Cyclic Tetrapeptide HDAC Inhibitors with Improved Plasmodium falciparum Selectivity and Killing Profile

Abstract

Cyclic tetrapeptide histone deacetylase inhibitors represent a promising class of antiplasmodial agents that epigenetically disrupt a wide range of cellular processes in Plasmodium falciparum. Unfortunately, certain limitations, including reversible killing effects and host cell toxicity, prevented these inhibitors from further development and clinical use as antimalarials. In this study, we present a series of cyclic tetrapeptide analogues derived primarily from the fungus Wardomyces dimerus that inhibit P. falciparum with low nanomolar potency and high selectivity. This cyclic tetrapeptide scaffold was diversified further via semisynthesis, leading to the identification of several key structural changes that positively impacted the selectivity, potency, and in vitro killing profiles of these compounds. We confirmed their effectiveness as HDAC inhibitors through the inhibition of PfHDAC1 catalytic activity, in silico modeling, and the hyperacetylation of histone H4. Additional analysis revealed the in vitro inhibition of the most active epoxide-containing analogue was plasmodistatic, exhibiting reversible inhibitory effects upon compound withdrawal after 24 or 48 h. In contrast, one of the new diacetyloxy semisynthetic analogues, CTP-NPDG 19, displayed a rapid and irreversible action against the parasite following compound exposure for 24 h.

Keywords: HDAC; PfHDAC1; Plasmodium; cyclic tetrapeptide; malaria; natural products.

Figure 1.

Activity of common hydroxamic acid and cyclic tetrapeptide HDAC inhibitors. Selectivity index (SI) for Apicidin B and SAHA based on HepG2 EC₅₀ / Dd2 and 3D7 EC₅₀ Average. Values for TSA and HC Toxin from reference 7, and SI based on 3D7 and W2 EC₅₀ average. Results are expressed as means from triplicate experiments +/− SEM.

Figure 2.

Linear regression of enzyme and in vitro inhibition values. Average in vitro pEC50s calculated as the negative log of the 3D7 and Dd2 average when converted to molar. Average enzyme pCI₅₀ calculated as the negative log of pfHDAC1 activity when converted to molar. Dotted lines represent the 99% confidence interval. Color key represents classification of R₂ group. Correlation as calculated by two-tailed Pearson test p < 0.0001. For purposes of analysis, if no IC₅₀ of EC₅₀ value below the maximum concentration tested was available, whole integers were substituted for greater than values (> 5 changed to 5, > 10 changed to 10).

Figure 3.

Rate of killing of compounds 6 and 27. Parasites were treated for 24 (A-B) or 48 h (C-D) prior to compound wash off. Flow cytometry with SYBR Green I and Mitotracker Deep Red FM (MTR) was performed after compound wash-off and every 24 h after for 6 days (100,000 events per sample) to track parasitemia (A, C). Parasites (SYBR+) were gated as either MTR+/MTR− and the results are expressed as the fraction of mitotracker positive parasites at each collection point, with the variance between all points expressed via box and whisker plot. Results expressed as means from duplicate experiments +/− SEM.

Figure 4.

Structure and properties of compounds 6, 27, and potential active metabolite 9. The microsomal stability of compounds 6 and 27 is reported as the percent of compound remaining after a 1 h incubation with the corresponding murine liver enzymes. Solubility studies were performed at room temperature in PBS, pH 7.4.

Figure 5.

(A, B) Hyperactylation of HDAC inhibitors after 4 h treatment with 5x EC₅₀. (B) Average band intensity of 3 western blots normalized to aldolase. Significance seen between DMSO and SAHA (p < 0.05), DMSO and cmpd. 2 (p < 0.05), DMSO and cmpd. 27 (p < 0.05), and DMSO and cmpd. 6 (p < 0.01). (C) Parasites treated in ring and collected in troph prior to incubation in chambered slides. Probing preformed with anti-acetyl histone H4 antibody in addition to anti-alpha tubulin and Hoechst.

Figure 6.

Stage specific inhibition following inhibitor treatment at 5 × EC₅₀ in synchronous Dd2 parasites at select timepoints. Parasites monitored every 12 h until second cycle (54 HPI) using YOYO-1 and Geimsa thin smears. (A) Treatment at 6 HPI with compounds 6, 27, or SAHA. Life cycle graph representative of intraerythrocytic stage in the DMSO vehicle control at selected times of 6, 18, 30, 42, and 54 HPI. (B) Treatment at 18 HPI. (C) Treatment at 30 HPI. (D) Treatment at 42 HPI. Images and flow for each timepoint shown taken simultaneous from the same replicate. Total results representative of 3 independent replicates, with the displayed results chosen from replicate 1 or 2.