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)
• 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
• The ample growth of InhA E V 7 d a y s tre a tm e n t m
n 6 In h A 7 d a y s tre a tm e n t 0
overexpressor strain was 0 6
In h A a t 0 d a y s
t a 4
observed e
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 !!
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