Transcript Antimycobacterial activity of synthetic analogues of bioactive Natural

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
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)
XDR cases
No XDR cases
9.6 million new TB cases
1.5 million deaths
480,000 MDR-TB cases reported in 2014
Natural products as a reservoir for novel
anti-infectives drugs
Rifampicin
Teixobactin
Streptomycin
Streptomyces mediterranei
Eleftheria terrae
Streptomyces griseus
Paclitaxel (Taxol)
Taxus brevifolia
The genus Allium - Allium stipitatum
MIC 0.5-8 µg/mL
Three Pyridine-N-oxides alkaloids were isolated:
(1) 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: 10.1021/np800572r
Generation of disulphide analogues
ES2
ES3
3-(benzylthio)-5-(methyldisulfanyl)-4H-1,2,4-triazol-4-amine
2-(methyldisulfanyl)thieno[2,3-d]pyrimidin-4-amine
ES4
7-fluoro-2-(methyldisulfanyl)benzo[d]thiazole
ES6
ES5
4-ethyl-5-(methyldisulfanyl)-4H-1,2,4-triazol-3-ol
(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
Biological evaluation: Antimycobacterial activity of
the synthesized analogues
Biological evaluation: Cytotoxicity assay
Macrophages RAW 264.7 cell line
Compound
ID
MICH37Rv
GIC50
Resazurin fluorescence assay
60
SI=GIC50/
MICH37Rv
50
62.50
40.93
0.65
Cpd 1
40
2 (ES3)
4.00
18.23
4.56
30
3 (ES4)
31.30
9.92
3.16
20
4 (ES5)
31.30
>250
>7.98
10
Relative fluorescence units
1 (ES2)
Cpd 2
Cpd 3
Cpd 4
Cpd 5
INH
0
5 (ES6)
31.30
>250
>7.98
INH
0
7.8
15.6
31.25
Conc (μg/mL)
SI - Selectivity Index
GIC – Growth Inhibitory Concentration
MIC – Minimum Inhibitory Concentration
62.5
125
250
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
Biological evaluation: Biofilm assay
Concentration dependent inhibition of biofilm formation by synthesized methyldisulphide ES3 (OD600 3.5)
Decreasing biofilm formation
Biofilm
Biofilm
Increasing concentration of ES3 (0.78-50µg/ml)
Quantification of biofilm: Crystal violet staining, absorbance measured
Microscopy: Biofilm assay
Light Microscope
Planktonic cells
Biofilm formed
1000X
1000X
Scanning Electron Microscope (SEM)
(c)
Mycobacterium smegmatis cells
Planktonic
Biofilm matrix
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.
Future work
Genes
Function
 Generation of mutants
Rv0538 (H37Rv)


Unknown function
Assumed to be a conserved protein
 Amplification of specific genes
/regions by PCR or
Whole genome sequencing
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)


Unknown function
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)


Unknown function
Assumed to be a conserved protein
Rv0359 (H37Rv)

Assumed to be a conserved integral membrane protein
Acknowledgements
Phytochemistry Laboratory
(UCL School of Pharmacy)
Prof. Simon Gibbons
Mycobacteria Research Laboratory
Dr. Sanjib Bhakta
Dr. Dimitrios Evangelopoulos
Dr. Proma Khondkhar
Ghana Education Trust Fund
Arundhati Maitra
British Society of Antimicrobial Chemotherapy
Thank you