Whole Cell Target Engagement Identifies Novel Inhibitors of Mycobacterium tuberculosis Decaprenylphosphoryl-β-d-ribose Oxidase.
We have targeted the Mycobacterium tuberculosis decaprenylphosphoryl-β-d-ribose oxidase (Mt-DprE1) for potential chemotherapeutic intervention of tuberculosis. A multicopy suppression strategy that overexpressed Mt-DprE1 in M. bovis BCG was used to profile the publically available GlaxoSmithKline antimycobacterial compound set, and one compound (GSK710) was identified that showed an 8-fold higher minimum inhibitory concentration relative to the control strain. Analogues of GSK710 show a clear relationship between whole cell potency and in vitro activity using an enzymatic assay employing recombinant Mt-DprE1, with binding affinity measured by fluorescence quenching of the flavin cofactor of the enzyme. M. bovis BCG spontaneous resistant mutants to GSK710 and a closely related analogue were isolated and sequencing of ten such mutants revealed a single point mutation at two sites, E221Q or G248S within DprE1, providing further evidence that DprE1 is the main target of these compounds. Finally, time-lapse microscopy experiments showed that exposure of M. tuberculosis to a compound of this series arrests bacterial growth rapidly followed by a slower cytolysis phase.
School of Biosciences, University of Birmingham , Birmingham B15 2TT, United Kingdom.
Sarah M
SM
Batt
Monica
M
Cacho Izquierdo
Julia
J
Castro Pichel
Christopher J
CJ
Stubbs
Laura
L
Vela-Glez Del Peral
Esther
E
Pérez-Herrán
School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL) , CH-1015 Lausanne, Switzerland.
Neeraj
N
Dhar
Bernadette
B
Mouzon
Mike
M
Rees
Jonathan P
JP
Hutchinson
Robert J
RJ
Young
School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL) , CH-1015 Lausanne, Switzerland.
John D
JD
McKinney
David
D
Barros Aguirre
Lluis
L
Ballell
School of Biosciences, University of Birmingham , Birmingham B15 2TT, United Kingdom.
Gurdyal S
GS
Besra
Argyrides
A
Argyrou
drug discovery; multicopy suppression; target overexpression; time-lapse microscopy