Exploring the structure-activity relationship of Mincle ligands

<p>Mincle is a C-type lectin that plays a critical role in the body’s innate immune response to bacteria and fungi. Compounds identified as ligands for Mincle include trehalose and glycerol esters of complex long chain fatty acids. Harnessing its ability to activate the immune system, trehalose dimycolate has been used as a vaccine adjuvant and for anti-cancer treatment, highlighting the potential use of Mincle immunomodulators in a medical context. To further understand the receptor-ligand interactions and optimise biological activity, the structure-activity relationship of Mincle ligands was investigated through the synthesis and biological evaluation of a series of modified ligands. The preparation of carbohydrate modified trehalose dibehenates was attempted in order to assess the importance of hydroxylprotein interactions, and despite the syntheses being incomplete, improvements on literature methodologies for the regioselective modification of α,α′-D-trehalose were developed. Simplified analogues of the glycerol based natural products identified as Mincle ligands containing straight chain and iso-branched lipids were prepared to evaluate the significance of the branch and establish whether there is a relationship between lipid length and Mincle activation. The corresponding trehalose diester analogues were also synthesised to gauge the capacity of the protein to tolerate changes in the carbohydrate portion of ligands.</p>

Exploring the structure-activity relationship of Mincle ligands

<p>Mincle is a C-type lectin that plays a critical role in the body’s innate immune response to bacteria and fungi. Compounds identified as ligands for Mincle include trehalose and glycerol esters of complex long chain fatty acids. Harnessing its ability to activate the immune system, trehalose dimycolate has been used as a vaccine adjuvant and for anti-cancer treatment, highlighting the potential use of Mincle immunomodulators in a medical context. To further understand the receptor-ligand interactions and optimise biological activity, the structure-activity relationship of Mincle ligands was investigated through the synthesis and biological evaluation of a series of modified ligands. The preparation of carbohydrate modified trehalose dibehenates was attempted in order to assess the importance of hydroxylprotein interactions, and despite the syntheses being incomplete, improvements on literature methodologies for the regioselective modification of α,α′-D-trehalose were developed. Simplified analogues of the glycerol based natural products identified as Mincle ligands containing straight chain and iso-branched lipids were prepared to evaluate the significance of the branch and establish whether there is a relationship between lipid length and Mincle activation. The corresponding trehalose diester analogues were also synthesised to gauge the capacity of the protein to tolerate changes in the carbohydrate portion of ligands.</p>

Structures of the mycobacterial membrane protein MmpL3 reveal its mechanism of lipid transport

The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.

Design of Potent Mincle Signalling Agonists Based on an Alkyl b- Glucoside Template

The innate immune receptor Mincle senses lipid-based molecules derived from pathogens, commensals and altered self. Based on emerging structure-activity relationships we design simple alkyl 6-<i>O</i>-acyl-b-D-glucosides that are effective agonists of Mincle and signal with potency on par with the prototypical ligand trehalose dimycolate.

Design of Potent Mincle Signalling Agonists Based on an Alkyl b- Glucoside Template

The innate immune receptor Mincle senses lipid-based molecules derived from pathogens, commensals and altered self. Based on emerging structure-activity relationships we design simple alkyl 6-<i>O</i>-acyl-b-D-glucosides that are effective agonists of Mincle and signal with potency on par with the prototypical ligand trehalose dimycolate.

Mycobacterium abscessus, an Emerging and Worrisome Pathogen among Cystic Fibrosis Patients

Nontuberculous mycobacteria (NTM) have recently emerged as important pathogens among cystic fibrosis (CF) patients worldwide. Mycobacterium abscessus is becoming the most worrisome NTM in this cohort of patients and recent findings clarified why this pathogen is so prone to this disease. M. abscessus drug therapy takes up to 2 years and its failure causes an accelerated lung function decline. The M. abscessus colonization of lung alveoli begins with smooth strains producing glycopeptidolipids and biofilm, whilst in the invasive infection, “rough” mutants are responsible for the production of trehalose dimycolate, and consequently, cording formation. Human-to-human M. abscessus transmission was demonstrated among geographically separated CF patients by whole-genome sequencing of clinical isolates worldwide. Using a M. abscessus infected CF zebrafish model, it was demonstrated that CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction seems to have a specific role in the immune control of M. abscessus infections only. This pathogen is also intrinsically resistant to many drugs, thanks to its physiology and to the acquisition of new mechanisms of drug resistance. Few new compounds or drug formulations active against M. abscessus are present in preclinical and clinical development, but recently alternative strategies have been investigated, such as phage therapy and the use of β-lactamase inhibitors.

Identification of New MmpL3 Inhibitors by Untargeted and Targeted Mutant Screens Defines MmpL3 Domains with Differential Resistance

ABSTRACT The Mycobacterium tuberculosis mycolate flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole-genome sequencing of isolated resistant mutants. Here, we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel M. tuberculosis growth inhibitors for differential growth inhibition of wild-type M. tuberculosis compared to the growth of a pool of 24 unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular M. tuberculosis. M. tuberculosis cells treated with these compounds were shown to accumulate trehalose monomycolates, have reduced levels of trehalose dimycolate, and displace an MmpL3-specific probe, supporting MmpL3 as the target. The inhibitors were mycobacterium specific, with several also showing activity against the nontuberculous mycobacterial species M. abscessus. Cluster analysis of cross-resistance profiles generated by dose-response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance-conferring substitutions to the MmpL3 crystal structure revealed clade-specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small-molecule inhibitors.

1H-Benzo[d]Imidazole Derivatives Affect MmpL3 in Mycobacterium tuberculosis

ABSTRACT 1H-benzo[d]imidazole derivatives exhibit antitubercular activity in vitro at a nanomolar range of concentrations and are not toxic to human cells, but their mode of action remains unknown. Here, we showed that these compounds are active against intracellular Mycobacterium tuberculosis. To identify their target, we selected drug-resistant M. tuberculosis mutants and then used whole-genome sequencing to unravel mutations in the essential mmpL3 gene, which encodes the integral membrane protein that catalyzes the export of trehalose monomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. The drug-resistant phenotype was also observed in the parental strain overexpressing the mmpL3 alleles carrying the mutations identified in the resistors. However, no cross-resistance was observed between 1H-benzo[d]imidazole derivatives and SQ109, another MmpL3 inhibitor, or other first-line antitubercular drugs. Metabolic labeling and quantitative thin-layer chromatography (TLC) analysis of radiolabeled lipids from M. tuberculosis cultures treated with the benzoimidazoles indicated an inhibition of trehalose dimycolate (TDM) synthesis, as well as reduced levels of mycolylated arabinogalactan, in agreement with the inhibition of MmpL3 activity. Overall, this study emphasizes the pronounced activity of 1H-benzo[d]imidazole derivatives in interfering with mycolic acid metabolism and their potential for therapeutic application in the fight against tuberculosis.

Synthetic mycobacterial molecular patterns partially complete Freund’s adjuvant

Complete Freund’s adjuvant (CFA) has historically been one of the most useful tools of immunologists. Essentially comprised of dead mycobacteria and mineral oil, we asked ourselves what is special about the mycobacterial part of this adjuvant, and could it be recapitulated synthetically? Here, we demonstrate the essentiality of N-glycolylated peptidoglycan plus trehalose dimycolate (both unique in mycobacteria) for the complete adjuvant effect using knockouts and chemical complementation. A combination of synthetic N-glycolyl muramyl dipeptide and minimal trehalose dimycolate motif GlcC14C18 was able to upregulate dendritic cell effectors, plus induce experimental autoimmunity qualitatively similar but quantitatively milder compared to CFA. This research outlines how to substitute CFA with a consistent, molecularly-defined adjuvant which may inform the design of immunotherapeutic agents and vaccines benefitting from cell-mediated immunity. We also anticipate using synthetic microbe-associated molecular patterns (MAMPs) to study mycobacterial immunity and immunopathogenesis.