Targeting FtsZ for antituberculosis drug discovery: Noncytotoxic taxanes as novel antituberculosis agents

Article abstract of DOI:10.1021/jm050920y

Screening of 120 taxanes identified a number of compounds that exhibited significant antituberculosis activity. Rational optimization of selected compounds led to the discovery that the C-seco-taxane-multidrug-resistance (MDR) reversal agents (C-seco-TRAs

Full text of DOI:10.1021/jm050920y

J. Med. Chem. 2006, 49, 463-466
463
Table 1. Antimicrobial Activities of Taxanes against Drug-Sensitive
Targeting FtsZ for Antituberculosis Drug
Discovery: Noncytotoxic Taxanes as Novel
Antituberculosis Agents
and Mutidrug-Resistant M. tuberculosisa
MIC
cytotoxicity
(µM)
(IC50, µM)
M. tuberculosis M. tuberculosis
entry
taxane
H37Rv
IMCJ946.K2
MCF7
0.019
A549
Qing Huang,† Fumiko Kirikae,§ Teruo Kirikae,§
Antonella Pepe,† Amol Amin,| Laurel Respicio,|
Richard A. Slayden,| Peter J. Tonge,†,‡ and Iwao Ojima†, ‡,
1
2
3
4
5
6
7
8
9
paclitaxel
40
5
5
5
5
10
2.5
5
20
10
10
20
10
2.5
2.5
2.5
2.5
40
0.028
0.65
15.7
14.0
80
12.5
4.5
11
2a
2b
4a
4b
4c
4d
6
1.25
2.5
2.5
5
10
2.5
5
10
10
5
5
0.65
4.5
7.6
5.4
9.3
3.4
5.3
14
7.0
3.9
>20
9.4
>80
>80
>80
>80
*
Department of Chemistry, State UniVersity of New York at
Stony Brook, Stony Brook, New York 11794-3400, Institute of
Chemical Biology & Drug DiscoVery, State UniVersity of New York
at Stony Brook, Stony Brook, New York 11794-3400, Department of
Infectious Diseases, Research Institute, International Medical
Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655,
Japan, and Department of Microbiology, Immunology and
Pathology Colorado State UniVersity,
5.0
N.D.
10.8
9.6
4.3
17.0
10 7a
11 7b
12 8a
13 8b
14 10a
15 10b
16 10c
17 10d
Fort Collins, Colorado 80523-1682
20
1.25
2.5
1.25
1.25
>80
ReceiVed September 16, 2005
>80
>80
>80
Abstract: Screening of 120 taxanes identified a number of compounds
that exhibited significant antituberculosis activity. Rational optimization
of selected compounds led to the discovery that the C-seco-taxane-
multidrug-resistance (MDR) reversal agents (C-seco-TRAs) are non-
cytotoxic at the upper limit of solubility and detection (>80 µM), while
maintaining MIC99 values of 1.25-2.5 µM against drug-resistant and
drug-sensitive strains of Mycobacterium tuberculosis (MTB). Treatment
of MTB cells with TRA 3aa and 10a at the MIC caused filamentation
and prolongation of the cells, a phenotypic response to FtsZ inactivation.
a M. tuberculosis (M. tb.) H37Rv is sensitive to all antibiotics tested.
M. tuberculosis IMCJ946.K2 is resistant to nine drugs including isoniazid
(INH), rifampicin (RFP), ethambutol (EB), streptomycin (SM), kanamycin
(KM), ethionamide (ETH), p-aminosalicylic acid (PAS), cycloserine (CS),
and enviomycin (EVM). MCF7 and A549 cells: human breast and non
small cell lung cancer cell lines, respectively.
Tuberculosis (TB) is the leading cause of death in the world
from a single infectious disease, claiming over three million
lives each year. The AIDS pandemic has led to an explosion of
HIV/TB coinfection, as TB is the most common opportunistic
infection for patients living with HIV/AIDS. Poor chemothera-
peutics and inadequate local-control programs contribute to the
inability to manage TB and lead to the emergence of drug
resistant strains of Mycobacterium tuberculosis (MTB).1 Con-
sequently, there is a pressing need for the development of novel
TB drugs for treating AIDS-related opportunistic infections that
are effective against both drug-sensitive and drug-resistant MTB
strains. To this end, we select FtsZ, a tubulin homologue in
MTB, as a novel target for anti-TB drug discovery.
FtsZ (filamentation temperature-sensitive protein Z) is an
essential cell division protein in bacteria and has been shown
to be a homologue of the mammalian cytoskeletal protein
tubulin. FtsZ and tubulin share extensive similarity in function.
In a process strongly reminiscent of microtubule formation by
tubulin, FtsZ polymerizes in a GTP-dependent manner into
filaments, which assemble into a highly dynamic structure
known as the Z ring on the inner membrane at the mid cell.2
Following recruitment of the other cell division proteins, the
Z-ring contracts, resulting in septation. Inactivation of FtsZ
results in the absence of septum formation. Accordingly, FtsZ
is a very promising target for new antimicrobial drug discovery.
Figure 1. Structures of DAB and TRA 2a.
A starting point for discovering inhibitors of FtsZ polymer-
ization or depolymerization is a library of compounds that are
known to affect the assembly of the FtZ homologue tubulin
into microtubules, since the latter protein has been a target for
anticancer chemotherapeutics for over 35 years. The fact that
the sequence homology between FtsZ and tubulin is low (<20%
identity) suggests that there is an excellent possibility in
discovering FtsZ specific taxanes that are noncytotoxic to human
host cells.
Taxanes were first screened for inhibitory activity by a real
time PCR-based (RT-PCR) assay.3 These taxanes represent two
diverse activities, highly cytotoxic taxoids (i.e., “taxol-like
compound”) that stabilize microtubules4-6 and noncytotoxic (or
very weakly cytotoxic) taxane-multidrug-resistance (MDR)
reversal agents (TRAs)7-14 which inhibit the efflux pumps of
ATP-binding cassette (ABC) transporters such as P-glycoprotein
(P-gp), multidrug resistant protein (MRP-1), and breast cancer
resistant protein (BCRP). Screening of 120 taxanes revealed
that a number of taxanes exhibited significant anti-TB activity.
The antibacterial activity of each compound was confirmed by
determining MIC99 values using the conventional microdilution
broth assay. Treatment of MTB cells with two TRAs at the MIC
caused filamentation and prolongation of the cells (see Sup-
porting Information for electron microscope images), a pheno-
typic response to FtsZ inactivation.
* To whom correspondence should be addressed. Phone (631) 632-7890;
Fax (631) 632-7942; Email iojima@notes.cc.sunysb.edu.
† Department of Chemistry, State University of New York at Stony
Brook.
‡ Institute of Chemical Biology & Drug Discovery, State University of
New York at Stony Brook.
In the MIC assay, it was found that TRA 2a,14 bearing a (E)-
3-(naphth-2-yl)acryloyl (2-NpCHdCHCO) group at the C-13
position possessed very promising anti-TB activity against drug-
§ Department of Infectious Diseases, Research Institute, International
Medical Center of Japan.
| Colorado State University.
10.1021/jm050920y CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/21/2005

464 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Scheme 1. Synthesis of Taxane-Based Anti-TB Agentsa
Letters
a Reagents and conditions: (i) RCOOH, DIC, DMAP, CH2Cl2; (ii) HF/pyridine, CH3CN/pyridine, room temperature, overnight; (iii) CeCl3, acid anhydride,
THF, room temperature, 4-6 h; (iv)TESCl, imidazole, room temperature; (v) acid chloride, LiHMDS, THF, -40 °C; (vi) RCOOH, EDC, DMAP, CH2Cl2,
room temperature.
sensitive as well as drug-resistant MTB strains (MIC99 ) 2.5-5
µM; Table 1). TRA 2a14 was selected as the lead compound
for further optimization, and a new library of taxanes was
prepared by modification of 10-deacetylbaccatin III (DAB)
(Figure 1 and Scheme 1).
For the FtsZ-interacting taxane-based anti-TB agents to be
useful as therapeutic drugs, these agents should not be cytotoxic
at the concentration required for their antibacterial activity.
Accordingly, it is necessary for the agents to distinguish human
â tubulin from the MTB FtsZ. It has been shown in the SAR

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 465
Figure 3. Structures of paclitaxel and 11.
growth inhibitory activity against drug-sensitive MTB strain
(H37Rv) and a multi-drug-resistant strain (IMCJ946K2), cul-
tured from clinical isolates of MDR-TB. The MTB strain
IMCJ946K2 is associated with nosocomial outbreaks in Japan
and is resistant to all the clinically prescribed anti-TB drugs
used in Japan (9 drugs; see Table 1 legend).
Paclitaxel (Figure 3), a microtubule-stabilizing anticancer
agent, exhibits modest antibacterial activity against both MTB
strains (MIC 40 µM), but its cytotoxicity against human cancer
cell lines (a benchmark for activity against human host cells)
is 3 orders of magnitude more potent (IC50 0.019-0.028 µM;
entry 1, Table 1). These data clearly indicate that paclitaxel is
highly specific for microtubules. Taxoid 11 (Figure 3) exhibits
1 order of magnitude higher antibacterial potency and 20-30
times reduced cytotoxicity compared to paclitaxel. Since it is
likely that the IC99 values would be at least 10 times larger
than the IC50 values (as the former measures complete cell
growth inhibition while the latter only measures 50% inhibition),
it appears that 11 has comparable affinities to microtubules and
FtsZ (entry 2, Table 1). TRA 2a and its congeners derived from
DAB (entries 3-13, Table 1) are clearly much less cytotoxic
than paclitaxel (200-1000 times less toxic) and 11, while
keeping the same level of antibacterial activity to that of 11.
These TRAs appear to have higher specificity to FtsZ than
microtubules. As entries 14-17, Table 1 clearly indicated,
C-seco-TRAs 10a-d are noncytotoxic so far at the upper limit
of solubility and detection, while keeping the MIC values of
1.25-2.5 µM against drug-resistant and drug-sensitive MTB
strains. Thus, we have now discovered noncytotoxic taxane lead
compounds to develop a novel class of anti-TB agents. The
specificity of these novel taxanes to microtubules as compared
to FtsZ appears to have been completely reversed through
systematic rational drug design. Moreover, we observed that
the treatment of MTB cells with TRA 10a at the MIC caused
filamentation and prolongation of the cells (see Supporting
Information), a phenotypic response to FtsZ inactivation. In
addition, a preliminary study on the effect of TRA 10a on the
polymerization-depolymerization, using the standard light-
scattering assay exhibited a dose-dependent stabilization of FtsZ
against depolymerization. The details will be reported elsewhere
in due course.
Figure 2. Structures of highly promising noncytotoxic anti-TB taxane
leads derived from C-seco-baccatin.
studies of paclitaxel (Taxol, Figure 3) and taxoids that substitu-
tion at the para-position of the C-2 benzoate6,15 substantially
diminishes the binding ability of the analogues. Furthermore,
the C-10 position may affect anti-TB activity. Therefore, we
synthesized C-2 and C-10 modified TRA 2a (Scheme 1, eq 1)
to examine the effects of those modifications on the cytotoxcity,
FtsZ binding ability, and anti-TB activity. Some C-10 modified
TRA 2a analogues show little or no anti-TB activity, while C-2
modification of TRA 2a results in slightly decreased cytotoxicity
and does not affect the anti-TB activity.
A variety of hydrophobic side chains were appended to the
C-13 position of DAB in order to generate a series of TRA 2a
analogues (Scheme 1, eq 2). Screening of these compounds
revealed several with activity as good as that of TRA 2a (entries
3, 5, 7, and 8, Table 1).
We also examined whether the attachment of the 3-(2-
naphthyl)acrylate side chain to the C-13 position is crucial for
its anti-TB activity through binding to FtsZ or not for the TRA
2a series. Accordingly, we attached the same side chain moiety
to the C-7 and C-10 position to see the effects of these changes
on the potency and profile of the resulting taxanes (Scheme 1,
eq 3). In fact, the 10-modified analogue 6 showed only slightly
reduced anti-TB activity (entry 9, Table 1).
In addition to the above modifications, we also introduced
functionalities to improve the water solubility of these TRAs.
Thus, N,N-dimethylglycine and N,N-diethyl-â-alanine esters
were introduced to TRA 2a as a pendant group at the C-7 or
C-10 position (Scheme 1, eq 4). This modification caused only
minor reduction in the anti-TB activity of these analogues (TRAs
7a, 7b, 8a, and 8b) as compared with TRA 2a (entries 3, 10,
11, 12, and 13, Table 1)
Although TRA 2a is certainly an excellent lead compound
for optimization, it will be even better if a noncytotoxic lead
compound, which does not bind to microtubules at all, is
identified. Recently, we have been investigating a novel
antiangiogenic taxoid (IDN5390),16,17 which bears a C-seco-
baccatin (i.e., C-ring-opened baccatin) skeleton and is much less
cytotoxic than paclitaxel. Accordingly, we prepared the C-seco-
analogue of TRA 2a, i.e., TRA 10a (Scheme 1, eq 5). Three
analogues of TRA 10a (Figure 2) were also prepared and
assayed for their anti-TB activity and cytotoxicity. Significantly,
TRA 10 series compounds (entries 14-17, Table 1) possessed
potent anti-TB activity (MIC 1.25-2.5 µM) against drug-
sensitive and drug-resistant MTB strains without appreciable
cytotoxicity (IC50 > 80 µM).
Further optimization and biological evaluation of these newly
discovered lead compounds are actively underway in these
laboratories.
Acknowledgment. This research is supported by grants from
the National Institutes of Health and the Japan Health Science
Foundation.
Supporting Information Available: Synthetic procedures and
characterization data for new TRAs; procedures for biological
evaluations; electron micrograph images. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
As Table 1 shows, paclitaxel, 10-hexanoylpaclitaxel 11
(Figure 3), TRA 2a, and its congeners were assayed for their
(1) Raviglione, M. C. Issues facing TB control (7). Multiple drug-resistant
tuberculosis. Scottish Med. J. 2000, 45, 52-55; discussion 56.

466 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Letters
(2) Bi, E.; Lutkenhaus, J. FtsZ ring structure associated with division in
Escherichia coli. Nature (London) 1991, 354, 161-164.
(3) Huang, Q.; Pepe, A.; Zanardi, I.; Tonge, P. J.; Slayden, R. A.; Kirikae,
F.; Kirikae, T. Ojima, I. Targeting FtsZ for anti-tuberculosis drug
discovery: noncytotoxic taxanes as novel anti-TB agents. Abstracts
of Papers, 229th American Chemical Society National Meeting, Mar
13-17, 2005, San Diego, CA; ACS: Washington, DC, 2005 MEDI-
386.
(12) Minderman, H.; Brooks, T. A.; O’Loughlin, K. L.; Ojima, I.;
Bernacki, R. J.; Baer, M. R. Modulation of ATP-Binding-Cassette
Transport Proteins by the Taxane Derivatives BAY 59-8862 and
tRA96023. Cancer Chemother. Pharm. 2004, 53, 363-369.
(13) Brooks, T. A.; Kennedy, D. R.; Gruol, D. J.; Ojima, I.; Baer, M. R.;
Bernacki, R. J. Structure-Activity Analysis of Taxane-Based Broad-
Spectrum Multidrug Resistance Modulators. Anticancer Res. 2004,
24, 409-415.
(14) Ojima, I. B., C. P.; Wu, X.; Bounaud, P.-Y.; Fumero-Oderda, C.;
Sturm, M.; Miller, M. L.; Chakravarty, S.; Chen, J.; Huang, Q.; Pera,
P.; Brooks, T. A.; Baer, M. R.; Bernacki, R. J. Design, Synthesis
and SAR of Novel Taxane-Based Multi-Drug Resistance Reversal
Agents. J. Med. Chem. 2005, 48, 2218-2228.
(15) Georg, G. I.; Harriman, G. C. B.; van der Velde, D. G.; Boge, T. C.;
Cheruvallath, Z. S.; Datta, A.; Hepperle, M.; Park, H.; Himes, R.
H.; Jayashinge, L. Medicinal Chemistry of paclitaxel: Chemistry,
structure-activity relationships, and conformational analysis. Taxane
Anticancer Agents: Basic Science and Current Status, Georg, G. I.,
Chen, T. T., Ojima, I., Wyas, D. M., Eds. ACS Symp. Ser., 1995,
583, 217-232.
(16) Appendino, G.; Danieli, B.; Jakupovic, J.; Belloro, E.; Scambia, G.;
Bombardelli, E. Synthesis and Evaluation of C-Seco Paclitaxel
Analogues. Tetrahedron Lett. 1997, 38, 4273-4276.
(4) Georg, G. I.; Chen, T. T.; Ojima, I.; Wyas, D. M.; Eds. Taxane
Anticancer Agents: Basic Science and Current Status. ACS Symp.
Ser. 1995, 583, 1-353.
(5) Ojima, I.; Kuduk, S. D.; Chakravarty, S. Recent Advances in the
Medicinal Chemistry of Taxoid Anticancer Agents. AdV. Med. Chem.
1998, 4, 69-124.
(6) Kingston, D. G. I.; Jagtap, P. G.; Yuan, H.; Samala, L. The Chemistry
of Paclitaxel (Taxol) and Related Taxoids. Prog. Chem. Org. Nat.
Prod. 2002, 84, 53-225.
(7) Ojima, I.; Bounaud, P.-Y.; Takeuchi, C. S.; Pera, P.; Bernacki, R. J.
New Taxanes as Highly Efficient Reversal Agents for Multi-Drug
Resistance in Cancer Cells. Bioorg. Med. Chem. Lett. 1998, 8, 189-
194.
(8) Ojima, I.; Bounaud, P.-Y.; Bernacki, R. J. New Weapons in the Fight
Against Cancer. CHEMTECH 1998, 28, 31-36.
(9) Ojima, I.; Bounaud, P.-Y.; Oderda, C. F. Recent Strategies for the
Treatment of Multi-drug Resistance in Cancer Cells. Expert Opin.
Ther. Pat. 1998, 8, 1587-1598.
(17) Taraboletti, G.; Micheletti, G.; Rieppi, M., Poli; M., T., M.; Rossi,
C., Borsotti, P.; Roccabianca, P.; Scanziani, E.; Nicoletti, M. I.;
Bombardelli, E.; Morazzoni, P.; Riva, A.; Giavazzi, R. Antiangiogenic
and Antitumor Activity of IDN 5390, a New Taxane Derivative. Clin.
Cancer Res. 2002, 8, 1182-1188.
(10) Ojima, I.; Bounaud, P.-Y.; Bernacki, R. J. Designing Taxanes to Treat
Multidrug-Resistant Tumors. Modern Drug DiscoVery 1999, 2 (3),
45-52.
(11) Brooks, T. A.; Minderman, H.; O’Loughlin, K. L.; Ojima, I.; Baer,
M. R.; Bernacki, R. J. Taxane-Based Reversal Agent Modulation of
P-glycoprotein-, Multidrug Resistance Protein- and Breast Cancer
Resistance Protein-Mediated Drug Transport. Mol. Cancer Ther.
2003, 2, 1195-1205.
JM050920Y