1. Cynthia Amaning Danquah UCL School of Pharmacy London, UK
Synthetic Analogues of Natural Product Disulfides from Allium stipitatum Demonstrate Anti-Tubercular Activities through Drug Efflux Pump and Biofilm Inhibition
Cynthia Amaning Danquah
UCL School of Pharmacy
London, UK

2. A rise in drug resistant tuberculosis (TB)
Countries that have reported at least one case of XDR by the end of 2014
(WHO Global Tuberculosis Report, 2015)
Every 20 seconds a person dies from TB
No XDR cases
XDR cases
9.6 million new TB cases
1.5 million deaths
480,000 MDR-TB cases reported in 2014

3. Natural products as a reservoir for novel anti-infectives drugs
Teixobactin Eleftheria terrae
Plants are a source of potential bioactive compounds
Bioassay guided isolation and characterisation of compounds from plants Efforts are been made to find new compounds with activity against pathogenic bacteria (Guzman, J.D. et al., ; Osman, K. et al., 2012 )
Rifampicin Streptomyces mediterranei
Natural
sources
Paclitaxel (Taxol) Taxus brevifolia
Streptomycin Streptomyces griseus

4. The genus Allium - Allium stipitatum
MIC µg/mL
Three Pyridine-N-oxides alkaloids were isolated:
2-(methyldithio)pyridine-N-oxide
(2) 2-[(methylthiomethyl)dithio]pyridine-N-oxide
(3) 2,2′-dithio-bis-pyridine-N-oxide
Bioactive Pyridine-N-oxide Disulfides from Allium stipitatum
Gemma O’Donnell, Rosemarie Poeschl, Oren Zimhony, Mekala Gunaratnam, Joao B. C. Moreira, Stephen Neidle,
Dimitrios Evangelopoulos, Sanjib Bhakta, John P. Malkinson, Helena I. Boshoff, Anne Lenaerts and Simon Gibbons
J. Nat. Prod., 2009, 72 (3), pp 360–365
Publication Date (Web): December 18, 2008 (Article)
DOI: /np800572r

5. Generation of disulphide analogues
3-(benzylthio)-5-(methyldisulfanyl)-4H-1,2,4-triazol-4-amine
ES3
2-(methyldisulfanyl)thieno[2,3-d]pyrimidin-4-amine
A series of synthetic analogues of the bioactive natural products were synthesized using the method of Kitson and Loomes(1985).
This was done to:
Enhance antibacterial activity
Reduce cytotoxicity
Increase permeability to the mycobacteria cell wall which is a highly hydrophobic barrier
Optimize drug ability of the compounds
ES4
7-fluoro-2-(methyldisulfanyl)benzo[d]thiazole
ES6
4-ethyl-5-(methyldisulfanyl)-4H-1,2,4-triazol-3-ol
ES5
(E)-3-(methyldisulfanyl)-5-styryl-4H-1,2,4-triazole
Kitson T.M. and Loomes K.M. (1985)
Synthesis of methyl 2- and 4-pyridyl disulfide from 2- and 4-thiopyridone and methylmethanethiosulfonate
Analytical Biochemistry Volume 146, Issue 2, 429–430

6. Biological evaluation: Antimycobacterial activity of the synthesized analogues

7. Biological evaluation: Cytotoxicity assay
Macrophages RAW cell line Resazurin fluorescence assay
Compound ID
MICH37Rv
GIC50
SI=GIC50/ MICH37Rv
1 (ES2)
62.50
40.93
0.65
2 (ES3)
4.00
18.23
4.56
3 (ES4)
31.30
9.92
3.16
4 (ES5)
>250
>7.98
5 (ES6)
SI - Selectivity Index
GIC – Growth Inhibitory Concentration
MIC – Minimum Inhibitory Concentration
Therapeutic window

8. Biological evaluation: Efflux Pump Inhibition Assay
(a, b and c) Efflux pump inhibition assay of ES compounds using M aurum. Ethidium bromide (EtBr) is an efflux pump substrate (used at a final concentration of 0.5 µg/mL) verapamil and chlorpromazine are known efflux pump inhibitors and are used as controls , (d) growth curve with optical density taken at 600nm

9. Biological evaluation: Biofilm assay
Concentration dependent inhibition of biofilm formation by synthesized methyldisulphide ES3 (OD )
Decreasing biofilm formation
Biofilm
Biofilm
Bar graph plate reader
Increasing concentration of ES3 ( µg/ml)
Quantification of biofilm: Crystal violet staining, absorbance measured

10. Microscopy: Biofilm assay
Light Microscope
Planktonic cells
Biofilm formed
1000X
1000X
Scanning Electron Microscope (SEM)
) Image of M. smegmatis cells (planktonic form) at early stationary phase (OD ) (b) Image of M. smegmatis () at early stationary phase (OD 3.5) taken with light microscope at 1000X magnification(a), (b), (c) images of M. smegmatis cells taken with at mid log phase (OD ) , early stationary phase (OD ) and formed biofilm matrix (OD ) respectively.
(c)
Mycobacterium smegmatis cells Planktonic Biofilm matrix
1000X
1000X

11. Conclusions
Synthesized analogues of bioactive natural product disulfides
They have shown anti-tubercular activity
Effective against other pathogenic bacteria
Efflux pump inhibitory effect
Biofilm inhibitory properties
Cytotoxicity profile of the compounds
These findings indicate that synthetic analogues of naturally occurring disulfides are bioactive and can serve as leads for the development of effective new treatment for Mycobacterial diseases and other pathogenic bacteria.

12. Future work Generation of mutants Amplification of specific genes
/regions by PCR or
Whole genome sequencing
Genes
Function
Rv0538 (H37Rv)
Unknown function
Assumed to be a conserved protein
Rv0539 (H37Rv)
A glycosyltransferase
Important for expression of mature GPLs.
Rv 3412 (H37Rv)
Unknown function but assumed to be a conserved protein
Rv 3413c (H37Rv)
Assumed to be an Alanine and Proline rich protein
Rv 3526 (H37Rv)
Assumed to be an oxidoreductase and involved in
cellular metabolism
Rv 0358 (H37Rv)
Rv0359 (H37Rv)
Assumed to be a conserved integral membrane protein
To find out specific genes responsible for activity

13. Acknowledgements Phytochemistry Laboratory
(UCL School of Pharmacy)
Prof. Simon Gibbons
Dr. Proma Khondkhar
Mycobacteria Research Laboratory
Dr. Sanjib Bhakta
Dr. Dimitrios Evangelopoulos
Arundhati Maitra
Ghana Education Trust Fund
British Society of Antimicrobial Chemotherapy

14. “No one is safe from tuberculosis until everyone is safe
“No one is safe from tuberculosis until everyone is safe the search continues’’
Thank you