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Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice.

The apicomplexan parasite Neospora caninum is an important causative agent of congenital neosporosis, resulting in abortion, birth of weak offspring and neuromuscular disorders in cattle, sheep, and many other species. Among several compound classes that are currently being developed, two have been reported to limit the effects of congenital neosporosis: (i) bumped kinase inhibitors (BKIs) target calcium dependent protein kinase 1 (CDPK1), an enzyme that is encoded by an apicoplast-derived gene and found only in apicomplexans and plants. CDPK1 is essential for host cell invasion and egress; (ii) endochin-like quinolones (ELQs) are inhibitors of the cytochrome bc1 complex of the mitochondrial electron transport chain and thus inhibit oxidative phosphorylation. We here report on the in vitro and in vivo activities of BKI-1748, and of ELQ-316 and its respective prodrugs ELQ-334 and ELQ-422, applied either as single-compounds or ELQ-BKI-combinations. In vitro, BKI-1748 and ELQ-316, as well as BKI-1748 and ELQ-334, acted synergistically, while this was not observed for the BKI-1748/ELQ-422 combination treatment. In a N. caninum-infected pregnant BALB/c mouse model, the synergistic effects observed in vitro were not entirely reproduced, but 100% postnatal survival and 100% inhibition of vertical transmission was noted in the group treated with the BKI-1748/ELQ-334 combination. In addition, the combined drug applications resulted in lower neonatal mortality compared to treatments with single drugs., Graphical abstract Image 1, Highlights • BKI-1748 is a bumped kinase inhibitor with anti-Neospora activity in vitro and in vivo • Endochin-like quinolones target the cytochrome bc1 complex and exhibit anti-Neospora properties. • Single and combined applications of these two compound classes do not interfere in pregnancy outcome in mice • BKI-1748/ELQ-334 combination treatment results in 100% postnatal survival and blocks vertical transmission in the neosporosis mouse model

A series of pyrrolo[2,3-d]pyrimidines were designed in silico as potential bumped kinase inhibitors targeting P. falciparum calcium dependent protein kinase 4 (PfCDPK4), with the potential to inhibit PfCDPK1 based on earlier studies of the two kinases. A small series of these compounds were prepared and assessed for inhibitory activity against PfCDPK4 and PfCDPK1 in vitro. Four of the compounds displayed promising inhibitory activity against either PfCDPK4 (IC

A phenotypic screen of the ReFRAME compound library was performed to identify cell-active inhibitors that could be developed as therapeutics for giardiasis. A primary screen against Giardia lamblia GS clone H7 identified 85 cell-active compounds at a hit rate of 0.72%. A cytotoxicity counterscreen against HEK293T cells was carried out to assess hit compound selectivity for further prioritization. Mavelertinib (PF-06747775), a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), was identified as a potential new therapeutic based on indication, activity, and availability after reconfirmation. Mavelertinib has

Giardiasis is a neglected parasitic diarrheal disease that is particularly associated with poverty. Current treatment options are limited in the face of growing resistance, but the reduced kinome of Giardia lamblia increases the likelihood of identifying nonredundant essential kinases as potential drug targets. Repurposing known and newly identified kinase inhibitors in drug development programs for novel giardiasis therapeutics could therefore be a cost-effective and time saving approach. Innovative improvements to physiologically-based pharmacokinetic modeling coupled with emerging imaging technologies and a CRISPR-interference method could accelerate progress toward the goal of more effective giardiasis therapeutics based on kinase inhibition.

Neospora caninum is an apicomplexan parasite closely related to Toxoplasma gondii, and causes abortions, stillbirths and/or fetal malformations in livestock. Target-based drug development has led to the synthesis of calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs). Previous studies have shown that several BKIs have excellent efficacy against neosporosis in vitro and in vivo. However, several members of this class of compounds impair fertility in pregnant mouse models and cause embryonic malformation in a zebrafish (Danio rerio) model. Similar to the first-generation antiprotozoal drug quinine, some BKIs have a quinoline core structure. To identify common targets in both organisms, we performed differential affinity chromatography with cell-free extracts from N. caninum tachyzoites and D. rerio embryos using the 5-aminopyrazole-4-carboxamide (AC) compound BKI-1748 and quinine columns coupled to epoxy-activated sepharose followed by mass spectrometry. BKI-binding proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from BKI-1748 as well as quinine columns. In N. caninum, 12 proteins were bound specifically to BKI-1748 alone, and 105 proteins, including NcCDPK1, were bound to both BKI-1748 and quinine. For D. rerio, the corresponding numbers were 13 and 98 binding proteins, respectively. In both organisms, a majority of BKI-1748 binding proteins was involved in RNA binding and modification, in particular, splicing. Moreover, both datasets contained proteins involved in DNA binding or modification and key steps of intermediate metabolism. These results suggest that BKI-1748 interacts with not only specific targets in apicomplexans, such as CDPK1, but also with targets in other eukaryotes, which are involved in common, essential pathways.

Bumped kinase inhibitors (BKIs) are a new class of antiprotozoal drugs that target calcium-dependent protein kinase 1 (CDPK1) in various apicomplexan parasites. A multiple dose regimen of BKI 1369 has been shown to be highly effective against Cystoisospora suis (syn. Isospora suis), the causative agent of neonatal porcine coccidiosis. However, multiple dosing may not be widely applicable in the field. The present study aimed to determine the efficacy of reduced treatment frequencies with BKI 1369 against porcine cystoisosporosis in vitro and in vivo. Pre-incubation of sporozoites with BKI 1369 completely failed to inhibit the infection in vitro unless treatment was prolonged post-infection. Notably, a single treatment of infected cell cultures 2 days post-infection (dpi) resulted in a significant reduction of merozoite replication. In an experimental infection model, treatment of suckling piglets experimentally infected with C. suis 2 and 4 dpi with 20 mg BKI 1369/kg body weight completely suppressed oocyst excretion. A single treatment on the day of infection or 2 dpi suppressed oocyst excretion in 50% and 82% of the piglets and reduced the quantitative excretion in those that shed oocysts by 95.2% and 98.4%, respectively. Moreover, a significant increase in body weight gain and reduced number of diarrhea days were observed in BKI 1369 treated piglets compared to the control piglets, irrespective of time points and frequencies of treatment. Given that reduced treatment frequencies with BKI 1369 are comparable in efficacy to repeated applications without any adverse effects, this could be considered as a practical therapeutic alternative against porcine cystoisosporosis., Graphical abstract Image 1, Highlights • BKI 1369 does not target host cell invasion by C. suis sporozoites but replication of merozoites. • Single treatment with BKI 1369 in parallel with experimental infection is not effective for the control of cystoisosporosis. • Two doses of BKI 1369 at 2 and 4 dpi completely suppressed oocyst excretion in piglets experimentally infected with C. suis.

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co-infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng-Ct co-infections. Development of a safe, effective, and inexpensive dual therapy for Ng-Ct co-infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X-ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high-throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4− parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4− parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4− late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCE Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.

Fungal infections are the leading cause of mortality by eukaryotic pathogens, with an estimated 150 million severe life-threatening cases and 1.7 million deaths reported annually. The rapid emergence of multidrug-resistant fungal isolates highlights the urgent need for new drugs with new mechanisms of action. In fungi, pantothenate phosphorylation, catalyzed by PanK enzyme, is the first step in the utilization of pantothenic acid and coenzyme A biosynthesis. In all fungi sequenced so far, this enzyme is encoded by a single PanK gene. Here, we report the crystal structure of a fungal PanK alone as well as with high-affinity inhibitors from a single chemotype identified through a high-throughput chemical screen. Structural, biochemical, and functional analyses revealed mechanisms governing substrate and ligand binding, dimerization, and catalysis and helped identify new compounds that inhibit the growth of several Candida species. The data validate PanK as a promising target for antifungal drug development.

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium dependent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4− parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4− parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild type and Plasmodium falciparum cdpk4− late gametocyte stages, to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events such as DNA replication, mRNA translation and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation, and thereby is critical for parasite transmission to the mosquito vector.IMPORTANCETransmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.

New drugs are critically needed to treat Cryptosporidium infections, particularly for malnourished children under 2 years old in the developing world and persons with immunodeficiencies. Bioactive compounds from the Tres-Cantos GSK library that have activity against other pathogens were screened for possible repurposing against Cryptosporidium parvum growth. Nineteen compounds grouped into nine structural clusters were identified using an iterative process to remove excessively toxic compounds and screen related compounds from the Tres-Cantos GSK library. Representatives of four different clusters were advanced to a mouse model of C. parvum infection, but only one compound, an imidazole-pyrimidine, led to significant clearance of infection. This imidazole-pyrimidine compound had a number of favorable safety and pharmacokinetic properties and was maximally active in the mouse model down to 30 mg/kg given daily. Though the mechanism of action against C. parvum was not definitively established, this imidazole-pyrimidine compound inhibits the known C. parvum drug target, calcium-dependent protein kinase 1, with a 50% inhibitory concentration of 2 nM. This compound, and related imidazole-pyrimidine molecules, should be further examined as potential leads for Cryptosporidium therapeutics.

Cystoisosporosis is a leading diarrheal disease in suckling piglets. With the confirmation of resistance against the only available drug toltrazuril, there is a substantial need for novel therapeutics to combat the infection and its negative effects on animal health. In closely related apicomplexan species, bumped kinase inhibitors (BKIs) targeting calcium-dependent protein kinase 1 (CDPK1) were shown to be effective in inhibiting host-cell invasion and parasite growth. Therefore, the gene coding for Cystoisospora suis CDPK1 (CsCDPK1) was identified and cloned to investigate activity and thermal stabilization of the recombinant CsCDPK1 enzyme by BKI 1369. In this comprehensive study, the efficacy, safety and pharmacokinetics of BKI 1369 in piglets experimentally infected with Cystoisospora suis (toltrazuril-sensitive, Wien-I and toltrazuril-resistant, Holland-I strains) were determined in vivo and in vitro using an established animal infection model and cell culture, respectively. BKI 1369 inhibited merozoite proliferation in intestinal porcine epithelial cells-1 (IPEC-1) by at least 50% at a concentration of 40 nM, and proliferation was almost completely inhibited (>95%) at 200 nM. Nonetheless, exposure of infected cultures to 200 nM BKI 1369 for five days did not induce structural alterations in surviving merozoites as confirmed by transmission electron microscopy. Five-day treatment with BKI 1369 (10 mg/kg BW twice a day) effectively suppressed oocyst excretion and diarrhea and improved body weight gains in treated piglets without obvious side effects for both toltrazuril-sensitive, Wien-I and resistant, Holland-I C. suis strains. The plasma concentration of BKI 1369 in piglets increased to 11.7 μM during treatment, suggesting constant drug accumulation and exposure of parasites to the drug. Therefore, oral applications of BKI 1369 could potentially be a therapeutic alternative against porcine cystoisosporosis. For use in pigs, future studies on BKI 1369 should be directed towards ease of drug handling and minimizing treatment frequencies., Graphical abstract Image 1, Highlights • Oral application of BKI 1369 effectively reduced oocyst excretion and diarrhea in Cystoisospora suis infected piglets. • 200 nM of BKI 1369 almost completely suppressed parasite proliferation in vitro. • IC50 and IC95 concentrations of BKI 1369 did not induce morphological alterations in in vitro cultured merozoites. • Cystoisospora suis CDPK1, the putative target of BKI 1369, has glycine as gatekeeper residue.

Cryptosporidium is a leading cause of pediatric diarrhea worldwide. Currently there is neither a vaccine nor a consistently effective drug available for this disease. Selective 5-aminopyrazole-4-carboxamide-based bumped-kinase inhibitors (BKIs) are effective in both in vitro and in vivo models of Cryptosporidium parvum. Potential cardiotoxicity in some BKIs led to the continued exploration of the 5-aminopyrazole-4-carboxamide scaffold to find safe and effective drug candidates for Cryptosporidium. A series of newly designed BKIs were tested for efficacy against C. parvum using in vitro and in vivo (mouse infection model) assays, and safety issues. Compound 6 (BKI 1708) was found to be efficacious at 8 mg/kg dosed once daily (QD) for 5 days with no observable signs of toxicity up to 200 mg/kg dosed QD for 7 days. Compound 15 (BKI 1770) was found to be efficacious at 30 mg/kg dosed twice daily (BID) for 5 days with no observable signs of toxicity up to 300 mg/kg dosed QD for 7 days. Compounds 6 and 15 are promising pre-clinical leads for cryptosporidiosis therapy with acceptable safety parameters and efficacy in the mouse model of cryptosporidiosis.

Bumped kinase inhibitors (BKIs) have been shown to be potent inhibitors of Toxoplasma gondii calcium-dependent protein kinase 1. Pyrazolopyrimidine and 5-aminopyrazole-4-carboxamide scaffold-based BKIs are effective in acute and chronic experimental models of toxoplasmosis. Through further exploration of these 2 scaffolds and a new pyrrolopyrimidine scaffold, additional compounds have been identified that are extremely effective against acute experimental toxoplasmosis. The in vivo efficacy of these BKIs demonstrates that the cyclopropyloxynaphthyl, cyclopropyloxyquinoline, and 2-ethoxyquinolin-6-yl substituents are associated with efficacy across scaffolds. In addition, a broad range of plasma concentrations after oral dosing resulted from small structural changes to the BKIs. These select BKIs include anti-Toxoplasma compounds that are effective against acute experimental toxoplasmosis and are not toxic in human cell assays, nor to mice when administered for therapy. The BKIs described here are promising late leads for improving anti-Toxoplasma therapy.

Graphical abstract, Calcium ions are used as important signals during various physiological processes. In malaria parasites, Plasmodium spp., calcium dependent protein kinases (CDPKs) have acquired the unique ability to sense and transduce calcium signals at various critical steps during the lifecycle, either through phosphorylation of downstream substrates or mediating formation of high molecular weight protein complexes. Calcium signaling cascades establish important crosstalk events with signaling pathways mediated by other secondary messengers such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). CDPKs play critical roles at various important physiological steps during parasite development in vertebrates and mosquitoes. They are also important for transmission of the parasite between the two hosts. Combined with the fact that CDPKs are not present in humans, they continue to be pursued as important targets for development of anti-malarial drugs.

New drugs are critically needed to treat

Bumped kinase inhibitors (BKIs) that target Cryptosporidium parvum calcium-dependent protein kinase 1 have been well established as potential drug candidates against cryptosporidiosis. Recently, BKI-1649, with a 7H-pyrrolo[2,3-d]pyrimidin-4-amine, or "pyrrolopyrimidine", central scaffold, has shown improved efficacy in mouse models of Cryptosporidium at substantially reduced doses compared to previously explored analogs of the pyrazolopyrimidine scaffold. Here, two pyrrolopyrimidines with varied substituent groups, BKI-1812 and BKI-1814, were explored in several in vitro and in vivo models and show improvements in potency over the previously utilized pyrazolopyrimidine bumped kinase inhibitors while maintaining equivalent results in other key properties, such as toxicity and efficacy, with their pyrazolopyrimidine isosteric counterparts.

This is a review of the development of bumped-kinase inhibitors (BKIs) for the therapy of One Health parasitic apicomplexan diseases. Many apicomplexan infections are shared between humans and livestock, such as cryptosporidiosis and toxoplasmosis, as well as livestock only diseases such as neosporosis. We have demonstrated proof-of-concept for BKI therapy in livestock models of cryptosporidiosis (newborn calves infected with Cryptosporidium parvum), toxoplasmosis (pregnant sheep infected with Toxoplasma gondii), and neosporosis (pregnant sheep infected with Neospora caninum). We discuss the potential uses of BKIs for the treatment of diseases caused by apicomplexan parasites in animals and humans, and the improvements that need to be made to further develop BKIs.

Bumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice, were assessed in pregnant mice. Drugs were emulsified in corn oil and applied by gavage for 5 days. Five BKIs did not affect pregnancy, 5 BKIs exhibited 15-35% of neonatal mortality, and 5 compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish (Danio rerio) embryo development was assessed by exposing freshly fertilized eggs to 0.2-50μM of BKIs and microscopical monitoring of embryo development in a blinded manner during 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows to calculate an impact score (Si) that indicates at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for 9 compounds no clear correlation between Si and pregnancy outcome was visible. However, those 3 BKIs affecting zebrafish embryos only at high concentrations (40μM or higher) did not impair mouse pregnancy at all, and those 3 compounds that inhibited zebrafish embryo development already at 0.2μM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has a limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude, that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects.

Transmission of human malaria parasites (Plasmodium spp.) by Anopheles mosquitoes is a continuous process that presents a formidable challenge for effective control of the disease. Infectious gametocytes continue to circulate in humans for up to four weeks after antimalarial drug treatment, permitting prolonged transmission to mosquitoes even after clinical cure. Almost all reported malaria cases are transmitted to humans by mosquitoes, and therefore decreasing the rate of Plasmodium transmission from humans to mosquitoes with novel transmission-blocking remedies would be an important complement to other interventions in reducing malaria incidence.

Bumped Kinase Inhibitors (BKIs), targeting Calcium-dependent Protein Kinase 1 (CDPK1) in apicomplexan parasites with a glycine gatekeeper, are promising new therapeutics for apicomplexan diseases. Here we will review advances, as well as challenges and lessons learned regarding efficacy, safety, and pharmacology, that have shaped our selection of pre-clinical candidates.

13 páginas, 2 tablas, 7 figuras., Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 mu M, and of 1 mu M in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after two applications, resulted in a 37-50% reduction in foetal mortality, and modulated immune responses; IFN. levels were increased early after infection and IgG levels were reduced subsequently. N. caninum was abundantly found in placental tissues; however, parasite detection in foetal brain tissue decreased from 94% in the infected/untreated group to 69-71% in the treated groups. In summary, BKI-1553 confers partial protection against abortion in a ruminant experimental model of N. caninum infection during pregnancy. In addition, reduced parasite detection, parasite load and lesions in foetal brains were observed., We gratefully acknowledge Jacobo Sampere from the SALUVETgroup (Complutense University of Madrid, Spain), Teresa Navarro andJosé María González (University of Zaragoza, Spain), Isabel Ayala,Lucas Troya, Clara Colmenero, Dolores González, Natalia Peral,Verónica Pérez, Ignacio Gómez and Isabel Santiago from the Clinical Veterinary Hospital (Complutense University of Madrid, Spain) for their excellent technical assistance, and also Rama Subba Rao Vidadala and Dustin Maly (Department of Chemistry, University of Washington) fortheir assistance in further purification of the study drug. The AnimalExperimentation Service (SEA) at the University of Zaragoza is ac-knowledged for providing their facilities to carry out the reproduction programme. Roberto Sánchez Sánchez is supported by a fellowship from the Spanish Ministry of Education, Culture and Sports (MECD), asa part of the Program of Training of University Teaching Staff (FPU,grant number FPU13/03438). Patricia Vázquez has a Juan de la Cierva-Formación post-doctoral contract (FJCI-2014-20982) from the Spanish Ministry of Economy and Competitiveness (MINECO). Andrew Hemphill is supported by the Swiss National Science Foundation grant(No. 310030 165782). This work was supported by the Public Health Service, National Institutes of Health, Bethesda, MD (grants R01 AI111341 and R01 HD 080670), the U.S. Department of Agriculture(grant 2014-67015-22106) and the Community of Madrid, Spain(PLATESA, S2013/ABI2906).

Selective inhibitors of Cryptosporidium calcium-dependent protein kinase 1 ( CpCDPK1) based on the 1 H-pyrazolo[3,4- d]pyrimidin-4-amine (pyrazolopyrimidine, PP) scaffold are effective in both in vitro and in vivo models of cryptosporidiosis. However, the search for distinct safety and pharmacokinetic (PK) properties has motivated our exploration of alternative scaffolds. Here, we describe a series of 7 H-pyrrolo[2,3- d]pyrimidin-4-amine (pyrrolopyrimidine, PrP)-based analogs of PP CpCDPK1 inhibitors. Most of the PrP-based inhibitors described potently inhibit the CpCDPK1 enzyme, demonstrate no toxicity against mammalian cells, and block proliferation of the C. parvum parasite in the low micromolar range. Interestingly, certain substituents that show reduced CpCDPK1 potency when displayed from a PP scaffold provided notably enhanced efficacy in the context of a PrP scaffold. PK studies on these paired compounds show that some PrP analogs have distinct physiochemical properties compared with their PP counterparts. These results demonstrate that inhibitors based on a PrP scaffold are distinct therapeutic alternatives to previously developed PP inhibitors.

Introduction Theileria equi, an etiologic agent of equine piroplasmosis, is a tick-transmitted hemoprotozoan of the phylum Apicomplexa. Recent outbreaks of piroplasmosis in the United States have renewed interest in safe and effective treatment options. Although imidocarb dipropionate (IMD) is the drug of choice for clearance of T. equi, adverse reactions and recently documented resistance support the need for alternative therapeutic strategies. The recently described bumped kinase inhibitors (BKIs) are a new class of compounds that could potentially be used as safe and effective alternatives to IMD. In an initial effort to evaluate this potential, herein we determined the T. equi growth inhibitory activity of 11 BKIs relative to that of IMD and the previously tested BKI 1294. Because some BKIs have known human ether-a-go-go related gene (hERG) channel activity, we also assessed the hERG activity of each compound with the goal to identify those with the highest potency against T. equi coupled with the lowest potential for cardiotoxicity. Results Six BKIs inhibited T. equi growth in vitro, including the previously evaluated BKI 1294 which was used as a positive control. All six compounds were significantly less potent (higher 50% effective concentration (EC50)) than IMD. Two of those compounds were more potent than BKI 1294 control but had similar hERG activity. Although the remaining three compounds had similar to lower potency than BKI 1294, hERG EC50 was higher for three of them (BKI 1735, BKI 1369 and BKI 1318). Conclusions The BKI compounds evaluated in this study inhibited T. equi in vitro and had diverse hERG activity. Based on these considerations, three compounds would be suitable for further evaluation. While these results provide a foundation for future work, in vivo pharmacokinetic, pharmacodynamics, and safety studies are needed before BKI compounds can be recommended for clinical use in T. equi infected horses.

Glycogen synthase kinase 3 has evolutionarily conserved roles in cell signaling and metabolism and is a recognized drug target in neurological pathologies, most prominently bipolar disorder. More recently it has been suggested that GSK3 may be a target for the treatment of trypanosomatid parasite infections, e.g. with T. brucei, due to the lethal phenotype observed in parasite GSK3 short RNAi knockdown experiments. Here we investigated the kinome selectivity of a library of pyrrolo[3,4-c]pyrazol inhibitors that were developed against T. brucei GSK3 but that also interact with the human orthologue and other protein kinases. We applied label-free MS-based kinome chemoproteomics profiling with kinobeads to obtain the selectivity profiles of all 39 library members against 217 human protein and lipid kinases. This allowed us to study the structure-activity relationship of the library members as well as the chemical genetic relationships between kinase targets. As a result, we identified a novel and highly selective HsGSK3 inhibitor containing a 2-chloroaniline-substituted squaric acid amide pharmacophore that confers low nanomolar (IC50 = 2.8 nM) and sub-micromolar potency against purified and cellular HsGSK3. The inhibitor will be useful as a new lead for GSK3 inhibitor development and as a chemical genetic probe to study roles of GSK3 in cell signaling.

Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans.

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.

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.