Identification and validation of mode of action of chalcones for their anti-mycobacterial activity Bhavani Anagani School of Life and Medical Sciences University of Hertfordshire, College Lane, Hatfield, UK. AL10 9AB 1 “The world is headed for a post-antibiotic era, in which common infections and minor injuries which have been treatable for decades can once again kill.” (WHO, 2015) 2 Tuberculosis • Top infectious killer disease – 1.6 Million people • Mycobacterium tuberculosis • Rifampicin, Isoniazid, Ethambutol, and Pyrazinamide • Multi drug treatment – 6 Months Rod shaped Mycobacterium tuberculosis 3 RR-TB; MDR-TB; XDR-TB; TDR-TB --- Treatment limited • New drugs • New classes • New targets • Understanding the resistance 4 Existing Antibiotics Streptomycin - Streptomyces griseus Rifampicin - Amycolatopsis rifamycinia 5 Chalcones Galenia africana Helichrysum melanacme Dalbergia odorifera 6 Synthesis of chalcones Scheme 1: Synthesis of chalcones 7 Physical data for the chalcones (1a-1o) Table1: In vitro preliminary screening data of Chalcones Substituents MIC (µg/ml) Compound M.wt Clogp m.smegmati J-774 (IC50) SI=IC50/MIC R R R m.bovis 1 2 3 s 1a H H H 270.33 3.35 50 6 50 8.4 1b H Br H 349.22 4.18 - - 25 - 1c Br H H 349.22 4.18 100 25 25 1 1d H H Br 349.22 4.18 200 12 25 2.08 1e H Cl H 304.77 3.91 - - 12 - 1f Cl H H 304.77 3.91 100 12 12 1 1g H H Cl 304.77 3.91 - 200 12 0.06 1h H H F 288.32 3.51 200 25 100 4 1i H H OCH3 300.35 3.23 200 50 100 2 1j H H CH3 284.35 3.84 100 25 25 1 1k H H C6 H5 346.43 5.03 200 100 25 0.25 1l H NO2 H 315.32 3.48 - 100 25 0.25 1m CH3 H H 284.35 3.84 200 50 25 0.5 1n H H NO2 315.32 3.48 - 200 50 0.25 1o Cl H Cl 339.21 4.47 - 200 6 0.03 8 MIC Determination against Mycobacteria • Colorimetric broth micro-dilution method according to CLSI guidelines • M. smegmatis and M. bovis (BCG) • Minimum inhibitory concentrations (MIC) < 6.25µg/ml B 200 100 50 25 12 6 3 1.5 0 .7 0.3 µg/ml B 1a R C Figure 1: Screen M. bovis BCG strain against chalcone (1a) and Rifampicin (R) and control (C) 9 Cytotoxicity assay • J774 macrophage cell line • IC50 value • Selectivity index (SI) • High SI value – low toxicity 10 Biological data for the chalcones (1a-1o) Table1: In vitro preliminary screening data of Chalcones (n=3) Substituents MIC (µg/ml) Compound M.wt Clogp M. bovis J-774 (IC50) SI=IC 50/MIC R R R M.smegmatis 1 2 3 (BCG) 1a H H H 270.33 3.35 50 6 50 8.4 1b H Br H 349.22 4.18 - - 25 - 1c Br H H 349.22 4.18 100 25 25 1 1d H H Br 349.22 4.18 200 12 25 2.08 1e H Cl H 304.77 3.91 - - 12 - 1f Cl H H 304.77 3.91 100 12 12 1 1g H H Cl 304.77 3.91 - 200 12 0.06 1h H H F 288.32 3.51 200 25 100 4 1i H H OCH3 300.35 3.23 200 50 100 2 1j H H CH3 284.35 3.84 100 25 25 1 1k H H C6H5 346.43 5.03 200 100 25 0.25 1l H NO2 H 315.32 3.48 - 100 25 0.25 1m CH3 H H 284.35 3.84 200 50 25 0.5 1n H H NO2 315.32 3.48 - 200 50 0.25 1o Cl H Cl 339.21 4.47 - 200 6 0.03 11 Analysis of MAMEs and FAMEs • dose dependent reduction of MAMEs FAMEs • overall abundance of FAMEs a-MAMEs • suggests that 1a targets Keto-MAMEs mycolic acid biosynthesis Origin (fatty acid synthase (FAS)-II inhibitors) Figure 3: TLC autoradiography of FAMEs and MAMEs from M. bovis BCG strains treated with 1a 12 Mycolic acid biosynthesis 13 Phenotypic MIC shift assay 8 E V a t 0 d a y s m • The ample growth of InhA n E V 7 d a y s tre a tm e n t 0 0 6 In h A 7 d a y s tre a tm e n t 6 overexpressor strain was t a In h A a t 0 d a y s e 4 observed c n a b r 2 o s • An increase in resistance and the b A MIC shift to the right 0 0 1 0 2 0 3 0 4 0 -1 [1 a ] ( g .m l ) Figure 5: Impact on the MICs of 1a up on the overexpression of members of FAS-II in M. bovis BCG (n=2) 14 InhA and ligand (1a) binding 4 0 0 0 D M S O c o rre c te d 1 a D M S O c o rre c te d tric lo s a n 3 0 0 0 U F R e c n e c 2 0 0 0 s e r o u l F 1 0 0 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 [1 a ] n M Figure 5: Saturation binding assay using intrinsic tryptophan fluorescence to quantify association of 1a with InhA. (n=3) Drug Bmax Kd 1a 3403 10 Triclosan 1915 8.89 15 Target Confirmation (Data pending) Generation of spontaneous resistant mutants Lead (5XMIC; 10XMIC; compound 20XMIC) (1a) Whole genome sequencing Identification of Single nucleotide polymorphisms (SNPs) Target validation And assignment 16 Overview • Synthesis of chalcones was successfully achieved Synthesis • The MIC values for the chalcones against M. bovis BCG showed MIC promising results • 1a was selected as a “Hit” candidate to further investigate MOA SI- "Hit" as it has higher SI value • Phenotypic MIC shift assay--- InhA as the target (FAS-II synthase Target identification inhibitors) Target • Intrinsic tryptophan fluorescence and WGS confirmation 17 Acknowledgements My Supervisors: Dr Madhu Goyal Dr Chris Benham Dr Paul Bassin Collaborators: Dr Jonathan Cox (Aston University) Prof Gurdyal Besra (University of Birmingham) THANK YOU !! 19.