Inhibition of Mycolic acid transport across the Mycobacterium tuberculosis plasma membrane

Transcription

1 Inhibition of Mycolic acid transport across the Mycobacterium tuberculosis plasma membrane Grzegorzewicz, A. E., H. Pham, V. A. K. B. Gundi, M. S. Scherman, E. J. North, T. Hess, V. Jones, V. Gruppo, S. E. M. Born, J. Korduláková, S. S. Chavadi, C. Morisseau, A. J. Lenaerts, R. E. Lee, M. R. McNeil, and M. Jackson Nat Chem Biol Feb Presented by: Seán McMahon 1

2 Tuberculosis (MTB) TB Common, and in many cases lethal infectious disease caused caused by the bacterium mycrobacteria tuberculosis. Researchers at the Swiss Tropical and Public Health Institute place its origin 70,000 years ago in Africa. "The evolutionary path of humans and the TB bacteria shows striking similarities, - Sebastien Gagneux (Swiss TPH) Nature Genetics, AOP, Sept

3 Nearly 20 years after the WHO declaration of TB as a global public health emergency, it still remains one of the worlds most dangerous diseases. In 2012, an estimated 8.6 million people developed TB and 1.3 million died from the disease Of those infected worldwide approx had been infected by multi drug resistant form of tuberculosis 3

4 Pathogenic Effects Typically attacks the lungs but can also affect other parts of the body. It is spread through the air when people who have an active TB infection cough, sneeze, or otherwise transmit respiratory fluids through the air. 4

5 Mycobacterium Tuberculosis M tuberculosis comes from the highly aerobic, gram + Mycobacterium genus. It has an unusually thick, waxy coating on its cell surface (primarily long chain Mycolic acids) they are naturally resistant to a number of antibiotics that disrupt cellwall biosynthesis, such as penicillin. This unique cell wall also enables them survive long exposure to unfavourable conditions and toxic chemicals. 5

6 Mycobacterium Cell Wall Two separate segments 1. mycolyl-arabinogalactan peptidoglycan (magp) complex. 2. Long and short free lipids, Interspersed with surface proteins and the glycolipids phosphatidylinositolmannosid es (PIM), the phthiocerolcontaining lipids, lipomannan (LM), and lipoarabinomannan(lam). 6

7 The Mycolic Acids Mycolic acids are very long chain α-alkyl-β-hydroxy fatty acids approx (C60-C90) Mycobacterium are known to synthesise α-, methoxy-, and keto-mycolic acids. 7

8 Mycolic acid Synthesis Mycolic acids are synthesised in the cytoplasm through the extension of lipid precursors generated by fatty acid Synthase I (FASI). The multienzyme complex FAS II is then responsible for the completion of this process. 8

9 Mycolic Acids in the Cell wall Occur in two forms 1. Covalently linked to the arabinogalactan. 2. Esterified to sugars, notably trehalose. These esters are termed Trehalose monomycholate TMM and Trehalose dimycholate TDM and are found in the outermost layer. 9

10 Trehalose monomycolate TMM Formed in the cytoplasm through the conjugation of fatty acids by FAS II and tailoring enzymes. Relocated to the cell wall by an unknown transporter. Is an essential precursor for TDM 10

11 Trehalose dimycolate TDM Is formed on the outer membrane of the cell wall. Produced by the mycolyl transferase Antigen 85 complex by catalyzing the transfer of mycolic acid from one trehalose 6-monomycolate to another, forming trehalose 6,6 -dimycolate (TDM). 11

12 Current first line drug treatment for Tuberculois Isoniazid-Inhibits cell wall synthesis. Rifampicin inhibits bacterial RNA synthesis by disrupting the activity RNA polymerase. Pyrazinamide disrupts fatty acid synthase which disrupts the cell membrane and disables energy production. Ethambutol disrupts arabinogalactan synthesis by inhibiting the enzyme arabinosyl transferase. 12

13 Multidrug-Resistant Tuberculosis (MDR TB) and Possible Effective Treatments 4-aminosalicylic acid (PAS) and Fluoroquinolones disrupt DNA replication. Aminoglycosides and cyclic peptides inhibit the synthesis of protein. Thioamides and Cyclosterine inhibit the cell wall synthesis. 13

14 Extensively Drug-Resistant Tuberculosis (XDR TB) Diminishing Options for Treatment 14

15 New Tuberculosis (TB) Drugs Under Development New treatments with novel mode of action urgently needed to combat drug resistance. The diarylquinoline antituberculosis drug Bedaquiline is the first anti Tuberculosis drug to be approved by the FDA in over 40 years. 15

16 Identification of the adamantyl urea inhibitor LeadScreen by Tripos to scan 12,000 compounds against M. Tuberculosis H37Rv grown in 7H9- OADC medium. 1-(2-Adamantyl)-3-(2,3,4- trifluorophenyl)urea (AU1235) AU1235 was shown to inhibit the growth of Tuberculosis H37Rv displaying a minimum inhibitory content of 0.1 μg ml 1 16

17 Ability of AU1235 to inhibit MDR isolates of M. Tuberculosis and other Mycobacterium Similarly active against MDR M. Tuberculosis Lack of cross resistance shown by MDR M. Tuberculosis indicating previously unknown biological target Also displayed inhibition of Mycobacterium Smegmatis and Mycobacterium Fortuitum. 17

18 Effect of AU1235 concentration on M. Tuberculosis growth Exposure of M. tb H37Rv in log-phase growth to 0.5 μg ml 1 AU1235 resulted in a reduction in viable CFUs of about 2 log units after 5 days indicating that this compound is bactericidal in vitro Increase in the concentration to 10xMIC shows no increase in killing effect in first 7 days Shows that the killing was time dependant. 18

19 Anaerobic model activity In an anaerobic model involving non-replicating M. tb H37Rv bacilli, AU1235 at10 μg ml 1 showed no detectable bactericidal activity. Suggesting that it acts on a biosynthetic pathway required for active bacterial multiplication. 19

20 AU1235 activity against other gram + and gram bacteria Shown to be completely ineffective against a broad panel of bacteria. Shows AU1235 specificity to Micobacteria genus with highest activity in M. Tuberculosis 20

21 Descovering the Macromolecular effects of AU1235 Metabolic labelling of untreated and AU1235-treated M. tuberculosis cells with various radiolabeld precursors was performed in order to monitor RNA, DNA, protein synthesis. 21

22 Effect of AU1235 on Protein,RNA and DNA synthesis No obvious inhibitory effect on Protein synthesis observed in the AU1235 treated cells when compared to control and Kanamycin treated M.tb. No inhibitory effect on RNA synthesis observed in the AU1235 treated cells when compared to a control and Rifampicin treated M.tb 22

23 Effect of AU1235 on Protein,RNA and DNA synthesis No significant DNA synthesis inhibition observed in the AU1235 treated cells in comparison to a standard and Ciprofloxacin treated. 23

24 Identification of Mycolic acid inhibition M. Tuberculosis was cultured with no inhibitor or with AU1235 at a concentration of 0.05 μg ml 1, 0.5 μg ml 1, or 1 μg ml 1 (0.5x to 10x MIC). [ 14 C]-acetate was added to the cultures at the same time as the inhibitor. (a) Bacterial cells (B) and culture filtrates (CF) were collected and the lipids and analysed by TLC. Mycolic acid methyl esters were prepared from delipidated cells as containing the total amount of α, methoxy- and keto- the three forms of mycolic acids produced by M. tb. The contents of these mycolic acids were compared by TLC analysis. 24

25 Analysis of resulting autoradiography 25

26 Effect of AU1235 on the export of TMM in M. Tuberculossis M. tb H37Ra Surface-exposed lipids were extracted from whole cells in water-saturated 1-butanol Butanol-treated cells were then re-extracted overnight with chloroform/methanol to recover all remaining extractable lipids. The lipids from the butanol and chloroform/methanol fractions were resuspended and loaded into the TLC lanes The TLC was developed in the solvent system revealed and semi-quantified using a PhosphorImager. 26

27 Relative Distribution results A 3-fold decrease in the proportion of TMM found in the butanol fraction was observed in the treated cells. In comparison, the decrease in the proportion of TDM found in the butanol fraction of the treated cells was 1.4 fold however. (TDM results affected by butanol pri treatment.) 27

28 Conclusion from these experiments From these experiments they were able to conclude AU1235 does not inhibit the biosynthesis of Mycolic acids. AU1235 is affecting their ability to be transferred onto their cell wall and outer membrane acceptors. This confirms that AU1235 has a mechanism of action not seen in any current treatment for M. Tuberculosis. Similar experiments performed in M. Smegmatis displayed the same results 28

29 In search of the lethal Target of AU1235 in M. Tuberculosis Is a general inhibition of the antigen 85 complex causing the collapse TDM formation and mycolic acid transfer onto the arabinogalactin? 29

30 Does AU1235 inhibit the mycolyltransferases FbpA, FbpB and FbpC in vitro? Mycolyltransferase activity of the purified FbpA FbpB and FbpC proteins in the presence of cold TMM was measured using a transesterification assay. 30

31 In search of the lethal Target of AU1235 in M. Tuberculosis A more comprehensible search was required. M. Tuberculosis H37Rv cultured in plates containing 2x and 4 x the MIC AU1235. Mutant M. Tuberculosis H37Rv with a noted resistance were isolated and checked for resistance to other anti tuberculosis agents. 31

32 In search of the lethal Target of AU1235 in M. Tuberculosis Whole genome sequencing followed by comparative analyses between Au1235 resistant M. Tuberculosis H37Rv and WT. A single gene was seen to be commonly affected, this gene encoded the putative inner membrane transporter Mmpl3. Mmpl3 is present in all known Mycobacteria with the exception of M. abscessus. 32

33 Effect of expressing the point-mutated mmpl3 gene on the susceptibility of M. smegmatis to AU1235 Recombinant M. Smegmatus used with the endrogenus mmpl3 replaced by plasmid encoded mmpl3-tb and mmpl-tb-g253e lead to a 2 fold increase in resistance Co expression of the wild type and mutant mmpl3 lead to the formation of hybrid forms of the protein which displayed a marked increase in resistance of 8 to 16 fold. 33

34 Accumulation of AU1235 in wild-type M. tb H37Ra cells and two spontaneous resistant mutants M. tb H37Ra cultures either wild-type or expressing the G253E mutated version of MmpL3tb isolates treated the inhibitor. And the results analysed by LC/MS. Showing that the mmpl3 mutation is not acting as an efflux pump for AU

35 MmpL3 is an essential gene of M. smegmatis. Of the 13 to 14 MmpL-type proteins encoded by the genome of M. tb, mmpl3 and mmpl11 are the only mmpl gene conserved across the Mycobacterium genus. It was also observed disruption on the mmpl3 gene was not achievable unless a wild type rescue copy of the gene was provided. 35

36 MmpL3 is an essential gene of M. smegmatis. Mutants of M. smagmitus were produced in which a rescue copy of mmpl3tb gene was placed under the control of a tetracyclineinducible promoter. It was shown that under non-permissive culture conditions where the mmpl3 gene was lost the merodiploid strains ceased growing. Thus, expression of mmpl3 is essential for the growth of M.smegmatis. The fact that mmpl3tb could rescue the viability of the M. smegmatis mmpl3 knock-out mutant further indicates that the two mmpl3 orthologs have analogous functions 36

37 Effect of repressing mmpl3 expression on the mycolic acid content of M. smegmatis. The loss of expression of mmpl3 in conditionally grown M.smegmatis is shown to reproduce the effects of AU1235 i.e significant decreases in TDM formation and Mycolic transfer onto arabinogalactan thereby reproducing the effects of AU1235 on whole mycobacterial cells. 37

38 Effect of repressing mmpl3 expression on the mycolic acid content of M. smegmatis. Analysis of cell wall bound mycolates of M. smegmatis WT and conditionally grown mutants was able to display the ability mmpl3 gene from M. tb to transfer forms of Mycolic acids produced by M. smegmatis, including certain forms specific to that bacteria. 38

39 Conclusion The potential role of mmpl3 as an efflux pump for AU1235 is refuted by the unchanged accumulation of AU1235 in the mutant mmpl3 expressing M. tb cells, along with the fact that decreased mmpl3 expression mimicked the phenotypic effects of treating mycobacteria with AU1235. This therefore confirms this transporter as the direct target of the prototype inhibitor. 39

40 Discussion AU1235 will be subjected to medicinal chemistry study's In an attempt to produce a drug like molecule for clinical development. 40

41 Discussion Significantly the discovery of MmpL3 has now revealed that it is in fact the target of two drugs currently in clinical trials. 41

42 Discussion Sequencing of mmpl3 from M. Tuberculosis mutants resistant to SQ109 and BM212 revealed a different mutation to those with AU1235 resistance. Future study's of cross resistant strains will help clarify the structure function relationship between theses compounds and this important lipid transporter, in turn helping to guide future drug development. 42

43 Thank you for your attention 43