases that plague this world. The efficiency by which the causative agent, Mycobacterium tuberculosis infects, is due to many different evolutionary functions that the bacilli employ. It is estimated that a quarter of the world’s population is infected with a form of a latent subpopulation of mycobacteria. This sustains the elevated incidences and death rates seen each year. One mechanism that the bacteria employ to overcome, or at the least tolerate, antibiotic attack and the human immune response, is the formation of a protective biofilm structure. A biofilm is an aggregation of bacteria within an enclosed extracellular polymeric matrix. Most, if not all Mycobacterium spp. form biofilms in nature and the clinical significance in human disease has been emphasized in the literature. There are currently no drugs or drug candidates that target biofilm formation or eradication of M. tuberculosis. One group of natural secondary metabolites that exhibit a broad range of biological activities as well as the inhibition of other bacterial biofilms are the coumarins. The position and type of substitution on the coumarin nucleus directly influence the pharmacological properties of the compound. This study attempted to synthesize twelve coumarin derivatives (CD) (6 x umbelliferone U, and 6 x 4-methylumbelliferone MU) and investigated their drug-susceptibility and biofilm inhibitory properties on mycobacterial planktonic and biofilm cultures. The aim was to identify a compound that targets biofilm but not necessarily has antimycobacterial activity through bactericidal or bacteriostatic effects. Twelve compounds (two base compounds and 12 derivatives) belonging to the coumarin class of compounds, of which two were novel (U5 and MU5), were synthesized. The samples were assessed for their antimycobacterial and antibiofilm activities on two mycobacterial organisms. Derivatives U1 and MUA showed moderate antimycobacterial activity against M. smegmatis mc2 155 with minimum inhibitory activity (MIC) values of 543 and 508 µM, respectively. Derivatives U2, MU2 and MU5 exhibited the highest selectivity towards M. smegmatis mc2 155 biofilm inhibition with sample MU2 showing a selectivity index of 64 towards biofilm inhibition when compared to its antimycobacterial activity. The structure-activity relationship indicates the importance of a methyl substitution on C-4 and an alkane with two free methyl groups on the end of the coumarin nucleus, for increased activity. These three derivatives (U2, MU2 and MU5) were shown to be the most active ones against M. tuberculosis H37Rv, with a MIC’s of approximately 100 µM. The intra-biofilm activity on M. tuberculosis mc2 7000 revealed that derivatives U3, U4 and MUA reduced that bacterial count within the mature biofilm by up to 90%. Owing to TB drug-induced liver toxicity in patients receiving conventional treatment, the derivatives were assessed for their hepatoprotective activities on acetaminophen-induced toxicity in a human hepatocyte cell line, HepG2. The derivatives showed moderate to negligible antiproliferative activities when tested for their cytotoxicity. All, except three derivatives (U2, MU1 and MU5), showed a protective effect at concentrations ranging from 100 to 200 µM. Compounds MU1 and MU5 had an antagonistic effect with the acetaminophen toxicity and this data was further substantiated by these compounds showing an increased inhibition of glutathione reductase in the mechanistic study, presenting an inhibitory concentration, where 50% of the enzymatic activity was inhibited (IC50), at 106.4±26.2 and 253.8±11.1 µM, respectively for MU1 and MU5. Glutathione reductase (Gtr) is an enzyme that reduces glutathione to the low molecular weight glutathiol and is produced by many eukaryotic and bacterial and fungi organisms. In the case of the Mycobacterium spp., mycothione reductase (Mtr) takes the role of Gtr. This enzyme has not only been identified as a possible drug-target for drug development, but the encoding gene has been shown to be upregulated during biofilm formation and the enzymatic product, glutathiol, has been found to be vital for the development of the biofilm. The mechanistic study involved the assessment of the derivatives as inhibitors of the mycobacterial analog Mtr and was compared to the activity with that on the human analog, Gtr. The derivative that showed the best activity and highest selectivity towards Mtr was MU2, with a selectivity index of 34. This inhibitory activity correlates with the biofilm activity and one can conclude that this could be a possible mechanism by which this compound exhibits its activity. An attempt was made to investigate the variation in activity between structurally similar coumarins (MU1 and MU2). Computational chemistry was employed through molecular docking and molecular dynamics simulations for evaluation. The only structural difference between these compounds is a double bond of the 3-methyl-2-butenyloxy moiety of MU1 vs. the saturated 3-methylbutoxy unit of MU2 at C-7 of the coumarin nucleus. The investigation through molecular docking revealed the most likely pose and orientation that these inhibitors/ligands will adopt in the active site of the enzyme. Further analysis with the use of molecular dynamics simulations revealed that the torsion angle, specifically at the aforementioned double bond position is important for the inhibition. This study is the first report of coumarins as mycobacterial biofilm inhibitors. Two new coumarin derivatives have been synthesized and their activity described. Additionally, this is the first report on the inhibition of mycothione reductase by coumarin derivatives, describing their possible molecular mechanism of inhibition at an atomic level, through the use of dynamics simulations. To date, the existing conventional TB-drugs have not been reported to have inhibitory activities on mycobacterial biofilms and the mycothione reductase enzyme. Derivative MU2 exhibited a selectivity index of 64 towards biofilm inhibition of M. smegmatis when compared to its antimycobacterial activity and showed significant antimycobacterial activity against M. tuberculosis H37Rv with an MIC value of 102 µM. Derivative MU2 also showed the highest selectivity towards Mtr (SI of 34) when compared to its Gtr inhibition. The preclinical and clinical assessment can be considered for future aspects to prove the efficacy of the identified CD, firstly, in an animal model followed by humans.