Aspartyl Protease Inhibitors as Anti-Filarial Drugs

Abstract

The current treatments for lymphatic filariasis and onchocerciasis do not effectively kill the adult parasitic nematodes, allowing these chronic and debilitating diseases to persist in millions of people. Thus, the discovery of new drugs with macrofilaricidal potential to treat these filarial diseases is critical. To facilitate this need, we first investigated the effects of three aspartyl protease inhibitors (APIs) that are FDA-approved as HIV antiretroviral drugs on the adult filarial nematode, Brugia malayi and the endosymbiotic bacteria, Wolbachia. From the three hits, nelfinavir had the best potency with an IC₅₀ value of 7.78 µM, followed by ritonavir and lopinavir with IC₅₀ values of 14.3 µM and 16.9 µM, respectively. The three APIs have a direct effect on killing adult B. malayi after 6 days of exposure in vitro and did not affect the Wolbachia titers. Sequence conservation and stage-specific gene expression analysis identified Bm8660 as the most likely primary aspartic protease target for these drug(s). Immunolocalization using antibodies raised against the Bm8660 ortholog of Onchocerca volvulus showed it is strongly expressed in female B. malayi, especially in metabolically active tissues such as lateral and dorsal/ventral chords, hypodermis, and uterus tissue. Global transcriptional response analysis using adult female B. pahangi treated with APIs identified four additional aspartic proteases differentially regulated by the three effective drugs, as well as significant enrichment of various pathways including ubiquitin mediated proteolysis, protein kinases, and MAPK/AMPK/FoxO signaling. In vitro testing against the adult gastro-intestinal nematode Trichuris muris suggested broad-spectrum potential for these APIs. This study suggests that APIs may serve as new leads to be further explored for drug discovery to treat parasitic nematode infections.

Keywords: Neglected tropical diseases (NTDs); Trichuris; anti-filarial drugs; filarial nematodes; gastro-intestinal nematodes; macrofilaricidal.

Figure 1

Aspartic protease inhibitor effects on motility in B. malayi. (A) Motility inhibition (% relative to DMSO) of adult female B. malayi treated in vitro with aspartic protease inhibitors over the course of 6 days. * All compounds treated at a concentration of 30 µM, except darunavir and pepstatin A, which were at concentrations of 50 µM. (B) After 6 days of treatment, the three APIs effective on B. malayi were also effective on B. pahangi. * Ritonavir was at 10 µM in B. pahangi; ** Results from Tyagi et al., 2021 [13]. IC₅₀ values of (C) lopinavir, (D) nelfinavir, and (E) ritonavir with adult female B. malayi, based on in vitro treatment for 6 days at six different concentrations, and four replicates per concentration.

Figure 2

Wsp/gst ratio of adult female Brugia malayi. Adult female B. malayi were treated with 100 µM lopinavir, 30 µM nelfinavir, and 30 µM ritonavir and collected on day 1 of the assay.

Figure 3

Sequence conservation and gene expression analysis of the B. malayi aspartic protease Bm8660. (A) Protein sequence-based clustering of the HIV-1 protease and all of the B. malayi aspartic proteases identified by MEROPS (sequence alignment using T-Coffee, clustering with Clustal Omega). The relative gene expression (Z score of log FPKM) and average absolute expression level (log FPKM, in green) of aspartic proteases across the life cycle are shown, based on RNA-seq data collected from Choi et al., 2011 [44]. Bm8660 was the closest B. malayi ortholog to HIV1 protease and had high expression in L4 and adult stages. (B) Sequence-based alignment and clustering of top-hit orthologs of Bm8660 among filarial worms, C. elegans (the lysosomal aspartic protease ASP-4), and human (Cathepsin D, CTSD). Arrows indicate two conserved aspartic protease catalytic triads (DTG).

Figure 4

Localization of Bm8660 in adult female B. malayi. (A) Immunohistological stain using an Ov-APR antibody of an entire cross-section shows strong red staining in the lateral and dorsal/ventral chords, the hypodermis, intrauterine stretched microfilariae, the uterine wall, and the intestinal wall. ((B–F) Immunogold TEM labeling of APR. (B) Section of the hypodermis showing parts of the cuticle, muscles, mitochondria, and a large vacuole. (C) Magnification of (B) showing multiple clusters of gold particles within the large vacuole. (D) Wolbachia in the lateral chord are not labeled by gold particles, but small clusters of particles are found in nearby cytoplasm. (E) Mitochondria are also not stained by the gold particles, but a large cluster of particles can be seen in moderately electron-dense structures in the cytoplasm. (F) A large cluster of gold particles is associated with a vacuole in the vicinity of vesicles released by Wolbachia endobacteria. Lc, lateral chord; dvc, dorsal/ventral chord; u, uterus; I, intestine; m, muscles; hy, hypodermis; va, vacuole; mi, mitochondrion; cu, cuticle; w, Wolbachia; ve, vesicles. Scale bar: (A) 25 µm, (B) 2 µm, (C–F) 500 nm.

Figure 5

RNA-seq analysis of adult female B. pahangi treated with 1% DMSO, 100 µM of lopinavir, nelfinavir, and ritonavir in vitro for 1 h. (A) Principal components analysis (PCA) of RNA-seq samples based on gene expression patterns across all genes. (B) Differential expression analysis using DESeq2 identified significantly (FDR ≤ 0.05) upregulated (↑) and downregulated (↓) genes in API treatments relative to 1% DMSO controls. (C) Significantly differentially expressed APs. “Class” refers to the MEROPS class annotation. Log2 fold change values are relative to DMSO control. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, after FDR adjustment, according to DESeq. NEL = nelfinavir; RIT = ritonavir; Bp_1278201 = BPAG_ 1278201; Bp_0201201 = BPAG_ 0201201; Bp_0106301 = BPAG_ 0106301; Bp_1342901 = BPAG_ 1342901.

Figure 6

Significant KEGG pathway enrichment among (A) genes upregulated and (B) genes downregulated by API treatments in adult female B. pahangi (100 µM for 1 h). The –log p value (FDR-corrected) for each pathway and each gene is represented by the color, and the number of significantly differentially expressed genes from each pathway is represented by the dot size.

Figure 7

Aspartic protease analysis using the whipworm Trichuris muris. (A) Protein sequence-based clustering of the aspartic proteases identified by MEROPS (sequence alignment using T-Coffee, clustering with Clustal Omega). The plot excludes 27 A11A and 38 A28B class aspartic proteases. The relative gene expression (Z score of log FPKM) and the average absolute expression level (log FPKM, in green) of aspartic proteases across the life cycle are shown, based on RNA-seq data collected from Foth et al., 2014 [45]. B. malayi orthologs of T. muris APs with a BLAST E value < 10⁻⁵ are indicated, along with the amino acid sequence similarity (%) of the match. (B) Motility inhibition (% relative to DMSO) of adult T. muris treated in vitro with APIs (50 µM over the course of 3 days).