Selective inhibition of PfA-M1, over PfA-M17, by an amino-benzosuberone derivative blocks malaria parasites development in vitro and in vivo

Background Plasmodium falciparum M1 family aminopeptidase is currently considered as a promising target for anti-malarial chemotherapy. Several series of inhibitors developed by various research groups display IC50/Ki values down to nM range on native PfA-M1 or recombinant forms and block the parasite development in culture at µM to sub-µM concentrations. A handful of these inhibitors has been tested on murine models of malaria and has shown anti plasmodial in vivo activity. However, most of these inhibitors do also target the other neutral malarial aminopeptidase, PfA-M17, often with lower Ki values, which questions the relative involvement and importance of each enzyme in the parasite biology. Results An amino-benzosuberone derivative from a previously published collection of chemicals targeting specifically the M1-aminopeptidases has been identified; it is highly potent on PfA-M1 (Ki = 50 nM) and devoid of inhibitory activity on PfA-M17 (no inhibition up to 100 µM). This amino-benzosuberone derivative (T5) inhibits, in the µM range, the in vitro growth of two P. falciparum strains, 3D7 and FcB1, respectively chloroquino-sensitive and resistant. Evaluated in vivo, on the murine non-lethal model of malaria Plasmodium chabaudi chabaudi, this amino-benzosuberone derivative was able to reduce the parasite burden by 44 and 40% in a typical 4-day Peters assay at a daily dose of 12 and 24 mg/kg by intraperitoneal route of administration. Conclusions The evaluation of a highly selective inhibitor of PfA-M1, over PfA-M17, active on Plasmodium parasites in vitro and in vivo, highlights the relevance of PfA-M1 in the biological development of the parasite as well as in the list of promising anti-malarial targets to be considered in combination with current or future anti-malarial drugs. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-2032-4) contains supplementary material, which is available to authorized users.


Pharmacological properties
Determination of pharmacological properties of T5 was performed by TechMed ILL , ESBS, Illkirch, France. Compounds are stored as 10 mM solution in DMSO at 4°C. After ultracentrifugation at 15000g for 10 min, the concentration in the supernatant was measured by a HPLC procedure on a kinetex 2.6µm C18 100A 50x4.6 mm column using a calibration line established for the compound by diluting the 10 mM DMSO stock solution to adapted concentrations.

Solubility assays
The injection volume was 20 µL, the mobile phase flow rate was 2 mL/min and the following program was applied for the elution: 0-0.1 min, 5% B; 0.1-2.6 min, 5-95% B; 2.6-3.1 min, 95% B; 3.1-3.3 min, 95-5% B and 3.3-6 min, 5% B. Solvent A was a mixture of 0.05% trifluoroacetic acid in water and solvent B was acetonitrile. The detection wavelength was 254 nm and the retention time was 1.64 min. Table S2. Thermodynamic solubilities of compound T5 in PBS with different excipients.

CHI -logD determination
Chromatographic Hydrophobicity Indices (CHI) were determined according to an original procedure, based on a reverse phase fast HPLC gradient on a Luna C18(2) 5µ 100A 50 x 4.6mm column. First, a solution with 10 reference compounds with known CHI values was injected onto the HPLC to generate a calibration line from their retention time (CHI = 59.049*t R -56.021, R 2 = 0.9932). The concentration of the mixture was 0.1 mg/mL for each compound and the injected volume was 5 µL. T5 was analysed on the same system. The 10 mM DMSO stock solution was diluted to 200 µM in CH 3 CN / 50 mM ammonium acetate pH 7.4 (3:7 v/v) and 5 µL were injected. The mobile phase flow rate was 2 mL/min and the following program was applied for the elution: 0-0.1 min, 0% B; 0.

Plasma protein binding protocol-Rapid Equilibrium Dialysis (RED) Assay Conditions
Single use RED device inserts were positioned in the PTFE RED base plate. Each insert consisted of 2 chambers separated by a vertically aligned semipermeable cellulose membrane with a molecular weight cut off (MWCO) of 12 kDa. Spiking solutions of T5 and warfarin were diluted in DMSO to 100 µM (10 µL of stock solution + 990 µL of DMSO). Human plasma was spiked with compound T5 or verapamil, which served as binding positive control, to achieve final concentrations of 1 µM. The final percentage of DMSO in the plasma incubation samples was 1%. Spiked plasma (200 µL) was added to the red chamber of the RED device insert and PBS (350 µL) was added to the white chamber. The samples were allowed to dialyze for 4 hours at 37°C in an orbitaler while agitating at a rate of 250 rpm. Each sample was incubated in duplicate. At the end of the incubation, 70 µL aliquots were removed from each chamber and matrix-matched, i.e., an equal volume of PBS was added to the plasma test incubation sample and an equal volume of plasma was added to the PBS test incubation sample. Samples where protein precipitated with 350 µL acetonitrile were freezed before analysis. After thawing, the samples were vortex-mixed for 3 min and sonicated for 3 min. The samples were then centrifuged at 15 000g for 5 min at 4°C to precipitate any proteins. The supernatants were analysed by UHPLC on a kinetex 2.6µm C18 100A 50x2.1 mm column coupled with a Shimadzu LCMS-8030 triple quadrupole using a calibration line established for the compound by diluting a 10 mM DMSO stock solution to adapted concentrations (0.05-2 µM). 1 µL of each sample was injected. The mobile phase flow rate was 0.5 mL/min and the following program was applied for the elution: 0 min, 5% B; 0-1.2 min, 5-95% B; 1.2-1.4 min, 95% B; 1.4-1.42 min, 95-5% B and 1.42-2.8 min, 5% B. Solvent A was a mixture of 0.05% formic acid in water and solvent B was acetonitrile. The detection wavelength was 254 nm. The unbound fraction was determined as the ratio of the peak area in buffer to that in plasma. Positive control was performed using verapamil (experimental F u 10%, literature value 8%).

CYP inhibition
The compound T5 (0.1−10 µM) was incubated with human liver microsomes (BD Biosciences, Switzerland) (0.05, 0.16 and 0.2 mg/mL, for 3A4, 2D6 and 2C9 inhibition assays, respectively) and NADPH (1 mM) in a 100 mM potassium phosphate buffer pH 7.4 in the presence of a cytochrome P450 isoform-specific probe substrate (diclofenac for CYP2C9, bufuralol hydrochloride for CYP2D6, and midazolam maleate for CYP3A4) in a 37 °C water bath for the appropriate incubation time (10, 30 and 15 min for 3A4, 2D6 and 2C9, respectively). The reactions were terminated by adding ice-cold acetonitrile containing an internal standard followed by vortex mixing. The samples were then centrifuged at 15 000g for 10 min at 4 °C to precipitate the microsomal proteins. After centrifugation, the supernatants were analyzed by an UHPLC on a kinetex 2.6µm C18 100A 50x2.1 mm column coupled with a Shimadzu LCMS-8030 triple quadrupole. For each isoform, a selective inhibitor (sulfaphenazole for CYP2C9, quinidine for CYP2D6, and ketoconazole for CYP3A4) was used as a positive control. The IC50 values were calculated by nonlinear regression analysis from the plotted remaining metabolic activity at each test compound concentration.

In vivo Pharmacokinetics
Female CD-1 mice were intravenously administered a 2 mL/kg solution of T5 (3mg/kg dose; 1.5mg/mL of T5 dissolved in PBS solution containing 10% w/v hydroxypropyl-β-cyclodextrin). In additional studies, mice were intraperitoneally administered a 10 mL/kg solution of T5 (3mg/kg dose; IV solution diluted 1:5 with PBS solution) or orally administered a 10 mL/kg solution of T5 (3mg/kg dose; IV solution diluted 1:5 with distilled water). The animals were sacrificed at 5, 15 and 30 minutes, 1, 2, 4, 6, and 8 hours after administration (n=3 per time), and blood samples were collected by cardiac puncture. The plasma was separated by centrifugation and stored frozen at -80°C. 400 µL of each plasma sample were mixed with 1 mL of acetonitrile, followed by vortex mixing for 3 min and sonication for 3 min. The samples were then centrifuged at 15 000g for 5 min at 4°C to precipitate any proteins. After centrifugation, the concentration in the supernatants were analyzed by UHPLC on a kinetex 2.6µm C18 100A 50x2.1 mm column coupled with a Shimadzu LCMS-8030 triple quadrupole using a calibration line established for the compound by diluting a 10 mM DMSO stock solution to adapted concentrations (0.05-2 µM). 1 µL was injected. The mobile phase flow rate was 0.5 mL/min and the following program was applied for the elution: 0 min, 5% B; 0-1.2 min, 5-95% B; 1.2-1.4 min, 95% B; 1.4-1.42 min, 95-5% B and 1.42-2.8 min, 5% B. Solvent A was a mixture of 0.05% formic acid in water and solvent B was acetonitrile. The detection wavelength was 254 nm. Pharmacokinetic parameters were derived from the blood concentration time curve using the non-compartmental analysis of PK Solver 2.0 [1].