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 and minor injuries which have been treatable for decades can once again kill.” (WHO, 2015)

2

• 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 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 () THANK YOU !!

19