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. 2019 Dec 12;24(24):4559.
doi: 10.3390/molecules24244559.

Coupling the Antimalarial Cell Penetrating Peptide TP10 to Classical Antimalarial Drugs Primaquine and Chloroquine Produces Strongly Hemolytic Conjugates

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Free PMC article

Coupling the Antimalarial Cell Penetrating Peptide TP10 to Classical Antimalarial Drugs Primaquine and Chloroquine Produces Strongly Hemolytic Conjugates

Luísa Aguiar et al. Molecules. .
Free PMC article

Abstract

Recently, we disclosed primaquine cell penetrating peptide conjugates that were more potent than parent primaquine against liver stage Plasmodium parasites and non-toxic to hepatocytes. The same strategy was now applied to the blood-stage antimalarial chloroquine, using a wide set of peptides, including TP10, a cell penetrating peptide with intrinsic antiplasmodial activity. Chloroquine-TP10 conjugates displaying higher antiplasmodial activity than the parent TP10 peptide were identified, at the cost of an increased hemolytic activity, which was further confirmed for their primaquine analogues. Fluorescence microscopy and flow cytometry suggest that these drug-peptide conjugates strongly bind, and likely destroy, erythrocyte membranes. Taken together, the results herein reported put forward that coupling antimalarial aminoquinolines to cell penetrating peptides delivers hemolytic conjugates. Hence, despite their widely reported advantages as carriers for many different types of cargo, from small drugs to biomacromolecules, cell penetrating peptides seem unsuitable for safe intracellular delivery of antimalarial aminoquinolines due to hemolysis issues. This highlights the relevance of paying attention to hemolytic effects of cell penetrating peptide-drug conjugates.

Keywords: Plasmodium; antimalarial; cell penetrating peptide; chloroquine; erythrocyte fluorescence; flow cytometry; hemolysis; microscopy; primaquine; red blood cell.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structures of CQ (1), PQ (2), PQ- CPP conjugates previously disclosed by us (3) [20], and the chloroquine analogue (Cq, 4) bearing a free primary amine to be linked to CPP (Table 1), and afford the first generation of Cq-CPP conjugates (Cq-C4-CPP, 5a-i).
Scheme 1
Scheme 1
Synthesis of conjugates 5a-5i: (i) 20% piperidine in dimethylformamide (DMF) containing 0.1 M of 1-hydroxybenzotriazole (HOBt), microwave irradiation (MWI) for 30 s at 24 W plus 3 min at 28 W; (ii) 5 eq, respective to the resin reactive groups, of the Fmoc-AA-OH in DMF (0.2 M), 5 eq of 0.5 M O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU)/HOBt in DMF, and 10 eq of 2 M N-ethyl-N,N-diisopropylamine (DIPEA) in N-methylpyrrolidone (NMP), MWI for 5 min at 35 W; (iii) trifluoroacetic acid (TFA)/water/triisopropylsilane (TIS) 95:2.5:2.5 v/v/v or TFA/thioanisole/ethane-1,2-dithiol/anisole 90:5:3:2 v/v/v/v cocktail (1 mL/100 mg of resin), room temperature (RT), 120 min; (iv) 5 eq of 6 and of (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 10 eq DIPEA in DMF, RT, overnight; (v) 1.2 eq of succinic anhydride and of DIPEA in DMF, RT, 120 min. Grey sphere stands for the Rink-amide resin used as polymer support used for solid-phase peptide synthesis (SPPS). PG, protecting group.
Figure 2
Figure 2
Structure of conjugate 14′, TP10-S-S-PQ.
Scheme 2
Scheme 2
Synthesis of conjugates 10–12: (i) 5 eq, respective to the resin reactive groups, of the fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc)-AA-OH in DMF (0.2 M), 5 eq of 0.5 M HBTU/HOBt in DMF, and 10 eq of 2 M DIPEA in N-NMP, MWI for 5 min at 35 W; (ii) 20% piperidine in DMF containing 0.1 M of HOBt, MWI for 30 s at 24 W plus 3 min at 28 W; (iii) 5 eq of 6 and of PyBOP, 10 eq DIPEA in DMF, RT, overnight; (iv) TFA/water/TIS 95:2.5:2.5 v/v/v (1 mL/100 mg of resin), RT, 120 min; (v) 1.2 eq of succinic anhydride and of DIPEA in DMF, RT, 120 min; (vi) 6-azido hexanoic acid (1.2 eq), HBTU (1.2 eq), DIPEA (2.4 eq) in DMF, 0 °C → RT, 3 h; (vii) copper(I) bromide (1.2 eq), 2,6-lutidine (10 eq), sodium ascorbate (3 eq), and DIPEA (10 eq), solubilized in a mixture of acetonitrile (ACN) and DMF (1:2 v/v), RT, overnight; (viii) Boc-Cys(Trt)-OH (1.2 eq), HBTU (1.2 eq), DIPEA (2.4 eq) in DMF, 0 °C → RT, 3 h; (ix) TFA:TIS:H2O 95:2.5:2.5 (v/v/v), 1 h, 0 °C → RT; (x) 1 M aqueous acetic acid, RT, 36 h. Grey sphere stands for the Rink-amide resin used as polymer support used for SPPS. Ph, phenyl group.
Scheme 3
Scheme 3
Synthesis of conjugates 13–17: (i) resin treated with 1% TFA in DCM, RT, until no yellow color detected in filtrate; (ii) 5 eq of 6 and of PyBOP, 10 eq DIPEA in DMF, RT, overnight; (iii) 1.2 eq of succinic anhydride and of DIPEA in DMF, RT, 120 min; (iv) 20% piperidine in DMF containing 0.1 M of HOBt, MWI for 30 s at 24 W plus 3 min at 28 W; (v) TFA/water/TIS 95:2.5:2.5 v/v/v (1 mL/100 mg of resin), RT, 120 min; (vi) 1 M aqueous acetic acid, RT, 36 h; (vii) Boc-Cys(Trt)-OH (1.2 eq), HBTU (1.2 eq), DIPEA (2.4 eq) in DMF, 0 °C → RT, 3 h; (viii) TFA:TIS:H2O 95:2.5:2.5 (v/v/v), 1 h, 0 °C → RT; (ix) 2,2′-dithiopyridine (2-PDS, 2.0 eq), glacial acetic acid (0.3 eq), anhydrous MeOH, 18 h, RT; (x) 5(6)-carboxyfluorescein (CF; 5.0 eq), PyBOP (5.0 eq), DIPEA (10 eq), DMF, 2 h, RT.
Figure 3
Figure 3
Flow cytometry analysis of the interaction between hRBC/PiRBC and CF-labelled (A) TP10, (B) Cq-C4-TP10, and (C) Cq-C4-TAT. Upper quadrants (Q1 and Q2) refer to compound-bound cells (green CF fluorescence) and right-hand quadrants (Q2 and Q4) refer to PiRBC (blue Hoechst fluorescence). Q3 includes hRBC, which were not internalized by the CF-labelled test compounds.
Figure 4
Figure 4
Microscopy fluorescence images showing the interaction between hRBC and (A) CF-labeled TP10 (15) and (B) CF-labeled Cq-C4-TP10 (16). Arrows depict “ghost” uninfected erythrocytes.
Figure 5
Figure 5
Microscopy fluorescence images showing the interaction between PiRBC (distinguished by the blue staining of parasites’ nuclei) and (A) CF-labeled TP10 (15) and (B) CF-labeled Cq-C4-TP10 (16). Arrows depict “ghost” Plasmodium-infected erythrocytes.
Figure 6
Figure 6
Microscopy fluorescence images showing an apparent co-localization of (A) CF-labeled TP10 (15) and DNA of parasites released after PiRBC disruption, or of (B) CF-labeled Cq-C4-TP10 (16) and what appears to be PiRBC debris surrounding nuclei of released parasites.

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