Functional studies of Plasmodium falciparum dipeptidyl aminopeptidase I using small molecule inhibitors and active site probes

Abstract

The widespread resistance of malaria parasites to all affordable drugs has made the identification of new targets urgent. Dipeptidyl aminopeptidases (DPAPs) represent potentially valuable new targets that are involved in hemoglobin degradation (DPAP1) and parasite egress (DPAP3). Here we use activity-based probes to demonstrate that specific inhibition of DPAP1 by a small molecule results in the formation of an immature trophozoite that leads to parasite death. Using computational methods, we designed stable, nonpeptidic covalent inhibitors that kill Plasmodium falciparum at low nanomolar concentrations. These compounds show signs of slowing parasite growth in a murine model of malaria, which suggests that DPAP1 might be a viable antimalarial target. Interestingly, we found that resynthesis and activation of DPAP1 after inhibition is rapid, suggesting that effective drugs would need to sustain DPAP1 inhibition for a period of 2-3 hr.

Figure 1. Selective inhibition of DPAP1 results in parasite death

(A) Structure of Ala-4(I)Phe-DMK. (B) DPAP and Falcipain (FP) inhibition by Ala-4(I)Phe-DMK. Trophozoites or merozoites lysates were incubated for 30 min with 10 µM of Ala-4(I)Phe-DMK followed by 1h labeling with FY01 or ¹²⁵I-DCG04. Labeling was analyzed by fluorescent scan of SDS-PAGE gels or autoradiography, respectively. Loss of labeling indicates inhibition of the indicated protease. (C) Ala-4(I)Phe-DMK acts as an irreversible inhibitor. Trophozoite lysates were incubated with inhibitor for 0.1–2h in acetate buffer. Residual activity was measured as the turnover rate of the DPAP1 specific substrate (Pro-Arg)₂-Rho. The k_i was obtained by fitting the data to eq 1. Standard errors of the fitted parameters are shown in parentheses. (D) Ala-4(I)Phe-DMK kills P. falciparum at low nM concentrations. Ring stage parasites were treated with inhibitor and cultured for ~75 h. Parasitemia was quantified by FACS, and the EC50_Pot determined by fitting the data to a dose response curve. (E) Phenotypic effects due to Ala-4(I)Phe-DMK treatment. Rings (upper pictures) or schizonts (lower pictures) were cultured for 24h in the presence of inhibitor. Phenotypic effects were visualized in Geimsa-stained thin blood smears. See also Figure S1.

Figure 2. Stability requirements for DPAP1 inhibitors

(A) Trophozoites are more susceptible to DPAP1 inhibition. Parasites at the indicated life stages were treated for 1h with Ala-4(I)Phe-DMK. Decrease in parasitemia was determined by FACS. Error bars show the standard error of 3 independent measurements. (B) Structure of SAK2 (C) Peptidic inhibitors are not stable in mouse serum. The stability of SAK2 and Ala-4(I)Phe-DMK for DPAP1 was determined by measuring their ability to block labeling of DPAP1 by FY01 either before (top) or after (bottom) overnight incubation in mouse serum. (D) DPAP1 activity is rapidly restored in living parasites. A culture of mixed-stage parasites was treated with 0.16–100 µM of SAK2 or 0.8–2500 nM of Ala-4(I)Phe-DMK for 0.5–7h. After cell lysis, DPAP1 activity was measured using the DPAP1 specific substrate (Pro-Arg)₂-Rho. Representative data are shown in the upper graph. The IC50_DPAP1 values for SAK2 and Ala-4(I)Phe-DMK at each treatment time are shown in the bar graph. The first two bars indicate the EC50_Pot for each compound measured in the standard 75 h replication assay. Error bars represent the standard deviation of the fitted parameter. See also Figure S2.

Figure 3. In silico design of non-peptidic inhibitors for DPAP1

(A) Docking of HN3019 and ML4118S into the active site of the DPAP1 model. HN3019 (green) and ML4118S (cyan) were docked as the final covalent product of the reaction between the inhibitor and the catalytic cysteine. The structure of the DPAP1 model bound to HN3019 is shown as an electrostatic surface. (B) Docking of ML4118S (cyan) and ML4118R (orange) into the active site of the DPAP1 model. See also Figure S3.

Figure 4. ML4118S is 500-fold more potent than ML4118R

(A) Structures. ML4118 is a mixture of the R and S diastereomers. (B) Specificity towards DPAPs and flacipains (FPs) measured using the competition assay. (C) DPAP1 Inhibition in intact parasites vs. lysates. Purified schizonts (filled symbols) or lysates (empty symbols) were treated with ML4118S (triangles), ML4118 (diamonds), or ML4118R (circles) followed by labeling with FY01 for 1h. (D) Potency of killing parasites determined using a 75h replication assay. (E) Specificity vs. potency vs. toxicity. IC50 values were obtained by quantifying the gel bands shown in B. EC50_Pot values were determined by FACS after treating schizonts for 1h with compound, followed by 3 washes with media, and culture of the parasites for another 48h. Cell toxicity was determined by incubating HFF cells for 24h with inhibitor and measuring ATP production using the CellTiter-Glo® assay. Error bars represent the standard deviation of the fitted parameter.

Figure 5. ML4118S is stable in mouse serum and sustains DPAP1 inhibition

(A) Serum stability of ML4118S. The stability was determined by measuring their ability of the ML41118S to block labeling of DPAP1 by FY01 either before (top) or after (bottom) overnight incubation in mouse serum. (B) Kinetics of DPAP1 inhibition by ML4118S in intact parasites. DPAP1 activity in a mix-stage parasite culture was measured after treatment with ML4118S over a range of times. DPAP1 activity was measured using the DPAP1 specific substrate (Pro-Arg)₂-Rho.

Figure 6. Non-peptidic DPAP1 inhibitors decrease parasite load in vivo

(A&B) Specificity of ML4118S (A) and KB16 (B) for PbDPAP1, BP1, and BP2 using competition assays in P. berghei lysates. (C) Parasite growth in mice after treatment with ML4118S and KB16. Treatment with 20 mg/Kg of compound via IP is indicated with black (KB16, n=4) or grey (ML4118S, n=2–5) dots. Parasitemia was quantified by Geimsa-stained thin blood smears (days 2–6) or by FACS (days 7–15). Control mice were treated either following the ML4118S (n=2) or the KB16 (n=3) treatment schedule. No difference in parasitemia was observed between the two sets of control mice. The value reported for the vehicle-treated mice is the average of the 5 control mice. Asterisks indicate significant differences between vehicle and drug-treated mice (p-values were determined using the Student T-test: *, p > 0.95; **, p > 0.99; ***, p > 0.999).