3of ART to heme, a process known as heme alkylation, was first observed by
Meshnick et al. ( 1991 ), who identified the ART-heme adduct by mass spectros-
copy. Later, Cazelles et al. ( 2001 ) also discovered that ART is capable of alkylat-
ing a model heme molecule at α, β, and δ carbon atoms. In mice with malarial
infection, Robert et al. ( 2005 ) characterized the ART-heme adduct in the spleen
and glucuro-conjugated ART-heme derivatives in the urine of ART-treated mice.
After a detailed investigation in tumor cells, on the other hand, Zhang and
Gerhard ( 2009 ) found that the acceleration of cellular heme biosynthesis increases
ART’s cytotoxicity, whereas the suppression of endogenous heme biosynthesis
decreases ART’s cytotoxic activity, underscoring that the heme entity serves as
an intracellular enhancer of ART attacking tumor cells. Afterwards, Zhang et al.
( 2010 ) observed that heme biosynthesis increases the cytotoxicity of ART-induced
free radicals, which are effectively suppressed by O 2 − scavengers. So these results
implied that O 2 − is most likely a cytotoxic mediator of ART.
Heme is commonly recognized in presence as a functional constituent of iron-
containing metalloproteins including hemoproteins, such as NOS, COX, catalase
(CAT), peroxidase (POX), hemoglobin, and myoglobin, etc. In theory, ART can
alkylate all hemoproteins and interfere with their functions without selectivity.
However, whether ART targets any specific hemoproteins and affects their func-
tions has not been reported until 2011. More strictfully, no individual hemopro-
tein that interacts with ART and alters its activity was identified before that time.
Therefore, no logical cues indicate ART being a NOS inhibitor.
In the first half of 2010, our eyes were absorbed on a published report in
Science, in which the authors wrote: “because bacterial pathogens use NOS pro-
tects them against antibiotics and immune attack, inhibition of NOS could serve
as an effective antibacterial intervention” (Gusarov et al. 2009 ). This startling
expectation on a novel antibacterial strategy encouraged us to rationally look for
and find out NOS inhibitors as antibiotic synergists. From the previous knowledge
of ART reactivity with hemozoin, a pigmented metabolite of hemoglobin in the
malarial parasite, we boldly assumed that ART may inhibit NOS by binding to its
heme, thereby blocking the inter-conversion between ferrous ion (Fe^2 +) and ferric
ion (Fe^3 +).
To testify the assumption, we immediately conducted a series of confirming
experiments to reveal whether a synergetic effect of ART exists as in combination
with common used antibiotics. As our expectation, ART substantially potentates
the antibacterial capacity of rifampicin (RIF) in the Gram-positive (G+) bacteria
Bacilus licheniformis and ampicillin (AMP) in the G− bacteria Escherichia coli.
Surprisingly, we found that ART synchronously inhibits NOS and CAT in B.
licheniformis. On combined treatment of bacteria by ART with either RIF or AMP,
we observed a correlation of suppressed bacterial proliferation with mitigated NO
production along with enhanced H 2 O 2 burst. So we concluded that ART as a syn-
ergist of antibiotics enables the most effective curation of bacterial infections upon
inhibiting NOS and CAT, which declines the protective NO level and elevates the
lethal H 2 O 2 level (Zeng et al. 2011 ).
1.1 A Brief Story About Discovery on the Pleiotropic Use of ART