Antimalarial activity of the anticancer and proteasome inhibitor bortezomib and its analog ZL3B
- Jennifer M Reynolds†1,
- Kamal El Bissati†1,
- Jens Brandenburg1,
- Arthur Günzl1 and
- Choukri Ben Mamoun1Email author
© Reynolds et al; licensee BioMed Central Ltd. 2007
Received: 10 July 2007
Accepted: 23 October 2007
Published: 23 October 2007
The high rate of mortality due to malaria and the worldwide distribution of parasite resistance to the commonly used antimalarial drugs chloroquine and pyrimethamine emphasize the urgent need for the development of new antimalarial drugs. An alternative approach to the long and uncertain process of designing and developing new compounds is to identify among the armamentarium of drugs already approved for clinical treatment of various human diseases those that may have strong antimalarial activity.
Proteasome inhibitor bortezomib (Velcade™: [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl) amino]propyl]amino]butyl] boronic acid), which has been approved for treatment of patients with multiple myeloma, and a second boronate analog Z-Leu-Leu-Leu-B(OH)2 (ZL3B), were tested against four different strains of P. falciparum (3D7, HB3, W2 and Dd2) that are either sensitive or have different levels of resistance to the antimalarial drugs pyrimethamine and chloroquine.
Bortezomib and ZL3B are equally effective against drug-sensitive and -resistant parasites and block intraerythrocytic development prior to DNA synthesis, but have no effect on parasite egress or invasion.
The identification of bortezomib and its analog as potent antimalarial drugs will set the stage for the advancement of this class of compounds, either alone or in combination therapy, for treatment of malaria, and emphasize the need for large-scale screens to identify new antimalarials within the library of clinically approved compounds.
Malaria is caused by intraerythrocytic protozoan parasites of the genus Plasmodium. It is responsible for more than 300 million clinical cases and over 2 million deaths annually . Plasmodium falciparum, the organism that causes the most lethal form of the disease, is becoming increasingly resistant to almost all available drugs in the antimalarial armamentarium . New chemotherapeutic strategies are therefore urgently needed to combat this disease.
During its intraerythrocytic life cycle, a single P. falciparum parasite undergoes multiple morphological and physiological changes and multiplies to produce up to 36 new daughter parasites in ~48 hours. Large-scale genomic and proteomic analyses revealed a coordinated program of gene and protein expression during parasite intraerythrocytic life cycle [2–7]. The first phase of this program occurs during parasite transition from ring to trophzoite stage and is marked by the induction of expression of enzymes required for biosynthesis of proteins and membranes, nutrient acquisition, and degradation of the host cytoplasm. The second phase occurs during transition from trophozoite to early schizont and is manifested by the induction of expression of enzymes required for biosynthesis of ribonucleotides and deoxyribonucleotides and for DNA replication. The third phase occurs during parasite schizogony and is marked by the induction of subunits of the proteasome. The last phase of this program occurs during late schizogony and immediately after invasion and becomes evident by the expression of specific proteins required for host cell invasion . The rise and fall of expression of subsets of proteins during specific stages of parasite intraerythrocytic life cycle suggest a coordinated control of protein turnover during parasite development. In eukaryotes, such regulation is controlled by the proteasome.
Proteasomes are multicatalytic protease complexes whose principle task is the selective degradation of proteins within the cell. Although a fully intact proteasome has not been isolated from P. falciparum, the sequencing of this organism revealed a complete set of ORFs encoding homologs of eukaryotic subunits of the proteasome [8–10]. The expression of seven α and six β subunits of the 20S particle and 16 subunits of the 19S regulatory particle of the putative P. falciparum proteasome suggest an important role for this multicatalytic complex in parasite intraerythrocytic cycle. Interestingly, this expression peaks during parasite transition from developmental, structural and metabolic functions to more specialized functions important for the generation of new daughter parasites capable of completing the cycle and invading new host cells [5, 6]. This suggests that the parasite proteasome could play an important role in protein turnover and parasite replication. Accordingly, the proteasome inhibitor lactacystin was found to inhibit erythrocytic schizogony of P. falciparum prior, but not subsequent, to DNA synthesis and parasite multiplication .
Several studies have highlighted the importance of proteasome inhibition as a possible approach for the treatment of cancer and parasitic diseases [11–13]. Lindenthal and colleagues showed that the boronate analog MLN-273 blocks the exoerythrocytic development of P. berghei and the intraerythrocytic development of P. falciparum .
Here we provide data indicating that the proteasome inhibitor and analog of MLN-273, bortezomib (Velcade™: [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl) amino]propyl]amino]butyl] boronic acid), which has been approved for treatment of patients with multiple myeloma, and a second boronate analog Z-Leu-Leu-Leu-B(OH)2 (ZL3B), which was found to be highly toxic to trypanosomatid parasites (IC50 of 0.32 nM in culture; ) are potent inhibitors of P. falciparum.
Bortezomib was the first proteasome inhibitor shown to have anti-cancer activity and to induce a marked and durable response in patients with multiple myeloma in clinical trials . We have tested bortezomib and ZL3B in different strains of P. falciparum including strains that are resistant to pyrimethamine and chloroquine. We found that both compounds are equally effective against drug-sensitive and -resistant parasites with inhibitory concentrations in the low nanomolar range. The compounds block intraerythrocytic development prior to DNA synthesis, but had no effect on parasite egress or invasion.
The clones 3D7, HB3, Dd2, and W2 of P. falciparum used in this study were obtained from the Malaria Research and Reference Reagent Resource Center (MR4).
Cell Culture and Materials
Parasites were cultured by the method of Trager and Jensen  by using a gas mixture of 3% O2, 3% CO2, and 94% N2. RPMI medium 1640 was supplemented with 30 mg/liter hypoxanthine (Sigma), 25 mM Hepes (Sigma), 0.225% NaHCO3 (Sigma), 0.5% Albumax I (Life Technologies, Grand Island, NY), and 10 μg/ml gentamycin (Life Technologies). Bortezomib was purchased from the University of Connecticut Health Center Pharmacy. ZL3B was purchased from Boston Biochemical Inc. (Cat# I-120). Parasite synchronization was obtained with three successive 5% sorbitol treatments . To determine visually the stage of the parasite life cycle, fixed smears of the P. falciparum-infected erythrocytes were stained with Giemsa stain and analyzed by bright-field microscopy.
Hypoxanthine incorporation assay
The susceptibility of parasites to different compounds was assessed by tritiated hypoxanthine uptake as described by Desjardins and colleagues . Briefly, infected erythrocytes (2% hematocrit, 3% rings) were washed and incubated with the appropriate drugs at the listed concentrations in hypoxanthine-free media for 48 hours. 200 μL of the mixture was then added to a 96 well plate with 3H-hypoxanthine at a concentration of 0.5 μCi/well. Following an incubation of 24 hours, the cells were washed on an ultrafilter and radioactivity was counted using a scintillation counter. IC50's are represented in nM. Values are means ± standard deviation of three independent experiments each performed in triplicate. These experiments were performed at least three times with similar results.
Results and Discussion
ZL3B and bortezomib inhibit the P. falciparum intraerythrocytic cycle
50% Inhibitory concentrations IC50 (nM) of ZL3 B, bortezomib, chloroquine and pyrimethamine in P. falciparum strains
ZL 3 B
40 ± 12
45 ± 5.8
34 ± 3.9
40 ± 11.1
31 ± 1.8
31 ± 2.7
43 ± 4
37 ± 5.1
6 ± 0.2
8 ± 1.4
90 ± 3.7
300 ± 21
5 ± 1.1
500 ± 45
1500 ± 5.8
2500 ± 97
Bortezomib and ZL3B antimalarial activities occur prior to DNA synthesis
The recommended adult dose of bortezomib for treatment of myeloma is 1.3 mg/m2; and in children the compound is used at a dose of 1.2 mg/m2 [22–24]. The mean peak plasma concentration (Cmax) determined 5 min after drug administration at doses between 1.3 and 1.7 mg/m2 was 63 ± 16 ng/ml and the mean area under the concentration-time curve extrapolated to infinity (AUCinf) was 27 h ng/ml . These values are 2 to 4-fold the IC50 observed with bortezomib in P. falciparum. In vivo studies to determine the dose and tolerability of this compound for treatment of malaria are warranted.
Our studies demonstrate that two boronates, ZL3B and its clinically-approved analog bortezomib, are potent inhibitors of the intraerythrocytic cycle of both drug-sensitive and resistant P. falciparum strains. These findings will set the stage for the evaluation of this new class of compounds for treatment and/or prophylaxis of falciparum malaria. Furthermore, our studies set the stage for large-scale screens to identify new antimalarials among clinically approved drugs. This approach could shorten the lengthy and expensive process of designing, developing and testing the potency, efficacy and safety of new drugs.
We are grateful to Harriett Zawistowski (General Clinical Research Center, University of Connecticut Health Center) for technical help. We thank Dr. David Sullivan for technical assistance. This research was supported by NIH and DOD grants AI51507, AI58962, PR033005 and BWF award 1006267 to CBM and NIH grant AI059377 to AG. UCHC General Clinical Research Center is supported by NIH Grant M01RR06192. CBM is a recipient of the Burroughs Wellcome Award, Investigators of Pathogenesis of Infectious Disease.
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