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Cite This: J. Med. Chem. 2017, 60, 9974−9975
Novel Molecules To Treat Chronic Central Nervous System Toxoplasmosis Sandhya Kortagere* Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, Pennsylvania 19129, United States ABSTRACT: A major challenge in treating toxoplasmosis in immunocompromised patients is lack of therapeutic agents to clear chronic infection and stop the recrudescence of infection after therapy. CDKP1 has emerged as a novel target for treating chronic infections and eliminating latent bradyzoites in the brain. In a mouse model of toxoplasmosis, pyrazolopyrimidine inhibitors of Toxoplasma gondii CDPK1 demonstrated in vitro and in vivo efficacy.
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inhibitory junction calcium activation domains. Sequencing of the apicomplexan genomes has led to the identification of CDPKs from several members of this phylum including Toxoplasma gondii, Plasmodium falciparum, Cryptosporidum parvum, and Sarcocystis neurona. Phylogenetic analysis of the genomes has suggested that apicomplexan CDPKs can be classified into several clades; however, the annotations of orthologs have been found to be inconsistent. The CDPK1 gene (TgCDPK1) in Toxoplasma gondii and Cryptosporidium parvum is annotated as CDPK4 in the Plasmodium genome.2 In addition, there are structural differences among the apicomplexan CDPKs that includes changes to the length of the Nterminal domain, variability in the number of EF hands, and their location with reference to the kinase domain. There are two major factors that have led to the nomination of TgCDPK1 as a unique target for treating toxoplasmosis. TgCDPK1 has been demonstrated in several studies to be essential for various functions of Toxoplasma gondii including adhesion, secretion, motility, cell invasion, and egress. Second, the crystal structure of TgCDPK1 showed the presence of an extended ATP binding pocket compared to the human kinases (Figure 1). Analysis of this feature suggested that the TgCDPK1 has a small glycine residue occupying the gate keeper position which is otherwise filled by large hydrophobic residues in the human host. In fact, TgCDPK1 is the only kinase in Toxoplasma gondii that exhibits this change at the gate keeper residue position, suggesting this target can be optimized to develop small molecule inhibitors with high specificity to the parasite. The extended ATP binding pocket also provides opportunities to design unique small molecules that could include signatures of ATP mimics coupled with other hydrophobic moieties such as the bump kinase inhibitors3 and the pyrazolopyrimidine (PP) compounds1 that can occupy the extended hydrophobic groove. The gate keeper residues at the ATP binding pocket are known to confer specificity to the human kinase inhibitors.4 The specificity of the PP molecules in this study suggests that this could be a universal phenomenon among all kinase inhibitors including the TgCDPK1.
urrent therapy for toxoplasmosis includes a combination dose of broad spectrum antiparasitic drugs such as pyrimethamine and sulfadiazine. These drugs inhibit the parasite growth by blocking the Toxoplasma gondii nuclei acid biosynthesis pathway. While they may be effective against actively replicating forms of the parasite such as the tachyzoites to clear the infection, they are ineffective against the latent bradyzoite form, which contributes to chronic disease. These bradyzoites form cysts in the tissues, remain dormant, and revert to active lytic forms in immunocompromised individuals, triggering the recurrence of the disease. Moreover, current therapeutics have not been effective against Toxoplasma gondii infections in the brain due to their limitations in blood−brain barrier penetrability. Hence, there is a significant unmet need to develop newer targeted therapies for chronic infections but also against dormant bradyzoites in tissues and to treat latent infections and prevent the recurrence of toxoplasmosis. A therapeutic solution to this unmet need will be a small molecule drug that has high specificity to Toxoplasma gondii and preferably at all stages of its life cycle including the cysts and with no toxicity to the human host. In the quest for such a drug target, calcium dependent protein kinase 1 (CDPK1) has emerged as a major player. The study published in the current issue of Journal of Medicinal Chemistry by Rutaganira et al.1 highlights the preclinical structure-based optimization of a novel series of pyrazolopyrimidine molecules for drug-like properties and in vivo efficacy to treat both acute and chronic infections in a mouse model of toxoplasmosis. The first reports of the activity and function of CDPKs were reported nearly 2 decades ago from investigations of soybean and Arabidopsis. Since these early discoveries, several other plant species ranging from green algae to angiosperms have been described to have CDPKs in their genomes. A search for the presence of CDPKs using genome comparison and sequence analysis methods has suggested that there exists a common evolutionary origin for these CDPKs from plants to protozoans but is conspicuously absent in higher eukaryotes. However, mammalian species have several calcium and calmodulin dependent kinases that are central to the myriad signaling cascades governing their activities. These are indeed distantly related to the CDPKs and share common structural elements such as the N-terminus, ATP binding sites, auto© 2017 American Chemical Society
Received: November 10, 2017 Published: December 4, 2017 9974
DOI: 10.1021/acs.jmedchem.7b01663 J. Med. Chem. 2017, 60, 9974−9975
Journal of Medicinal Chemistry
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Figure 1. Structural differences between the ATP binding pocket of T. gondii and human Src kinase. (A) Crystal structure of TgCDPK1 depicted in orange ribbons and in complex with the inhibitor (compound 1) bound to ATP binding pocket and the gate keeper residue glycine 128 shown as licorice sticks and colored atom type (C, cyan; O, red; N, blue; S, yellow). (B) Close-up view of compound 1 in the ATP binding pocket shown as surface model and colored atom type. (C) Close-up view of bosutinib bound to a human Src kinase (PDB code 4mxo) at the ATP binding pocket with the gate keeper residue threonine 341 which directly interacts with the inhibitor.
in vitro parasite killing assays and ex vivo assays and in treating acute and chronic infections in mouse models of toxoplasmosis. It is clear from this study that pyrazolopyrimidine or pyrrolopyrimidine molecules exemplified by compound 24 have a good potential to be advanced to a clinical candidate; however, more research needs to be done to improve its pharmacokinetics in plasma and brain and the safety profile of this series of molecules.
One of the major challenges in designing and developing small molecule inhibitors is the simultaneous optimization of the lead molecule for efficacy and drug-like properties. In this study, the pyrazolopyrimidine or pyrrolopyrimidine core containing molecules were tested for specificity to TgCDPK1 and hSrc kinase and were optimized for metabolic stability and efficacy by sampling several methylene and ether linkages. It was clear from the optimization studies that the ether linkages provided both the metabolic stability and parasite specificity/ efficacy. Designing a kinase inhibitor requires a right balance of size, hydrophobicity, and the presence of favorable hydrogen bonding partners such as protonatable amines in the molecules or water molecules that can bridge these interactions.4 This optimization often leads to a trade-off between potency and permeability (increase in clogP), and stability of the molecules. New methods to include multiple end points such as parasite efficacy, CDPK inhibition, microsomal stability while maintaining specificity to the parasite CDPK1 described in Figures 1−3 of Rutaganira et al.1 are essential in developing an optimal lead candidate. Several compounds have been shown to treat the acute infection using either cultured parasites or ex vivo preparations, but none have progressed into clinical trials for treating toxoplasmosis. In the Rutaganira et al. study,1 the lead molecule designated as compound 24 was chosen to be tested in acute and chronic toxoplasmosis in a mouse model. Pharmacokinetic studies for compound 24 using oral dosing indicated modest half-life (1.5 h) and clearance rate and good oral bioavailability. While the brain levels of compound 24 were not directly assessed, they were modeled based on the plasma parameters and the brain to plasma ratio was found to be 1.3. Compound 24 demonstrated significant efficacy in treating both acute and chronic infections in mice including a complete cure in one animal. In addition, treatment with compound 24 also prolonged the survival of immunocompromised mice and a complete cure in some of the treated mice. This is a very exciting finding from this study as no other compounds previously tested have demonstrated such consistent efficacy in
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: 215-991-8135. Fax: 215848-2271. ORCID
Sandhya Kortagere: 0000-0002-3532-8669
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REFERENCES
(1) Rutaganira, F. U.; Barks, J.; Dhason, M. S.; Wang, Q.; Lopez, M. S.; Long, S.; Radke, J. B.; Jones, N. G.; Maddirala, A. R.; Janetka, J. W.; El Bakkouri, M.; Hui, R.; Shokat, K. M.; Sibley, L. D. Inhibition of calcium dependent protein kinase 1 (CDPK1) by pyrazolopyrimidine analogs decreases establishment and reoccurrence of central nervous system disease by Toxoplasma gondii. J. Med. Chem. 2017, DOI: 10.1021/acs.jmedchem.7b01192. (2) Hui, R.; El Bakkouri, M.; Sibley, L. D. Designing selective inhibitors for calcium-dependent protein kinases in apicomplexans. Trends Pharmacol. Sci. 2015, 36, 452−460. (3) Ojo, K. K.; Dangoudoubiyam, S.; Verma, S. K.; Scheele, S.; DeRocher, A. E.; Yeargan, M.; Choi, R.; Smith, T. R.; Rivas, K. L.; Hulverson, M. A.; Barrett, L. K.; Fan, E.; Maly, D. J.; Parsons, M.; Dubey, J. P.; Howe, D. K.; Van Voorhis, W. C. Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy. Int. J. Parasitol. 2016, 46, 871− 880. (4) Levinson, N. M.; Boxer, S. G. A conserved water-mediated hydrogen bond network defines bosutinib’s kinase selectivity. Nat. Chem. Biol. 2014, 10, 127−132.
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DOI: 10.1021/acs.jmedchem.7b01663 J. Med. Chem. 2017, 60, 9974−9975
