The structure of PknB in complex with mitoxantrone, an ATP-competitive
inhibitor, suggests a mode of protein kinase regulation in mycobacteria
Annemarie Wehenkela,1, Pablo Fernandeza,1, Marco Bellinzonia, Vincent Catherinotb,
Nathalie Barilonec, Gilles Labesseb, Mary Jacksonc, Pedro M. Alzaria,*
aUnite ´ de Biochimie Structurale and CNRS URA2185, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France
bCentre de Biochimie Structurale, INSERM U414, CNRS UMR5048, Universite ´ Montpellier 1, 15 Avenue Charles Flahault,
34060 Montpellier, France
cUnite ´ de Ge ´ne ´tique Mycobacte ´rienne, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France
Received 2 March 2006; revised 15 April 2006; accepted 18 April 2006
Available online 27 April 2006
Edited by Hans Eklund
receptor-like protein kinase involved in cell growth control.
Here, we demonstrate that mitoxantrone, an anthraquinone
derivative used in cancer therapy, is a PknB inhibitor capable
of preventing mycobacterial growth. The structure of the com-
plex reveals that mitoxantrone partially occupies the adenine-
binding pocket in PknB, providing a framework for the design
of compounds with potential therapeutic applications. PknB
crystallizes as a ‘back-to-back’ homodimer identical to those ob-
served in other structures of PknB in complex with ATP analogs.
This organization resembles that of the RNA-dependent protein
kinase PKR, suggesting a mechanism for kinase activation in
? ? 2006 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Mycobacterium tuberculosis PknB is an essential
Keywords: Drug design; Back-to-back dimerization; Crystal
structure; Ser/Thr protein kinase-inhibitor complex;
The ability of Mycobacterium tuberculosis, the pathogen
responsible for tuberculosis (TB), to adapt to changing envi-
ronmental conditions requires an efficient way of sensing and
transducing extracellular signals. One of the mechanisms used
in mycobacteria to assure a tight regulation of cell growth and
division involves the reversible phosphorylation on serine/thre-
onine residues, a well-established process for eukaryotic signal-
ing networks .
M. tuberculosis PknB is a trans-membrane Ser/Thr protein
kinase (STPK) highly conserved in Gram-positive bacteria
and apparently essential for mycobacterial viability . The
crystal structure of the kinase domain of PknB in complex with
an ATP analogue [3,4] showed a striking conservation of both
protein fold and catalytic mechanism between eukaryotic and
prokaryotic STPKs. We have previously shown that PknB is
regulated by autophosphorylation and dephosphorylation by
the Ser/Thr protein phosphatase PstP [5,6] and recent work
showed that PknB is predominantly expressed during exponen-
tial growth, where its overexpression causes morphological
changes linked to defects in cell wall synthesis and cell division
Aberrant kinase activity is implicated in numerous human
diseases and, not surprisingly, protein kinases represent today
one of the most important groups of drug targets [8,9]. Here
we report that mitoxantrone, a compound used in cancer treat-
ment, is a PknB inhibitor capable of preventing mycobacterial
cell growth, suggesting that bacterial kinases may also repre-
sent a potential target for drug design. The crystal structure
of the complex demonstrates that mitoxantrone is an ATP-
competitive inhibitor of PknB and suggests a mode of regula-
tion of protein kinases in mycobacteria.
2. Materials and methods
2.1. In silico screening
Over 40000 compounds from different chemical libraries, including
the Comprehensive Medicinal Chemistry database, were docked into
the nucleotide-binding pocket of the M. tuberculosis PknB structure
(pdb ID 1O6Y ) using the program FlexX .
2.2. Kinase assays
The kinase assays were carried out in 15 ll kinase buffer (50 mM
HEPES, pH 7.0, 1 mM DTT, 0.01% Brij35, 5% glycerol, and 2 mM
MnCl2) using GarA as a substrate  (kinase:substrate molar ratio
1:2000). The reactions were started with the addition of 2.25 lM final
ATP (containing 1 lCi of [c-33P]ATP), and carried out for 20 min at
30 ?C. For the inhibition experiments, each compound was pre-incu-
bated for 30 min at 4 ?C with the reaction mixture (without ATP).
The reactions were stopped by heat inactivation and the mixture trans-
ferred onto P81 paper (phosphocellulose, Whatman). The paper was
washed with 1% phosphoric acid, rinsed with acetone and allowed to
dry. Radiolabelled spots were analyzed with a PhosphorImager
(Storm, Molecular Dynamics). IC50 values were determined using
KaleidaGraph (Synergy Software).
2.3. Determination of MIC values
Minimal inhibitory concentration (MIC) values for mitoxantrone
against different mycobacteria (Fig. 1c) were determined using the col-
orimetric resazurin microtiter assay in 7H9-OADC broth (Difco) at
37 ?C, as described .
*Corresponding author. Fax: +33 145688604.
E-mail address: firstname.lastname@example.org (P.M. Alzari).
1These authors contributed equally to this work.
0014-5793/$32.00 ? 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 580 (2006) 3018–3022