Two key cathepsins, TgCPB and TgCPL, are targeted by the vinyl sulfone inhibitor K11777 in in vitro and in vivo models of toxoplasmosis

RESEARCH ARTICLE Two key cathepsins, TgCPB and TgCPL, are targeted by the vinyl sulfone inhibitor K11777 in in vitro and in vivo models of toxoplasmosis Juan D. Chaparro1¤a, Timmy Cheng2¤b, Uyen Phuong Tran2¤c, Rosa M. Andrade3, Sara B. T. Brenner2, Grace Hwang2¤d, Shara Cohn2¤e, Ken Hirata2, James H. McKerrow4, Sharon L. Reed2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Chaparro JD, Cheng T, Tran UP, Andrade RM, Brenner SBT, Hwang G, et al. (2018) Two key cathepsins, TgCPB and TgCPL, are targeted by the vinyl sulfone inhibitor K11777 in in vitro and in vivo models of toxoplasmosis. PLoS ONE 13(3): e0193982. https://doi.org/10.1371/journal. pone.0193982 Editor: Marcelo U. Ferreira, University of Sao Paulo, BRAZIL Received: August 9, 2017 Accepted: February 19, 2018 Published: March 22, 2018 Copyright: © 2018 Chaparro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. 1 Department of Pediatrics, Division of Infectious Diseases, Rady Children’s Hospital, University of California, San Diego, School of Medicine, La Jolla, California, United States of America, 2 Department of Pathology, University of California, San Diego School of Medicine, La Jolla, California, United States of America, 3 Department of Medicine, Division of Infectious Diseases, University of California, Irvine School of Medicine, Irvine, California, United States of America, 4 Department of Pharmacy, Skaggs School of Pharmacy and Pharmaceutical Science, University of California, San Diego, La Jolla, California, United States of America ¤a Current address: Nationwide Children’s Hospital, 700 Children’s Dr., Columbus, Ohio, United States of America ¤b Current address: Department of Medicine, Division of Pulmonary Medicine and Critical Care, University of California Irvine, Irvine, California, United States of America ¤c Current address: Department of Pathology, University of California Irvine School of Medicine, Irvine, California, United States of America ¤d Current address: Illumina, Inc., San Diego, California, United States of America ¤e Current address: Department of Anesthesiology, Stanford University School of Medicine, Stanford, California, United States of America * Abstract Although toxoplasmosis is one of the most common parasitic infections worldwide, therapeutic options remain limited. Cathepsins, proteases that play key roles in the pathogenesis of toxoplasmosis and many other protozoan infections, are important potential therapeutic targets. Because both TgCPB and TgCPL play a role in T. gondii invasion, we evaluated the efficacy of the potent, irreversible vinyl sulfone inhibitor, K11777 (N-methyl-piperazine-PhehomoPhe-vinylsulfone-phenyl). The inhibitor’s toxicity and pharmacokinetic profile have been well-studied because of its in vitro and in vivo activity against a number of parasites. We found that it inhibited both TgCPB (EC50 = 114 nM) and TgCPL (EC50 = 71 nM) in vitro. K11777 also inhibited invasion of human fibroblasts by RH tachyzoites by 71% (p = 0.003) and intracellular replication by >99% (p<0.0001). In vivo, a single dose of K11777 led to 100% survival of chicken embryos in an model of acute toxoplasmosis (p = 0.015 Cox regression analysis). Therefore, K11777 shows promise as a novel therapeutic agent in the treatment of toxoplasmosis, and may prove to be a broadly effective anti-parasitic agent. Funding: This work was supported by funds from NIAID grant AI41093 (to SLR), the University of California University-wide AIDS Research Program ID04-SD-079 (to SLR), the Rockefeller Brothers Fund (to SLR), Harold Amos Minority Medical Faculty Development Program (to RMA), UCSD Academic Senate Grant 07427A (to RMA), Robert PLOS ONE | https://doi.org/10.1371/journal.pone.0193982 March 22, 2018 1 / 11 A cathepsin inhibitor blocks TgCPB, TgCPL, and invasion by T. gondii Wood Johnson Foundation Grant RWJF 70642 (to RMA), and NIH 2T32A1007036-31AI (to RMA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Toxoplasma gondii is an intracellular protozoan parasite recognized as a pathogen more than 100 years ago. Humans acquire the parasite mainly through ingestion of contaminated undercooked meat, food or water contaminated with feline feces, vertical transmission from mother to fetus, or through blood transfusions or organ transplants [1]. An estimated 22.5% of the population 12 years and older or 60,000,000 people in the US have been infected with toxoplasmosis, and the seroprevalence is markedly higher in developing countries [2,3]. Although acute infection of an immunocompetent host is usually clinically asymptomatic, it leads to lifelong, latent infection. Furthermore, primary infection of the fetus (approximately 1 in 1000 live births in the US) can cause devastating and even fatal disease [4,5]. Additionally, reactivation of latent infection in immunosuppressed individuals, particularly AIDS patients, can manifest as Toxoplasma encephalitis, a uniformly fatal condition if left untreated [2]. The first line of therapy for toxoplasmosis includes a combination of pyrimethamine and sulfadiazine, a regimen more than 50 years old with frequent toxic side effects. Pyrimethamine, a folic acid antagonist, is considered the most effective anti-Toxoplasma agent, but it requires monitoring during therapy for bone marrow suppression and is contraindicated during pregnancy due to teratogenicity. Access to pyrimethamine may also be limited by recent significant increases in the retail price [6]. Sulfadiazine, which acts synergistically with pyrimethamine, is a major cause of drug reactions. This is particularly prevalent in the HIV-infected population, where up to 34% of patients receiving prophylactic trimethoprim-sulfamethoxazole experience fever and rash [7]. These percentages rise to as high as 50% in the setting of active AIDS and Pneumocystis jirovecii pneumonia [8]. These factors led the CDC to designate toxoplasmosis as one of the neglected parasitic infections in the US in 2014. Thus, drug development to treat toxoplasmosis is an important priority. T. gondii tachyzoites can invade any nucleated cell in a process mediated by the sequential release of specialized secretory organelles in an apical complex: micronemes, rhoptries and dense granules. Many of these key proteins require proteolytic processing; more than half of microneme proteins and the majority of rhoptry proteins [9,10] are synthesized as preproproteins that undergo enzymatic maturation before storage and secretion. (...truncated)