Discovery of 1,4-didydroxy-2-naphthoate prenyltransferase inhibitors: New drug leads for multidrug-resistant gram-positive pathogens

Article abstract of DOI:10.1021/jm070638m

Since utilization of menaquinone in the electron transport system is a characteristic of Gram-positive organisms, the 1,4-dihydroxy-2-naphthoate prenyltransferase (MenA) inhibitors 1a and 2a act as selective antibacterial agents against organisms such as methicillin-resistant Stapylococcus aureus (MRSA), Staphylococcus epidermidis (MRSE), and Mycobacterium spp. Growth of drug-resistant Gram-positive organisms was sensitive to the MenA inhibitors, indicating that menaquinone synthesis is a valid new drug target in Gram-positive organisms.

Full text of DOI:10.1021/jm070638m

J. Med. Chem. 2007, 50, 3973-3975
3973
Discovery of 1,4-Didydroxy-2-naphthoate
Prenyltransferase Inhibitors: New Drug Leads
for Multidrug-Resistant Gram-Positive Pathogens
Michio Kurosu,* Prabagaran Narayanasamy,
Kallolmay Biswas, Rakesh Dhiman, and Dean C. Crick*
Department of Microbiology, Immunology, and Pathology,
College of Veterinary Medicine and Biomedical Sciences,
Colorado State UniVersity, 1682 Campus DeliVery,
Fort Collins, Colorado 80523-1682
Figure 1. Schematic bacterial electron transport chain and menaquino-
ne biosynthesis.
ReceiVed June 4, 2007
thesis is essential for survival of nonfermenting Gram-positive
bacteria.6 On the other hand, Gram-negative organisms such as
E. coli utilize ubiquinone (CoQ) under aerobic conditions and
utilize menaquinone under anaerobic conditions. Moreover, the
electron transport chain in humans does not utilize menaquino-
ne.7 Therefore, inhibitors of menaquinone biosynthesis have
great potential for the development of novel and selective drugs
against MDR Gram-positive pathogens.8 However, no study on
the development of inhibitors for menaquinone biosynthetic
enzymes has been reported. In this communication, we report
that inhibition of 1,4-dihydroxy-2-naphthoate prenyltransferase
(MenA), which catalyzes a formal decarboxylative prenylation
of 1,4-dihydroxy-2-napthoate (DHNA) (Figure 1),9 showed
significant growth inhibitory activities against drug-resistant
Gram-positive bacteria.
The MenA activity was characterized using membrane
fractions prepared from M. tuberculosis as previously de-
scribed.11 MenA is predicted to have five transmembrane
segments, and there are highly conserved Asp residues that
would be located in the inner-plasma membrane.12 The activity
is absolutely dependent on the presence of the divalent cations
such as Mg2+. Thus, it is likely that such divalent cations form
ion pairs with Asp residues existing in the catalytic site of
MenA. On the basis of the observation of this enzymatic activity
and the structure of the MenA product, demethylmenaquinone
(DMMK), we designed tertiary or secondary amine or hydra-
zine-containing DMMK mimics (1) in hope that the amine
moiety would interact with Asp residue(s) directly or through
the divalent cation(s) in the active site and (2) in which the
chemically unstable 1,4-quinone system is replaced with the
hydrophobicly substituted benzophenones. As illustrated in
Scheme 1, the designed DMMK mimics were synthesized
efficiently in four to six steps including (1) Friedel-Crafts
acylation, (2) deprotection, (3) alkylation(s), (4) bromination,
and (5) amination reactions.
Abstract: Since utilization of menaquinone in the electron transport
system is a characteristic of Gram-positive organisms, the 1,4-
dihydroxy-2-naphthoate prenyltransferase (MenA) inhibitors 1a and 2a
act as selective antibacterial agents against organisms such as methi-
cillin-resistant Stapylococcus aureus (MRSA), Staphylococcus epider-
midis (MRSE), and Mycobacterium spp. Growth of drug-resistant Gram-
positive organisms was sensitive to the MenA inhibitors, indicating
that menaquinone synthesis is a valid new drug target in Gram-positive
organisms.
Antimicrobial resistance of pathogens is a global problem.
Each year worldwide, more than 11 million people die from
the major infectious killers (i.e., MDRa tuberculosis, malaria,
HIV, diarrhea diseases, and pneumonia).1 The increasing drug
resistance among Gram-positive bacteria is a significant problem
because they are responsible for one-third of nosocomial
infections; drug resistance in Gram-positive organisms (i.e.,
staphylococci, pneumococci, vancomycin resistance in entero-
cocci, and mycobacteria) have achieved prominence in the past
15 years. Methicillin-resistant S. aureus (MRSA) is one of most
frequent nosocomial pathogens in developed countries.2 In
addition, Mycobacterium tuberculosis (Mtb) is responsible for
nearly 2 million deaths annually and one-third of the world
population is infected with latent Mtb. In particular, people who
are malnourished or have HIV-AIDS are susceptible to TB
infection. Moreover, the emergence multidrug-resistant strains
of Mtb (MDR-TB) seriously threatens TB control and prevention
efforts.3 The results of over 10 years of screening of strains
and molecular targets (existing and new) from traditional product
sources (randomly generated library molecules, secondary
metabolites, and drug libraries) have been disappointing.4
Therefore, identification of new molecular targets and mecha-
nisms of action that involved identifying essential, ubiquitous
bacterial genes in pathogens that are prokaryote and eukaryote
selective to prevent side effects in the host has been studied.
The lipid-soluble electron carriers (lipoquinones) occupy a
central and essential role in electron transport coupled ATP
synthesis. The lipoquinones involved in the respiratory chains
of bacteria consist of menaquinones and ubiquinones. From the
taxonomic studies it is evident that a majority of Gram-positive
bacteria including Mycobacterium spp. utilize only menaquinone
in their electron transport systems,5 and menaquinone biosyn-
We have synthesized 100 molecules in solution, and the
library of molecules was evaluated in enzymatic assays in vitro
(IC50) against Mtb MenA11 and in mycobacterial growth assays
(MIC). More than 18 molecules exhibited MenA IC50 and MIC
values of less than 20 µM, and in all cases the MIC value was
in good agreement with the IC50 value. From these preliminary
screenings it was shown that the shorter length of linker (C5-
C7 in 1) between the phenolic oxygen and the nitrogen atom
decreased the ability to inhibit MenA and the efficacy of growth
inhibition. In addition, the structure of amine or hydrazine
significantly influences the activity; R-substituted amine or bulky
tertiary amine containing molecules did not show MenA
inhibitory activity at lower concentrations.10 Identification of
the effective substitution pattern (R1, R2, R3, and R4) in
* To whom correspondence should be addressed. For M.K.: phone, 970-
491-7628; fax, 970-491-1815; e-mail, michio.kurosu@colostate.edu. For
D.C.C.: phone, 970-491-3308; fax, 970-491-1815; e-mail, dean.crick@
colostate.edu.
a Abbreviations: MenA, 1,4-dihydroxy-2-naphthoate prenyltransferase;
MDR, multidrug-resistant; MRSA, methicillin-resistant Stapylococcus au-
reus; MRSE, methicillin-resistant Staphylococcus epidermidis; Mtb, My-
cobacterium tuberculosis; CoQ, ubiquinone; DHNA, 1,4-dihydroxy-2-
napthoate; DMMK, demethylmenaquinone.
10.1021/jm070638m CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/21/2007

3974 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Letters
Table 1. MICs of 1a, 2a, and Representative Antibacterial Agents (Clinically Used) for Gram-Positive Bacteria Including Mycobacterium spp.
MIC (µg/mL)a
entry
species and strain
1ab
2ac
RFPd
INHd
VCMd
LZDd
1
2
3
4
5
6
7
8
9
M. tuberculosis H37Rv
M. tuberculosis H37Rv INHr
M. tuberculosis H37Rv RFPr
M. boVis BCG Tokyo
M. aVium Flamingo
1.5
1.5
1.5
3.1
6.3
6.3
6.3
12.5
12.5
12.5
0.2
0.2
>25
0.1
>25
0.05
0.1
>25
12.5
6.3
0.1
3.1
3.1
M. intracellulare ATCC15984
M. aurum
0.78
M. fortuitum NIHJ1615
M. smegmatis Takeo
>25
>25
6.3
12.5
10
11
12
13
14
15
16
17
18
19
MRSA high-resistance
VRSA 70
MRSA 92-1191
4
4
4
8
8
4
8
2
4
8
1
0.5
1
1
1
1
2
2
2
2
32
2
2
4
2
2
MRSA Mu50
MRSA LDZ-resistance06
S. aureus macrolide-resistance
E. faecalis NCTC12201 (VanA)
E. faecalis NCTC12203 (VanA)
S. aureus FDA209P
S. aureus Smith
>128
>128
1
1
20
21
22
23
E. coli NIHJ JC-2
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
128
K. pneumoniae NCTN9632
P. mirabilis IFO3849
P. aeruginosa 46001
>128
a The agar plate dilution method was used (see Supporting Information). b MICs against Gram-positive bacteria were >12.5. c MICs of 2a against
Mycobacterium spp. were >10-fold higher than those of 1a. d VCM: vancomycin. LZD: linezolid. RFP: rifampicin. INH: isoniazid.
Scheme 1. Generation of a Library of Molecules in Solution10,a
negative bacteria (entries 20-23 in Table 1). As summarized
in Table 1, only Gram-positive bacterial growth was inhibited
by 1a (for Mtb) and 2a (others), supporting the hypothesis that
menaquinone synthesis is the target of these molecules.14 In
addition, growth of drug-resistant Gram-positive organisms was
sensitive to the MenA inhibitors (entries 2, 3, 14, 10-17),
indicating that MenA is likely to be a valid drug target in Gram-
positive pathogens involved in emerging diseases.
In conclusion, we have shown, for the first time, that MenA
inhibitors 1a and 2a inhibited growth of drug-resistant Myco-
bacterium spp. and other Gram-positive bacteria at low con-
centrations. The MenA inhibitors described here can be
synthesized cost-effectively, and structural modifications to
improve the inhibitory activity in vitro can be achieved in a
time efficient manner. The results are expected to be of
significance in terms of discovering new lead molecules that
can be developed into new drugs to combat Gram-positive
pathogens.
Acknowledgment. This paper is dedicated to Professor
Yoshito Kishi (Harvard University) on the occasion of his 70th
birthday. We thank the National Institutes of Health (NIAID
Grants AI049151, AI018357, and AI06357) for generous
financial support. We are grateful to Drs. Yamada and Hanaki
(Kitasato Institute Research Center) and Dr. Matsumoto (Otsuka
Pharmaceutical) for conducting MIC assays.
a Reagents and conditions: (a) AlCl3, PhNO2 (75-90%); (b) (i) 48%
HBr, AcOH (90%); (ii) 1,5-dibromopentane or 1,6-dibromohexane or 1,7-
dibromoheptane or 1,8-dibromooctane, K2CO3, DMF (for 1) (80-95%);
1,4-dibromobutane, K2CO3, DMF; 1,3-propanediol, NaH, DMF; CBr4, PPh3,
CH2Cl2 (for 2) (65%); 1,4-dibromobutene, K2CO3, DMF; 1,3-propanediol,
NaH, DMF; CBr4, PPh3, CH2Cl2 (for 3) (65%); 1,4-dibromobutyne, K2CO3,
DMF; 1,3-propanediol, NaH, DMF; CBr4, PPh3, CH2Cl2 (for 4) (65%); (iii)
R5 (primary or secondary amines or hydrazines), NaHCO3, DMF (50-
98%); (iv) TFA, CH2Cl2 (for Boc-protected R5) (100%).
Supporting Information Available: Experimental procedures
and results from characterization of compounds and MenA inhibi-
tory assay. This material is available free of charge via the Internet
at http://pubs.acs.org.
benzophenone moiety requires extensive SAR studies; however,
the hydroxy group on R3 (R3 ) OH in 1 and 3) seems to be
superior to the others (R3 ) H or Cl) regardless of the structure
of linker.13
Two molecules 1a and 2a, named allylaminomethanone-A
and phenethylaminomethanone-A, respectively, showed MIC
values of 1.5 and 12.5 µg/mL against Mtb (H37Rv, a common
laboratory strain), respectively. We resynthesized 1a and 2a and
determined MICs (µg/mL) against a variety of species and
strains of Gram-positive (entries 1-19 in Table 1) and Gram-
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JM070638M