Screening of the 'Stasis Box' identifies two kinase inhibitors under pharmaceutical development with activity against Haemonchus contortus

Abstract

Background: In partnership with the Medicines for Malaria Venture (MMV), we screened a collection ('Stasis Box') of 400 compounds (which have been in clinical development but have not been approved for illnesses other than neglected infectious diseases) for inhibitory activity against Haemonchus contortus, in order to attempt to repurpose some of the compounds to parasitic nematodes.

Methods: We assessed the inhibition of compounds on the motility and/or development of exsheathed third-stage (xL3s) and fourth-stage (L4) larvae of H. contortus using a whole-organism screening assay.

Results: In the primary screen, we identified compound MMV690767 (also known as SNS-032) that inhibited xL3 motility by ~70% at a concentration of 20 μM after 72 h as well as compound MMV079840 (also known as AG-1295), which induced a coiled xL3 phenotype, with ~50% inhibition on xL3 motility. Subsequently, we showed that SNS-032 (IC₅₀ = 12.4 μM) and AG-1295 (IC₅₀ = 9.92 ± 1.86 μM) had a similar potency to inhibit xL3 motility. Although neither SNS-032 nor AG-1295 had a detectable inhibitory activity on L4 motility, both compounds inhibited L4 development (IC₅₀ values = 41.24 μM and 7.75 ± 0.94 μM for SNS-032 and AG-1295, respectively). The assessment of the two compounds for toxic effects on normal human breast epithelial (MCF10A) cells revealed that AG-1295 had limited cytotoxicity (IC₅₀ > 100 μM), whereas SNS-032 was quite toxic to the epithelial cells (IC₅₀ = 1.27 μM).

Conclusions: Although the two kinase inhibitors, SNS-032 and AG-1295, had moderate inhibitory activity on the motility or development of xL3s or L4s of H. contortus in vitro, further work needs to be undertaken to chemically alter these entities to achieve the potency and selectivity required for them to become nematocidal or nematostatic candidates.

Keywords: Anthelmintic; Haemonchus contortus; Medicines for Malaria Venture (MMV); Nematodes; Repurposing; Stasis Box; Whole-organism screen.

Fig. 1

Primary screen of 400 individual compounds from the ‘Stasis Box’ from the Medicines for Malaria Venture (MMV) at a concentration of 20 μM identified compound SNS-032 (MMV690767) to inhibit the motility of exsheathed third-stage larvae (xL3) of Haemonchus contortus (at 72 h) by ≥ 70% compared with negative (LB* + 0.5% dimethyl sulfoxide; DMSO) and positive controls (monepantel). Another compound, AG-1295 (MMV079840), was found to inhibit xL3 motility by ~50%, displaying a “coiled” phenotype based on visual inspection of video recordings (see Additional file 1). Other compounds with apparent inhibition of ≥ 50% did not exhibit a characteristic phenotype by visual inspection. Each data point represents the mean of a triplicate (± standard error of the mean, SEM). Chemical structures and physiochemical properties of ‘hit’ compounds SNS-032 and AG-1295 are indicated. Abbreviations: Mw, molecular weight; PSA, polar surface area; FRB, freely rotating bonds; HBD, hydrogen bond donor; HBA, hydrogen bond acceptor; ARC, aromatic ring count; cLogP, calculated partition coefficient

Fig. 2

Dose-response curves for compounds SNS-032 (MMV690767) and AG-1295 (MMV079840) on larval stages of Haemonchus contortus in vitro with reference to the positive-control compound monepantel. Inhibition of motility of (a) third-stage (xL3) and (b) fourth-stage (L4) larvae at 72 h and of development of fourth-stage larvae (L4s) at 7 days (c) of exposure to each of the compound. Assessment of the toxicity of compounds SNS-032 (MMV690767) and AG-1295 (MMV079840) on breast epithelial (MCF10A) cells after 48 h of exposure to each compound in vitro (d). Each data point represents the mean of two to five experiments repeated in triplicate on different days (± standard error of the mean, SEM)

Fig. 3

Multiple-sequence alignment showing levels of similarity in the kinase catalytic domains (Pfam identifier: PF00069) of CDK9 homologs between Haemonchus contortus (Hc-PK-236.1) and human (UniProt accession no.: P50750). a The pairwise sequence alignment was constructed using the program MUSCLE [39]. b Three-dimensional model of CDK9 homolog of H. contortus (Hc-PK-236.1; orange) superimposed on to the crystal structure of human CDK9 (protein data bank (PDB) identifier: 4or5A; blue). The TM-score and root-mean-square deviation (RMSD) values indicate a high-confidence prediction. Conformational differences predicted in G-rich loop, activation loop and α-C helix between the two structures are indicated. The three-dimensional structure for Hc-PK-236.1 was predicted using the program I-TASSER [40] using default parameters