Assessment ofAntimycobacterial Activity ofa Series ofMainly Marine Derived Natural Products
7 8C. After removal of extracellular bacilli by washing in PBS,
Planta Med. 66 (2000)
341
3
fortunately, at concentrations up to 16mg/ml it was found to be
devoid of activity in these tests. The most likely explanations
for this are that the compound is metabolised to an inactive
form or is simply not taken up by the macrophage. Either of
these possibilities could also explain the observed lack of cyto-
toxicity in the KBand Vero cells.
coverslips were transferred to fresh culture medium and incu-
bated for 24 h to allow complete phagocytosis of any extracel-
lular organisms adhering to the macrophage cell surface. Ini-
3
tial levels of M. tuberculosis were approximately 10 bacilli/
macrophage monolayer.
Test compounds were dissolved in DMSO and then diluted
Compound 36 is a lichen derived triterpene. Plant derived tri-
terpenes have previously been described as having antimyco-
bacterial properties in the same concentration range (MIC
8mg/ml) as observed for compound 36 (9). Structural require-
ments for biological activity of these cycloartane derivatives
seemed to be the presence of cycylopropyl, epoxy, and hy-
droxy functionalities. Compound 36 only contains hydroxy
functionality but still demonstrates significant in vitro anti-
mycobacterial activity without cytotoxicity.
1
:500 in culture medium. Fourfold dilutions were made in
culture medium and 1 ml of each concentration was added to
macrophage monolayers in triplicate one day post infection
resulting in final concentrations of 1, 4, 16 and 32 mg/ml.
Negative control wells received culture medium containing
diluted DMSO. Rifampicin served as a positive drug control
and was added at final concentrations of 1.2, 0.3, 0.075, and
0
.02 mg/ml. After 7 days of incubation at 37 8C in moist 5%
CO macrophages were lysed by transferring the coverslips to
2
a 24 well tissue culture plate containing 0.5 ml/well 0.25%
SDS. After 10 min incubation at room temperature, 0.5 ml of
RPMI 1640 (Gibco) +20% FBS was added and the lysates were
transferred to a sealed tube, sonicated, serially diluted (10-
fold) in RPMI 1640 + 10% FBS and plated on 7H11 agar plates.
Of the thirty-nine mainly marine derived compounds tested
in this study about one third exhibited biological activity in
the antimycobacterial test system employed. The broad diver-
sity in the structures of the isolates made assignment of
structure-activity relationships impossible. The presence of
an isonitrile group, e.g., in compound 26, however, clearly has
a significant influence and seems to be important for the ac-
tivity of the compounds 20±26. The identification of antimy-
cobacterial activity, albeit weak, in a substantial percentage of
divergent compounds isolated from a range of taxonomically
different samples substantiates the potential of marine natu-
ral products to serve as an important resource for the discov-
ery of novel antimycobacterial candidates. The best example
of this being axisonitrile-3 (26) which has been identified as a
very good antimycobacterial lead compound for the first time.
Additionally, the non-cytotoxic compounds 20, 23 and 36, and
possibly 17 can also be regarded as potentially good lead com-
pounds for the development of antimycobacterial agents. Any
of these compounds are sufficiently novel in terms of structure
and/or biological activity to warrant further investigation. As a
result of these findings the value of exploring natural products
as potential sources of drugs is further strengthened.
The plates were incubated at 37 8C in moist 5% CO for 18±21
2
days, and then CFU were enumerated. Using the best curve fit
(
(
CurveExpert, Microsoft), concentrations effecting a 90%
EC90) and 99% (EC99) inhibition of M. tuberculosis growth in
macrophages were determined.
Cytotoxicity testing: The cytotoxicity testing with KBcells was
performed as described previously (4), (9), (10). Cytotoxicity
towards Vero cells (ATCC CCL-81) was assessed using the Cell-
Titer 96 aqueous non-radioactive cell proliferation assay
(
Promega Corp., Madison, WI, USA).
Results and Discussion
The antimycobacterial activities of the thirty-nine selected
compounds were assessed against M. tuberculosis and where
material was sufficient against M. avium. From the test results
(see Table 1) it is evident that out of the thirty-nine compounds
tested fifteen demonstrated moderate activity (MIC <32mg/ml)
against M. tuberculosis, eleven of which (5, 17, 20±26, 35 and
Acknowledgements
3
6), gave MICs of 16mg/ml or less. Closer inspection of the data
in Table 1 revealed that of these eleven four compounds (21,
2, 24 and 25), also had significant cytotoxic properties which
The antimicrobial testing was supported in part by intra-
agency agreement Y1-A1-5016 from the US National Institute
of Allergy and Infectious Disease. Financial support from the
German Research Foundation (DFG KO 902/2-2) is gratefully
acknowledged.
2
may to some extent explain their antimycobacterial properties.
Of the other seven compounds (5, 17, 20, 23, 26, 25 and 36), on-
ly 35 was cytotoxic towards KBcells at the 20 mg/ml or less lev-
el. For compounds 5 and 17 no cytotoxicity measurements
were performed due to a lack of material. The active com-
pounds 20 and 23, from the sponge Cymbastela hooperi, are
closely related structurally, whereas the Acanthella klethra de-
rived metabolite 26 is of a totally different structural class, but
all three compounds are isonitriles. From the data presented in
Table 1 it is evident that the molecules with an isonitrile group
are the most active. This observation permits the assumption
that this functional group may play a role in the observed anti-
mycobacterial activity. Axisonitrile-3 (26) was not toxic to Vero
cells at concentrations <200mg/ml. As axisonitrile-3 was the
most active compound detected in this study as well as being
not cytotoxic it was the only one, after reisolation, to be further
tested for its ability to inhibit the growth of M. tuberculosis re-
siding within cultured mouse peritoneal macrophages. Un-
References
1
Dolin PJ, Raviglione MC, Kochi A. Global tuberculosis incidence
and mortality during 1990±2000. Bulletin of the World Health
Organisation 1994; 72: 213±20
Raviglione MC, Snider DE, Kochi A. Global epidemiology of tuber-
2
culosis. Morbidity and mortality of a worldwide epidemic. Journal
of the American Medical Association 1995; 273: 220±6
Cohn DL, Bustreo F, Raviglione MC. Drug-resistant tuberculosis:
3
Review of the worldwide situation and the WHO/IUATLD Global
Surveillance Project. Clinical Infectious Diseases 1997; 24 (Suppl.
1): S121±30
4
Pablos-Mendez A, Raviglione MC, Laszlo A, Binkin N, Rieder HL,
Bustreo F et al. Global surveillance for antituberculosis-drug re-
sistance. Tuberculosis. New England Journal of Medicine 1998;
23: 1641±9, and references therein
Koenig, Gabriele M.
