In vitro efficacy of bumped kinase inhibitors against Besnoitia besnoiti tachyzoites

• Published in: International journal for parasitology 2017-10, Vol.47 (12), p.811-821
• Main Authors: Jiménez-Meléndez, Alejandro, Ojo, Kayode K., Wallace, Alexandra M., Smith, Tess R., Hemphill, Andrew, Balmer, Vreni, Regidor-Cerrillo, Javier, Ortega-Mora, Luis M., Hehl, Adrian B., Fan, Erkang, Maly, Dustin J., Van Voorhis, Wesley C., Álvarez-García, Gema
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Besnoitia besnoiti; Bumped kinase inhibitors; CDPK1; Cell Line; Chemotherapy; Cloning, Molecular; DNA, Protozoan - chemistry; DNA, Protozoan - isolation & purification; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Fibroblasts - cytology; Fibroblasts - parasitology; Fluorescent Antibody Technique; Humans; In vitro assay; Male; Microscopy, Electron, Transmission; Protein Kinase Inhibitors - pharmacology; Protein Kinases - chemistry; Protein Kinases - drug effects; Protein Kinases - genetics; Protein Kinases - isolation & purification; Real-Time Polymerase Chain Reaction; Sarcocystidae - drug effects; Sarcocystidae - genetics; Sarcocystidae - growth & development; Sarcocystidae - ultrastructure; Serial Passage
• ISSN: 0020-7519; 1879-0135
• DOI: 10.1016/j.ijpara.2017.08.005

[Display omitted] •The Besnoitia besnoiti CDPK1 orthologue was identified and cloned.•Activity of recombinant BbCDPK1 was greatly inhibited by a panel of bumped kinase inhibitors (BKIs).•BKIs 1294, 1517, 1553 and 1571 inhibited tachyzoite invasion and proliferation with a parasitostatic effect.•BKIs may be effective candidates to control B. besnoiti infection. Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.