University of Kentucky UKnowledge Veterinary Science Faculty Publications Veterinary Science 9-2017 Extended-Spectrum Antiprotozoal Bumped Kinase Inhibitors: A Review Wesley C. Van Voorhis University of Washington J. Stone Doggett Portland VA Medical Center Marilyn Parsons University of Washington Matthew A. Hulverson University of Washington Ryan Choi University of Washington Follow this and additional works at: https://uknowledge.uky.edu/gluck_facpub See next page for additional authors Part of the Animal Sciences Commons, Immunology of Infectious Disease Commons, and the Parasitology Commons Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Repository Citation Van Voorhis, Wesley C.; Doggett, J. Stone; Parsons, Marilyn; Hulverson, Matthew A.; Choi, Ryan; Arnold, Samuel L. M.; Riggs, Michael W.; Hemphill, Andrew; Howe, Daniel K.; Mealey, Robert H.; Lau, Audrey O. T.; Merritt, Ethan A.; Maly, Dustin J.; Fan, Erkang; and Ojo, Kayode K., "Extended-Spectrum Antiprotozoal Bumped Kinase Inhibitors: A Review" (2017). Veterinary Science Faculty Publications. 45. https://uknowledge.uky.edu/gluck_facpub/45 This Article is brought to you for free and open access by the Veterinary Science at UKnowledge. It has been accepted for inclusion in Veterinary Science Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact [email protected]. Authors Wesley C. Van Voorhis, J. Stone Doggett, Marilyn Parsons, Matthew A. Hulverson, Ryan Choi, Samuel L. M. Arnold, Michael W. Riggs, Andrew Hemphill, Daniel K. Howe, Robert H. Mealey, Audrey O. T. Lau, Ethan A. Merritt, Dustin J. Maly, Erkang Fan, and Kayode K. Ojo Extended-Spectrum Antiprotozoal Bumped Kinase Inhibitors: A Review Notes/Citation Information Published in Experimental Parasitology, v. 180, p. 71-83. © 2017 Elsevier Inc. All rights reserved. This manuscript version is made available under the CC‐BY‐NC‐ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/. The document available for download is the author's post-peer-review final draft of the article. Digital Object Identifier (DOI) https://doi.org/10.1016/j.exppara.2017.01.001 This article is available at UKnowledge: https://uknowledge.uky.edu/gluck_facpub/45 HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Exp Parasitol Manuscript Author . Author manuscript; Manuscript Author available in PMC 2018 September 01. Published in final edited form as: Exp Parasitol. 2017 September ; 180: 71–83. doi:10.1016/j.exppara.2017.01.001. Extended-Spectrum Antiprotozoal Bumped Kinase Inhibitors: A Review Wesley C. Van Voorhisa,b,*, J. Stone Doggettc, Marilyn Parsonsb,d, Matthew A. Hulversona, Ryan Choia, Samuel Arnolda, Michael W. Riggse, Andrew Hemphillf, Daniel K. Howeg, Robert H. Mealeyh, Audrey O. T. Laui, Ethan A. Merrittj, Dustin J. Malyk, Erkang Fanj, and Kayode K. Ojoa,* aDepartment of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, Seattle, WA 98109 USA bDepartment of Global Health, University of Washington, Seattle, WA 98195 USA cPortland VA Medical Center, Portland, OR 97239, United States dCenter for Infectious Disease Research, Seattle, WA 98109 USA eSchool of Animal and Comparative Biomedical Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, Arizona 85721 USA fInstitute of Parasitology, Vetsuisse Faculty, University of Berne, Berne, Switzerland gDepartment of Veterinary Science, University of Kentucky, Lexington, KY 40546 USA hDepartment of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040 iThe National Institutes of Health, NIAID, DEA, 5601 Fishers Lane, Rockville, MD 20892 USA jDepartment of Biochemistry, University of Washington, Seattle, WA 98195 USA kDepartment of Chemistry, University of Washington, Seattle, WA 98195 USA Abstract Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites’ survival and proliferation. In this review, we review efficacy against the kinase target, the parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped-kinase inhibitors. Graphical abstract *Corresponding authors. Wesley C. Van Voorhis, Kayode K. Ojo, Division of Allergy and Infectious Disease, Center for Emerging and Reemerging Infectious Disease (CERID), University of Washington, 750 Republican Street, Box 358061, Seattle, WA 98109 USA. [email protected] (Wesley C. Van Voorhis), [email protected] (Kayode K. Ojo). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Van Voorhis et al. Page 2 Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author Keywords Bumped-kinase inhibitors; Calcium-dependent protein kinase; Gatekeeper residue 1. Introduction Protein kinase inhibitors have been sought for therapy of parasitic diseases, but the challenge of specific inhibition of parasite kinases versus mammalian kinases has limited their practical application. Bumped-kinase inhibitors (BKIs) were originally generated in Prof. Kevan Shokat’s laboratory to build specificity into protein kinase inhibitors that compete with ATP for the kinase active site (Bishop, et al., 1998, Bishop and Shokat, 1999) The “bump” on BKIs precludes their binding to almost all mammalian kinases, since these have a bulky gatekeeper residue in the ATP-binding pocket (see Fig. 1, below). Dr. Shokat’s group pioneered the use of BKIs in genetically-altered mammalian kinases, in which the bulky gatekeeper residue was mutated to glycine or alanine, rendering the mutant kinase susceptible to BKIs (Bishop, et al., 2000, Liu, et al., 1999) If the mutant protein kinase was active, the mutation could be introduced into the corresponding gene in mice, allowing the function of that kinase to be probed anytime during the life of the mice by inhibiting that kinase specifically with BKIs. BKIs had no discernable effects on wild-type mice in control experiments, providing evidence that these compounds as a class have low activity against mammalian kinases and hence low toxicity. We and Dr. Hui’s group determined the structure of Toxoplasma gondii and Cryptosporidium parvum calcium-dependent protein kinase 1 (CDPK1) and immediately noticed that these parasite proteins contain a naturally occurring glycine gatekeeper residue in the ATP binding site (Ojo, et al., 2010, Wernimont, et al., 2010). We reasoned that this active site should therefore be sensitive to BKI inhibition and found that to be the case experimentally. Given the safety and specificity of BKIs demonstrated by Shokat’s group, we embarked on a medicinal chemistry project to optimize BKIs for use against parasites that have CDPKs, primarily apicomplexans. This review describes progress in this area. 2. Structural Basis of Cross-Parasite CDPK inhibition by BKIs CDPKs have no closely related orthologs in vertebrates, but the CDPK kinase domain is similar in sequence and structure to other members of the large family of serine threonine kinases. As with many protein kinases, CDPKs have conformationally distinct active and inactive states that differ in their competence to bind to and act on their protein substrates. CDPK activity is not regulated through phosphorylation or interaction with a partner protein. Instead, regulation is accomplished via a radical reorganization of the calcium-binding Exp Parasitol. Author manuscript; available in PMC 2018 September 01. Van Voorhis et al. Page 3 domain such that in the Ca-bound active state, substrate proteins have unobstructed access to Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author the face of the CDPK containing the active site, while in the inactive state, access to this face of the protein is occluded (Ojo, et al., 2010, Wernimont, et al., 2010). The internal conformation of the active site pocket is unchanged between the active and inactive state. Even the “inactive” state is catalytically competent to phosphorylate small peptide substrates, and crystal structures show that the binding pose of ATP, ATP analogs, and ATP- competitive inhibitors is maintained in both conformations (Murphy, et al., 2010, Wernimont, et al., 2010). Thus, both the active and inactive states of CDPKs are targeted by the BKIs discussed here. The overall ATP binding pocket comprises three areas necessarily shared by all kinases: a region adjacent to the ATP β and 7gamma; phosphates containing the catalytic residues, a relatively hydrophilic